[med-svn] r87 - in trunk/packages: . perlprimer perlprimer/branches
perlprimer/branches/upstream perlprimer/branches/upstream/current
perlprimer/branches/upstream/current/tutorial_files
Charles Plessy
charles-guest at costa.debian.org
Mon Aug 7 13:27:12 UTC 2006
Author: charles-guest
Date: 2006-08-07 13:20:50 +0000 (Mon, 07 Aug 2006)
New Revision: 87
Added:
trunk/packages/perlprimer/
trunk/packages/perlprimer/branches/
trunk/packages/perlprimer/branches/upstream/
trunk/packages/perlprimer/branches/upstream/current/
trunk/packages/perlprimer/branches/upstream/current/Changelog
trunk/packages/perlprimer/branches/upstream/current/ReadMe.txt
trunk/packages/perlprimer/branches/upstream/current/copying
trunk/packages/perlprimer/branches/upstream/current/gcg.603
trunk/packages/perlprimer/branches/upstream/current/perlprimer.pl
trunk/packages/perlprimer/branches/upstream/current/todo
trunk/packages/perlprimer/branches/upstream/current/tutorial.html
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/egfp_c1_mcs.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/egfp_n2_mcs.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/ensembl_20.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img1.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img10.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img2.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img3.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img4.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img5.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img6.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img7.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img8.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/img9.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/magnify.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/open_dna_20.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/preferences_20.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/report_20.png
trunk/packages/perlprimer/branches/upstream/current/tutorial_files/sflogo.png
trunk/packages/perlprimer/tags/
Log:
[svn-inject] Installing original source of perlprimer
Added: trunk/packages/perlprimer/branches/upstream/current/Changelog
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/Changelog 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/Changelog 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,306 @@
+22/6/06 (PerlPrimer-1.1.13)
+- Ensembl compatibility restored again (back to searchview)
+- Added code to check for the presence of Genomic and mRNA seqs before running spidey (error message that resulted otherwise was confusing to users)
+
+5/6/06 (PerlPrimer-1.1.12)
+- Retrieval of un-annotated genes from Ensembl (using the Ensembl Gene ID) is now possible
+- BLAST search results compatibility restored
+- ORF calculation re-written to allow for transciption initiation from TTG, GTG and CTG codons, in the absence of an initiating ATG
+
+31/5/06 (PerlPrimer-1.1.11)
+- Ensembl compatibility restored yet again (Ensembl now apparently back to using a modified text view rather than searchview)
+- Better error notification if the Ensembl format changes yet again
+- Fixed a small bug related to finding the GCG restriction enzyme file in the user's home directory
+
+20/3/06 (PerlPrimer-1.1.10)
+- Ensembl compatibility yet again restored
+- Button in Ensembl transcripts dialogue to allow user to open a browser
+window to view the different transcripts (on the Ensembl gene web page)
+- Option in Preferences->Network to specify web browser to use (may not work
+under Win32 - seems to use default system browser regardless)
+- List of Ensembl genome databases updated
+- Translation from forward primer to ORF start is now shown in a lighter
+colour
+- Active buttons in dialogues are now invoked by pressing <Return>
+- Update to checkbutton referencing code to support later versions of Perl/Tk
+under *nix
+- Slight cosmetic changes to GUI colours
+
+8/9/05 (PerlPrimer-1.1.9)
+- Ensembl compatibility restored once again
+- Slight change to Spidey error dialogue to reflect the possibility that the error might be Ensembl-related
+
+9/8/05 (PerlPrimer-1.1.8)
+- Ensembl compatibility is restored, and now allows the user to select the desired transcripts from genes
+- View intron/exon structure now provides a indication of sequence length
+- Minor code tidying
+
+30/05/05 (PerlPrimer-1.1.7)
+- Added "Span Intron/Exon Boundary" option for QPCR - this is selected by default and was the previous default behaviour; however, it is now possible to design primers for sequences without introns (e.g. 18S)
+- Added GC% information for Primer information tab, and adjusted the layout
+- Ensembl genome databases updated (added Canis_familiaris, Gallus_gallus, Pan_troglodytes, Saccharomyces_cerevisiae, Tetraodon_nigroviridis, Xenopus_tropicalis; removed Caenorhabditis_briggsae)
+- Retieve gene from Ensembl function now always prompts user to select from the list of matching gene names, even if only one name matches - this ensures that the correct gene has been identified (Ensembl occasionally has some unexpected single matches to queries)
+- Some Ensembl search matches were not detected correctly due to a slight change in the HTML output from Ensembl - this is now adjusted for
+- Added word-size option to blast searching - BLAST now uses the more correct word size of 7 bases by default
+- Blast search now uses real clock measurments to show elapsed time, rather than just calculating the number of times the routine had been called
+- Default interface font on Windows is now Arial instead of MS Sans Serif - using a TrueType font such as Arial allows nice sub-pixel rendering on LCD displays
+- Updated Rebase restriction enzyme file to gcg.505
+- BUGFIX: Sequence right-click menu works again
+- BUGFIX: Save FASTA dialogue now works correctly
+- BUGFIX: DNA graphic and primers are now resized when window is resized
+
+01/04/05 (PerlPrimer-1.1.6)
+- BUGFIX: Problems in Tm and dG calculation that prevented program startup when [dNTPs] > [Mg++] - quick fix provided to prevent this by keeping the value of sqrt([Mg++] - [dNTPs]) as 0. A warning prompt is displayed if this situation is set in the preferences
+- BUGFIX: Oligo concentration in the preferences was incorrectly described as being in mM when it is in fact calculated as nM
+- Acknowledgment of Rebase files provided in the acknowledgements dialogue
+- Tutorial html file from http://perlprimer.sf.net added to distribution package
+
+21/12/04 (PerlPrimer-1.1.5)
+- Old preferences are automatically copied to the new home directory location when upgrading
+- Win32 executable built with ActivePerl 5.8.6 and PAR 0.86
+
+20/12/04
+- PerlPrimer now translates in frame from the forward primer if this starts upstream of the ORF (allows easy identification of stop codons when designing cloning primers)
+- Better handling of information from FASTA files - FASTA header now automatically becomes the file name
+- Several minor bug fixes and code-tidying
+
+03/11/04
+- Home and temporary directory fixes - directories are now set from Perl environmental variables if possible; tmp directory set to home if user does not have correct read/write permissions or if directory does not exist
+- Full primer-dimer values are calculated as well as extensible primer-dimers, and results are sorted based on both values
+
+01/09/04
+- BLAST search can now use a local blast database and server
+- "View intron/exon structure" button in QPCR tab graphically draws the exon/intron structure of the genomic DNA (thus showing the size of the introns)
+- New browse directory code (requires a recent version of Perl/Tk to run correctly)
+- BUGFIX: Saving .ppr files did not work correctly under some Perl distributions
+- Complete re-write of the internal GUI code (now made more extensible and more comprehensible)
+
+13/07/04 (PerlPrimer-1.1.4a)
+- BUGFIX: "Open Sequence" buttons did not work correctly when opening sequences in FASTA format (or genomic DNA sequence for QPCR tab)
+- BUGFIX: Bisulphite PCR tab did not display primer information in the status bar when dimer dG == 0
+- BUGFIX: Finding sequencing primers without a dG cutoff did not calculate dG for primers
+- Minor GUI changes for Win32 to work with ActivePerl v5.8.4 (some cosmetic Perl/Tk changes)
+
+21/06/04 (PerlPrimer-1.1.4)
+- View spidey output command now runs spidey directly
+- Win32::FileOp code disabled - causes redraw problems with Perl/Tk
+- Minor code tidying
+
+17/06/04
+- BUGFIX: Fixed a bug added in the last version that did not reverse the 3' restriction enzyme site and anchor when adding cloning sequences
+
+28/04/04
+- Work begun on unequal loops primer-dimer routines (not fully implemented as yet)
+
+22/04/04
+- Code to support Win32::FileOp if it exists under windows for selecting directories, as DirTree seems flakey at best with some extremely eratic behaviour. This is not currently an ActivePerl module, so will only be relevant for the Windows installer version.
+
+21/04/04 (PerlPrimer-1.1.3a)
+- Windows installer version only
+- PerlPrimer now searches for Spidey in the PerlPrimer directory on startup
+- Minor code tidying
+
+19/04/04 (PerlPrimer-1.1.3)
+- Trial self-executable format for Windows (warning: >3 Mb download) created using PAR (http://par.perl.org)
+- Save DNA sequence dialogue now has options for both .fasta and .txt formats (default is .fasta)
+- Saving DNA sequence in FASTA format now prompts the user for a description line (default is the open file name, if any)
+- Better processing of FASTA files when opening (whitespace and some illegal characters are removed from the description line when taking the file name)
+
+18/04/04
+- Buttons to choose home, tmp, and spidey-location directories in the Preferences dialogue
+- Rearranged Preferences dialogue: Separate sections for directories and behaviour when opening new files in the "General" tab; defer-to-capitalised-regions option is now in the "Bioinformatics" tab
+
+15/04/04
+- Checkbox now present to control whether QPCR primers need to span an intron/exon boundary or not (old method from 02/04/04 still works as well)
+- When QPCR data are retrieved from Ensembl, Spidey is run automatically and intron/exon boundaries are shown on the DNA graphic
+- QPCR exon limits can now be selected with the mouse on the DNA graphic - use the left button to select the 5' range and the middle button (or Ctrl-Button 1) to select the 3' range (i.e. just like setting the range for other PCR methods)
+- Species name is now added to the default file name when a sequence is retrieved from Ensembl
+- Path to system temporary directory can be set in the Preferences
+- BUGFIX: Generate report did not save a report if the perlprimer file had not been saved
+- BUGFIX: PerlPrimer now handles degenerate sequences better (including gapped sequences) when finding ORFs
+- BUGFIX: If default values were 0 they were not restored to default when opening a new file
+- Code tidying and new balloon help messages
+
+14/04/04
+- QPCR can now select primers targeting a range of exons or a single exon (suggested by Alf Eaton)
+- BUGFIX: temporary dna and mrna files for Spidey are now saved in the system tmp directory, rather than the directory where Spidey is located (suggested by Alf Eaton)
+
+02/04/04
+- QPCR can now design primers that surround an intron/exon boundary but do not span the boundary (delete the value in the "Minimum Intron/Exon boundary" box for this to work (now deprecated - see above)) (suggested by John Luckey)
+
+01/04/04
+- Changes to dialogue box code
+- Minor code tidying
+
+31/03/04
+- Preferences option to automatically find primers when receiving data from external applications through the tcp socket
+- Opening a new file when previous data exists now prompts the user to save (may be overridden in the preferences)
+- Opening a file now explicitly clears all data before opening
+
+30/03/04
+- TCP socket reading added for interaction between PerlPrimer and Contig Viewer
+
+29/03/04 (PerlPrimer-1.1.2)
+- BUGFIX: After finding sequencing primers, primer-dimer finding routine was not reset to only search for extensible dimers
+- Minor changes to DNA sequence opening routines to make use of the improved FASTA header functions
+
+24/03/04
+- BUGFIX: a simple bug in the CpG island detection algorithm prevented small islands from being reported
+- BUGFIX: PerlPrimer under MacOS X now correctly detects it is running under a unix environment
+- Better routines for finding the Spidey executable on all platforms (perlprimer now uses a *pidey.* glob and picks the largest file from all matches, in case the archive file is present in the same directory)
+- Perlprimer now screens each primer for degenerate/non-DNA symbols and will not calculate Tm if these are present
+
+22/03/04
+- All fonts (main gui font, primer list font and fixed font for sequences) can now be specified in the preferences (suggested by Alf Eaton)
+
+16/03/04 (PerlPrimer-1.1.1)
+- Added the .fasta file type to the Open File dialogue box
+
+15/03/04
+- New feature: PerlPrimer can save current parameters as defaults for each PCR tab (Use the Tools menu option "Save default values for this page"). Values are saved to files in the user's home directory, ".perlprimer.[name_of_page]". (Use the Tools menu option "Restore in-built default values" to delete this file)
+
+11/03/04
+- New feature: PerlPrimer can now open FASTA files with range information included in the FASTA header - syntax for the first line is ">Name of DNA sequence 5prime_region[?-?] 3prime_region[?-?] page[?]" where the page argument is optional (defaults to the standard PCR page) and where page 1 = standard PCR, page 2 = bisulphite PCR, etc. Suggested by Alexander Kozik, for use by Contig Viewer (http://www.atgc.org/Py_ContigViewer/)
+
+09/03/04 (PerlPrimer-1.1)
+- Minor code tidying
+
+02/03/04
+- New icons for opening and saving DNA sequences
+- BUGFIX: a workaround for a very weird Perl/Tk bug - if the Preferences dialogue was opened and closed twice, PerlPrimer stopped doing floating point arithmetic (?!).
+
+27/02/04
+- If multiple genes match the expression used to search the ensembl database, PerlPrimer now displays the complete list and allows the user to choose any of the descriptions (or to cancel and refine the search)
+- Primer-dimer routines revised again - previous method was not considering that initial/terminal mismatches will not contribute to dimer instability and thus should not be included when calculating dG values
+- New feature: Users can select the temperature at which to calculate primer-dimer dG in the preferences
+- Default MRU file list is now 8 files
+
+23/02/04
+- New feature: Users can disable repeat/run checking in the Preferences window
+- Completed abstraction of the add_cloning subroutine
+- BUGFIX: Preference writing routine now saves the MRU file list when closing preferences dialogue (previously the MRU file list was lost if the preferences were changed and then PerlPrimer was killed)
+- Several minor bug fixes
+- General code tidying
+
+18/02/04
+- New feature: "Find primers for cloning" menu option (in the tools menu) - only available in standard PCR mode, reduces the 5' outer range by 10 bp increments and the 3' inner range by 10 bp increments until primers are found
+- Renamed "Auto +", "Auto -" buttons to "Find outwards" and "Find inwards"
+
+05/02/04
+- New feature: "Add cloning sequences" dialogue. PerlPrimer now uses the GCG restriction enzyme database file from REBASE to list the non-cutting restriction enzymes within a sequence when in Standard PCR mode. To use, select "Add cloning sequences" from the Tools menu. (note: the file 'gcg.###' - included with the distribution - will need to be installed in either your home directory or in the same directory as PerlPrimer for this to work)
+- Minor bug fixes
+
+02/02/04 (PerlPrimer-1.0.2)
+- Added a Recently Opened Files menu (number of files is customisable through the preferences)
+- Added bindings for left and right arrow keys when focussed in the primer lists - allows the user to quickly switch to the primer-dimer tab (with the right arrow key) and back again (with the left arrow key). Note that up and down arrow keys will still move through the primer list as before
+- PerlPrimer now deletes the dna and mrna temporary files required by Spidey after use
+- The %GC above and below which to exclude primers automatically can now be set in the preferences (before it was between 40% and 60%)
+- Several cosmetic changes:
+- look-and-feel of menubar and toolbar is now OS dependent
+- fonts are consistent across all widgets (exception under windows is the HList font - set to be verdana at one point less than the user assigned font size)
+- most fonts may be overridden by system defaults if desired
+- the notebook widget on Win32 now has shaded inactive tabs
+- minor interface tidying
+- If sequencing primers cannot be found within the specified read length between primers, a dialogue is now displayed and the run is aborted (previously, the program would find the next best primer, which could be at a distance much greater than that specified)
+- Slight modifications to the preference reading/writing routines to allow array data to be saved (needed for the recently used files list)
+- Open and Save file functions now list the percentage opened/saved on the status line
+- The dG hash for calculating primer-dimer stability is now calculated from the current salt conditions and the enthalpy and entropy hashes - meaning that primer-dimer calculations are much more accurate. (note that if changing salt concentrations in the preferences, OK needs to be pressed before the hash is recalculated)
+- Reworked menus in the new (and more correct) Perl/Tk format
+- Re-ordering of File menu items
+- "Sort primers" popup menu is now a sub-menu
+- Minor bugfixes and code tidying
+
+23/1/04 (PerlPrimer-1.0.1)
+- PerlPrimer now uses eval routines to check for all perl modules, and either exits with a useful message or disables some functions if a module is not found.
+- Several minor bugs fixed/general code tidying.
+
+- NB: Previous public release (1.0) had an older copy of perlprimer (Beta 5) by mistake - please upgrade if using that release as several important bugs have been fixed.
+
+18/12/03 (PerlPrimer-1.0)
+- BUGFIX: Generate Report (and copy printable) now includes the last line of sequence
+- Minor code tidying
+
+16/12/03 (PerlPrimer-1.0_Beta_6)
+- Rehashed primer-dimer routine (again!) - routine now only searches for *extensible* 3' primer-dimers when searching for primer pairs, and pd_full now searches for all other primer-dimers (3' and 5'). This equates to approximately a 100% increase in speed when searching for primers, and provides more useful information to the end user.
+- BUGFIX: Generate Report now provides the correct information for amplicon size and primer positions.
+- Partially abstracted the added-cloning-sequence routines to a subroutine (only implemented for the dna_magnify routine as yet)
+
+12/12/03
+- BUGFIX: removed a few extraneous bitwise ands that were lying around (caused problems with some Perl distributions) - suggested by Chris Vega
+- BUGFIX: made all Tk calls use a dash for each option key (caused problems on some systems) - suggested by Chris Vega
+
+11/12/03 (PerlPrimer-1.0_Beta_5)
+
+10/12/03
+- BLAST search now has a (semi-fake) elapsed time display while searching (counts 15 seconds per search interval, which should be approximately correct!)
+- BUGFIX: Many minor bugs squashed, including several problems that occured when range values were defined but 0
+- Open sequence will accept and work with multi-lined entries (preventing slowdowns, although ORF finding will still convert to a single lined entry - this needs to be changed)
+- Many other minor changes/tidying
+- Balloon help texts updated for the new options (sequencing page, pref options, etc)
+
+
+07/12/03
+- New feature: Sequencing page (finds primers spaced at a user-defined interval along a sequence)
+- Gui packing system greatly reworked, now uses far less reliance on the grid geometry manager and less frames to achieve layout. A new subroutine, nr() (=newrow) is used to create new frame rows, and all widgets are packed into the last row created from the left - simpler and easier. Should also be slight faster when redrawing on old systems.
+- BUGFIX: QPCR tab was not assigned a "File not saved" description, thus not displaying the open file name correctly.
+- Reworked the primer windowing subroutines to now use a two dimensional array to store primer details (slightly faster and much neater)
+- New feature: PerlPrimer no longer defers to capitalised regions by default but uses the ORF/CpG finding algorithms instead (less confusing and more useful) Defer to capitalised regions is now a preferences option.
+- New feature: PerlPrimer no longer emulates cpgplot behaviour by default, but uses the "correct" algorithm instead. Emulate cpgplot behaviour is now a preferences option.
+- Dialogues are now unique (for non-static dialogues the old is destroyed before the new one is created);
+- Main window now has a minimum size (= the default packing size when the program is started) to prevent widgets disappearing when the window is resized too small.
+- General code tidying and far too many small changes to list independantly.
+
+27/11/03 (perlprimer-1.0_Beta_4)
+- Redesigned Tm and Primer Size widgets, and amplicon size widgets in QPCR tab
+
+26/11/03
+- File saving reworked: PerlPrimer now has standard "Save"/"Save as" functionality and behaves as expected
+- Title of program now displays the name of the file being worked on, even if multiple files are open at the same time
+- Default extention of saved sequences and reports is now .txt
+
+25/11/03
+- New feature: proxy server support (enter in preferences) - will still use env_proxy if box unchecked
+- File open/save now allows multiple line DNA sequences - prevents slowdown for large genomic sequences used for QPCR
+- Default exclude_ie value is now 7 (was 5, which generates far too many primers for most genes and is not as specific)
+- BUGFIX: intron-exon boundary checking was not quite right with exclude_ie - this is now fixed.
+
+24/11/03 (perlprimer-1.0_Beta_3)
+- New feature: retrieve genes from Ensembl - which also will automatically retrieve genomic and cdna sequence for QPCR
+- New QPCR option: minimum intron/exon boundary - sets minimum overlap of primers across intron/exon boundary
+- Minor code tidying/fixes
+
+20/11/03 (perlprimer-1.0_Beta_2)
+- BUGFIX: fixed infinite loop that occurred when no primers were found using the "Auto +" button
+- pp_open_file routine now checks that a PerlPrimer file has in fact been selected and prompts if not
+- "Generate Report" now includes Amplicon size and primer position information
+- Minor code tidying
+
+13/11/03 (perlprimer-1.0_Beta_1)
+- Preferences dialogue now in NoteBook format
+- Preference options for BLAST searching (and associated modification of BLAST subroutine)
+- "Generate Report" now includes modified primers, file name and date
+- pack_gui subroutine now supports BrowseEntry widgets (needed for BLAST preferences)
+- Minor code tidying
+
+11/11/03 (perlprimer-0.99.95)
+- BUGFIX: primer Tm was not calculated for lower-case primers
+- get_tm subroutine is now specific for a single primer if only one is entered
+- Minor code changes/tidying
+
+28/10/03 (perlprimer-0.99.9)
+- "New File" feature added (see new_file subroutine)
+- Changes to dialogue subroutine to allow different dialogue types
+- New button-packing code for toolbars
+- Code tidying (especially GUI packing routines)/bug fixes
+
+26/10/03
+- "Generate Report" feature added
+- Toolbar added
+- Magnify button created next to canvas display
+- "New File" feature added to menus, but not yet implemented
+- Several icons added
+- General code tidying
+- "Reset" buttons removed - functionality taken over by "Set from ..." buttons
+
+24/10/03
+- Initial public release (perlprimer-0.99.8)
Added: trunk/packages/perlprimer/branches/upstream/current/ReadMe.txt
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/ReadMe.txt 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/ReadMe.txt 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,91 @@
+PerlPrimer
+----------
+
+Copyright © 2003-2006, Owen Marshall (owenjm at users.sourceforge.net)
+
+
+Contents
+--------
+
+1. Introduction
+2. Installation / Using the program
+3. Links to external programs
+4. Suggestions/patches/bugs/support
+
+
+1. Introduction
+---------------
+
+Perlprimer is a GUI application that designs primers for standard PCR, bisulphite PCR, Real-time PCR (QPCR) and sequencing.
+
+Perlprimer's current features include the following:
+
+* Calculation of possible primer-dimers
+* Retrieval of genomic or cdna sequences from Ensembl (including both sequences automatically for QPCR)
+* Ability to BLAST search primers using either the NCBI server or a local server
+* Results can be saved or optionally exported in a tab-delimited format that is compatible with most spreadsheet applications.
+* ORF and CpG island detection algorithms
+* Ability to add cloning sequences to primers, automatically adjusted to be in-frame
+* QPCR primer design without manual intron-exon boundary entry
+
+Perlprimer calculates primer melting temperature using J. SantaLucia's extensive nearest-neighbour thermodynamic parameters. To adjust for the salt conditions of the PCR, PerlPrimer uses the empirical formula derived by von Ahsen, et al. (2001) and allows the user to specify the concentration of Mg2+, dNTPs and primers, or use default, standard, PCR conditions. The result is a highly accurate prediction of primer melting temperature, giving rise to a maximum yeild of product when amplified.
+
+Perlprimer is written in Perl and requires Perl/Tk. In addition, for QPCR functionality perlprimer requires the open-source Spidey executable from NCBI. The program is designed to be cross-platform and has been tested on both Microsoft Windows and GNU/Linux-based operating systems. Users have also had success using the program under Mac OS X.
+
+Restriction enzyme data is provided by the REBASE project (http://rebase.neb.com/)
+
+
+2. Installation / Using the program
+-----------------------------------
+
+PerlPrimer requires Perl and Perl/Tk. These should be provided by your distribution if using a UNIX-based operating system; if using Windows I recommend using ActiveState's distribution of Perl 5.8, which includes Perl/Tk, freely available from
+
+ http://www.activestate.com/Products/ActivePerl/
+
+After installing ActivePerl under Windows, the file "perlprimer.pl" should run if double-clicked.
+
+PerlPrimer can also be started from the commandline, and this would be the normal mode of operation for UNIX-based OSes (including Mac OS X):
+
+ $ perl perlprimer.pl [file to open]
+
+(the file to open parameter is optional)
+
+
+Users under UNIX-based OSes may need to install Perl/Tk manually if it is not included in your distribution. Perl/Tk can be downloaded from
+
+ http://search.cpan.org/~ni-s/Tk/
+
+and can be installed by following the instructions included in the archive. Users may also need the Perl modules HTTP::Request and LWP::UserAgent for BLAST searching and gene retieval from Ensembl; these are part of libwww-perl-5.76 which can be found at
+
+ http://search.cpan.org/~gaas/libwww-perl-5.76/
+
+In addition to installing Perl/Tk (and libwww-perl if required), Mac OS X users will generally require OS 10.3 (Panther) or later and an X-server. (Please note that as I do not have access to a Mac OS X system I cannot guarantee compatibility, although I will try to fix any issues that are reported).
+
+
+Using PerlPrimer should be fairly self-explanatory, with extensive "balloon help" (turned on by default) and a separate help window detailing the operation of the graphical display of the DNA sequence, selection ranges and primers. A tutorial is provided with this distribution, and is also available at
+
+ http://perlprimer.sourceforge.net/tutorial.html
+
+which covers the most commonly used features of the program.
+
+
+3. Links to external programs
+-----------------------------
+
+For Real-time PCR (QPCR) functionality, PerlPrimer uses the program Spidey, released freely by the NCBI, to calculate intron/exon boundaries. This may be obtained from
+
+ http://www.ncbi.nlm.nih.gov/spidey/
+
+The program by default expects the spidey executable to be in your home directory on UNIX-based systems, or at C:\Spidey.exe for Windows. The location may be changed in the preferences window.
+
+
+4. Suggestions/patches/bugs/support
+------------------------------------
+
+The latest release of PerlPrimer can be obtained from
+
+ http://perlprimer.sourceforge.net/
+
+Please check the lastest version before reporting bugs.
+
+Bugs, feature suggestions and support requests can be placed on the web forums provided at the above address, or alternatively by emailing me directly.
Added: trunk/packages/perlprimer/branches/upstream/current/copying
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/copying 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/copying 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,281 @@
+ GNU GENERAL PUBLIC LICENSE
+ Version 2, June 1991
+
+ Copyright (C) 1989, 1991 Free Software Foundation, Inc.
+ 59 Temple Place, Suite 330, Boston, MA 02111-1307 USA
+ Everyone is permitted to copy and distribute verbatim copies
+ of this license document, but changing it is not allowed.
+
+ Preamble
+
+ The licenses for most software are designed to take away your
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+License is intended to guarantee your freedom to share and change free
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+Foundation's software and to any other program whose authors commit to
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+ When we speak of free software, we are referring to freedom, not
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+ 8. If the distribution and/or use of the Program is restricted in
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+ NO WARRANTY
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+ 11. BECAUSE THE PROGRAM IS LICENSED FREE OF CHARGE, THERE IS NO WARRANTY
+FOR THE PROGRAM, TO THE EXTENT PERMITTED BY APPLICABLE LAW. EXCEPT WHEN
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+PROGRAMS), EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE
+POSSIBILITY OF SUCH DAMAGES.
+
+ END OF TERMS AND CONDITIONS
+
Added: trunk/packages/perlprimer/branches/upstream/current/gcg.603
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/gcg.603 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/gcg.603 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,746 @@
+
+REBASE version 603 gcg.603
+
+ =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
+ REBASE, The Restriction Enzyme Database http://rebase.neb.com
+ Copyright (c) Dr. Richard J. Roberts, 2006. All rights reserved.
+ =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
+
+Rich Roberts Mar 01 2006
+
+
+REBASE codes for commercial sources of enzymes
+
+ A GE Healthcare (8/05)
+ B Invitrogen Corporation (8/05)
+ C Minotech Biotechnology (9/05)
+ E Stratagene (9/05)
+ F Fermentas International Inc. (2/06)
+ G Qbiogene (9/05)
+ H American Allied Biochemical, Inc. (9/05)
+ I SibEnzyme Ltd. (2/06)
+ J Nippon Gene Co., Ltd. (8/05)
+ K Takara Bio Inc. (9/05)
+ M Roche Applied Science (8/05)
+ N New England Biolabs (2/06)
+ O Toyobo Biochemicals (9/05)
+ Q Molecular Biology Resources (8/05)
+ R Promega Corporation (9/05)
+ S Sigma Chemical Corporation (9/05)
+ U Bangalore Genei (9/05)
+ V Vivantis Technologies (1/06)
+ X EURx Ltd. (9/05)
+ Y CinnaGen Inc. (9/05)
+
+..
+AarI 11 CACCTGCnnnn'nnnn_ 4 ! >F
+;AasI 7 GACnn_nn'nnGTC -2 ! DrdI,DseDI >F
+;AatI 3 AGG'CCT 0 ! StuI,Eco147I,PceI,SseBI >O
+AatII 5 G_ACGT'C -4 ! ZraI >AFGIKMNORV
+AccI 2 GT'mk_AC 2 ! FblI,XmiI >ABGJKMNORSU
+;AccII 2 CG'CG 0 ! Bsh1236I,BstFNI,BstUI,MvnI >AJK
+;AccIII 1 T'CCGG_A 4 ! Aor13HI,BlfI,BseAI,Bsp13I,BspEI,Kpn2I,MroI >GJKR
+;Acc16I 3 TGC'GCA 0 ! AviII,FspI,NsbI >IV
+;Acc36I 10 ACCTGCnnnn'nnnn_ 4 ! BspMI,BfuAI,BveI >I
+Acc65I 1 G'GTAC_C 4 ! KpnI,Asp718I >FGINRV
+;AccB1I 1 G'GyrC_C 4 ! BanI,BshNI,BspT107I >IV
+;AccB7I 7 CCAn_nnn'nTGG -3 ! PflMI,Van91I >IRV
+;AccBSI 3 CCG'CTC 0 ! BsrBI,MbiI >IV
+;AceIII 13 CAGCTCnnnnnnn'nnnn_ 4 ! >
+AciI 1 C'CG_C 2 ! BspACI,SsiI >N
+AclI 2 AA'CG_TT 2 ! Psp1406I >INV
+;AclWI 9 GGATCnnnn'n_ 1 ! AlwI,BspPI >I
+AcoI 1 y'CCGG_r 4 ! >I
+;AcsI 1 r'AATT_y 4 ! ApoI,XapI >IMV
+AcuI 22 CTGAAGnnnnnnnnnnnnnn_nn' -2 ! Eco57I >IN
+;AcvI 3 CAC'GTG 0 ! PmaCI,BbrPI,Eco72I,PmlI,PspCI >QX
+;AcyI 2 Gr'CG_yC 2 ! BsaHI,BssNI,BstACI,Hin1I,Hsp92I >JM
+;AdeI 6 CAC_nnn'GTG -3 ! DraIII >F
+;AfaI 2 GT'AC 0 ! RsaI,Csp6I >AK
+AfeI 3 AGC'GCT 0 ! Eco47III,Aor51HI >IN
+;AfiI 7 CCnn_nnn'nnGG -3 ! BsiYI,Bsc4I,BseLI,BslI >V
+AflII 1 C'TTAA_G 4 ! BfrI,BspTI,Bst98I,MspCI,Vha464I >AJKNO
+AflIII 1 A'CryG_T 4 ! >GMNS
+AgeI 1 A'CCGG_T 4 ! AsiGI,BshTI,CspAI,PinAI >GJNR
+;AhaIII 3 TTT'AAA 0 ! DraI >
+AhdI 6 GACnn_n'nnGTC -1 ! Eam1105I,AspEI,BmeRI,DriI,EclHKI >GN
+;AhlI 1 A'CTAG_T 4 ! SpeI,BcuI >IV
+;AjiI 3 CAC'GTC 0 ! BtrI,BmgBI >F
+;AjnI 0 'CCwGG_ 5 ! EcoRII,BptI,BseBI,BstNI,BstOI,Bst2UI,MvaI,Psp6I,PspGI >I
+AjuI 25 GAAnnnnnnnTTGGnnnnnn_nnnnn' -5 ! >F
+AjuI 26 CCAAnnnnnnnTTCnnnnnnn_nnnnn' -5 ! >F
+AleI 5 CACnn'nnGTG 0 ! OliI >N
+AlfI 24 GCAnnnnnnTGCnnnnnnnnnn_nn' -2 ! >F
+AloI 25 GAACnnnnnnTCCnnnnnnn_nnnnn' -5 ! >F
+AloI 25 GGAnnnnnnGTTCnnnnnnn_nnnnn' -5 ! >F
+AluI 2 AG'CT 0 ! >ABCFGHIJKMNOQRSUVXY
+AlwI 9 GGATCnnnn'n_ 1 ! AclWI,BspPI >N
+;Alw21I 5 G_wGCw'C -4 ! Bbv12I,BsiHKAI >F
+;Alw26I 6 GTCTCn'nnnn_ 4 ! BsmAI,BstMAI >FR
+;Alw44I 1 G'TGCA_C 4 ! ApaLI,VneI >FJMORS
+;AlwFI 6 GAAAynnnnnrTG 0 ? ! >
+AlwNI 6 CAG_nnn'CTG -3 ! CaiI >N
+;Ama87I 1 C'yCGr_G 4 ! AvaI,BmeT110I,BsiHKCI,BsoBI,Eco88I >IV
+;Aor13HI 1 T'CCGG_A 4 ! AccIII,BlfI,BseAI,Bsp13I,BspEI,Kpn2I,MroI >K
+;Aor51HI 3 AGC'GCT 0 ! Eco47III,AfeI >AK
+ApaI 5 G_GGCC'C -4 ! Bsp120I,PspOMI >ABFGIJKMNOQRSUVX
+;ApaBI 8 GCA_nnnnn'TGC -5 ! BstAPI >
+ApaLI 1 G'TGCA_C 4 ! Alw44I,VneI >AKNU
+;ApeKI 1 G'CwG_C 3 ! TseI >N
+ApoI 1 r'AATT_y 4 ! AcsI,XapI >N
+AscI 2 GG'CGCG_CC 4 ! PalAI,SgsI >GN
+AseI 2 AT'TA_AT 2 ! VspI,PshBI >JNO
+;AsiGI 1 A'CCGG_T 4 ! AgeI,BshTI,CspAI,PinAI >IV
+AsiSI 5 GCG_AT'CGC -2 ! SgfI,RgaI >N
+;AspI 4 GACn'n_nGTC 1 ! Tth111I,PflFI,PsyI >M
+;Asp700I 5 GAAnn'nnTTC 0 ! XmnI,MroXI,PdmI >M
+;Asp718I 1 G'GTAC_C 4 ! KpnI,Acc65I >M
+;AspA2I 1 C'CTAG_G 4 ! AvrII,BlnI,XmaJI >IV
+;AspCNI 2 GCCGC 0 ? ! >
+;AspEI 6 GACnn_n'nnGTC -1 ! Eam1105I,AhdI,BmeRI,DriI,EclHKI >M
+;AspLEI 3 G_CG'C -2 ! HhaI,BstHHI,CfoI,Hin6I,HinP1I,HspAI >IV
+;AspS9I 1 G'GnC_C 3 ! BmgT120I,Cfr13I,Sau96I >IV
+;AssI 3 AGT'ACT 0 ! ScaI,BmcAI,ZrmI >U
+;AsuI 1 G'GnC_C 3 ! AspS9I,BmgT120I,Cfr13I,Sau96I >
+;AsuII 2 TT'CG_AA 2 ! Bpu14I,Bsp119I,BspT104I,BstBI,Csp45I,NspV,SfuI >C
+;AsuC2I 2 CC's_GG 1 ! BcnI,BpuMI,NciI >I
+;AsuHPI 13 GGTGAnnnnnnn_n' -1 ! HphI >IV
+;AsuNHI 1 G'CTAG_C 4 ! NheI,BmtI >IV
+AvaI 1 C'yCGr_G 4 ! Ama87I,BmeT110I,BsiHKCI,BsoBI,Eco88I >ABGJKMNORSUX
+AvaII 1 G'GwC_C 3 ! Bme18I,Eco47I,SinI,VpaK11BI >AGJKMNRSY
+;AvaIII 3 ATGCAT 0 ? ! EcoT22I,Mph1103I,NsiI,Zsp2I >
+;AviII 3 TGC'GCA 0 ! Acc16I,FspI,NsbI >M
+AvrII 1 C'CTAG_G 4 ! AspA2I,BlnI,XmaJI >N
+;AxyI 2 CC'TnA_GG 3 ! Bse21I,Bsu36I,Eco81I >J
+BaeI 23 ACnnnnGTAyCnnnnnnn_nnnnn' -5 ! >N
+BaeI 26 GrTACnnnnGTnnnnnnnnnn_nnnnn' -5 ! >N
+;BalI 3 TGG'CCA 0 ! MlsI,MluNI,MscI,Msp20I >AJKR
+BamHI 1 G'GATC_C 4 ! >ABCFGHIJKMNOQRSUVXY
+BanI 1 G'GyrC_C 4 ! AccB1I,BshNI,BspT107I >NORU
+BanII 5 G_rGCy'C -4 ! Eco24I,EcoT38I,FriOI >AGKMNOQRSX
+;BanIII 2 AT'CG_AT 2 ! ClaI,Bsa29I,BseCI,BshVI,BspDI,BspXI,Bsu15I,BsuTUI >O
+;BauI 1 C'ACGA_G 4 ! BssSI,Bst2BI >F
+BbeI 5 G_GCGC'C -4 ! NarI,DinI,EgeI,EheI,KasI,Mly113I,SfoI >AK
+;Bbr7I 13 GAAGACnnnnnnn'nnnn_ 4 ! BbsI,BpiI,BpuAI,BstV2I >
+;BbrPI 3 CAC'GTG 0 ! PmaCI,AcvI,Eco72I,PmlI,PspCI >MO
+BbsI 8 GAAGACnn'nnnn_ 4 ! BpiI,BpuAI,BstV2I >N
+;BbuI 5 G_CATG'C -4 ! SphI,PaeI >R
+BbvI 13 GCAGCnnnnnnnn'nnnn_ 4 ! BseXI,BstV1I >N
+;BbvII 8 GAAGACnn'nnnn_ 4 ! BbsI,BpiI,BpuAI,BstV2I >
+;Bbv12I 5 G_wGCw'C -4 ! Alw21I,BsiHKAI >IV
+BbvCI 2 CC'TCA_GC 3 ! >N
+BccI 9 CCATCnnnn'n_ 1 ! >N
+;Bce83I 22 CTTGAGnnnnnnnnnnnnnn_nn' -2 ! BpuEI >
+BceAI 17 ACGGCnnnnnnnnnnnn'nn_ 2 ! >N
+;BcefI 17 ACGGCnnnnnnnnnnnn'n_ 1 ! BceAI >
+BcgI 24 CGAnnnnnnTGCnnnnnnnnnn_nn' -2 ! >N
+BcgI 24 GCAnnnnnnTCGnnnnnnnnnn_nn' -2 ! >N
+BciVI 12 GTATCCnnnnn_n' -1 ! BfuI >N
+BclI 1 T'GATC_A 4 ! FbaI,Ksp22I >CFGJMNORSUY
+;BcnI 2 CC's_GG 1 ! AsuC2I,BpuMI,NciI >FK
+;BcuI 1 A'CTAG_T 4 ! SpeI,AhlI >F
+BdaI 24 TGAnnnnnnTCAnnnnnnnnnn_nn' -2 ! >F
+;BetI 1 w'CCGG_w 4 ! BsaWI >
+BfaI 1 C'TA_G 2 ! MaeI,FspBI,XspI >N
+;BfiI 11 ACTGGGnnnn_n' -1 ! BmrI,BmuI >F
+;BfmI 1 C'TryA_G 4 ! BpcI,BstSFI,SfcI >F
+;BfrI 1 C'TTAA_G 4 ! AflII,BspTI,Bst98I,MspCI,Vha464I >MO
+;BfuI 12 GTATCCnnnnn_n' -1 ! BciVI >F
+;BfuAI 10 ACCTGCnnnn'nnnn_ 4 ! BspMI,Acc36I,BveI >N
+;BfuCI 0 'GATC_ 4 ! MboI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >N
+BglI 7 GCCn_nnn'nGGC -3 ! >ACFGHIJKMNOQRSUVXY
+BglII 1 A'GATC_T 4 ! >ABCFGHIJKMNOQRSUVXY
+;BinI 9 GGATCnnnn'n_ 1 ! AclWI,AlwI,BspPI >
+BisI 2 GC'n_GC 1 ! >I
+;BlfI 1 T'CCGG_A 4 ! AccIII,Aor13HI,BseAI,Bsp13I,BspEI,Kpn2I,MroI >U
+;BlnI 1 C'CTAG_G 4 ! AvrII,AspA2I,XmaJI >AKMS
+BlpI 2 GC'TnA_GC 3 ! Bpu1102I,Bsp1720I,CelII >N
+;BmcAI 3 AGT'ACT 0 ! ScaI,AssI,ZrmI >V
+;Bme18I 1 G'GwC_C 3 ! AvaII,Eco47I,SinI,VpaK11BI >IV
+;Bme1390I 2 CC'n_GG 1 ! ScrFI,BmrFI,BssKI,BstSCI,MspR9I,StyD4I >F
+Bme1580I 5 G_kGCm'C -4 ! BseSI >N
+;BmeRI 6 GACnn_n'nnGTC -1 ! Eam1105I,AhdI,AspEI,DriI,EclHKI >V
+BmeT110I 2 Cy'CG_rG 2 ! AvaI,Ama87I,BsiHKCI,BsoBI,Eco88I >K
+;BmgI 3 GkGCCC 0 ? ! >
+BmgBI 3 CAC'GTC 0 ! BtrI,AjiI >N
+BmgT120I 2 GG'n_CC 1 ! AspS9I,Cfr13I,Sau96I >K
+;BmiI 3 GGn'nCC 0 ! NlaIV,BspLI,PspN4I >V
+BmrI 11 ACTGGGnnnn_n' -1 ! BfiI,BmuI >N
+;BmrFI 2 CC'n_GG 1 ! ScrFI,Bme1390I,BssKI,BstSCI,MspR9I,StyD4I >V
+BmtI 5 G_CTAG'C -4 ! NheI,AsuNHI >INV
+;BmuI 11 ACTGGGnnnn_n' -1 ! BfiI,BmrI >I
+;BoxI 5 GACnn'nnGTC 0 ! PshAI,BstPAI >F
+;BpcI 1 C'TryA_G 4 ! BfmI,BstSFI,SfcI >U
+;BpiI 8 GAAGACnn'nnnn_ 4 ! BbsI,BpuAI,BstV2I >F
+BplI 24 GAGnnnnnCTCnnnnnnnn_nnnnn' -5 ! >F
+BpmI 22 CTGGAGnnnnnnnnnnnnnn_nn' -2 ! GsuI >IN
+;BptI 2 CC'w_GG 1 ! EcoRII,AjnI,BseBI,BstNI,BstOI,Bst2UI,MvaI,Psp6I,PspGI >U
+Bpu10I 2 CC'TnA_GC 3 ! >FINV
+;Bpu14I 2 TT'CG_AA 2 ! AsuII,Bsp119I,BspT104I,BstBI,Csp45I,NspV,SfuI >IV
+;Bpu1102I 2 GC'TnA_GC 3 ! BlpI,Bsp1720I,CelII >AFK
+;BpuAI 8 GAAGACnn'nnnn_ 4 ! BbsI,BpiI,BstV2I >M
+BpuEI 22 CTTGAGnnnnnnnnnnnnnn_nn' -2 ! >N
+;BpuMI 2 CC's_GG 1 ! AsuC2I,BcnI,NciI >V
+;BpvUI 4 CG_AT'CG -2 ! PvuI,MvrI,Ple19I >V
+BsaI 7 GGTCTCn'nnnn_ 4 ! Eco31I,Bso31I,BspTNI >N
+;Bsa29I 2 AT'CG_AT 2 ! ClaI,BanIII,BseCI,BshVI,BspDI,BspXI,Bsu15I,BsuTUI >I
+BsaAI 3 yAC'GTr 0 ! BstBAI,Ppu21I >N
+BsaBI 5 GATnn'nnATC 0 ! Bse8I,BseJI,MamI >N
+BsaHI 2 Gr'CG_yC 2 ! AcyI,BssNI,BstACI,Hin1I,Hsp92I >N
+BsaJI 1 C'CnnG_G 4 ! BseDI,BssECI >N
+;BsaMI 7 GAATG_Cn' -2 ! BsmI,Mva1269I,PctI >GR
+BsaWI 1 w'CCGG_w 4 ! >N
+BsaXI 21 ACnnnnnCTCCnnnnnnn_nnn' -3 ! >N
+BsaXI 23 GGAGnnnnnGTnnnnnnnnn_nnn' -3 ! >N
+;BsbI 3 CAACAC 0 ? ! >
+;Bsc4I 7 CCnn_nnn'nnGG -3 ! BsiYI,AfiI,BseLI,BslI >I
+;BscAI 9 GCATCnnnn'nn_ 2 ! SfaNI,LweI >
+;BscGI 2 CCCGT 0 ? ! >
+;Bse1I 6 ACTG_Gn' -2 ! BsrI,BseNI,BsrSI >IV
+;Bse8I 5 GATnn'nnATC 0 ! BsaBI,BseJI,MamI >IV
+;Bse21I 2 CC'TnA_GG 3 ! AxyI,Bsu36I,Eco81I >IV
+;Bse118I 1 r'CCGG_y 4 ! Cfr10I,BsrFI,BssAI >IV
+;BseAI 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,Bsp13I,BspEI,Kpn2I,MroI >CM
+;BseBI 2 CC'w_GG 1 ! EcoRII,AjnI,BptI,BstNI,BstOI,Bst2UI,MvaI,Psp6I,PspGI >C
+;BseCI 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BshVI,BspDI,BspXI,Bsu15I,BsuTUI >C
+;BseDI 1 C'CnnG_G 4 ! BsaJI,BssECI >F
+;Bse3DI 8 GCAATG_nn' -2 ! BsrDI,BseMI >IV
+;BseGI 7 GGATG_nn' -2 ! FokI,BstF5I,BtsCI >F
+;BseJI 5 GATnn'nnATC 0 ! BsaBI,Bse8I,MamI >F
+;BseLI 7 CCnn_nnn'nnGG -3 ! BsiYI,AfiI,Bsc4I,BslI >F
+;BseMI 8 GCAATG_nn' -2 ! BsrDI,Bse3DI >F
+BseMII 15 CTCAGnnnnnnnn_nn' -2 ! BspCNI >F
+;BseNI 6 ACTG_Gn' -2 ! BsrI,Bse1I,BsrSI >FG
+;BsePI 1 G'CGCG_C 4 ! BssHII,PauI >IV
+BseRI 16 GAGGAGnnnnnnnn_nn' -2 ! >N
+;BseSI 5 G_kGCm'C -4 ! Bme1580I >F
+;BseXI 13 GCAGCnnnnnnnn'nnnn_ 4 ! BbvI,BstV1I >F
+;BseX3I 1 C'GGCC_G 4 ! BstZI,EagI,EclXI,Eco52I >IV
+BseYI 1 C'CCAG_C 4 ! >N
+BsgI 22 GTGCAGnnnnnnnnnnnnnn_nn' -2 ! >N
+;Bsh1236I 2 CG'CG 0 ! AccII,BstFNI,BstUI,MvnI >F
+;Bsh1285I 4 CG_ry'CG -2 ! BsiEI,BstMCI >F
+;BshFI 2 GG'CC 0 ! HaeIII,BsnI,BspANI,BsuRI,PhoI >C
+;BshNI 1 G'GyrC_C 4 ! AccB1I,BanI,BspT107I >F
+;BshTI 1 A'CCGG_T 4 ! AgeI,AsiGI,CspAI,PinAI >F
+;BshVI 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BseCI,BspDI,BspXI,Bsu15I,BsuTUI >V
+;BsiI 1 C'ACGA_G 4 ! BauI,BssSI,Bst2BI >
+BsiEI 4 CG_ry'CG -2 ! Bsh1285I,BstMCI >N
+BsiHKAI 5 G_wGCw'C -4 ! Alw21I,Bbv12I >N
+;BsiHKCI 1 C'yCGr_G 4 ! AvaI,Ama87I,BmeT110I,BsoBI,Eco88I >QX
+;BsiSI 1 C'CG_G 2 ! HpaII,HapII,MspI >C
+BsiWI 1 C'GTAC_G 4 ! Pfl23II,PspLI >MNO
+;BsiYI 7 CCnn_nnn'nnGG -3 ! AfiI,Bsc4I,BseLI,BslI >M
+BslI 7 CCnn_nnn'nnGG -3 ! BsiYI,AfiI,Bsc4I,BseLI >GN
+;BslFI 15 GGGACnnnnnnnnnn'nnnn_ 4 ! BsmFI,FaqI >I
+BsmI 7 GAATG_Cn' -2 ! BsaMI,Mva1269I,PctI >JMNOS
+BsmAI 6 GTCTCn'nnnn_ 4 ! Alw26I,BstMAI >N
+BsmBI 7 CGTCTCn'nnnn_ 4 ! Esp3I >N
+BsmFI 15 GGGACnnnnnnnnnn'nnnn_ 4 ! BslFI,FaqI >N
+;BsnI 2 GG'CC 0 ! HaeIII,BshFI,BspANI,BsuRI,PhoI >V
+;Bso31I 7 GGTCTCn'nnnn_ 4 ! Eco31I,BsaI,BspTNI >IV
+;BsoBI 1 C'yCGr_G 4 ! AvaI,Ama87I,BmeT110I,BsiHKCI,Eco88I >N
+;Bsp13I 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,BseAI,BspEI,Kpn2I,MroI >IV
+;Bsp19I 1 C'CATG_G 4 ! NcoI >IV
+;Bsp24I 24 GACnnnnnnTGGnnnnnnn_nnnnn' -5 ! >
+;Bsp24I 25 CCAnnnnnnGTCnnnnnnnn_nnnnn' -5 ! >
+;Bsp68I 3 TCG'CGA 0 ! NruI,BtuMI >F
+;Bsp119I 2 TT'CG_AA 2 ! AsuII,Bpu14I,BspT104I,BstBI,Csp45I,NspV,SfuI >F
+;Bsp120I 1 G'GGCC_C 4 ! ApaI,PspOMI >FG
+;Bsp143I 0 'GATC_ 4 ! MboI,BfuCI,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >F
+;Bsp143II 5 r_GCGC'y -4 ! HaeII,BstH2I >F
+Bsp1286I 5 G_dGCh'C -4 ! SduI,MhlI >JKNR
+;Bsp1407I 1 T'GTAC_A 4 ! BsrGI,BstAUI,SspBI >FK
+;Bsp1720I 2 GC'TnA_GC 3 ! BlpI,Bpu1102I,CelII >IV
+;BspACI 1 C'CG_C 2 ! AciI,SsiI >I
+;BspANI 2 GG'CC 0 ! HaeIII,BshFI,BsnI,BsuRI,PhoI >X
+BspCNI 14 CTCAGnnnnnnn_nn' -2 ! BseMII >N
+;BspDI 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BseCI,BshVI,BspXI,Bsu15I,BsuTUI >N
+BspEI 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,BseAI,Bsp13I,Kpn2I,MroI >N
+;BspGI 3 CTGGAC 0 ? ! >
+BspHI 1 T'CATG_A 4 ! PagI,RcaI >N
+;BspLI 3 GGn'nCC 0 ! NlaIV,BmiI,PspN4I >F
+;BspLU11I 1 A'CATG_T 4 ! PciI,PscI >M
+BspMI 10 ACCTGCnnnn'nnnn_ 4 ! Acc36I,BfuAI,BveI >N
+;BspMII 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,BseAI,Bsp13I,BspEI,Kpn2I,MroI >
+;BspMAI 5 C_TGCA'G -4 ! PstI >X
+;BspNCI 2 CCAGA 0 ? ! >
+;BspPI 9 GGATCnnnn'n_ 1 ! AclWI,AlwI >F
+;BspTI 1 C'TTAA_G 4 ! AflII,BfrI,Bst98I,MspCI,Vha464I >F
+;BspT104I 2 TT'CG_AA 2 ! AsuII,Bpu14I,Bsp119I,BstBI,Csp45I,NspV,SfuI >K
+;BspT107I 1 G'GyrC_C 4 ! AccB1I,BanI,BshNI >K
+;BspTNI 7 GGTCTCn'nnnn_ 4 ! Eco31I,BsaI,Bso31I >X
+;BspXI 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BseCI,BshVI,BspDI,Bsu15I,BsuTUI >G
+BsrI 6 ACTG_Gn' -2 ! Bse1I,BseNI,BsrSI >N
+BsrBI 3 CCG'CTC 0 ! AccBSI,MbiI >N
+BsrDI 8 GCAATG_nn' -2 ! Bse3DI,BseMI >N
+BsrFI 1 r'CCGG_y 4 ! Cfr10I,Bse118I,BssAI >N
+BsrGI 1 T'GTAC_A 4 ! Bsp1407I,BstAUI,SspBI >N
+;BsrSI 6 ACTG_Gn' -2 ! BsrI,Bse1I,BseNI >R
+;BssAI 1 r'CCGG_y 4 ! Cfr10I,Bse118I,BsrFI >C
+;BssECI 1 C'CnnG_G 4 ! BsaJI,BseDI >I
+BssHII 1 G'CGCG_C 4 ! BsePI,PauI >AJKMNOQRSX
+;BssKI 0 'CCnGG_ 5 ! ScrFI,Bme1390I,BmrFI,BstSCI,MspR9I,StyD4I >N
+;BssMI 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BstKTI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >V
+;BssNI 2 Gr'CG_yC 2 ! AcyI,BsaHI,BstACI,Hin1I,Hsp92I >V
+;BssNAI 3 GTA'TAC 0 ! Bst1107I,BstZ17I >IV
+BssSI 1 C'ACGA_G 4 ! BauI,Bst2BI >N
+;BssT1I 1 C'CwwG_G 4 ! StyI,Eco130I,EcoT14I,ErhI >IV
+;Bst6I 7 CTCTTCn'nnn_ 3 ! Ksp632I,Eam1104I,EarI >IV
+;Bst98I 1 C'TTAA_G 4 ! AflII,BfrI,BspTI,MspCI,Vha464I >R
+;Bst1107I 3 GTA'TAC 0 ! BssNAI,BstZ17I >FKM
+;BstACI 2 Gr'CG_yC 2 ! AcyI,BsaHI,BssNI,Hin1I,Hsp92I >I
+BstAPI 7 GCAn_nnn'nTGC -3 ! >IN
+;BstAUI 1 T'GTAC_A 4 ! Bsp1407I,BsrGI,SspBI >IV
+BstBI 2 TT'CG_AA 2 ! AsuII,Bpu14I,Bsp119I,BspT104I,Csp45I,NspV,SfuI >N
+;Bst2BI 1 C'ACGA_G 4 ! BauI,BssSI >IV
+;BstBAI 3 yAC'GTr 0 ! BsaAI,Ppu21I >IV
+;Bst4CI 3 AC_n'GT -1 ! HpyCH4III,TaaI >IV
+;BstC8I 3 GCn'nGC 0 ! Cac8I >I
+;BstDEI 1 C'TnA_G 3 ! DdeI,HpyF3I >IV
+;BstDSI 1 C'CryG_G 4 ! BtgI >IV
+BstEII 1 G'GTnAC_C 5 ! BstPI,Eco91I,EcoO65I,PspEI >GHJMNORSU
+;BstENI 5 CCTnn'n_nnAGG 1 ! EcoNI,XagI >IV
+BstF5I 7 GGATG_nn' -2 ! FokI,BseGI,BtsCI >INV
+;BstFNI 2 CG'CG 0 ! AccII,Bsh1236I,BstUI,MvnI >IV
+;BstH2I 5 r_GCGC'y -4 ! HaeII,Bsp143II >IV
+;BstHHI 3 G_CG'C -2 ! HhaI,AspLEI,CfoI,Hin6I,HinP1I,HspAI >IV
+BstKTI 3 G_AT'C -2 ! MboI,BfuCI,Bsp143I,BssMI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >I
+;BstMAI 6 GTCTCn'nnnn_ 4 ! BsmAI,Alw26I >IV
+;BstMBI 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,DpnII,Kzo9I,NdeII,Sau3AI >IV
+;BstMCI 4 CG_ry'CG -2 ! Bsh1285I,BsiEI >IV
+;BstMWI 7 GCnn_nnn'nnGC -3 ! MwoI,HpyF10VI >I
+BstNI 2 CC'w_GG 1 ! EcoRII,AjnI,BptI,BseBI,BstOI,Bst2UI,MvaI,Psp6I,PspGI >N
+;BstNSI 5 r_CATG'y -4 ! NspI,XceI >IV
+;BstOI 2 CC'w_GG 1 ! EcoRII,AjnI,BptI,BseBI,BstNI,Bst2UI,MvaI,Psp6I,PspGI >R
+;BstPI 1 G'GTnAC_C 5 ! BstEII,Eco91I,EcoO65I,PspEI >K
+;BstPAI 5 GACnn'nnGTC 0 ! PshAI,BoxI >IV
+;BstSCI 0 'CCnGG_ 5 ! ScrFI,Bme1390I,BmrFI,BssKI,MspR9I,StyD4I >I
+;BstSFI 1 C'TryA_G 4 ! BfmI,BpcI,SfcI >I
+;BstSNI 3 TAC'GTA 0 ! SnaBI,Eco105I >IV
+BstUI 2 CG'CG 0 ! AccII,Bsh1236I,BstFNI,MvnI >N
+;Bst2UI 2 CC'w_GG 1 ! EcoRII,AjnI,BptI,BseBI,BstNI,BstOI,MvaI,Psp6I,PspGI >IV
+;BstV1I 13 GCAGCnnnnnnnn'nnnn_ 4 ! BbvI,BseXI >I
+;BstV2I 8 GAAGACnn'nnnn_ 4 ! BbsI,BpiI,BpuAI >IV
+BstXI 8 CCAn_nnnn'nTGG -4 ! >AFGHIJKMNOQRVX
+;BstX2I 1 r'GATC_y 4 ! XhoII,BstYI,MflI,PsuI >IV
+BstYI 1 r'GATC_y 4 ! XhoII,BstX2I,MflI,PsuI >N
+;BstZI 1 C'GGCC_G 4 ! BseX3I,EagI,EclXI,Eco52I >R
+BstZ17I 3 GTA'TAC 0 ! BssNAI,Bst1107I >N
+;Bsu15I 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BseCI,BshVI,BspDI,BspXI,BsuTUI >F
+Bsu36I 2 CC'TnA_GG 3 ! AxyI,Bse21I,Eco81I >NR
+;BsuRI 2 GG'CC 0 ! HaeIII,BshFI,BsnI,BspANI,PhoI >FI
+;BsuTUI 2 AT'CG_AT 2 ! ClaI,BanIII,Bsa29I,BseCI,BshVI,BspDI,BspXI,Bsu15I >X
+BtgI 1 C'CryG_G 4 ! BstDSI >N
+BtgZI 16 GCGATGnnnnnnnnnn'nnnn_ 4 ! >N
+;BthCI 4 G_CnG'C -3 ! Fnu4HI,Fsp4HI,ItaI,SatI >
+;BtrI 3 CAC'GTC 0 ! AjiI,BmgBI >IV
+BtsI 8 GCAGTG_nn' -2 ! >N
+;BtsCI 7 GGATG_nn' -2 ! FokI,BseGI,BstF5I >N
+;BtuMI 3 TCG'CGA 0 ! NruI,Bsp68I >V
+;BveI 10 ACCTGCnnnn'nnnn_ 4 ! BspMI,Acc36I,BfuAI >F
+Cac8I 3 GCn'nGC 0 ! BstC8I >N
+;CaiI 6 CAG_nnn'CTG -3 ! AlwNI >F
+;CauII 2 CC's_GG 1 ! AsuC2I,BcnI,BpuMI,NciI >
+;CciNI 2 GC'GGCC_GC 4 ! NotI >IV
+;CdiI 4 CATC'G 0 ! >
+;CelII 2 GC'TnA_GC 3 ! BlpI,Bpu1102I,Bsp1720I >M
+;CfoI 3 G_CG'C -2 ! HhaI,AspLEI,BstHHI,Hin6I,HinP1I,HspAI >GMRS
+;CfrI 1 y'GGCC_r 4 ! EaeI >F
+;Cfr9I 1 C'CCGG_G 4 ! SmaI,XmaI,XmaCI >FO
+;Cfr10I 1 r'CCGG_y 4 ! Bse118I,BsrFI,BssAI >FGKO
+;Cfr13I 1 G'GnC_C 3 ! AspS9I,BmgT120I,Sau96I >AFKO
+;Cfr42I 4 CC_GC'GG -2 ! SacII,KspI,Sfr303I,SgrBI,SstII >F
+;ChaI 4 _GATC' -4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >
+;CjeI 26 CCAnnnnnnGTnnnnnnnnn_nnnnnn' -6 ! >
+;CjeI 25 ACnnnnnnTGGnnnnnnnn_nnnnnn' -6 ! >
+;CjeNII 5 GAGnnnnnGT 0 ? ! >
+;CjePI 26 CCAnnnnnnnTCnnnnnnnn_nnnnnn' -6 ! >
+;CjePI 25 GAnnnnnnnTGGnnnnnnn_nnnnnn' -6 ! >
+;CjuI 5 CAynnnnnrTG 0 ? ! >
+;CjuII 5 CAynnnnnCTC 0 ? ! >
+ClaI 2 AT'CG_AT 2 ! BanIII,Bsa29I,BseCI,BshVI,BspDI,BspXI,Bsu15I,BsuTUI >ABHKMNRSU
+;CpoI 2 CG'GwC_CG 3 ! RsrII,CspI,Rsr2I >AFK
+;CseI 10 GACGCnnnnn'nnnnn_ 5 ! HgaI >F
+;CspI 2 CG'GwC_CG 3 ! RsrII,CpoI,Rsr2I >OR
+Csp6I 1 G'TA_C 2 ! RsaI,AfaI >F
+;Csp45I 2 TT'CG_AA 2 ! AsuII,Bpu14I,Bsp119I,BspT104I,BstBI,NspV,SfuI >OR
+;CspAI 1 A'CCGG_T 4 ! AgeI,AsiGI,BshTI,PinAI >C
+CspCI 24 CAAnnnnnGTGGnnnnnnnnnn_nn' -2 ! >N
+CspCI 25 CCACnnnnnTTGnnnnnnnnnnn_nn' -2 ! >N
+;CstMI 26 AAGGAGnnnnnnnnnnnnnnnnnn_nn' -2 ! >
+CviAII 1 C'AT_G 2 ! NlaIII,FaeI,FatI,Hin1II,Hsp92II >N
+CviJI 2 rG'Cy 0 ! >VX
+;CviRI 2 TG'CA 0 ! HpyCH4V >
+DdeI 1 C'TnA_G 3 ! BstDEI,HpyF3I >BGMNORS
+;DinI 3 GGC'GCC 0 ! NarI,BbeI,EgeI,EheI,KasI,Mly113I,SfoI >V
+DpnI 2 GA'TC 0 ! MalI >BEFGMNRS
+;DpnII 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,Kzo9I,NdeII,Sau3AI >N
+DraI 3 TTT'AAA 0 ! >ABFGIJKMNOQRSUVXY
+;DraII 2 rG'GnC_Cy 3 ! EcoO109I >GM
+DraIII 6 CAC_nnn'GTG -3 ! AdeI >GIMNV
+DrdI 7 GACnn_nn'nnGTC -2 ! AasI,DseDI >N
+;DrdII 3 GAACCA 0 ? ! >
+;DriI 6 GACnn_n'nnGTC -1 ! Eam1105I,AhdI,AspEI,BmeRI,EclHKI >I
+;DsaI 1 C'CryG_G 4 ! BstDSI,BtgI >
+;DseDI 7 GACnn_nn'nnGTC -2 ! DrdI,AasI >I
+EaeI 1 y'GGCC_r 4 ! CfrI >AKMN
+EagI 1 C'GGCC_G 4 ! BseX3I,BstZI,EclXI,Eco52I >GN
+;Eam1104I 7 CTCTTCn'nnn_ 3 ! Ksp632I,Bst6I,EarI >F
+;Eam1105I 6 GACnn_n'nnGTC -1 ! AhdI,AspEI,BmeRI,DriI,EclHKI >FK
+EarI 7 CTCTTCn'nnn_ 3 ! Ksp632I,Bst6I,Eam1104I >N
+EciI 17 GGCGGAnnnnnnnnn_nn' -2 ! >N
+;Ecl136II 3 GAG'CTC 0 ! SacI,EcoICRI,Psp124BI,SstI >F
+;EclHKI 6 GACnn_n'nnGTC -1 ! Eam1105I,AhdI,AspEI,BmeRI,DriI >R
+;EclXI 1 C'GGCC_G 4 ! BseX3I,BstZI,EagI,Eco52I >MS
+;Eco24I 5 G_rGCy'C -4 ! BanII,EcoT38I,FriOI >F
+;Eco31I 7 GGTCTCn'nnnn_ 4 ! BsaI,Bso31I,BspTNI >F
+;Eco32I 3 GAT'ATC 0 ! EcoRV >F
+;Eco47I 1 G'GwC_C 3 ! AvaII,Bme18I,SinI,VpaK11BI >FO
+;Eco47III 3 AGC'GCT 0 ! AfeI,Aor51HI >FGMOR
+;Eco52I 1 C'GGCC_G 4 ! BseX3I,BstZI,EagI,EclXI >FKO
+;Eco57I 22 CTGAAGnnnnnnnnnnnnnn_nn' -2 ! AcuI >F
+;Eco72I 3 CAC'GTG 0 ! PmaCI,AcvI,BbrPI,PmlI,PspCI >F
+;Eco81I 2 CC'TnA_GG 3 ! AxyI,Bse21I,Bsu36I >AFKO
+;Eco88I 1 C'yCGr_G 4 ! AvaI,Ama87I,BmeT110I,BsiHKCI,BsoBI >F
+;Eco91I 1 G'GTnAC_C 5 ! BstEII,BstPI,EcoO65I,PspEI >F
+;Eco105I 3 TAC'GTA 0 ! SnaBI,BstSNI >FO
+;Eco130I 1 C'CwwG_G 4 ! StyI,BssT1I,EcoT14I,ErhI >F
+;Eco147I 3 AGG'CCT 0 ! StuI,AatI,PceI,SseBI >F
+;EcoHI 0 'CCsGG_ 5 ! AsuC2I,BcnI,BpuMI,NciI >
+EcoICRI 3 GAG'CTC 0 ! SacI,Ecl136II,Psp124BI,SstI >IRV
+Eco57MI 22 CTGrAGnnnnnnnnnnnnnn_nn' -2 ! >F
+EcoNI 5 CCTnn'n_nnAGG 1 ! BstENI,XagI >N
+;EcoO65I 1 G'GTnAC_C 5 ! BstEII,BstPI,Eco91I,PspEI >K
+EcoO109I 2 rG'GnC_Cy 3 ! DraII >AFJKN
+EcoRI 1 G'AATT_C 4 ! >ABCFGHIJKMNOQRSUVXY
+;EcoRII 0 'CCwGG_ 5 ! AjnI,BptI,BseBI,BstNI,BstOI,Bst2UI,MvaI,Psp6I,PspGI >FJMOS
+EcoRV 3 GAT'ATC 0 ! Eco32I >ABCGHIJKMNOQRSUVXY
+;EcoT14I 1 C'CwwG_G 4 ! StyI,BssT1I,Eco130I,ErhI >K
+;EcoT22I 5 A_TGCA'T -4 ! Mph1103I,NsiI,Zsp2I >AKO
+;EcoT38I 5 G_rGCy'C -4 ! BanII,Eco24I,FriOI >J
+;EgeI 3 GGC'GCC 0 ! NarI,BbeI,DinI,EheI,KasI,Mly113I,SfoI >I
+;EheI 3 GGC'GCC 0 ! NarI,BbeI,DinI,EgeI,KasI,Mly113I,SfoI >FO
+;ErhI 1 C'CwwG_G 4 ! StyI,BssT1I,Eco130I,EcoT14I >IV
+;EsaBC3I 2 TC'GA 0 ! TaqI >
+;EspI 2 GC'TnA_GC 3 ! BlpI,Bpu1102I,Bsp1720I,CelII >
+;Esp3I 7 CGTCTCn'nnnn_ 4 ! BsmBI >F
+;FaeI 4 _CATG' -4 ! NlaIII,CviAII,FatI,Hin1II,Hsp92II >I
+FalI 24 AAGnnnnnCTTnnnnnnnn_nnnnn' -5 ! >I
+;FaqI 15 GGGACnnnnnnnnnn'nnnn_ 4 ! BslFI,BsmFI >F
+FatI 0 'CATG_ 4 ! NlaIII,CviAII,FaeI,Hin1II,Hsp92II >IN
+FauI 9 CCCGCnnnn'nn_ 2 ! SmuI >IN
+;FauNDI 2 CA'TA_TG 2 ! NdeI >IV
+;FbaI 1 T'GATC_A 4 ! BclI,Ksp22I >AK
+;FblI 2 GT'mk_AC 2 ! AccI,XmiI >IV
+;FinI 2 GGGAC 0 ? ! BslFI,BsmFI,FaqI >
+;FmuI 4 G_GnC'C -3 ! AspS9I,BmgT120I,Cfr13I,Sau96I >
+;FnuDII 2 CG'CG 0 ! AccII,Bsh1236I,BstFNI,BstUI,MvnI >
+Fnu4HI 2 GC'n_GC 1 ! Fsp4HI,ItaI,SatI >N
+FokI 14 GGATGnnnnnnnnn'nnnn_ 4 ! BseGI,BstF5I,BtsCI >AGIJKMNRV
+;FriOI 5 G_rGCy'C -4 ! BanII,Eco24I,EcoT38I >IV
+FseI 6 GG_CCGG'CC -4 ! >AKN
+FspI 3 TGC'GCA 0 ! Acc16I,AviII,NsbI >JNO
+FspAI 4 rTGC'GCAy 0 ! >F
+;FspBI 1 C'TA_G 2 ! MaeI,BfaI,XspI >F
+;Fsp4HI 2 GC'n_GC 1 ! Fnu4HI,ItaI,SatI >I
+;GdiII 1 C'GGCC_r 4 ! >
+;GsuI 22 CTGGAGnnnnnnnnnnnnnn_nn' -2 ! BpmI >F
+;HaeI 3 wGG'CCw 0 ! >
+HaeII 5 r_GCGC'y -4 ! Bsp143II,BstH2I >GJKMNORS
+HaeIII 2 GG'CC 0 ! BshFI,BsnI,BspANI,BsuRI,PhoI >ABGHIJKMNOQRSUXY
+;HaeIV 25 GAynnnnnrTCnnnnnnnnn_nnnnn' -5 ! >
+;HaeIV 24 GAynnnnnrTCnnnnnnn_nnnnnn' -6 ! >
+;HapII 1 C'CG_G 2 ! HpaII,BsiSI,MspI >AK
+HgaI 10 GACGCnnnnn'nnnnn_ 5 ! CseI >IN
+;HgiAI 5 G_wGCw'C -4 ! Alw21I,Bbv12I,BsiHKAI >
+;HgiCI 1 G'GyrC_C 4 ! AccB1I,BanI,BshNI,BspT107I >
+;HgiEII 6 ACCnnnnnnGGT 0 ? ! >
+;HgiJII 5 G_rGCy'C -4 ! BanII,Eco24I,EcoT38I,FriOI >
+HhaI 3 G_CG'C -2 ! AspLEI,BstHHI,CfoI,Hin6I,HinP1I,HspAI >ABFGJKNORUY
+;Hin1I 2 Gr'CG_yC 2 ! AcyI,BsaHI,BssNI,BstACI,Hsp92I >FKO
+;Hin1II 4 _CATG' -4 ! NlaIII,CviAII,FaeI,FatI,Hsp92II >F
+Hin4I 24 GAynnnnnvTCnnnnnnnn_nnnnn' -5 ! >F
+Hin4I 24 GAbnnnnnrTCnnnnnnnn_nnnnn' -5 ! >F
+;Hin4II 2 CCTTC 0 ? ! >
+;Hin6I 1 G'CG_C 2 ! HhaI,AspLEI,BstHHI,CfoI,HinP1I,HspAI >F
+HinP1I 1 G'CG_C 2 ! HhaI,AspLEI,BstHHI,CfoI,Hin6I,HspAI >N
+HincII 3 GTy'rAC 0 ! HindII >ABFGHJKNOQRUXY
+;HindII 3 GTy'rAC 0 ! HincII >IMSV
+HindIII 1 A'AGCT_T 4 ! >ABCFGHIJKMNOQRSUVXY
+HinfI 1 G'AnT_C 3 ! >ABCFGHIJKMNOQRUVXY
+HpaI 3 GTT'AAC 0 ! KspAI >ABCGHIJKMNOQRSUVX
+HpaII 1 C'CG_G 2 ! BsiSI,HapII,MspI >BFGIMNOQRSUVX
+HphI 13 GGTGAnnnnnnn_n' -1 ! AsuHPI >FN
+Hpy8I 3 GTn'nAC 0 ! >F
+Hpy99I 5 _CGwCG' -5 ! >N
+;Hpy178III 2 TC'nn_GA 2 ! Hpy188III >
+Hpy188I 3 TC_n'GA -1 ! >N
+Hpy188III 2 TC'nn_GA 2 ! >N
+;HpyAV 11 CCTTCnnnnn_n' -1 ! >
+HpyCH4III 3 AC_n'GT -1 ! Bst4CI,TaaI >N
+HpyCH4IV 1 A'CG_T 2 ! MaeII,TaiI >N
+HpyCH4V 2 TG'CA 0 ! >N
+;HpyF3I 1 C'TnA_G 3 ! DdeI,BstDEI >F
+;HpyF10VI 7 GCnn_nnn'nnGC -3 ! MwoI,BstMWI >F
+;Hsp92I 2 Gr'CG_yC 2 ! AcyI,BsaHI,BssNI,BstACI,Hin1I >R
+;Hsp92II 4 _CATG' -4 ! NlaIII,CviAII,FaeI,FatI,Hin1II >R
+;HspAI 1 G'CG_C 2 ! HhaI,AspLEI,BstHHI,CfoI,Hin6I,HinP1I >IV
+;ItaI 2 GC'n_GC 1 ! Fnu4HI,Fsp4HI,SatI >M
+KasI 1 G'GCGC_C 4 ! NarI,BbeI,DinI,EgeI,EheI,Mly113I,SfoI >N
+KpnI 5 G_GTAC'C -4 ! Acc65I,Asp718I >ABCFGHIJKMNOQRSUVXY
+;Kpn2I 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,BseAI,Bsp13I,BspEI,MroI >F
+;KspI 4 CC_GC'GG -2 ! SacII,Cfr42I,Sfr303I,SgrBI,SstII >MS
+;Ksp22I 1 T'GATC_A 4 ! BclI,FbaI >IV
+;Ksp632I 7 CTCTTCn'nnn_ 3 ! Bst6I,Eam1104I,EarI >M
+;KspAI 3 GTT'AAC 0 ! HpaI >F
+;Kzo9I 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,NdeII,Sau3AI >I
+;LguI 8 GCTCTTCn'nnn_ 3 ! SapI,PciSI >F
+;LpnI 3 rGC'GCy 0 ! HaeII,Bsp143II,BstH2I >
+;LweI 10 GCATCnnnnn'nnnn_ 4 ! SfaNI >F
+;MabI 1 A'CCwGG_T 5 ! SexAI >I
+;MaeI 1 C'TA_G 2 ! BfaI,FspBI,XspI >M
+;MaeII 1 A'CG_T 2 ! HpyCH4IV,TaiI >M
+MaeIII 0 'GTnAC_ 5 ! >M
+;MalI 2 GA'TC 0 ! DpnI >I
+;MamI 5 GATnn'nnATC 0 ! BsaBI,Bse8I,BseJI >M
+;MbiI 3 CCG'CTC 0 ! BsrBI,AccBSI >F
+MboI 0 'GATC_ 4 ! BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,NdeII,Sau3AI >ABCFGKNQRUXY
+MboII 13 GAAGAnnnnnnn_n' -1 ! >AFGIJKNOQRVX
+;McrI 4 CG_ry'CG -2 ! Bsh1285I,BsiEI,BstMCI >
+MfeI 1 C'AATT_G 4 ! MunI >N
+;MflI 1 r'GATC_y 4 ! XhoII,BstX2I,BstYI,PsuI >K
+;MhlI 5 G_dGCh'C -4 ! SduI,Bsp1286I >IV
+;MjaIV 3 GTnnAC 0 ? ! Hpy8I >
+;MlsI 3 TGG'CCA 0 ! BalI,MluNI,MscI,Msp20I >F
+MluI 1 A'CGCG_T 4 ! >ABFGHIJKMNOQRSUVX
+;MluNI 3 TGG'CCA 0 ! BalI,MlsI,MscI,Msp20I >MS
+MlyI 10 GAGTCnnnnn' 0 ! PleI,PpsI,SchI >N
+;Mly113I 2 GG'CG_CC 2 ! NarI,BbeI,DinI,EgeI,EheI,KasI,SfoI >I
+MmeI 26 TCCrACnnnnnnnnnnnnnnnnnn_nn' -2 ! >NX
+MnlI 11 CCTCnnnnnn_n' -1 ! >FGINQVX
+;Mph1103I 5 A_TGCA'T -4 ! EcoT22I,NsiI,Zsp2I >F
+;MroI 1 T'CCGG_A 4 ! AccIII,Aor13HI,BlfI,BseAI,Bsp13I,BspEI,Kpn2I >MO
+;MroNI 1 G'CCGG_C 4 ! NaeI,NgoMIV,PdiI >IV
+;MroXI 5 GAAnn'nnTTC 0 ! XmnI,Asp700I,PdmI >IV
+MscI 3 TGG'CCA 0 ! BalI,MlsI,MluNI,Msp20I >BNO
+MseI 1 T'TA_A 2 ! Tru1I,Tru9I >BN
+MslI 5 CAynn'nnrTG 0 ! SmiMI >N
+;MspI 1 C'CG_G 2 ! HpaII,BsiSI,HapII >AFGHIJKMNOQRSUVXY
+;Msp20I 3 TGG'CCA 0 ! BalI,MlsI,MluNI,MscI >IV
+MspA1I 3 CmG'CkG 0 ! >INRV
+;MspCI 1 C'TTAA_G 4 ! AflII,BfrI,BspTI,Bst98I,Vha464I >C
+;MspR9I 2 CC'n_GG 1 ! ScrFI,Bme1390I,BmrFI,BssKI,BstSCI,StyD4I >I
+;MssI 4 GTTT'AAAC 0 ! PmeI >F
+;MstI 3 TGC'GCA 0 ! Acc16I,AviII,FspI,NsbI >
+;MunI 1 C'AATT_G 4 ! MfeI >FKM
+;MvaI 2 CC'w_GG 1 ! EcoRII,AjnI,BptI,BseBI,BstNI,BstOI,Bst2UI,Psp6I,PspGI >AFGKMOS
+;Mva1269I 7 GAATG_Cn' -2 ! BsmI,BsaMI,PctI >F
+;MvnI 2 CG'CG 0 ! AccII,Bsh1236I,BstFNI,BstUI >M
+;MvrI 4 CG_AT'CG -2 ! PvuI,BpvUI,Ple19I >U
+MwoI 7 GCnn_nnn'nnGC -3 ! BstMWI,HpyF10VI >N
+NaeI 3 GCC'GGC 0 ! MroNI,NgoMIV,PdiI >ACKMNORU
+NarI 2 GG'CG_CC 2 ! BbeI,DinI,EgeI,EheI,KasI,Mly113I,SfoI >GJMNOQRUX
+NciI 2 CC's_GG 1 ! AsuC2I,BcnI,BpuMI >GJNORS
+NcoI 1 C'CATG_G 4 ! Bsp19I >ABCFGHJKMNOQRSUXY
+NdeI 2 CA'TA_TG 2 ! FauNDI >ABFGJKMNRSXY
+;NdeII 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,Sau3AI >GJMRS
+NgoMIV 1 G'CCGG_C 4 ! NaeI,MroNI,PdiI >NR
+NheI 1 G'CTAG_C 4 ! AsuNHI,BmtI >ABFGJKMNORSU
+NlaIII 4 _CATG' -4 ! CviAII,FaeI,FatI,Hin1II,Hsp92II >GN
+NlaIV 3 GGn'nCC 0 ! BmiI,BspLI,PspN4I >GN
+;Nli3877I 5 C_yCGr'G -4 ! AvaI,Ama87I,BmeT110I,BsiHKCI,BsoBI,Eco88I >
+;NmuCI 0 'GTsAC_ 5 ! Tsp45I >F
+NotI 2 GC'GGCC_GC 4 ! CciNI >ABCFGHJKMNOQRSUXY
+NruI 3 TCG'CGA 0 ! Bsp68I,BtuMI >ABCGIJKMNOQRSUX
+;NsbI 3 TGC'GCA 0 ! Acc16I,AviII,FspI >FK
+NsiI 5 A_TGCA'T -4 ! EcoT22I,Mph1103I,Zsp2I >BGHJMNRSU
+NspI 5 r_CATG'y -4 ! BstNSI,XceI >MN
+;NspV 2 TT'CG_AA 2 ! AsuII,Bpu14I,Bsp119I,BspT104I,BstBI,Csp45I,SfuI >JO
+;NspBII 3 CmG'CkG 0 ! MspA1I >
+;OliI 5 CACnn'nnGTG 0 ! AleI >F
+;PabI 3 G_TA'C -2 ! RsaI,AfaI,Csp6I >
+PacI 5 TTA_AT'TAA -2 ! >GNO
+;PaeI 5 G_CATG'C -4 ! SphI,BbuI >F
+;PaeR7I 1 C'TCGA_G 4 ! XhoI,Sfr274I,SlaI,StrI,TliI >N
+;PagI 1 T'CATG_A 4 ! BspHI,RcaI >F
+;PalAI 2 GG'CGCG_CC 4 ! AscI,SgsI >I
+PasI 2 CC'CwG_GG 3 ! >F
+;PauI 1 G'CGCG_C 4 ! BsePI,BssHII >F
+;PceI 3 AGG'CCT 0 ! StuI,AatI,Eco147I,SseBI >IV
+PciI 1 A'CATG_T 4 ! BspLU11I,PscI >IN
+;PciSI 8 GCTCTTCn'nnn_ 3 ! SapI,LguI >I
+;PctI 7 GAATG_Cn' -2 ! BsmI,BsaMI,Mva1269I >IV
+;PdiI 3 GCC'GGC 0 ! NaeI,MroNI,NgoMIV >F
+;PdmI 5 GAAnn'nnTTC 0 ! XmnI,Asp700I,MroXI >F
+;PfeI 1 G'AwT_C 3 ! TfiI >F
+;Pfl23II 1 C'GTAC_G 4 ! BsiWI,PspLI >F
+;Pfl1108I 3 TCGTAG 0 ? ! >
+;PflFI 4 GACn'n_nGTC 1 ! Tth111I,AspI,PsyI >N
+PflMI 7 CCAn_nnn'nTGG -3 ! AccB7I,Van91I >N
+PfoI 1 T'CCnGG_A 5 ! >F
+;PhoI 2 GG'CC 0 ! HaeIII,BshFI,BsnI,BspANI,BsuRI >N
+;PinAI 1 A'CCGG_T 4 ! AgeI,AsiGI,BshTI,CspAI >BM
+PleI 9 GAGTCnnnn'n_ 1 ! MlyI,PpsI,SchI >N
+;Ple19I 4 CG_AT'CG -2 ! PvuI,BpvUI,MvrI >I
+;PmaCI 3 CAC'GTG 0 ! AcvI,BbrPI,Eco72I,PmlI,PspCI >AK
+PmeI 4 GTTT'AAAC 0 ! MssI >GN
+PmlI 3 CAC'GTG 0 ! PmaCI,AcvI,BbrPI,Eco72I,PspCI >N
+PpiI 25 GAACnnnnnCTCnnnnnnnn_nnnnn' -5 ! >F
+PpiI 24 GAGnnnnnGTTCnnnnnnn_nnnnn' -5 ! >F
+;PpsI 9 GAGTCnnnn'n_ 1 ! PleI,MlyI,SchI >I
+;Ppu10I 1 A'TGCA_T 4 ! EcoT22I,Mph1103I,NsiI,Zsp2I >
+;Ppu21I 3 yAC'GTr 0 ! BsaAI,BstBAI >F
+PpuMI 2 rG'GwC_Cy 3 ! Psp5II,PspPPI >NO
+;PscI 1 A'CATG_T 4 ! BspLU11I,PciI >F
+PshAI 5 GACnn'nnGTC 0 ! BoxI,BstPAI >AKN
+;PshBI 2 AT'TA_AT 2 ! VspI,AseI >K
+PsiI 3 TTA'TAA 0 ! >IN
+;Psp03I 4 G_GwC'C -3 ! AvaII,Bme18I,Eco47I,SinI,VpaK11BI >
+;Psp5II 2 rG'GwC_Cy 3 ! PpuMI,PspPPI >F
+;Psp6I 0 'CCwGG_ 5 ! EcoRII,AjnI,BptI,BseBI,BstNI,BstOI,Bst2UI,MvaI,PspGI >I
+;Psp1406I 2 AA'CG_TT 2 ! AclI >FKM
+;Psp124BI 5 G_AGCT'C -4 ! SacI,Ecl136II,EcoICRI,SstI >IV
+;PspCI 3 CAC'GTG 0 ! PmaCI,AcvI,BbrPI,Eco72I,PmlI >IV
+;PspEI 1 G'GTnAC_C 5 ! BstEII,BstPI,Eco91I,EcoO65I >IV
+PspGI 0 'CCwGG_ 5 ! EcoRII,AjnI,BptI,BseBI,BstNI,BstOI,Bst2UI,MvaI,Psp6I >N
+;PspLI 1 C'GTAC_G 4 ! BsiWI,Pfl23II >I
+;PspN4I 3 GGn'nCC 0 ! NlaIV,BmiI,BspLI >I
+PspOMI 1 G'GGCC_C 4 ! ApaI,Bsp120I >INV
+;PspPPI 2 rG'GwC_Cy 3 ! PpuMI,Psp5II >I
+PspXI 2 vC'TCGA_Gb 4 ! >IN
+PsrI 25 GAACnnnnnnTACnnnnnnn_nnnnn' -5 ! >I
+PsrI 25 GTAnnnnnnGTTCnnnnnnn_nnnnn' -5 ! >I
+;PssI 5 rG_GnC'Cy -3 ! DraII,EcoO109I >
+PstI 5 C_TGCA'G -4 ! BspMAI >ABCFGHIJKMNOQRSUVXY
+;PsuI 1 r'GATC_y 4 ! XhoII,BstX2I,BstYI,MflI >F
+;PsyI 4 GACn'n_nGTC 1 ! Tth111I,AspI,PflFI >F
+PvuI 4 CG_AT'CG -2 ! BpvUI,MvrI,Ple19I >ABFGKMNOQRSUXY
+PvuII 3 CAG'CTG 0 ! >ABCFGHIJKMNOQRSUVXY
+;RcaI 1 T'CATG_A 4 ! BspHI,PagI >M
+;RgaI 5 GCG_AT'CGC -2 ! SgfI,AsiSI >I
+;RleAI 18 CCCACAnnnnnnnnn_nnn' -3 ! >
+RsaI 2 GT'AC 0 ! AfaI,Csp6I >BCFGHIJMNOQRSVXY
+RsrII 2 CG'GwC_CG 3 ! CpoI,CspI,Rsr2I >MNQX
+;Rsr2I 2 CG'GwC_CG 3 ! RsrII,CpoI,CspI >I
+SacI 5 G_AGCT'C -4 ! Ecl136II,EcoICRI,Psp124BI,SstI >AFGHJKMNOQRSUX
+SacII 4 CC_GC'GG -2 ! Cfr42I,KspI,Sfr303I,SgrBI,SstII >AGHJKNOQRX
+SalI 1 G'TCGA_C 4 ! >ABCFGHIJKMNOQRSUVXY
+SanDI 2 GG'GwC_CC 3 ! >E
+SapI 8 GCTCTTCn'nnn_ 3 ! LguI,PciSI >N
+;SatI 2 GC'n_GC 1 ! Fnu4HI,Fsp4HI,ItaI >F
+;SauI 2 CC'TnA_GG 3 ! AxyI,Bse21I,Bsu36I,Eco81I >
+Sau96I 1 G'GnC_C 3 ! AspS9I,BmgT120I,Cfr13I >GJMNOU
+;Sau3AI 0 'GATC_ 4 ! MboI,BfuCI,Bsp143I,BssMI,BstKTI,BstMBI,DpnII,Kzo9I,NdeII >AGHJKMNOQRSUX
+SbfI 6 CC_TGCA'GG -4 ! Sse8387I,SdaI >INV
+ScaI 3 AGT'ACT 0 ! AssI,BmcAI,ZrmI >ABCFGJKMNOQRSX
+;SchI 10 GAGTCnnnnn' 0 ! PleI,MlyI,PpsI >F
+;SciI 3 CTC'GAG 0 ! XhoI,PaeR7I,Sfr274I,SlaI,StrI,TliI >
+ScrFI 2 CC'n_GG 1 ! Bme1390I,BmrFI,BssKI,BstSCI,MspR9I,StyD4I >JMNOS
+;SdaI 6 CC_TGCA'GG -4 ! Sse8387I,SbfI >F
+;SduI 5 G_dGCh'C -4 ! Bsp1286I,MhlI >F
+;SecI 1 C'CnnG_G 4 ! BsaJI,BseDI,BssECI >
+;SelI 0 'CGCG_ 4 ! AccII,Bsh1236I,BstFNI,BstUI,MvnI >
+SexAI 1 A'CCwGG_T 5 ! MabI >MN
+SfaNI 10 GCATCnnnnn'nnnn_ 4 ! LweI >IN
+SfcI 1 C'TryA_G 4 ! BfmI,BpcI,BstSFI >N
+;SfeI 1 C'TryA_G 4 ! BfmI,BpcI,BstSFI,SfcI >
+SfiI 8 GGCCn_nnn'nGGCC -3 ! >ACFGIJKMNOQRSUVX
+SfoI 3 GGC'GCC 0 ! NarI,BbeI,DinI,EgeI,EheI,KasI,Mly113I >N
+;Sfr274I 1 C'TCGA_G 4 ! XhoI,PaeR7I,SlaI,StrI,TliI >IV
+;Sfr303I 4 CC_GC'GG -2 ! SacII,Cfr42I,KspI,SgrBI,SstII >IV
+;SfuI 2 TT'CG_AA 2 ! AsuII,Bpu14I,Bsp119I,BspT104I,BstBI,Csp45I,NspV >M
+;SgfI 5 GCG_AT'CGC -2 ! AsiSI,RgaI >R
+SgrAI 2 Cr'CCGG_yG 4 ! >MN
+;SgrBI 4 CC_GC'GG -2 ! SacII,Cfr42I,KspI,Sfr303I,SstII >C
+;SgrDI 4 CGTCGACG 0 ? ! >
+;SgsI 2 GG'CGCG_CC 4 ! AscI,PalAI >F
+;SimI 2 GG'GTC_ 3 ! >
+;SinI 1 G'GwC_C 3 ! AvaII,Bme18I,Eco47I,VpaK11BI >GR
+;SlaI 1 C'TCGA_G 4 ! XhoI,PaeR7I,Sfr274I,StrI,TliI >C
+SmaI 3 CCC'GGG 0 ! Cfr9I,XmaI,XmaCI >ABCFGHIJKMNOQRSUVXY
+;SmiI 4 ATTT'AAAT 0 ! SwaI >FIV
+;SmiMI 5 CAynn'nnrTG 0 ! MslI >I
+SmlI 1 C'TyrA_G 4 ! SmoI >N
+;SmoI 1 C'TyrA_G 4 ! SmlI >F
+;SmuI 9 CCCGCnnnn'nn_ 2 ! FauI >F
+;SnaI 3 GTATAC 0 ? ! BssNAI,Bst1107I,BstZ17I >
+SnaBI 3 TAC'GTA 0 ! BstSNI,Eco105I >ACKMNR
+SpeI 1 A'CTAG_T 4 ! AhlI,BcuI >ABGHJKMNOQRSUX
+SphI 5 G_CATG'C -4 ! BbuI,PaeI >ABCGHIJKMNOQRSVX
+;SplI 1 C'GTAC_G 4 ! BsiWI,Pfl23II,PspLI >
+SrfI 4 GCCC'GGGC 0 ! >EO
+;Sse9I 0 'AATT_ 4 ! TspEI,TasI,Tsp509I >IV
+;Sse232I 2 CG'CCGG_CG 4 ! >
+;Sse8387I 6 CC_TGCA'GG -4 ! SbfI,SdaI >AK
+;Sse8647I 2 AG'GwC_CT 3 ! >
+;SseBI 3 AGG'CCT 0 ! StuI,AatI,Eco147I,PceI >C
+;SsiI 1 C'CG_C 2 ! AciI,BspACI >F
+SspI 3 AAT'ATT 0 ! >ABCFGIJKMNOQRSUVX
+;SspBI 1 T'GTAC_A 4 ! Bsp1407I,BsrGI,BstAUI >M
+;SspD5I 13 GGTGAnnnnnnnn' 0 ! HphI,AsuHPI >
+;SstI 5 G_AGCT'C -4 ! SacI,Ecl136II,EcoICRI,Psp124BI >BC
+;SstII 4 CC_GC'GG -2 ! SacII,Cfr42I,KspI,Sfr303I,SgrBI >B
+;Sth132I 8 CCCGnnnn'nnnn_ 4 ! >
+;Sth302II 2 CC'GG 0 ! HpaII,BsiSI,HapII,MspI >
+;StrI 1 C'TCGA_G 4 ! XhoI,PaeR7I,Sfr274I,SlaI,TliI >U
+;StsI 15 GGATGnnnnnnnnnn'nnnn_ 4 ! FokI,BseGI,BstF5I,BtsCI >
+StuI 3 AGG'CCT 0 ! AatI,Eco147I,PceI,SseBI >ABJKMNQRSUX
+StyI 1 C'CwwG_G 4 ! BssT1I,Eco130I,EcoT14I,ErhI >CJMNRS
+StyD4I 0 'CCnGG_ 5 ! ScrFI,Bme1390I,BmrFI,BssKI,BstSCI,MspR9I >N
+SwaI 4 ATTT'AAAT 0 ! SmiI >GKMNS
+;TaaI 3 AC_n'GT -1 ! Bst4CI,HpyCH4III >F
+TaiI 4 _ACGT' -4 ! MaeII,HpyCH4IV >F
+TaqI 1 T'CG_A 2 ! >ABCFGIJKMNOQRSUVXY
+TaqII 17 GACCGAnnnnnnnnn_nn' -2 ! >VX
+TaqII 17 CACCCAnnnnnnnnn_nn' -2 ! >VX
+;TasI 0 'AATT_ 4 ! TspEI,Sse9I,Tsp509I >F
+TatI 1 w'GTAC_w 4 ! >F
+TauI 4 G_CsG'C -3 ! >F
+TfiI 1 G'AwT_C 3 ! PfeI >N
+;TliI 1 C'TCGA_G 4 ! XhoI,PaeR7I,Sfr274I,SlaI,StrI >N
+;Tru1I 1 T'TA_A 2 ! MseI,Tru9I >F
+;Tru9I 1 T'TA_A 2 ! MseI,Tru1I >GIMRV
+TseI 1 G'CwG_C 3 ! ApeKI >N
+TsoI 17 TArCCAnnnnnnnnn_nn' -2 ! >F
+Tsp45I 0 'GTsAC_ 5 ! NmuCI >N
+Tsp509I 0 'AATT_ 4 ! TspEI,Sse9I,TasI >N
+;Tsp4CI 3 AC_n'GT -1 ! Bst4CI,HpyCH4III,TaaI >
+TspDTI 16 ATGAAnnnnnnnnn_nn' -2 ! >VX
+;TspEI 0 'AATT_ 4 ! Sse9I,TasI,Tsp509I >O
+TspGWI 16 ACGGAnnnnnnnnn_nn' -2 ! >VX
+TspRI 9 _nnCAsTGnn' -9 ! >GN
+;TssI 4 GAGnnnCTC 0 ? ! >
+TstI 24 CACnnnnnnTCCnnnnnnn_nnnnn' -5 ! >F
+TstI 25 GGAnnnnnnGTGnnnnnnnn_nnnnn' -5 ! >F
+;TsuI 2 GCGAC 0 ? ! >
+Tth111I 4 GACn'n_nGTC 1 ! AspI,PflFI,PsyI >GIKNQRVX
+;Tth111II 17 CAArCAnnnnnnnnn_nn' -2 ! >
+;UbaF9I 6 TACnnnnnrTGT 0 ? ! >
+;UbaF11I 2 TCGTA 0 ? ! >
+;UbaPI 3 CGAACG 0 ? ! >
+;UnbI 0 'GGnCC_ 5 ! AspS9I,BmgT120I,Cfr13I,Sau96I >
+;Van91I 7 CCAn_nnn'nTGG -3 ! PflMI,AccB7I >AFGKM
+;Vha464I 1 C'TTAA_G 4 ! AflII,BfrI,BspTI,Bst98I,MspCI >IV
+;VneI 1 G'TGCA_C 4 ! ApaLI,Alw44I >IV
+;VpaK11AI 0 'GGwCC_ 5 ! AvaII,Bme18I,Eco47I,SinI,VpaK11BI >
+;VpaK11BI 1 G'GwC_C 3 ! AvaII,Bme18I,Eco47I,SinI >K
+;VspI 2 AT'TA_AT 2 ! AseI,PshBI >FIRV
+;XagI 5 CCTnn'n_nnAGG 1 ! EcoNI,BstENI >F
+;XapI 1 r'AATT_y 4 ! ApoI,AcsI >F
+XbaI 1 T'CTAG_A 4 ! >ABCFGHIJKMNOQRSUVXY
+;XceI 5 r_CATG'y -4 ! NspI,BstNSI >F
+XcmI 8 CCAnnnn_n'nnnnTGG -1 ! >N
+XhoI 1 C'TCGA_G 4 ! PaeR7I,Sfr274I,SlaI,StrI,TliI >ABFGHJKMNOQRSUXY
+;XhoII 1 r'GATC_y 4 ! BstX2I,BstYI,MflI,PsuI >GMR
+XmaI 1 C'CCGG_G 4 ! SmaI,Cfr9I,XmaCI >INRUV
+;XmaIII 1 C'GGCC_G 4 ! BseX3I,BstZI,EagI,EclXI,Eco52I >
+;XmaCI 1 C'CCGG_G 4 ! SmaI,Cfr9I,XmaI >M
+;XmaJI 1 C'CTAG_G 4 ! AvrII,AspA2I,BlnI >F
+;XmiI 2 GT'mk_AC 2 ! AccI,FblI >F
+XmnI 5 GAAnn'nnTTC 0 ! Asp700I,MroXI,PdmI >GNRU
+;XspI 1 C'TA_G 2 ! MaeI,BfaI,FspBI >K
+ZraI 3 GAC'GTC 0 ! AatII >INV
+;ZrmI 3 AGT'ACT 0 ! ScaI,AssI,BmcAI >I
+;Zsp2I 5 A_TGCA'T -4 ! EcoT22I,Mph1103I,NsiI >IV
+
Added: trunk/packages/perlprimer/branches/upstream/current/perlprimer.pl
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/perlprimer.pl 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/perlprimer.pl 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,10380 @@
+#!/usr/bin/perl -w
+
+# PerlPrimer
+# Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
+
+# version 1.1.13 (22/6/2006)
+# Copyright © 2003-2006, Owen Marshall
+
+# This program is free software; you can redistribute it and/or modify
+# it under the terms of the GNU General Public License as published by
+# the Free Software Foundation; either version 2 of the License, or (at
+# your option) any later version.
+#
+# This program is distributed in the hope that it will be useful, but
+# WITHOUT ANY WARRANTY; without even the implied warranty of
+# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU
+# General Public License for more details.
+#
+# You should have received a copy of the GNU General Public License
+# along with this program; if not, write to the Free Software
+# Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA 02111-1307
+# USA
+
+use strict;
+
+#---------------#
+# Usage message #
+#---------------#
+
+my ($version, $commandline, $win_exe);
+BEGIN {
+ $version = "1.1.13";
+ $win_exe = 0;
+
+ ($commandline) = @ARGV;
+
+ if ($commandline && $commandline =~ /^-[\w-]+/) {
+ print <<EOT;
+PerlPrimer v$version
+Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
+
+Copyright © 2003-2006 Owen Marshall\n
+Usage: perlprimer.pl [file.ppr]\n
+EOT
+ exit 0;
+ }
+
+ # Usage message for the Win32 dos box for the exe version:
+ if ($win_exe) {
+ print <<EOT;
+PerlPrimer v$version
+Copyright © 2003-2006 Owen Marshall
+Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
+
+This window is required for PerlPrimer to run -
+you may minimise it but please do not close it
+
+Please wait while PerlPrimer loads ...
+EOT
+ }
+}
+
+
+#---------------#
+# Load packages #
+#---------------#
+
+my $failed_packages;
+BEGIN {
+ # A modular package checking routine ...
+ # ($failed_packages is used by the check_packages() subroutine)
+
+ $failed_packages = " "; # check_packages gives errors if undefined
+ my $warning;
+ my $eval_sub = sub {
+ eval $_;
+ if ($@) {
+ /(use|require)\s*([\w:-]+)\;/;
+ my $package = $2;
+
+ $failed_packages .= " $package ";
+
+ # Special circumstances!
+ return if $package eq "Benchmark";
+ return if $package eq "File::Copy";
+ return if $package eq "Win32::GUI";
+
+ # Print warning header if not already printed
+ unless ($warning) {
+ print "PerlPrimer v$version\nCopyright © 2003-2005 Owen Marshall\n\n";
+ $warning = 1;
+ }
+
+ # Print specific warning messages to alert the user that modules are missing
+ if ($package eq "Tk") {
+ print "Error: Perl/Tk not found!\nPerlPrimer requires Perl/Tk to run - please download from CPAN (http://cpan.org) and install before running.\n\n";
+ exit 0;
+ } elsif ($1 eq "require") {
+ print "Error: $package not found!\nPerlPrimer requires $package to run - please download from CPAN (http://cpan.org) and install before running.\n\n";
+ exit 0;
+ }
+
+ print "Warning: PerlPrimer requires $package for full functionality\n- some features may be disabled.\n$package should be obtainable from CPAN (http://cpan.org)\n\n";
+ }
+ };
+
+ foreach (split("\n","
+use Tk;
+use Benchmark;
+use HTTP::Request;
+use LWP::UserAgent;
+use IO::Socket;
+use Win32::GUI;
+use File::Copy;
+use File::Glob ':glob';
+require Tk::NoteBook;
+require Tk::HList;
+require Tk::ItemStyle;
+require Tk::Dialog;
+require Tk::LabFrame;
+require Tk::ROText;
+require Tk::Balloon;
+require Tk::BrowseEntry;
+require Tk::DirTree;
+ ")) {&$eval_sub($_)};
+}
+
+
+#------------------------------------------------------------------#
+# Primer pair storage code for two dimensional array @primer_pair: #
+#------------------------------------------------------------------#
+
+# @primer_pair[index][see below]:
+#
+# 0. primer_f
+# 1. pos_f
+# 2. window_length_f
+# 3. Tm_f
+#
+# 4. primer_r
+# 5. pos_r
+# 6. window_length_r
+# 7. Tm_r
+# 8. real_pos_r
+#
+# 9. amplicon_size
+# 10. deltaG of most stable extensible primer-dimer
+# 11. [primer_f sequence pre-conversion for Bisulphite PCR]
+# 12. [primer_r sequence pre-conversion for Bisulphite PCR]
+# 13. deltaG of most stable non-extensible primer-dimer
+
+
+# Load thermodynamic data and genetic code...
+my (%oligo_dH, %oligo_dH_full, %oligo_dS, %oligo_dS_full, %genetic_code);
+load_data();
+
+
+# Starting ionic concentration variables
+my $oligo_conc = 200; #in nM
+my $mg_conc=1.5; #in mM
+my $monovalent_cation_conc=50; #in mM
+my $dntp_conc=0.2; #in mM
+
+
+# Default primer variables
+my $max_tm_pr=63; # standard PCR
+my $min_tm_pr=57;
+my $max_diff_pr=3;
+my $pri_win_min_pr=20;
+my $pri_win_max_pr=24;
+my $exclude_gc=1;
+my $exclude_clamp=1;
+
+my $max_tm_bs=65; # bisulphite PCR
+my $min_tm_bs=55;
+my $max_diff_bs=5;
+my $pri_win_min_bs=25;
+my $pri_win_max_bs=30;
+my $bisul_min_c=30;
+my $pre_bs=1;
+my $exclude_3c=1;
+my $exclude_cpg=0;
+
+my $max_tm_q=62; # QPCR
+my $min_tm_q=58;
+my $max_diff_q=2;
+my $pri_win_min_q=20;
+my $pri_win_max_q=24;
+my $min_ampsize_q=100;
+my $max_ampsize_q=300;
+my $ie_overlap=1;
+my $exclude_ie=7;
+my $ie_span=1;
+
+my $min_tm_seq = 56; # Sequencing
+my $max_tm_seq = 64;
+my $pri_win_min_seq = 20;
+my $pri_win_max_seq = 24;
+my $seq_spacing_min = 500;
+my $seq_spacing_max = 700;
+my $exclude_gc_seq = 1;
+my $exclude_clamp_seq = 1;
+my $exclude_pd_seq = 1;
+my $seq_pd_min = 5;
+
+
+# Establish default varibles for opening a new file - perhaps a bit verbose
+# but it is rather neat ...
+my %default_variables = (
+ pd => {
+ \$min_tm_pr => $min_tm_pr,
+ \$max_tm_pr => $max_tm_pr,
+ \$max_diff_pr => $max_diff_pr,
+ \$pri_win_min_pr => $pri_win_min_pr,
+ \$pri_win_max_pr => $pri_win_max_pr,
+ \$exclude_gc => $exclude_gc,
+ \$exclude_clamp => $exclude_clamp,
+ },
+ seq => {
+ \$min_tm_seq => $min_tm_seq,
+ \$max_tm_seq => $max_tm_seq,
+ \$pri_win_min_seq => $pri_win_min_seq,
+ \$pri_win_max_seq => $pri_win_max_seq,
+ \$seq_spacing_min => $seq_spacing_min,
+ \$seq_spacing_max => $seq_spacing_max,
+ \$exclude_gc_seq => $exclude_gc_seq,
+ \$exclude_clamp_seq => $exclude_clamp_seq,
+ \$exclude_pd_seq => $exclude_pd_seq,
+ \$seq_pd_min => $seq_pd_min,
+ },
+ bis => {
+ \$min_tm_bs => $min_tm_bs,
+ \$max_tm_bs => $max_tm_bs,
+ \$max_diff_bs => $max_diff_bs,
+ \$pri_win_min_bs => $pri_win_min_bs,
+ \$pri_win_max_bs => $pri_win_max_bs,
+ \$exclude_cpg => $exclude_cpg,
+ \$pre_bs => $pre_bs,
+ \$exclude_3c => $exclude_3c,
+ \$bisul_min_c => $bisul_min_c,
+ },
+ qpcr => {
+ \$min_tm_q => $min_tm_q,
+ \$max_tm_q, => $max_tm_q,
+ \$max_diff_q => $max_diff_q,
+ \$pri_win_min_q => $pri_win_min_q,
+ \$pri_win_max_q => $pri_win_max_q,
+ \$min_ampsize_q => $min_ampsize_q,
+ \$max_ampsize_q => $max_ampsize_q,
+ \$exclude_gc => $exclude_gc,
+ \$exclude_clamp => $exclude_clamp,
+ \$ie_overlap => $ie_overlap,
+ \$exclude_ie => $exclude_ie,
+ \$ie_span => $ie_span,
+ },
+);
+
+# Home environment variables and OS-specific tweaking:
+
+# These variables are the total changes required for nice cross-platform compatibility
+# (and most of these are cosmetic - strange that the Tk look&feel is not consistent with
+# things such as widget spacing, most notably the checkbuttons)
+my $HOME = $ENV{HOME} || $ENV{HOMEPATH}; # HOMEPATH may not work correctly on Win32 ...
+my $tmp = $ENV{TEMP} || $ENV{TMP};
+my ($dir_sep, $os, $gui_font_face, $gui_font_size, $text_font_face, $text_font_size, $list_font_face, $list_font_size, $font_override, $menu_relief, $frame_pady, $check_pady, $button_pady, $button_pack_padx, $button_pack_pady, $browser);
+
+if ($^O =~ /mswin/i) {
+ # MS Windows OS
+ $os = 'win';
+ $browser = 'c:\Program Files\Internet Explorere\iexplore';
+ $HOME ||= 'c:\\'; # only if not set by $ENV above
+ $tmp ||= 'c:\temp\\'; # only if not set by $ENV above
+ $dir_sep = '\\';
+ $gui_font_face = "Arial";
+ $gui_font_size = 8;
+ $list_font_face = "Verdana";
+ $list_font_size = 7;
+ $text_font_face = "Courier";
+ $text_font_size = 8;
+ $menu_relief = 'flat';
+ $button_pady = 1;
+ $button_pack_padx = 2;
+ $button_pack_pady = 2;
+ $frame_pady = 2;
+ $check_pady = 0;
+} else {
+ # *nix OS
+ $os = 'nix';
+ $browser = 'firefox';
+ $HOME ||= (getpwuid($<))[7].'/'; # only if not set above
+ $tmp = '/tmp/'; # only if not set above
+ $dir_sep = '/';
+ $gui_font_face = "Helvetica";
+ $gui_font_size = 10;
+ $list_font_face = "Helvetica";
+ $list_font_size = 10;
+ $text_font_face = "Courier";
+ $text_font_size = 10;
+ $menu_relief = 'raised';
+ $button_pady = 2;
+ $button_pack_padx = 0;
+ $button_pack_pady = 0;
+ $frame_pady = 1;
+ $check_pady = 3;
+}
+
+# check tmp directory exists, is user readable and writable,
+# else use the home directory
+$tmp = $HOME unless (-e $tmp && -r $tmp && -w $tmp);
+
+# check directories have trailing slash ...
+$tmp = check_path($tmp);
+$HOME = check_path($HOME);
+
+# directory the program has been run in (for RE enzyme database)
+my $program_directory = $0;
+$program_directory =~ s/([^\\\/]*$)//;
+
+# flags
+my $bs=0;
+my $qpcr_flag=0;
+my $cancel=0;
+my $defer_to_caps=0;
+
+# benchmarking - for code optimisation
+my $benchmark=0;
+
+# DNA canvas sizing:
+my $dna_canvas_offset=15;
+my $dna_canvas_height=21;
+my $dna_canvas_middle=int($dna_canvas_height/2)+2;
+
+my $half_dna_size=3;
+my $dc_dna_y1 = $dna_canvas_middle - $half_dna_size;
+my $dc_dna_y2 = $dna_canvas_middle + $half_dna_size;
+
+my $dc_selection_offset = 3;
+my $dc_sel_y1 = $dc_dna_y1-$dc_selection_offset;
+my $dc_sel_y2 = $dc_dna_y2+$dc_selection_offset;
+my $dc_sel_offset2 = 2;
+
+# Repeats/runs
+my $exclude_rr = 1;
+my $repeat = 3;
+my $run = 4;
+
+my $exclude_rr_bs = 1;
+my $repeat_bs = 3;
+my $run_bs = 4;
+
+# %GC exclusions
+my $max_gc = 60;
+my $min_gc = 40;
+
+# CpG prediction variables
+my $cpg_window = 200;
+my $min_cpg_island = 200;
+my $cpg_gc = 50;
+my $cpg_oe = 0.6;
+my $cpgplot_method = 0;
+
+# Http proxy
+my $use_proxy = 0;
+my ($http_proxy, $http_port);
+
+# Ensembl data
+my $ensembl; # GUI page
+my $ensembl_gene;
+my $ensembl_organism = 'Homo_sapiens';
+my $ensembl_type = 'cdna';
+my @ensembl_species = split("\n",
+"Homo_sapiens
+Mus_musculus
+-------------------
+Anopheles_gambiae
+Apis_mellifera
+Bos_taurus
+Caenorhabditis_elegans
+Canis_familiaris
+Ciona_intestinalis
+Danio_rerio
+Drosophila_melanogaster
+Fugu_rubripes
+Gallus_gallus
+Macaca_mulatta
+Monodelphis_domestica
+Pan_troglodytes
+Rattus_norvegicus
+Saccharomyces_cerevisiae
+Tetraodon_nigroviridis
+Xenopus_tropicalis");
+
+my @ensembl_types = split("\n",
+"genomic
+cdna
+coding
+utr5
+utr3");
+
+# BLAST search parameters
+my $blast_expect = 10;
+my $blast_word_size = 7;
+my $blast_database = 'nr';
+my @blast_database_array = split("\n",
+"nr
+est
+est_human
+est_mouse
+est_others
+gss
+htgs
+pat
+yeast
+mito
+vector
+pdb
+month
+alu
+dbsts
+chromosome
+E. coli
+Drosophila genome");
+
+my $blast_entrez_query = 'none';
+my @blast_entrez_array = split("\n",
+"none
+-------------------
+Viruses [ORGN]
+Archaea [ORGN]
+Bacteria [ORGN]
+Eukaryota [ORGN]
+Viridiplantae [ORGN]
+Fungi [ORGN]
+Metazoa [ORGN]
+Arthropoda [ORGN]
+Vertebrata [ORGN]
+Mammalia [ORGN]
+Rodentia [ORGN]
+Primates [ORGN]
+-------------------
+Aeropyrum pernix [ORGN]
+Aquifex aeolicus [ORGN]
+Arabidopsis thaliana [ORGN]
+Bacillus subtilis [ORGN]
+Bos taurus [ORGN]
+Caenorhabditis elegans [ORGN]
+Danio rerio [ORGN]
+Dictyostelium discoideum [ORGN]
+Drosophila melanogaster [ORGN]
+Escherichia coli [ORGN]
+Gallus gallus [ORGN]
+Homo sapiens [ORGN]
+Human immunodeficiency virus type 1 [ORGN]
+Methanococcus jannaschii [ORGN]
+Mus musculus [ORGN]
+Oryctolagus cuniculus [ORGN]
+Oryza sativa [ORGN]
+Ovis aries [ORGN]
+Plasmodium falciparum [ORGN]
+Rattus norvegicus [ORGN]
+Saccharomyces cerevisiae [ORGN]
+Schizosaccharomyces pombe [ORGN]
+Simian immunodeficiency virus [ORGN]
+Synechocystis PCC6803 [ORGN]
+Takifugu rubripes [ORGN]
+Xenopus laevis [ORGN]
+Zea mays [ORGN]");
+
+my $local_blast = 0;
+my $local_blast_directory = $HOME;
+my $local_blast_database;
+
+my $blast_count;
+my $http_abort;
+
+# Primer-dimer parameters
+# my $pd_full=0;
+my $pd_extensible=1;
+my $pd_temperature=37;
+
+# Restriction enzyme cloning sequence parameters
+my $cloning_anchor = "GCGCGC";
+my $simple_sites = 1;
+my $exclude_found_sites = 1;
+
+# IPC using TCP sockets (with Contig Viewer)
+my $file_data_overwrite = 1;
+my $ipc_autofind = 0;
+my $tcp_port = 2500;
+my $socket_polling_interval = 1000; # msec
+
+# Header columns
+my @header_list_primers = split("\n",
+"Forward Primer
+Pos
+Len
+Tm
+Reverse Primer
+Pos
+Len
+Tm
+Amp
+Ext. dimer dG
+Full dimer dG");
+
+# Mouse wheel
+my $scroll_factor = 2;
+
+# Win32 only - for button pre-lights
+my $activebackground_color="#ffffff";
+
+# Spidey details
+my (%spidey_exec);
+my $spidey_path = "$HOME";
+
+# search for spidey in program directory
+my @spidey_files = glob("$program_directory*pidey.*");
+ at spidey_files = glob("$program_directory*pidey*") unless @spidey_files;
+if (@spidey_files) {
+ $spidey_path = "$program_directory";
+}
+
+# my $spidey_out;
+
+# More global variables
+my (
+ $primer_f, $primer_r, $pos,
+ $rprimer_r,
+ $pd, @score_sort, $reverse,
+ $pfkeys, $pkeys, @PF, @PR, %primer_hash,
+ $gc_exclude, $gc_percent_ex, @primer_pairs,
+ $prl, $pfl, $pl, @bind_string, %rating_hash, @score,
+ %packed_widgets,
+
+ $save_seq, $save_seq2,
+ @primer_pairs_pr_s, @primer_pairs_seq_s, @primer_pairs_bs_s, @primer_pairs_q_s, @save_selection,
+ @intron_exon_bounds, $ie_limit, $ie_limit_5p, $ie_limit_3p,
+
+ $primer_seq_5f, $primer_seq_5r, $primer_seq_5f_frame, $primer_seq_5r_frame,
+ $primer_seq_5f_atg, $primer_seq_5r_atg,
+);
+
+my (
+ $min_ampsize_pr, $max_ampsize_pr, $max_range_5p_pr, $min_range_pr, $max_range_pr, $max_range_3p_pr,
+ $min_range_seq, $max_range_seq,
+ $min_ampsize_bs, $max_ampsize_bs, $max_range_5p_bs, $min_range_bs, $max_range_bs, $max_range_3p_bs,
+ $max_range_5p_q, $min_range_q, $max_range_q, $max_range_3p_q,
+);
+
+# GUI globals
+my (
+ %prr, %prc, %pre, %prb, %prl, %prf,
+ $fprimer, $fprimer_tm, $fprimer_len, $fprimer_ds, $fprimer_dh, $fprimer_dg, $fprimer_gc,
+ $rprimer, $rprimer_tm, $rprimer_len, $rprimer_ds, $rprimer_dh, $rprimer_dg, $rprimer_gc,
+ $old_reference, $text_widget_ref,
+ $style_primer, $style_tm,
+ %min_amp_canvas, %max_amp_canvas, %min_range_canvas, %max_range_canvas,
+ $flag, $rid_get, @blast_results_1, @blast_results_2, @blast_results_3, $rptid, $blast_status,
+ $popup_sort, $popup_sort_seq,
+ $stored_page,
+ $forward_re_site, $reverse_re_site,
+);
+
+# GUI dialogues
+my (
+ $blast_d, $view_base, $prefs, $ack_d, $canvas_info_d, $info_d,
+ $cloning_d, $view_ie,
+);
+
+my $balloon_help = 1;
+
+# Recently used files
+my @mru;
+my $mru_number = 8;
+
+
+# Define the perlprimer recognised filetypes
+my $file_types = [
+ ['PerlPrimer Files', '.ppr'],
+ ['Fasta Files', '.fasta'],
+ ['All Files', '*']
+ ];
+
+my $file_types_dna = [
+ ['Fasta Files', '.fasta'],
+ ['Text Files', '.txt'],
+ ['All Files', '*']
+ ];
+
+my $file_types_text = [
+ ['Text Files', '.txt'],
+ ['All Files', '*']
+ ];
+
+
+
+# Open file hash (for writing reports)
+my %open_file;
+for my $i (qw(pd seq bis qpcr)) {
+ $open_file{$i} = "File not saved"
+}
+
+# Variable/array hashes for opening and saving
+my %variables = (
+ pd => {
+ mintm => \$min_tm_pr,
+ maxtm => \$max_tm_pr,
+ maxdiff => \$max_diff_pr,
+ minwin => \$pri_win_min_pr,
+ maxwin => \$pri_win_max_pr,
+ minamp => \$min_ampsize_pr,
+ maxamp => \$max_ampsize_pr,
+ maxrange5p => \$max_range_5p_pr,
+ minrange => \$min_range_pr,
+ maxrange => \$max_range_pr,
+ maxrange3p => \$max_range_3p_pr,
+ exclude_gc => \$exclude_gc,
+ gc_clamp => \$exclude_clamp,
+ seq => \$save_seq,
+ primer_seq_5f => \$primer_seq_5f,
+ primer_seq_5f_frame => \$primer_seq_5f_frame,
+ primer_seq_5r => \$primer_seq_5r,
+ primer_seq_5r_frame => \$primer_seq_5r_frame,
+ },
+ seq => {
+ mintm => \$min_tm_seq,
+ maxtm => \$max_tm_seq,
+ minwin => \$pri_win_min_seq,
+ maxwin => \$pri_win_max_seq,
+ minrange => \$min_range_seq,
+ maxrange => \$max_range_seq,
+ seq_spacing_min => \$seq_spacing_min,
+ seq_spacing_max => \$seq_spacing_max,
+ exclude_gc => \$exclude_gc_seq,
+ gc_clamp => \$exclude_clamp_seq,
+ exclude_pd => \$exclude_pd_seq,
+ seq_pd_min => \$seq_pd_min,
+ seq => \$save_seq,
+ },
+ bis => {
+ mintm => \$min_tm_bs,
+ maxtm => \$max_tm_bs,
+ maxdiff => \$max_diff_bs,
+ minwin => \$pri_win_min_bs,
+ maxwin => \$pri_win_max_bs,
+ minamp => \$min_ampsize_bs,
+ maxamp => \$max_ampsize_bs,
+ maxrange5p => \$max_range_5p_bs,
+ minrange => \$min_range_bs,
+ maxrange => \$max_range_bs,
+ maxrange3p => \$max_range_3p_bs,
+ exclude_cpg => \$exclude_cpg,
+ rrpostconv => \$pre_bs,
+ exclude_cs => \$exclude_3c,
+ min_c => \$bisul_min_c,
+ seq => \$save_seq,
+ },
+ qpcr => {
+ mintm => \$min_tm_q,
+ maxtm => \$max_tm_q,
+ maxdiff => \$max_diff_q,
+ minwin => \$pri_win_min_q,
+ maxwin => \$pri_win_max_q,
+ minamp => \$min_ampsize_q,
+ maxamp => \$max_ampsize_q,
+ exclude_gc => \$exclude_gc,
+ gc_clamp => \$exclude_clamp,
+ mrna_seq => \$save_seq,
+ dna_seq => \$save_seq2,
+ ie_overlap => \$ie_overlap,
+ exclude_ie => \$exclude_ie,
+ ie_limit => \$ie_limit,
+ ie_limit_5p => \$ie_limit_5p,
+ ie_limit_3p => \$ie_limit_3p,
+ ie_span => \$ie_span,
+ },
+);
+
+my %arrays = (
+ pd => {
+ res => \@primer_pairs_pr_s,
+ selection => \@save_selection,
+ },
+ seq => {
+ res => \@primer_pairs_seq_s,
+ selection => \@save_selection,
+ },
+ bis => {
+ res => \@primer_pairs_bs_s,
+ selection => \@save_selection,
+ },
+ qpcr => {
+ res => \@primer_pairs_q_s,
+ selection => \@save_selection,
+ intron_exon => \@intron_exon_bounds,
+ },
+);
+
+# Hash for opening/saving prefs
+my %pref_variables = (
+ # home => \$HOME,
+ browser => \$browser,
+ tmp => \$tmp,
+ spidey_path => \$spidey_path,
+ repeats => \$repeat,
+ runs => \$run,
+ exclude_rr => \$exclude_rr,
+ repeats_bs => \$repeat_bs,
+ runs_bs => \$run_bs,
+ exclude_rr_bs => \$exclude_rr_bs,
+ max_gc => \$max_gc,
+ min_gc => \$min_gc,
+ mg_conc => \$mg_conc,
+ monovalent_cation_conc => \$monovalent_cation_conc,
+ dNTP_conc => \$dntp_conc,
+ oligo_conc => \$oligo_conc,
+ cpg_window => \$cpg_window,
+ min_cpg_island => \$min_cpg_island,
+ cpg_gc_percent => \$cpg_gc,
+ cpg_observed_expected => \$cpg_oe,
+ balloon_help => \$balloon_help,
+ blast_database => \$blast_database,
+ blast_entrez_query => \$blast_entrez_query,
+ blast_expect => \$blast_expect,
+ blast_word_size => \$blast_word_size,
+ local_blast => \$local_blast,
+ local_blast_directory => \$local_blast_directory,
+ local_blast_database => \$local_blast_database,
+ gui_font_face => \$gui_font_face,
+ gui_font_size => \$gui_font_size,
+ list_font_face => \$list_font_face,
+ list_font_size => \$list_font_size,
+ text_font_face => \$text_font_face,
+ text_font_size => \$text_font_size,
+ font_override => \$font_override,
+ scroll_factor => \$scroll_factor,
+ ensembl_organism => \$ensembl_organism,
+ ensembl_type => \$ensembl_type,
+ use_proxy => \$use_proxy,
+ http_proxy => \$http_proxy,
+ http_proxy_port => \$http_port,
+ cpgplot_method => \$cpgplot_method,
+ defer_to_caps => \$defer_to_caps,
+ mru_number => \$mru_number,
+ re_simple_sites => \$simple_sites,
+ cloning_anchor_sequence => \$cloning_anchor,
+ pd_temperature => \$pd_temperature,
+ exclude_found_sites => \$exclude_found_sites,
+ file_overwrite => \$file_data_overwrite,
+ ipc_autofind => \$ipc_autofind,
+ tcp_port => \$tcp_port,
+);
+
+my %pref_arrays = (
+ mru => \@mru,
+);
+
+
+# Hash for variable references
+my $null = undef;
+my %page_specific_vars = (
+ pd => {
+ min_tm => \$min_tm_pr,
+ max_tm => \$max_tm_pr,
+ max_diff => \$max_diff_pr,
+ pri_win_win => \$pri_win_min_pr,
+ pri_win_max => \$pri_win_max_pr,
+ min_ampsize => \$min_ampsize_pr,
+ max_ampsize => \$max_ampsize_pr,
+ max_range_5p => \$max_range_5p_pr,
+ min_range => \$min_range_pr,
+ max_range => \$max_range_pr,
+ max_range_3p => \$max_range_3p_pr,
+ seq => \$packed_widgets{seq},
+ primers => \@primer_pairs_pr_s,
+ hlist => \$packed_widgets{res},
+ canvas => \$packed_widgets{primer_canvas},
+ subroutine => \&get_gene,
+ find_sub => \&find_orf,
+ primer_sub => \&get_primers,
+ popup => \$popup_sort,
+ },
+ seq => {
+ min_tm => \$min_tm_seq,
+ max_tm => \$max_tm_seq,
+ pri_win_win => \$pri_win_min_seq,
+ pri_win_max => \$pri_win_max_seq,
+ min_ampsize => \$null,
+ max_ampsize => \$null,
+ max_range_5p => \$null,
+ min_range => \$min_range_seq,
+ max_range => \$max_range_seq,
+ max_range_3p => \$null,
+ seq => \$packed_widgets{seq_seq},
+ primers => \@primer_pairs_seq_s,
+ hlist => \$packed_widgets{seq_res},
+ canvas => \$packed_widgets{seq_canvas},
+ subroutine => \&get_gene,
+ find_sub => \&find_orf,
+ primer_sub => \&get_seq_primers,
+ popup => \$popup_sort_seq,
+ },
+ bis => {
+ min_tm => \$min_tm_bs,
+ max_tm => \$max_tm_bs,
+ max_diff => \$max_diff_bs,
+ pri_win_win => \$pri_win_min_bs,
+ pri_win_max => \$pri_win_max_bs,
+ min_ampsize => \$min_ampsize_bs,
+ max_ampsize => \$max_ampsize_bs,
+ max_range_5p => \$max_range_5p_bs,
+ min_range => \$min_range_bs,
+ max_range => \$max_range_bs,
+ max_range_3p => \$max_range_3p_bs,
+ seq => \$packed_widgets{bisul_seq},
+ primers => \@primer_pairs_bs_s,
+ hlist => \$packed_widgets{bisul_res},
+ canvas => \$packed_widgets{bisul_canvas},
+ subroutine => \&get_cpg,
+ find_sub => \&find_cpg,
+ primer_sub => \&get_bisulphite,
+ popup => \$popup_sort,
+ },
+ qpcr => {
+ min_tm => \$min_tm_q,
+ max_tm => \$max_tm_q,
+ max_diff => \$max_diff_q,
+ pri_win_win => \$pri_win_min_q,
+ pri_win_max => \$pri_win_max_q,
+ min_ampsize => \$min_ampsize_q,
+ max_ampsize => \$max_ampsize_q,
+ max_range_5p => \$null,
+ min_range => \$null,
+ max_range => \$null,
+ max_range_3p => \$null,
+ seq => \$packed_widgets{qmrna_seq},
+ primers => \@primer_pairs_q_s,
+ hlist => \$packed_widgets{qpcr_res},
+ canvas => \$packed_widgets{qprimer_canvas},
+ find_sub => \&find_orf,
+ popup => \$popup_sort,
+ },
+);
+
+# prefs_file
+my $pref_file = $HOME.'.perlprimer';
+
+# check for upgrading users under win32 who have a different home environment from c:\
+if ($os eq 'win' && !check_packages('File::Copy') && -e "c:\.perlprimer" && !-e $pref_file) {
+ print "\n\nPlease Note: as of PerlPrimer v1.1.5 the preferences file is now stored in the user's home directory\nCopying old PerlPrimer preferences to $pref_file ...\n";
+ copy("c:\.perlprimer",$pref_file);
+}
+
+read_prefs();
+
+# Balloon help messages
+my %balloonmsg = (
+ 'primer_getgene', "Find the longest ORF within the sequence and set the selected range",
+ 'primer_reset', "Reset the range to the ORF boundaries",
+ 'primer_stepin', "Reduce the inner range by 10bp on each side",
+ 'primer_stepout', "Increase the outer range by 10bp on each side",
+ 'exclude_gc', "Exclude primers with a GC content outside the range specified in the preferences\n(default is 40-60% GC)",
+ 'gc_clamp', "Require primers to have a 3' GC clamp\n(two of the last three bases G or C)",
+ 'primer_seq_5f', "Sequence to add to the 5' end of the forward primer\nPlace an underscore (_) before restriction enzyme site\n\nUse the 'Add cloning sequences' menu option to configure this automatically",
+ 'primer_seq_5r', "Sequence to add to the 5' end of the reverse primer\nPlace an underscore (_) before restriction enzyme site\n\nUse the 'Add cloning sequences' menu option to configure this automatically",
+ 'primer_seq_5f_frame', "Frame of the restriction enzyme site in the cloning plasmid\n0 = in frame\n1 = +1 base\n2 = +2 bases",
+ 'primer_seq_5r_frame', "Frame of the restriction enzyme site in the cloning plasmid\n0 = in frame\n1 = +1 base\n2 = +2 bases",
+ 'seq', "DNA sequence: right-click for menu with options\nincluding opening and saving files",
+ 'res', "Matching primer pairs are displayed here:\nDouble click to see primer-dimers\nRight-click for option menu",
+ 'primerbutton', "Find primer pairs",
+ 'autobuttonin', "Successively reduce the inner range by 10bp increments\nuntil primer pairs are found",
+ 'autobuttonout', "Successively increase the outer range by 10bp increments\nuntil primer pairs are found",
+ 'primer_cancel', "Cancel the current task",
+ 'primer_view', "Copy the selected primer pairs to the clipboard\n(format is tab-delimited text)",
+ 'primer_canvas', "Primer canvas",
+
+ 'bisul_getcpg', "Find CpG islands within the sequence and set the selected range",
+ 'bisul_reset', "Reset the range to the ORF boundaries",
+ 'bisul_stepin', "Reduce the inner range by 10bp on each side",
+ 'bisul_stepout', "Increase the outer range by 10bp on each side",
+ 'bisul_seq', "DNA sequence: right-click for menu with options\nincluding opening and saving files",
+ 'bisul_res', "Matching primer pairs are displayed here:\nDouble click to see primer-dimers\nRight-click for option menu",
+ 'bisul_exclude_cpg', "Exclude primers with CpG residues",
+ 'bisul_pre_bs', "Exclude primers with repeats / runs after bisulphite conversion",
+ 'bisul_exclude_cs', "Exclude primers without 3' C content\n(either last base as C, or two of last three bases C)",
+ 'bisul_min_c', "Minimum \%C content for primers",
+ 'bisul_button', "Find primer pairs",
+ 'bisul_autobuttonin', "Successively reduce the inner range by 10bp increments\nuntil primer pairs are found",
+ 'bisul_autobuttonout', "Successively increase the outer range by 10bp increments\nuntil primer pairs are found",
+ 'bisul_cancel', "Cancel the current task",
+ 'bisul_view', "Copy the selected primer pairs to the clipboard\n(format is tab-delimited text)",
+
+ 'qexclude_gc', "Exclude primers with a GC content outside the range specified in the preferences\n(default is 40-60% GC)",
+ 'qgc_clamp', "Require primers to have a 3' GC clamp\n(two of the last three bases G or C)",
+ 'qdna_seq', "Genomic DNA sequence: right-click for menu with options\nincluding opening and saving files",
+ 'qmrna_seq', "mRNA sequence: right-click for menu with options\nincluding opening and saving files",
+ 'qpcr_res', "Matching primer pairs are displayed here:\nDouble click to see primer-dimers\nRight-click for option menu",
+ 'qprimerbutton', "Find primer pairs\n(at least one primer must span an intron-exon boundary)",
+ 'qprimer_cancel', "Cancel the current task",
+ 'qprimer_view', "Copy the selected primer pairs to the clipboard\n(format is tab-delimited text)",
+ 'qprimer_spidey', "Detailed intron/exon boundary information",
+ 'qexclude_ie', "Minimum number of bases within a primer that should overlap an intron/exon boundary",
+ 'qie_limit', "Limit primer search to this range of exons",
+ 'qie_limit_5p', "Leave blank to represent the first exon\nThe exon can also be selected by left-clicking on the DNA graphic",
+ 'qie_limit_3p', "Leave blank to represent the last exon\nThe exon can also be selected by middle-clicking on the DNA graphic",
+ 'qie_overlap', "Require at least one primer to overlap an intron/exon boundary",
+ 'qie_span', "Require primers to span an intron/exon boundary",
+
+ 'sspacingmin', "Minimum distance between sequencing primers",
+ 'sspacingmax', "Maximum distance between sequencing primers",
+ 'seqminrange', "The region of the DNA to be sequenced",
+ 'seqmaxrange', "The region of the DNA to be sequenced",
+ 'seq_getgene', "Set the sequencing region to the boundaries of the largest ORF present",
+ 'exclude_gc_seq', "Exclude primers with a GC content outside the range specified in the preferences\n(default is 40-60% GC)",
+ 'gc_clamp_seq', "Require primers to have a 3' GC clamp\n(two of the last three bases G or C)",
+ 'exclude_pd_seq', "Do not consider primers which can form dimers\nwith a stability greater than this value",
+ 'spdmin', "Do not consider primers which can form dimers\nwith a stability greater than this value",
+ 'seq_button', "Find primer pairs",
+ 'seq_cancel', "Cancel the current task",
+ 'seq_view', "Copy the selected primer pairs to the clipboard\n(format is tab-delimited text)",
+
+ 'tmbutton', "Calculate Tm, thermodynamic properties and primer-dimers from the primer sequences",
+ 'blastbutton', "Perform a BLAST search on the primers\n(requires an internet connection)",
+
+ 'prefs_defer', "Do not attempt to find an ORF or CpG Islands if\na captalised region is already present in the\nDNA sequence - use that region to set the range instead",
+ 'prefs_cpgplot_method', "Emulate the behaviour of the program cpgplot when\nfinding CpG islands (please note that this will cause the \%GC and O/E\nto be an overestimate - see the documentation for details)",
+ 'prefs_gui_override', "Use system default fonts for most widgets\n(requires program restart)",
+ 'prefs_gui_family', "Changes will not take effect until program restart",
+ 'prefs_gui_list_family', "Changes will not take effect until program restart",
+ 'prefs_gui_text_family', "Please use a fixed-width font\nChanges will not take effect until program restart",
+ 'prefs_simple_sites', "Limit restriction enzyme database to 6-base cutting enzymes\n(Also excludes degenerate cutting enzymes)",
+ 'prefs_exclude_found_sites', "When displaying the list of restriction enzymes,\nonly list those that will not cut the input sequence\n(Highly recommended!)",
+
+ 'cloning_anchor', "Sequence to add 5' of the restiction enzyme sites\n(recommended for successful digestion after amplification)",
+
+ 'blast_f', "Display results for the forward primer",
+ 'blast_r', "Display results for the reverse primer",
+ 'blast_search_string', "Enter a string to limit results",
+ 'blast_search', "Limit results",
+
+ 'view_base_copy', "Copies the layout to the clipboard\nas 80-column wrapped text",
+ 'view_base_pf', "Jump to the forward primer",
+ 'view_base_pr', "Jump to the reverse primer",
+);
+
+
+#--------------------#
+# deltaG (kcal/mol) #
+#--------------------#
+
+# Recalculate oligo_dG (kcal/mol) for PCR salt conditions
+# (the initiation values are salt independent)
+my %oligo_dG=(
+ qw(initC 0.98 initG 0.98
+ initA 1.03 initT 1.03),
+);
+recalculate_dG();
+
+
+# Load pixmap icon data
+my (
+ $perlprimer_icon, $icon_open, $icon_open_small, $icon_save, $icon_clear,
+ $icon_info, $icon_magnify, $icon_new, $dna_canvas_pixmap,
+ $icon_separator, $icon_prefs, $icon_report, $icon_save_as,
+ $icon_ensembl, $icon_dna_open, $icon_dna_save,
+ $info_pixmap, $error_pixmap,
+);
+load_icon_data();
+
+
+ #-----------------#
+ # #
+ # GUI Interface #
+ # #
+ #-----------------#
+
+#-------------#
+# Main Window #
+#-------------#
+
+my $top = MainWindow->new(-title=>"PerlPrimer v$version");
+$top->withdraw();
+
+# configure fonts
+my $gui_font = "{$gui_font_face} $gui_font_size";
+my $font = $gui_font;
+my $gui_font_bold = $gui_font . ' bold';
+my $text_font = "{$text_font_face} $text_font_size";
+my $list_font = "{$list_font_face} $list_font_size";
+
+unless ($font_override) {
+ $top->optionAdd("*font", $gui_font);
+ $top->optionAdd("*ROText.font", $text_font);
+ $top->optionAdd("*Text.font", $text_font);
+ $top->optionAdd("*HList.font", $list_font);
+}
+
+# general options
+$top->optionAdd("*Entry.relief", 'groove');
+$top->optionAdd("*Entry.background", '#eeeeee');
+
+$top->optionAdd("*Text.relief", 'groove');
+$top->optionAdd("*Text.background", '#eeeeee');
+
+$top->optionAdd("*ROText.relief", 'groove');
+$top->optionAdd("*ROText.background", '#eeeeee');
+
+$top->optionAdd("*HList.relief", 'groove');
+$top->optionAdd("*HList.background", '#eeeeee');
+$top->optionAdd("*HList.header", 1);
+$top->optionAdd("*HList.selectmode", 'extended');
+
+$top->optionAdd("*Label.justify", 'left');
+
+$top->optionAdd("*Button.padY", $button_pady);
+$top->optionAdd("*Button.padX", 5);
+
+$top->optionAdd("*Checkbutton.padY", $check_pady);
+
+$top->optionAdd("*LabFrame.LabelSide", 'acrosstop');
+# $top->optionAdd("*LabFrame.background", '#eeeeee');
+
+# $top->optionAdd("*BrowseEntry.background", '#eeeeee');
+
+
+#--------------#
+# Balloon help #
+#--------------#
+
+# Set the balloon background colour (nobody actually *likes* the default sick yellow
+# colour, do they??)
+$top->optionAdd("*Balloon.Background", "#ececec");
+
+my $Balloon = $top->Balloon();
+$Balloon->configure(-state => 'none') if $balloon_help == 0;
+$Balloon->configure(-state => 'balloon') if $balloon_help == 1;
+
+# Notebook reference hash
+my %nb_page_ref = (
+ 1 => 'pd',
+ 2 => 'bis',
+ 3 => 'qpcr',
+ 4 => 'seq',
+ );
+
+# Load user-defined default variables
+load_defaults();
+
+
+#---------#
+# Menubar #
+#---------#
+
+# It seems impossible to get the same menu behaviour across platforms with the
+# same code! This is a compromise which seems to work on my systems. Hopefully
+# this will hold for other systems too ...
+
+my $menu = $top->Menu(-bd => 1, -relief => $menu_relief, -type=>'menubar');
+
+# setting $menu as the -menu for $top causes geometry problems under *nix ...
+# packing the menubar as a widget causes alignment problems under Win32 ...
+# (sometimes I think it was easier with menubuttons ....)
+$menu->pack(-fill=>'x') if $os eq 'nix';
+$top->configure(-menu => $menu) if $os eq 'win';
+
+# recently used items
+my $menu_mru = $menu->Menu(-title => "Recently opened", -menuitems => [
+ [command => "- none available -", -state => "disabled" ]
+ ]);
+recently_used_files();
+
+my $menu_file = $menu->cascade(-label => "File", -menuitems => [
+ [command => "New file", -command =>\&new_file, -accelerator=>'Ctrl-N' ],
+ [command => "Open ...", -command =>\&pp_file_open, -accelerator=>'Ctrl-O' ],
+ [cascade => "Open Previous", -menu=>$menu_mru ],
+ "-",
+ [command => "Save ...", -command =>\&pp_file_save, -accelerator=>'Ctrl-S' ],
+ [command => "Save as ...", -command =>[sub {pp_file_save(1)}], ],
+ "-",
+ [command => "Retrieve gene from Ensembl", -command =>\&get_ensembl, -accelerator=>'Ctrl-E' ],
+ "-",
+ [command => "Restart", -command => \&restart, ],
+ [command => "Exit", -command => \&end_prog, -accelerator=>'Ctrl-Q' ]
+ ]);
+
+my $menu_tools = $menu->cascade(-label => 'Tools', -menuitems => [
+ [command => "Find primers for cloning", -command => \&get_primers_cloning, ],
+ [command => "Add cloning sequences", -command => \&find_re_sites, ],
+ "-",
+ [command => "Generate report", -command => \&generate_report, -accelerator=>'Ctrl-R' ],
+ "-",
+ [command => "Save default values for this page", -command => \&save_defaults, ],
+ [command => "Restore in-built default values", -command => \&restore_defaults, ],
+ "-",
+ [command => "Preferences", -command => \&prefs, -accelerator=>'Ctrl-P' ],
+ ]);
+
+my $menu_help = $menu->cascade(-label => "Help", -menuitems => [
+ ['Checkbutton' => "Balloon help", -variable => \$balloon_help, -command => \&balloon_toggle],
+ "-",
+ [command => "Acknowledgements", -command => \&acknowledgements ],
+ [command => "About ...", -command => \&info, -accelerator=>'Ctrl-Shift-A' ],
+ ]);
+
+
+#--------------#
+# Key bindings #
+#--------------#
+
+# If you're wondering about the anonymous sub for file opening - it's because
+# Perl/Tk sends the calling widget as the first argument ...
+
+$top->bind('<Control-n>' => \&new_file);
+$top->bind('<Control-o>' => [sub {pp_file_open()}]);
+$top->bind('<Control-s>' => [sub {pp_file_save()}]);
+$top->bind('<Control-q>' => \&end_prog);
+$top->bind('<Control-r>' => \&generate_report);
+$top->bind('<Control-p>' => \&prefs);
+$top->bind('<Control-A>' => \&info);
+$top->bind('<Control-e>' => \&get_ensembl);
+
+
+#---------#
+# Toolbar #
+#---------#
+
+my $separator_menu = $top->Frame(-relief => "groove", -height=>2, -bd => 2);
+$separator_menu->pack(-fill=> 'x') if $os eq 'win';
+
+ my $toolbar = $top->Frame(-relief => $menu_relief, -bd => 1) ->pack(-fill=> 'x');
+ my $tool_new = pack_button($toolbar, $top->Pixmap(-data => $icon_new), \&new_file)->pack(-side=>'left');
+ $Balloon->attach($tool_new, -balloonposition => 'mouse', -balloonmsg => "New file");
+ my $tool_open = pack_button($toolbar, $top->Pixmap(-data => $icon_open), \&pp_file_open)->pack(-side=>'left');
+ $Balloon->attach($tool_open, -balloonposition => 'mouse', -balloonmsg => "Open PerlPrimer file");
+ my $tool_save = pack_button($toolbar, $top->Pixmap(-data => $icon_save), \&pp_file_save)->pack(-side=>'left');
+ $Balloon->attach($tool_save, -balloonposition => 'mouse', -balloonmsg => "Save file");
+ my $tool_save_as = pack_button($toolbar, $top->Pixmap(-data => $icon_save_as), [sub {pp_file_save(1)}])->pack(-side=>'left');
+ $Balloon->attach($tool_save_as, -balloonposition => 'mouse', -balloonmsg => "Save file as ...");
+
+my $tool_sep1 = $toolbar->Label(-image => $top->Pixmap(-data => $icon_separator))->pack(-side=>'left', -padx=>2);
+
+ my $tool_ensembl = pack_button($toolbar, $top->Pixmap(-data => $icon_ensembl), \&get_ensembl)->pack(-side=>'left');
+ $Balloon->attach($tool_ensembl, -balloonposition => 'mouse', -balloonmsg => "Retrieve gene from Ensembl");
+
+my $tool_sep2 = $toolbar->Label(-image => $top->Pixmap(-data => $icon_separator))->pack(-side=>'left', -padx=>2);
+
+ my $tool_prefs = pack_button($toolbar, $top->Pixmap(-data => $icon_prefs), \&prefs)->pack(-side=>'left');
+ $Balloon->attach($tool_prefs, -balloonposition => 'mouse', -balloonmsg => "Preferences");
+
+ my $tool_report = pack_button($toolbar, $top->Pixmap(-data => $icon_report), \&generate_report)->pack(-side=>'left');
+ $Balloon->attach($tool_report, -balloonposition => 'mouse', -balloonmsg => "Generate report");
+
+my $separator_tools = $top->Frame(-relief => "groove", -height=>2, -bd => 2);
+$separator_tools->pack(-fill=> 'x') if $os eq 'win';
+
+#---------------#
+# Draw Notebook #
+#---------------#
+
+# notebook inactivebackground calculation
+# (uses a 90% shade of the default widget colour - as per Perl/Tk *nix defaults.
+# Win32 does not use this by default, but I feel it improves usability)
+my $nb_colour = $top->cget(-bg);
+my ($red, $green, $blue) = $top->rgb($nb_colour);
+$red *= 0.9;
+$green *= 0.9;
+$blue *= 0.9;
+$nb_colour = sprintf "%lx%lx%lx", $red, $green, $blue;
+
+my $nb = $top->NoteBook(
+ -relief => 'raised',
+ -inactivebackground => "#$nb_colour",
+ -bd => 1,
+ )->pack(
+ -expand=>1,
+ -padx=>2,
+ -pady=>4,
+ -fill=>'both',
+ );
+
+# Notebook pages
+my $page_primer_design = $nb->add("pd", -label=>"Standard PCR", -anchor=>"nw");
+my $page_bisul_seq = $nb->add("bis", -label=>"Bisulphite PCR", -anchor=>"nw");
+my $page_qpcr = $nb->add("qpcr", -label=>"Real-time PCR", -anchor=>'nw');
+my $page_seq = $nb->add("seq", -label=>"Sequencing", -anchor=>"nw");
+my $page_primer = $nb->add("primer", -label=>"Primers", -anchor=>"nw");
+
+# Event binding to allow name display
+$page_primer_design->bind('<Visibility>' => \&update_title);
+$page_bisul_seq->bind('<Visibility>' => \&update_title);
+$page_qpcr->bind('<Visibility>' => \&update_title);
+$page_seq->bind('<Visibility>' => \&update_title);
+
+# Have to use frames to get the nw anchor - grid plonks everything in the centre ...
+my $page_primerf = $page_primer->Frame()->pack(-anchor=>'nw', -expand=>'1', -fill=>'both');
+my $page_primerfb = $page_primer->Frame()->pack(-side=>'bottom', -anchor=>'sw');
+
+my $page_primer_designf = $page_primer_design->Frame()->pack(-anchor=>'nw', -expand=>'1', -fill=>'both');
+my $page_primer_designfb = $page_primer_design->Frame()->pack(-side=>'bottom', -anchor=>'sw');
+
+my $page_sequencingf = $page_seq->Frame()->pack(-anchor=>'nw', -expand=>'1', -fill=>'both');
+my $page_sequencingfb = $page_seq->Frame()->pack(-side=>'bottom', -anchor=>'sw');
+
+my $page_bisul_seqf = $page_bisul_seq->Frame()->pack(-anchor=>'nw', -expand=>'1', -fill=>'both');
+my $page_bisul_seqfb = $page_bisul_seq->Frame()->pack(-side=>'bottom', -anchor=>'sw');
+
+my $page_qpcrf = $page_qpcr->Frame()->pack(-anchor=>'nw', -expand=>'1', -fill=>'both');
+my $page_qpcrfb = $page_qpcr->Frame()->pack(-side=>'bottom', -anchor=>'sw');
+
+#------------#
+# Status bar #
+#------------#
+
+my $status_bar_f = $top->Frame()->pack(-side=>'bottom', -fill=>'x', -padx=>0,);
+my $status_bar = $status_bar_f->ROText(-height=>1,
+ -bg=>'#eeeeee',
+ -relief=>'sunken',
+ )->pack(-fill=>'x');
+
+$status_bar->tagConfigure('red',
+ -foreground => 'red');
+$status_bar->tagConfigure('blue',
+ -foreground => 'blue');
+$status_bar->tagConfigure('grey',
+ -foreground => 'grey20');
+
+tie (*SBAR, 'Tk::Text', $status_bar);
+
+# Packing system
+#
+# IMPORTANT UPDATE: reliance on grid() has now been greatly reduced (only used for labframe
+# widgets) - each widget row is now separately defined by the nr() subroutine and widgets
+# are packed from the left until nr() is called again. Much simpler and easier!
+# (NB - GUI code still has lots of relic options in the pack_gui() calls - needs to be
+# tidied up a lot!)
+#
+# FURTHER UPDATE: as of versions > 1.1.4a, the system has been completely re-written, providing
+# a simpler and more straightforward system with the ability to include any number of custom arguments.
+# It should be slightly faster, too, at the expense of some code verbosity.
+#
+# New format is:
+# <widget type> <Text/variable> <widget hash reference> [more specific args] [generic Perl/Tk options]
+#
+# Widgets are packed from the left into horizontal frames defined by the nr() call
+
+my @row_counter;
+
+#-------------#
+# Primer page #
+#-------------#
+
+pack_gui('LabFrame', 'Forward primer', 'forward_l', \$page_primerf);
+ nr(\$packed_widgets{forward_l});
+ pack_gui('Label', 'Sequence', 'fprimerseql');
+ pack_gui('Entry', \$fprimer, 'fprimerseq', 40);
+ nr();
+ my $col_ref = $row_counter[-1];
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'Tm: ', 'fprimertm', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_tm, 'fprimertm');
+ pack_gui('Label', '°C', 'fprimertm');
+ nr('',0);
+ pack_gui('Label', 'dS°: ', 'fprimerds', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_ds, 'fprimerds');
+ pack_gui('Label', 'eu', 'fprimerds');
+ # nr('',0);
+ # pack_gui('Label', "dG°$pd_temperature: ", 'fprimerds');
+ # pack_gui('Label', \$fprimer_dg, 'fprimerds');
+ # pack_gui('Label', 'kcal/mol', 'fprimerds');
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'Length: ', 'fprimerlen', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_len, 'fprimerlen');
+ pack_gui('Label', 'bases', 'fprimerlen');
+ nr('',0);
+ pack_gui('Label', 'dH°: ', 'fprimerdh', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_dh, 'fprimerdh');
+ pack_gui('Label', 'kcal/mol', 'fprimerdh');
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'GC: ', 'fprimergc', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_gc, 'fprimergc');
+ pack_gui('Label', '%', 'fprimergc');
+ nr('',0);
+ pack_gui('Label', "dG°$pd_temperature: ", 'fprimerds', -font=>$gui_font_bold);
+ pack_gui('Label', \$fprimer_dg, 'fprimerds');
+ pack_gui('Label', 'kcal/mol', 'fprimerds');
+
+
+pack_gui('LabFrame', 'Reverse primer', 'reverse_l', \$page_primerf);
+ nr(\$packed_widgets{reverse_l});
+
+ pack_gui('Label', 'Sequence', 'rprimerseq');
+ pack_gui('Entry', \$rprimer, 'rprimerseq', 40);
+ nr();
+ $col_ref = $row_counter[-1];
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'Tm: ', 'rprimertm', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_tm, 'rprimertm');
+ pack_gui('Label', '°C', 'rprimertm');
+ nr('',0);
+ pack_gui('Label', 'dS°: ', 'rprimerds', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_ds, 'rprimerds');
+ pack_gui('Label', 'eu', 'rprimerds');
+ # nr('',0);
+ # pack_gui('Label', "dG°$pd_temperature: ", 'rprimerds');
+ # pack_gui('Label', \$rprimer_dg, 'rprimerds');
+ # pack_gui('Label', 'kcal/mol', 'rprimerds');
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'Length: ', 'rprimerlen', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_len, 'rprimerlen');
+ pack_gui('Label', 'bases', 'rprimerlen');
+ nr('',0);
+ pack_gui('Label', 'dH°: ', 'rprimerdh', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_dh, 'rprimerdh');
+ pack_gui('Label', 'kcal/mol', 'rprimerdh');
+ nc(\$col_ref);
+ nr(\$row_counter[-1],0);
+ pack_gui('Label', 'GC: ', 'fprimergc', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_gc, 'fprimergc');
+ pack_gui('Label', '%', 'fprimergc');
+ nr('',0);
+ pack_gui('Label', "dG°$pd_temperature: ", 'fprimerds', -font=>$gui_font_bold);
+ pack_gui('Label', \$rprimer_dg, 'fprimerds');
+ pack_gui('Label', 'kcal/mol', 'fprimerds');
+
+pack_gui('LabFrame', 'Dimers', 'dim_l', \$page_primerf);
+ nr(\$packed_widgets{dim_l}, $frame_pady, 1);
+ pack_gui('ROText', 'Dimers', 'dim', 60, 15, -scrollbars=>'oe');
+ $packed_widgets{dim}->bind('<Key-Right>' => \&jump_to_tm);
+ $packed_widgets{dim}->bind('<Key-Left>' => \&jump_back);
+ $packed_widgets{dim}->bind('<Return>' => \&jump_back);
+
+nr(\$page_primerfb, 0);
+ pack_gui('Button', 'Calculate Tm', 'tmbutton', \&get_tm, 'active');
+ pack_gui('Button', 'BLAST primers', 'blastbutton', \&blast_primers);
+
+# Grid layout
+pack_grid(qw(forward_l));
+pack_grid(qw(reverse_l));
+pack_grid(qw(dim_l));
+
+$packed_widgets{dim}->configure(-fg=>'grey30');
+$packed_widgets{dim}->tagConfigure('blue',
+ -foreground => 'midnightblue');
+$packed_widgets{dim}->tagConfigure('red',
+ -foreground => 'red');
+$packed_widgets{dim}->tagConfigure('black',
+ -foreground => 'black');
+
+# Column and row weights
+$page_primerf->gridColumnconfigure(0,-weight=>1);
+$page_primerf->gridRowconfigure(2,-weight=>1);
+
+
+tie (*DIMER, 'Tk::Text',$packed_widgets{'dim'});
+
+#--------------------#
+# Primer Design page #
+#--------------------#
+
+pack_gui('LabFrame', 'Primer Tm', 'primer_tm_l', \$page_primer_designf);
+ nr(\$packed_widgets{primer_tm_l});
+ pack_gui('Entry', \$min_tm_pr, 'mintm', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_tm_pr, 'maxtm', 3);
+ pack_gui('Label', '°C Difference');
+ pack_gui('Entry', \$max_diff_pr, 'maxdiff', 3);
+ pack_gui('Label', '°C');
+
+pack_gui('LabFrame', 'Primer Length', 'primer_len_l', \$page_primer_designf);
+ nr(\$packed_widgets{primer_len_l});
+ pack_gui('Entry', \$pri_win_min_pr, 'minwin', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$pri_win_max_pr, 'maxwin', 3);
+ pack_gui('Label', 'bases');
+
+pack_gui('LabFrame', 'Amplified range', 'primer_range_l', \$page_primer_designf);
+ nr(\$packed_widgets{primer_range_l}, 2);
+ pack_gui('Label', "5'");
+ pack_gui('Entry', \$max_range_5p_pr, 'maxrange5p', 5);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$min_range_pr, 'minrange', 5);
+ pack_gui('Label', " 3'");
+ pack_gui('Entry', \$max_range_pr, 'maxrange', 5);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_range_3p_pr, 'maxrange3p', 5);
+ nr('');
+ pack_gui('Label', 'Amplicon size: ');
+ pack_gui('Label', \$min_ampsize_pr);
+ pack_gui('Label', '-');
+ pack_gui('Label', \$max_ampsize_pr);
+ pack_gui('Label', 'bases');
+ nr('', 0);
+ pack_gui('Button', 'Set from ORF', 'primer_getgene', \&reset_bounds);
+ pack_gui('Button', '-10', 'primer_stepin', \&step_in);
+ pack_gui('Button', '+10', 'primer_stepout', \&step_out);
+
+pack_gui('LabFrame', 'Options', 'primer_options_l', \$page_primer_designf);
+ nr(\$packed_widgets{primer_options_l});
+ pack_gui('Checkbutton', 'Exclude %GC', 'exclude_gc', \$exclude_gc);
+ pack_gui('Checkbutton', 'GC clamp', 'gc_clamp', \$exclude_clamp);
+ nr();
+ pack_gui('Label', "Add 5' F seq ");
+ pack_gui('Entry', \$primer_seq_5f, 'primer_seq_5f', 12);
+ pack_gui('Label', 'Frame');
+ pack_gui('Entry', \$primer_seq_5f_frame, 'primer_seq_5f_frame', 2);
+ # not currently implemented and may never be ...
+ # pack_gui('ATG', $primer_seq_5f_atg, 'primer_seq_5f_atg', '', 'c', 0, 1);
+ nr();
+ pack_gui('Label', "Add 5' R seq ");
+ pack_gui('Entry', \$primer_seq_5r, 'primer_seq_5r', 12);
+ pack_gui('Label', 'Frame');
+ pack_gui('Entry', \$primer_seq_5r_frame, 'primer_seq_5r_frame', 2);
+ # pack_gui('ATG', $primer_seq_5r_atg, 'primer_seq_5r_atg', '', 'c', 0, 1);
+
+pack_gui('LabFrame', 'Sequence', 'seq_l', \$page_primer_designf);
+ nr(\$packed_widgets{seq_l});
+ pack_gui('Text', 'Sequence', 'seq', 60, 6, -scrollbars=>'oe');
+ pack_menu('seq');
+ pack_osc_buttons('seq');
+
+pack_gui('LabFrame', 'Results', 'res_l', \$page_primer_designf);
+ nr(\$packed_widgets{res_l}, $frame_pady, 1);
+ pack_gui('HList', '', 'res', 60, 10, 11, \&browse_primer);
+ header_create(\$packed_widgets{'res'}, @header_list_primers);
+
+ nr();
+ pack_gui('Canvas', '', 'primer_canvas', 1, 1);
+
+nr(\$page_primer_designfb, 0);
+pack_gui('Button', 'Find primers', 'primerbutton', \&get_primers, 'active');
+pack_gui('Button', 'Find inwards', 'autobuttonin', \&get_primers_auto_in);
+pack_gui('Button', 'Find outwards', 'autobuttonout', \&get_primers_auto_out);
+pack_gui('Button', 'Cancel', 'primer_cancel', \&cancel);
+pack_gui('Button', 'Copy selected', 'primer_view', \©_selected_primers);
+
+# Grid layout
+pack_grid(qw(primer_tm_l primer_len_l));
+pack_grid(qw(primer_range_l primer_options_l));
+pack_grid(qw(seq_l -));
+pack_grid(qw(res_l -));
+
+# Column and row weights
+$page_primer_designf->gridColumnconfigure(0,-weight=>1);
+$page_primer_designf->gridColumnconfigure(1,-weight=>1);
+$page_primer_designf->gridRowconfigure(3,-weight=>1);
+
+
+#-----------------#
+# Sequencing page #
+#-----------------#
+
+pack_gui('LabFrame', 'Primer Tm', 'seq_tm_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_tm_l});
+ pack_gui('Entry', \$min_tm_seq, 'smintm', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_tm_seq, 'smaxtm', 3);
+ pack_gui('Label', '°C');
+
+pack_gui('LabFrame', 'Primer Length', 'seq_len_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_len_l});
+ pack_gui('Entry', \$pri_win_min_seq, 'sminwin', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$pri_win_max_seq, 'smaxwin', 3);
+ pack_gui('Label', 'bases');
+
+pack_gui('LabFrame', 'Spacing / Coverage', 'seq_amp_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_amp_l}, 2);
+ pack_gui('Label', 'Primers every');
+ pack_gui('Entry', \$seq_spacing_min, 'sspacingmin', 4);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$seq_spacing_max, 'sspacingmax', 4);
+ pack_gui('Label', 'bases');
+ nr('');
+ pack_gui('Label', "Range: 5'");
+ pack_gui('Entry', \$min_range_seq, 'seqminrange', 5);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_range_seq, 'seqmaxrange', 5);
+ pack_gui('Label', "3'");
+ nr('', 0);
+ pack_gui('Button', 'Set range from ORF', 'seq_getgene', \&reset_bounds);
+
+pack_gui('LabFrame', 'Options', 'seq_options_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_options_l}, 0);
+ pack_gui('Checkbutton', 'Exclude %GC', 'exclude_gc_seq', \$exclude_gc_seq);
+ pack_gui('Checkbutton', 'GC clamp', 'gc_clamp_seq', \$exclude_clamp_seq);
+ nr('', 0);
+ pack_gui('Checkbutton', 'Exclude self-complimentarity > -', 'exclude_pd_seq', \$exclude_pd_seq);
+ pack_gui('Entry', \$seq_pd_min, 'spdmin', 3);
+ pack_gui('Label', 'dG°37');
+
+pack_gui('LabFrame', 'Sequence', 'seq_seq_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_seq_l});
+ pack_gui('Text', 'Sequence', 'seq_seq', 60, 6, -scrollbars=>'oe');
+ pack_menu('seq_seq');
+ pack_osc_buttons('seq_seq');
+
+pack_gui('LabFrame', 'Results', 'seq_res_l', \$page_sequencingf);
+ nr(\$packed_widgets{seq_res_l}, $frame_pady, 1);
+ pack_gui('HList', '', 'seq_res', 60, 10, 11, \&browse_primer);
+ header_create(\$packed_widgets{'seq_res'}, @header_list_primers);
+
+ nr();
+ pack_gui('Canvas', '', 'seq_canvas', 1, 1);
+
+nr(\$page_sequencingfb, 0);
+pack_gui('Button', 'Find primers', 'seq_button', \&get_seq_primers, 'active');
+pack_gui('Button', 'Cancel', 'seq_cancel', \&cancel);
+pack_gui('Button', 'Copy selected', 'seq_view', \©_selected_primers);
+
+# Grid layout
+pack_grid(qw(seq_tm_l seq_len_l));
+pack_grid(qw(seq_amp_l seq_options_l));
+pack_grid(qw(seq_seq_l -));
+pack_grid(qw(seq_res_l -));
+
+# Column and row weights
+$page_sequencingf->gridColumnconfigure(0,-weight=>1);
+$page_sequencingf->gridColumnconfigure(1,-weight=>1);
+$page_sequencingf->gridRowconfigure(3,-weight=>1);
+
+
+#----------------------------#
+# Bisulphite sequencing page #
+#----------------------------#
+
+pack_gui('LabFrame', 'Primer Tm', 'bisul_tm_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_tm_l});
+ pack_gui('Entry', \$min_tm_bs, 'bisul_mintm', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_tm_bs, 'bisul_maxtm', 3);
+ pack_gui('Label', '°C Difference');
+ pack_gui('Entry', \$max_diff_bs, 'bisul_maxdiff', 3);
+ pack_gui('Label', '°C');
+
+pack_gui('LabFrame', 'Primer Length', 'bisul_len_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_len_l});
+ pack_gui('Entry', \$pri_win_min_bs, 'minwin', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$pri_win_max_bs, 'maxwin', 3);
+ pack_gui('Label', 'bases');
+
+pack_gui('LabFrame', 'Amplified range', 'bisul_range_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_range_l}, 2);
+ pack_gui('Label', "5'");
+ pack_gui('Entry', \$max_range_5p_bs, 'bisul_maxrange5p', 5);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$min_range_bs, 'bisul_minrange', 5);
+ pack_gui('Label', " 3'");
+ pack_gui('Entry', \$max_range_bs, 'bisul_maxrange', 5);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_range_3p_bs, 'bisul_maxrange3p', 5);
+ nr('');
+ pack_gui('Label', 'Amplicon size: ');
+ pack_gui('Label', \$min_ampsize_bs);
+ pack_gui('Label', '-');
+ pack_gui('Label', \$max_ampsize_bs);
+ pack_gui('Label', 'bases');
+ nr('', 0);
+ pack_gui('Button', 'Set from CpG island', 'bisul_getcpg', \&reset_bounds);
+ pack_gui('Button', '-10', 'primer_stepin', \&step_in);
+ pack_gui('Button', '+10', 'primer_stepout', \&step_out);
+
+pack_gui('LabFrame', 'Options', 'bisul_options_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_options_l}, 0);
+ pack_gui('Checkbutton', 'Exclude CpG', 'bisul_exclude_cpg', \$exclude_cpg);
+ pack_gui('Checkbutton', "Require 3' C", 'bisul_exclude_cs', \$exclude_3c);
+ nr('', 0);
+ pack_gui('Checkbutton', 'Repeats / runs post conversion', 'bisul_pre_bs', \$pre_bs);
+ nr();
+ pack_gui('Label', 'Minimum primer C content ');
+ pack_gui('Entry', \$bisul_min_c, 'bisul_min_c', 4);
+ pack_gui('Label', '%');
+
+pack_gui('LabFrame', 'Sequence', 'bisul_seq_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_seq_l});
+ pack_gui('Text', 'Sequence', 'bisul_seq', 60, 6, -scrollbars=>'oe');
+ pack_menu('bisul_seq');
+ pack_osc_buttons('bisul_seq');
+
+pack_gui('LabFrame', 'Results', 'bisul_res_l', \$page_bisul_seqf);
+ nr(\$packed_widgets{bisul_res_l}, $frame_pady, 1);
+ pack_gui('HList', '', 'bisul_res', 60, 10, 11, \&browse_bisulphite);
+ header_create(\$packed_widgets{'bisul_res'}, @header_list_primers);
+
+ nr();
+ pack_gui('Canvas', '', 'bisul_canvas', 1, 1);
+
+nr(\$page_bisul_seqfb, 0);
+pack_gui('Button', 'Find primers', 'bisul_button', \&get_bisulphite, 'active');
+pack_gui('Button', 'Find inwards', 'bisul_autobuttonin', \&get_primers_auto_in);
+pack_gui('Button', 'Find outwards', 'bisul_autobuttonout', \&get_primers_auto_out);
+pack_gui('Button', 'Cancel', 'bisul_cancel', \&cancel);
+pack_gui('Button', 'Copy selected', 'bisul_view', \©_selected_primers);
+
+# Grid layout
+pack_grid(qw(bisul_tm_l bisul_len_l));
+pack_grid(qw(bisul_range_l bisul_options_l));
+pack_grid(qw(bisul_seq_l -));
+pack_grid(qw(bisul_res_l -));
+
+# Column and row weights
+$page_bisul_seqf->gridColumnconfigure(0,-weight=>1);
+$page_bisul_seqf->gridColumnconfigure(1,-weight=>1);
+$page_bisul_seqf->gridRowconfigure(3,-weight=>1);
+
+
+#-----------#
+# QPCR page #
+#-----------#
+
+pack_gui('LabFrame', 'Primer Tm', 'qprimer_tm_l', \$page_qpcrf);
+ nr(\$packed_widgets{qprimer_tm_l});
+ pack_gui('Entry', \$min_tm_q, 'qmintm', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_tm_q, 'qmaxtm', 3);
+ pack_gui('Label', '°C Difference');
+ pack_gui('Entry', \$max_diff_q, 'qmaxdiff', 3);
+ pack_gui('Label', '°C');
+
+pack_gui('LabFrame', 'Primer Length', 'qprimer_len_l', \$page_qpcrf);
+ nr(\$packed_widgets{qprimer_len_l});
+ pack_gui('Entry', \$pri_win_min_q, 'qminwin', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$pri_win_max_q, 'qmaxwin', 3);
+ pack_gui('Label', 'bases');
+
+pack_gui('LabFrame', 'Amplicon size', 'qprimer_amp_l', \$page_qpcrf);
+ nr(\$packed_widgets{qprimer_amp_l});
+ pack_gui('Entry', \$min_ampsize_q, 'qminamp', 4);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_ampsize_q, 'qmaxamp', 4);
+ pack_gui('Label', 'bases');
+ nr();
+ pack_gui('Checkbutton', 'Limit primers to exon(s)', 'qie_limit', \$ie_limit);
+ pack_gui('Entry', \$ie_limit_5p, 'qie_limit_5p', 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$ie_limit_3p, 'qie_limit_3p', 3);
+
+pack_gui('LabFrame', 'Options', 'qprimer_options_l', \$page_qpcrf);
+ nr(\$packed_widgets{qprimer_options_l}, 0);
+ pack_gui('Checkbutton', 'Exclude %GC', 'qexclude_gc', \$exclude_gc);
+ pack_gui('Checkbutton', 'GC clamp', 'qgc_clamp', \$exclude_clamp);
+ nr();
+ pack_gui('Checkbutton', 'Span intron/exon boundary', 'qie_span', \$ie_span);
+ nr();
+ pack_gui('Checkbutton', 'Overlap intron/exon boundary by', 'qie_overlap', \$ie_overlap);
+ pack_gui('Entry', \$exclude_ie, 'qexclude_ie', 3);
+ pack_gui('Label', 'bases');
+
+pack_gui('LabFrame', 'Genomic sequence', 'qdna_seq_l', \$page_qpcrf);
+ nr(\$packed_widgets{qdna_seq_l});
+ pack_gui('Text', '', 'qdna_seq', 25, 6, -scrollbars=>'oe');
+ pack_menu('qdna_seq');
+ pack_osc_buttons('qdna_seq');
+
+pack_gui('LabFrame', 'mRNA sequence', 'qmrna_seq_l', \$page_qpcrf);
+ nr(\$packed_widgets{qmrna_seq_l});
+ pack_gui('Text', '', 'qmrna_seq', 25, 6, -scrollbars=>'oe');
+ pack_menu('qmrna_seq');
+ pack_osc_buttons('qmrna_seq');
+
+pack_gui('LabFrame', 'Results', 'qpcr_res_l', \$page_qpcrf);
+ nr(\$packed_widgets{qpcr_res_l}, $frame_pady, 1);
+ pack_gui('HList', '', 'qpcr_res', 60, 10, 11, \&browse_primer);
+ header_create(\$packed_widgets{'qpcr_res'}, @header_list_primers);
+
+ nr();
+ pack_gui('Canvas', '', 'qprimer_canvas', 1, 1);
+
+nr(\$page_qpcrfb, 0);
+pack_gui('Button', 'Find primers', 'qprimerbutton', \&get_qprimers, 'active');
+pack_gui('Button', 'Cancel', 'qprimer_cancel', \&cancel);
+pack_gui('Button', 'Copy selected', 'qprimer_view', \©_selected_primers);
+pack_gui('Button', 'View intron/exon structure', 'qprimer_view_ie', \&view_intron_exon_structure);
+pack_gui('Button', 'Spidey output', 'qprimer_spidey', \&view_spidey_out);
+
+# Grid layout
+pack_grid(qw(qprimer_tm_l qprimer_len_l));
+pack_grid(qw(qprimer_amp_l qprimer_options_l));
+pack_grid(qw(qdna_seq_l qmrna_seq_l));
+pack_grid(qw(qpcr_res_l -));
+
+# Column and row weights
+$page_qpcrf->gridColumnconfigure(0,-weight=>1);
+$page_qpcrf->gridColumnconfigure(1,-weight=>1);
+$page_qpcrf->gridRowconfigure(3,-weight=>1);
+
+
+#-------------#
+# Window icon #
+#-------------#
+
+my $pixmap = $top->Pixmap(-data => $perlprimer_icon);
+
+# set icon for main window ...
+$top->Icon(-image => $pixmap);
+
+
+#-------------#
+# Popup Menus #
+#-------------#
+
+# A big problem with perl/Tk:
+# HList does not have any way to bind to column headers or even
+# to identify in which column a click was - this is the only way to
+# provide sorting ... It's very clumsy!
+
+# (update: after using this for a while, I actually think it works rather well
+# for this system - since not all the Hlist column headers are generally visible
+# at any one time, it's nice to have this system in place)
+
+my $sort_reverse=0;
+my $sort_prev = 10;
+
+$popup_sort = $top->Menu(-menuitems => [
+ [cascade => 'Sort primers by ...', -menuitems => [
+ [command => 'Forward position', -command => [\&sort_primers, 1]],
+ [command => ' ... length', -command => [\&sort_primers, 2]],
+ [command => ' ... Tm', -command => [\&sort_primers, 3]],
+ '-',
+ [command => 'Reverse position', -command => [\&sort_primers, 8]],
+ [command => ' ... length', -command => [\&sort_primers, 6]],
+ [command => ' ... Tm', -command => [\&sort_primers, 7]],
+ '-',
+ [command => 'Amplicon size', -command => [\&sort_primers, 9]],
+ [command => 'Extensible Dimer dG', -command => [\&sort_primers, 10]],
+ [command => 'Full Dimer dG', -command => [\&sort_primers, 13]],
+ '-',
+ [Checkbutton => 'Reversed', -variable => \$sort_reverse, -command =>[\&sort_primers, "r"]],
+ ]],
+ '-',
+ [command => 'Select all', -command => [\&select_all_primers]],
+ [command => 'Copy', -command => [\©_selected_primers]],
+ '-',
+ [command => 'Set range from selected', -command => [\&primer_take_range]],
+ ]);
+
+$popup_sort_seq = $top->Menu(-menuitems => [
+ [Button => 'Select all', -command => [\&select_all_primers]],
+ '-',
+ [Button => 'Copy', -command => [\©_selected_primers]],
+ ]);
+
+# Text menu
+my $popup_text = $top->Menu(-menuitems => [
+ [Button => 'Open sequence ...', -command => [\&open_seq, \$text_widget_ref]],
+ [Button => 'Save sequence ...', -command => [\&save_seq, \$text_widget_ref]],
+ '-',
+ [Button => 'Select All', -command => [\&select_all_text, \$text_widget_ref]],
+ [Button => 'Clear', -command => [\&clear_text, \$text_widget_ref]],
+ [Button => 'Lower case', -command => [\&lc_text, \$text_widget_ref]],
+ '-',
+ [Button => 'Reverse complement', -command => [\&reverse_complement, \$text_widget_ref]],
+ ]);
+
+# Minimize the dosbox if we're running as a standalone executable ...
+if ($win_exe && !check_packages("Win32::GUI")) {
+ # Use GUI::Hide if you want to hide the box, but personally I'd rather leave
+ # the console visible in case of debugging info.
+ Win32::GUI::Minimize(scalar(Win32::GUI::GetPerlWindow()));
+}
+
+# Show the main window
+$top->deiconify();
+
+# prevent the user from resizing the window any smaller (which makes things hidden/messy)
+my $min_width = $top->geometry;
+my ($min_x, $min_y) = ($min_width =~ /(\d.*)x(\d.*?)\+/);
+$top->minsize($min_x, $min_y);
+
+# Open file if specified on the command line
+if ($commandline) {
+ $top->update;
+ pp_file_open($commandline);
+}
+
+# Override HList bindings for left/right and other commands...
+my $class = "Tk::HList";
+$top->bind($class,'<Right>', \&jump_to_tm);
+$top->bind($class,'<Left>', \&jump_back);
+$top->bind($class,'<2>', \©_selected_primers);
+$top->bind($class,'<Control-a>', \&select_all_primers);
+$top->bind($class,'<Control-c>', \©_selected_primers);
+$top->bind($class,'<3>', \&menu_popup);
+
+# Socket code for contig viewer
+my ($sock, $sel, $sock_repeat_id);
+setup_sock();
+
+if ($win_exe) {
+ print "\nPerlPrimer started successfully\n";
+}
+
+MainLoop();
+
+# Program end
+exit_program();
+
+ #------------------------------#
+ # #
+ # Subroutines start here ... #
+ # #
+ #------------------------------#
+
+
+#------------------#
+# Post-GUI routine #
+#------------------#
+
+sub exit_program {
+ # Save updated prefs, (including Balloon help)
+ my $file_data = "";
+ foreach my $i (keys %pref_variables) {
+ my $pointer = $pref_variables{$i};
+ $file_data .= "$i = $$pointer\n";
+ }
+
+ foreach my $i (keys %pref_arrays) {
+ my $pointer = $pref_arrays{$i};
+ $file_data .= "$i = [".join(",",@$pointer)."]\n";
+ }
+
+ open (PREFS, ">$pref_file") || die "Error: could not open $pref_file for writing: $!\n";
+ print PREFS $file_data;
+ close (PREFS);
+ exit 0;
+}
+
+
+#----------------#
+# Socket reading #
+#----------------#
+
+sub setup_sock {
+ $sock = IO::Socket::INET->new(
+ Listen => SOMAXCONN,
+ Reuse => 1,
+ LocalPort => $tcp_port,
+ Proto => 'tcp',
+ );
+
+ if (defined($sock)) {
+ if ($os eq 'win') {
+ # Fileevent with sockets does not work with Win32 -
+ # here we check by manually polling the socket every second
+ use IO::Select;
+ $sel = IO::Select->new;
+ $sel->add($sock);
+ $sock_repeat_id = $top->repeat($socket_polling_interval => \&read_sock);
+ } else {
+ $top->fileevent($sock, 'readable' => \&read_sock);
+ }
+ } else {
+ # no socket available
+ print "Could not open socket at port $tcp_port\n";
+ }
+}
+
+sub read_sock {
+ # Read the socket
+ my $hand = $sock;
+
+ if ($os eq 'win') {
+ # direct polling ...
+ my(@ready) = $sel->can_read(0);
+ return if $#ready == -1; # Nothing to read ... move along ...
+ $hand = $ready[0];
+ }
+
+ my $new_sock = $hand->accept();
+
+ my $numbytes = 2048;
+ my $data = "";
+ my $count;
+ my $num = $numbytes;
+
+ while ($num==$numbytes) {
+ my $buf;
+ $num = sysread $new_sock, $buf, $numbytes;
+ unless (defined($num)) {
+ last;
+ }
+ $data .= $buf;
+ }
+
+ my (@lines) = split("\n", $data);
+
+ if ($#lines == -1) {
+ # connection has been broken or something has gone wrong
+ return;
+ } else {
+ # Open the socket data as a FASTA file
+ pp_file_open("[data from external application]", '', @lines);
+ my ($sub) = get_variables(qw(primer_sub));
+ &$sub() if $ipc_autofind;
+ }
+}
+
+
+
+#-------------------------#
+# GUI building subroutine #
+#-------------------------#
+
+# The messy guts that allow all the other GUI parts of the program to be so neat
+# and tidy. It's nothing fancy - in fact, it's downright inelegant - but it gets
+# the job done, keeps things consistent, and writing new gui code (eg, the
+# preferences dialogue) is a matter of minutes to do ...
+
+
+sub nr {
+ # New row signal
+ my ($reference, $pady, $expand) = @_;
+ $reference ||= $old_reference;
+
+ $expand ||= 0;
+ my $fill = $expand ? 'both' : 'x';
+
+ $pady = $frame_pady unless defined($pady);
+
+ push @row_counter, $$reference->Frame(
+ )->pack(-side=>'top', -anchor=>'nw', -expand=>$expand, -fill=>$fill, -pady=>$pady, -padx=>3);
+ $old_reference = $reference;
+}
+
+
+sub nc {
+ # New column signal
+ my ($reference, $padx, $pady) = @_;
+ $reference ||= $old_reference;
+
+ $padx = 4 unless defined($padx);
+ $pady = $frame_pady unless defined($pady);
+
+ push @row_counter, $$reference->Frame(
+ )->pack(-side=>'left', -anchor=>'nw', -pady=>$pady, -padx=>$padx);
+ $old_reference = $reference;
+}
+
+
+sub pack_gui {
+ my ($widget_type, $textvariable, $widget_name, @args) = @_;
+ my $top_reference = $row_counter[-1];
+ $widget_name||='null'; # makes it easy for labels, etc, that don't need balloon messages
+ my $widget_ref = \$packed_widgets{$widget_name};
+
+ # Switch on $widget_type ...
+ for ($widget_type) {
+ /^Entry/ && do {
+ my ($width, @widget_args) = @args;
+ $$widget_ref = $top_reference->$widget_type(
+ -textvariable=>$textvariable,
+ -width=>$width,
+ @widget_args,
+ )->pack(-side=>'left');
+ last; };
+
+ /^Label/ && do {
+ my (@widget_args) = @args;
+ my $text_option = ref($textvariable) ? "textvariable" : "text";
+ $$widget_ref = $top_reference->$widget_type(
+ -$text_option=>$textvariable,
+ @widget_args,
+ )->pack(-side=>'left');
+ last; };
+
+ /^Checkbutton/ && do {
+ my ($variable, @widget_args) = @args;
+ $$widget_ref = $top_reference->$widget_type(
+ -text=>$textvariable,
+ -variable=>$variable,
+ -onvalue=>1,
+ -offvalue=>0,
+ @widget_args,
+ )->pack(-side=>'left', -anchor=>'w', -pady=>0, -ipady=>0);
+ last; };
+
+ /^BrowseEntry/ && do {
+ my ($array_ref, $listwidth, @widget_args) = @args;
+ $listwidth||=20;
+ $$widget_ref = $top_reference->$widget_type(
+ -variable=>$textvariable,
+ -choices=>[@$array_ref],
+ -listwidth=>$listwidth*7,
+ -width => $listwidth,
+ @widget_args,
+ )->pack(-side=>'left', -padx=>0, -ipadx=>0);
+ last; };
+
+ /^Button/ && do {
+ my ($command, $default, $state, @widget_args) = @args;
+ $state ||= 'normal';
+ $default ||= 'normal';
+
+ $$widget_ref = $top_reference->$widget_type(
+ -text=>$textvariable,
+ -command=>\$command,
+ -state=>$state,
+ -default=>$default,
+ @widget_args,
+ )->pack(-side=>'left', -anchor=>'w', -pady=>$button_pack_pady, -padx=>$button_pack_padx);
+
+ # bind return to default button
+ if ($default eq 'active') {
+ my $parent = $$old_reference->parent;
+ $parent->bind('<Return>' => sub{$$widget_ref->invoke()});
+ }
+
+ last; };
+
+ /^LabFrame/ && do {
+ my ($frame_reference, @widget_args) = @args;
+ $$widget_ref=$$frame_reference->$widget_type(
+ # -background=>'grey50',
+ -label=>$textvariable,
+ -labelside=>'acrosstop',
+ @widget_args,
+ );
+ last; };
+
+ /Text/ && do {
+ my ($width, $height, @widget_args) = @args;
+ $$widget_ref=$top_reference->Scrolled($widget_type,
+ -width=>$width,
+ -height=>$height,
+ @widget_args,
+ )->pack(-side=>'left', -expand=>1, -fill=>'both');
+ bind_mousewheel($$widget_ref);
+ last; };
+
+ /^HList/ && do {
+ my ($width, $height, $columns, $browse_command, @widget_args) = @args;
+ $$widget_ref=$top_reference->Scrolled($widget_type,
+ -scrollbars=>'osoe',
+ -width=>$width,
+ -height=>$height,
+ -columns=>$columns,
+ -browsecmd=>\&$browse_command,
+ -command=>\&hlist_command,
+ @widget_args,
+ )->pack(-side=>'left', -expand=>1, -fill=>'both');
+ bind_mousewheel($$widget_ref);
+ last; };
+
+ /^Canvas/ && do {
+ my ($buttons, $bindings, @widget_args) = @args;
+ canvas_buttons($top_reference, $widget_name) if $buttons;
+ $$widget_ref=$top_reference->$widget_type(
+ -height=>$dna_canvas_height,
+ @widget_args,
+ )->pack(-side=>'left', -expand=>1, -fill=>'x', -padx=>6, -pady=>2);
+ bind_canvas($widget_ref) if $bindings;
+ last; };
+
+ /^Radio/ && do {
+ my (@widget_args) = @args;
+ $$widget_ref=$top_reference->$widget_type(
+ -text=>$textvariable,
+ @widget_args,
+ )->pack(-side=>'left', -anchor=>'w');
+ last; };
+
+ # Warning - an undefined widget type ...
+ print "Undefined widget: @_\n";
+ }
+
+ #--------------#
+ # Balloon help #
+ #--------------#
+
+ # This is a very easy way to attach balloons to widgets, while keeping all the help messages together
+ # (in the %balloonmsg hash above)
+
+ if ($balloonmsg{$widget_name}) {
+ $Balloon->attach($$widget_ref, -balloonposition => 'mouse', -balloonmsg => $balloonmsg{$widget_name});
+ }
+}
+
+sub pack_grid {
+ my @widgets = map {/[\-\+\^]/ ? $_ : $packed_widgets{$_}} @_;
+ Tk::grid(@widgets, -sticky=>'nsew', -padx=>4);
+}
+
+sub pack_button {
+ my ($widget, $image, $sub_ref) = @_;
+ return $widget->Button(
+ -relief => 'flat',
+ -image => $image,
+ -command=> $sub_ref,
+ -activebackground=>$activebackground_color,
+ ) if $os eq 'win';
+ return $widget->Button(
+ -relief => 'flat',
+ -image => $image,
+ -command=> $sub_ref,
+ )->pack(-side=>'left');
+}
+
+sub pack_osc_buttons {
+ #open/save/clear button stuff ...
+ my ($widget_name) = @_;
+ $packed_widgets{"$widget_name b"} = $row_counter[-1]->Frame()->pack(-side=>'left', -anchor=>'nw');
+
+ $packed_widgets{"$widget_name open"}=pack_button($packed_widgets{"$widget_name b"}, $top->Pixmap(-data => $icon_dna_open), [\&open_seq, \$packed_widgets{$widget_name}])
+ ->pack(-side=>'top', -anchor=>'w', -fill=>'x');
+ $Balloon->attach($packed_widgets{"$widget_name open"}, -balloonposition => 'mouse', -balloonmsg => "Open DNA sequence");
+
+ $packed_widgets{"$widget_name save"}=pack_button($packed_widgets{"$widget_name b"}, $top->Pixmap(-data => $icon_dna_save), [\&save_seq, \$packed_widgets{$widget_name}])
+ ->pack(-side=>'top', -anchor=>'w', -fill=>'x');
+ $Balloon->attach($packed_widgets{"$widget_name save"}, -balloonposition => 'mouse', -balloonmsg => "Save DNA sequence");
+
+ $packed_widgets{"$widget_name clear"}=pack_button($packed_widgets{"$widget_name b"}, $top->Pixmap(-data => $icon_clear), [\&clear_text, \$packed_widgets{$widget_name}])
+ ->pack(-side=>'top', -anchor=>'w', -fill=>'x');
+ $Balloon->attach($packed_widgets{"$widget_name clear"}, -balloonposition => 'mouse', -balloonmsg => "Clear sequence");
+}
+
+sub pack_menu {
+ my $name = shift;
+ $packed_widgets{$name}->menu(undef);
+ $packed_widgets{$name}->bind('<3>', \&menu_text);
+}
+
+sub canvas_buttons {
+ my ($widget_ref, $widget_name) = @_;
+
+ $packed_widgets{"$widget_name info"}=pack_button($widget_ref, $top->Pixmap(-data => $icon_info), \&canvas_info)
+ ->pack(-side=>'right', -expand=>0, -anchor=>'e', -fill=>'none');
+ $Balloon->attach($packed_widgets{"$widget_name info"}, -balloonposition => 'mouse', -balloonmsg => "Graphical display help");
+ $packed_widgets{"$widget_name magnify"}=pack_button($widget_ref, $top->Pixmap(-data => $icon_magnify), [\&dna_magnify, 0])
+ ->pack(-side=>'right', -expand=>0, -anchor=>'e', -fill=>'none');
+ $Balloon->attach($packed_widgets{"$widget_name magnify"}, -balloonposition => 'mouse', -balloonmsg => "Magnified view");
+
+}
+
+
+sub bind_canvas {
+ # Bindings for DNA canvas
+ my ($widget_ref) = @_;
+ $$widget_ref->CanvasBind('<B1-Motion>' => sub {items_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<B2-Motion>' => sub {amplicon_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<1>' => sub {items_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<2>' => sub {amplicon_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<Control-B1-Motion>' => sub {amplicon_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<Control-1>' => sub {amplicon_drag($Tk::event->x, $widget_ref)});
+ $$widget_ref->CanvasBind('<3>' => sub {dna_magnify($Tk::event->x)});
+ $$widget_ref->CanvasBind('<Control-3>', \&draw_dna);
+ $$widget_ref->CanvasBind('<Configure>' => \&draw_dna);
+}
+
+#---------------------#
+# MouseWheel bindings #
+#---------------------#
+
+sub bind_mousewheel {
+ # Thanks to Slaven Rezic, Steve Liddie and "Mastering Perl/Tk" for this routine (slightly modified) ...
+ my ($w) = @_;
+
+ if ($os eq 'win') {
+ # Windows bindings
+ # Apparently mousewheel support is now built in .... ?!
+ # $w->bind('<MouseWheel>' => [ sub {
+ # $_[0]->yview('scroll', -($_[1] / 120) * $scroll_factor, 'units')
+ # }, Ev('D') ]
+ # );
+ } else {
+ # *nix bindings
+ $w->bind('<4>' => sub {
+ $_[0]->yview('scroll', -$scroll_factor, 'units');
+ });
+ $w->bind('<5>' => sub {
+ $_[0]->yview('scroll', $scroll_factor, 'units');
+ });
+ }
+}
+
+sub update_title {
+ my $nb_page = which_nb_page();
+ if ($open_file{$nb_page}) {
+ if ($open_file{$nb_page} eq 'File not saved') {
+ $top->configure(-title=>"PerlPrimer v$version");
+ return;
+ } else {
+ my $file = $open_file{$nb_page};
+ $top->configure(-title=>"PerlPrimer v$version - $file");
+ }
+ }
+ return;
+}
+
+#-------------------#
+# Read DNA sequence #
+#-------------------#
+
+sub read_windows {
+ my ($sub_ref, $dnaseq_f, $dnaseq_f_len, $dnaseq_r) = @_;
+
+ $reverse=0;
+ sbarprint("\nWindowing forward seq ...");
+ @PF = &$sub_ref($dnaseq_f, $dnaseq_f_len);
+ $pfkeys=@PF;
+
+ if ($dnaseq_r) {
+ $reverse=1;
+ sbarprint("\nWindowing reverse seq ...");
+ @PR = &$sub_ref($dnaseq_r, $dnaseq_f_len);
+ }
+}
+
+
+#-------------------------------------------------#
+# basic complementation and bisulphite conversion #
+#-------------------------------------------------#
+
+sub complement {
+ $_ = shift;
+ tr/AGCTagct/TCGAtcga/;
+ return $_;
+}
+
+sub bisulphite {
+ $_ = shift;
+ tr/C/T/;
+ return $_;
+}
+
+
+#----------------------------#
+# primer window calculations #
+#----------------------------#
+
+sub primer_window {
+ my ($primer_seq, $dnaseq_len) = @_;
+ my $key;
+ my @primer_list=();
+ my $repeat_real = $repeat-1;
+
+ my ($min_tm, $max_tm, $max_diff, $pri_win_min, $pri_win_max, $min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_tm max_tm max_diff pri_win_win pri_win_max min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ # old method - using amplicon range only ...
+ my ($seqbound5,$seqbound3)=($$min_range,$$max_range);
+
+ $seqbound5 ||= 0;
+ $seqbound5 = $$max_range-$$max_ampsize if ($$max_range) && ($$max_ampsize) && ($reverse==0) && ($$max_range-$$max_ampsize>0);
+ $seqbound5 = $dnaseq_len-$$min_range-$$max_ampsize if ($$min_range) && ($reverse==1) && ($dnaseq_len-$$min_range-$$max_ampsize > 0);
+
+ if (($$max_ampsize) && ($$min_ampsize)) {
+ if ($reverse==0) {
+ unless ($$min_range) {
+ $seqbound3 = $dnaseq_len-$$min_ampsize;
+ } else {
+ $seqbound3 = ($dnaseq_len-$$min_ampsize>$$min_range ? $$min_range : $dnaseq_len-$$min_ampsize);
+ }
+ } else {
+ $seqbound3 = ($$max_range ? $dnaseq_len-$$max_range+$$pri_win_max : $dnaseq_len)
+ }
+ }
+
+ # my $i;
+ # foreach (@intron_exon_bounds) {
+ # print $i++," $_\n";
+ # }
+
+ # print "[$reverse] bounds: $seqbound5 - $seqbound3\n";
+
+ # QPCR specific (limiting range to specific exons)
+ if ($ie_limit) {
+ unless ($reverse) {
+ if ($ie_limit_5p) {
+ my $limit = $ie_limit_5p-2;
+ if ($limit>=0) {
+ $seqbound5 = $intron_exon_bounds[$limit]-$$pri_win_max;
+ }
+ }
+ if ($ie_limit_3p) {
+ my $limit = $ie_limit_3p-1;
+ $limit = 0 if $limit < 0;
+ if (defined($limit)) {
+ unless ($limit > $#intron_exon_bounds) {
+ $seqbound3 = $intron_exon_bounds[$limit]+$$pri_win_max;
+ }
+ }
+ }
+ } else {
+ # reverse sequence
+ if ($ie_limit_3p) {
+ #### in progress
+ my $limit = $ie_limit_3p-1;
+ $limit = 0 if $limit < 0;
+ if (defined($limit)) {
+ unless ($limit > $#intron_exon_bounds) {
+ $seqbound5 = $dnaseq_len - ($intron_exon_bounds[$limit]+$$pri_win_max);
+ }
+ }
+ }
+ if ($ie_limit_5p) {
+ my $limit = $ie_limit_5p-2;
+ if ($limit>=0) {
+ $seqbound3 = $dnaseq_len - ($intron_exon_bounds[$limit]-$$pri_win_max);
+ }
+ }
+ }
+ }
+
+ # print "[$reverse] bounds: $seqbound5 - $seqbound3\n";
+
+
+ # the above covers the unlikely possibility that $max_ampsize is set
+ # and $max_range_5p and $max_range_3p are not. It might be desirable at some
+ # point, and it was the original way of doing things. It doesn't hurt!
+ # ... and is now used for qpcr!
+
+ # To use without specific range boundaries but only with a minimum and
+ # maximum amplicon distance, keep $max_range_5p and $min_range equal,
+ # and likewise at the 3' end
+
+ if (defined($$max_range_5p)&&defined($$max_range_3p)) {
+ unless (($$max_range_5p==$$min_range)&&($$max_range_3p==$$max_range)) {
+ if ($reverse==0) {
+ $seqbound5 = $$max_range_5p;
+ $seqbound3 = $$min_range + $$pri_win_max;
+ }
+
+ if ($reverse==1) {
+ $seqbound5 = $dnaseq_len-$$max_range_3p-1;
+ $seqbound3 = $dnaseq_len-$$max_range + $$pri_win_max;
+ }
+ }
+ }
+
+ return unless defined($seqbound5) && defined($seqbound3);
+
+
+ for my $i ($seqbound5 .. $seqbound3) {
+ for my $primer_win ($$pri_win_min .. $$pri_win_max) {
+ next if $i+$primer_win>$dnaseq_len;
+
+ my $currwindow=substr($primer_seq, $i, $primer_win);
+
+ next if check_degenerate($currwindow);
+
+ if ($exclude_gc) {
+ # exclude based on %GC content
+ my $gc=gc($currwindow);
+ next if $gc < $min_gc or $gc > $max_gc;
+ }
+
+ if ($exclude_clamp) {
+ # calculate GC clamp at 5' end: two out of last three residues required...
+ my $gc_clamp=gc(substr($currwindow, -3, 3));
+ next unless $gc_clamp > 50;
+ }
+
+ if ($exclude_rr) {
+ # simple run exclusion:
+ # ($run is number of consecutive bases to exclude)
+ next if ($currwindow =~ /(C{$run,}|A{$run,}|G{$run,}|T{$run,})/);
+
+ # simple repeat exclusion:
+ # ($repeat is number of consecutive repeats of two or more bases)
+ next if ($currwindow =~ /(.{2,})\1{$repeat_real,}/);
+ }
+
+ # All possible primers have now been excluded
+ # Get the tm and check this against the accepted range:
+ my ($tm)=tm($currwindow);
+ if (($$min_tm < $tm) && ($tm < $$max_tm)) {
+ # Looks good, so let's add the primer to the array
+ push @primer_list, [$i, $primer_win, $currwindow, $tm];
+ }
+ }
+ }
+ return @primer_list;
+}
+
+
+sub check_degenerate {
+ $_ = shift;
+ if (/[^ATGC]/i) {
+ dialogue("One of your primer sequences has a degenerate or non-DNA character. PerlPrimer cannot calculate the Tm of degenerate sequences") if shift;
+ return 1;
+ }
+}
+
+#---------------------------------#
+# bisulphite window calculations #
+#---------------------------------#
+
+sub bisul_window {
+ # See the primer_window subroutine above for general comments
+ my ($primer_seq, $dnaseq_len)=@_;
+ my $key;
+ my $repeat_bs_real = $repeat_bs-1;
+
+ my ($seqbound5,$seqbound3) = 0;
+ my @primer_list=();
+
+ my ($min_tm, $max_tm, $max_diff, $pri_win_min, $pri_win_max, $min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_tm max_tm max_diff pri_win_win pri_win_max min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+
+ $seqbound5 = 0;
+ $seqbound5 = $$max_ampsize-$$max_range if ($$max_range) && ($reverse==0) && ($$max_ampsize-$$max_range>0);
+ $seqbound5 = $dnaseq_len-$$min_range-$$max_ampsize if ($$min_range) && ($reverse==1) && ($dnaseq_len-$$min_range-$$max_ampsize > 0);
+
+ if (defined($$max_range_5p)&&defined($$max_range_3p)) {
+ unless (($$max_range_5p==$$min_range)&&($$max_range_3p==$$max_range)) {
+ if ($reverse==0) {
+ $seqbound5 = $$max_range_5p;
+ $seqbound3 = $$min_range + $$pri_win_max;
+ }
+
+ if ($reverse==1) {
+ $seqbound5 = $dnaseq_len-$$max_range_3p-1;
+ $seqbound3 = $dnaseq_len-$$max_range + $$pri_win_max;
+ }
+ }
+ } else {
+ if ($reverse==0) {
+ unless ($$min_range) {
+ $seqbound3 = $dnaseq_len-$$min_ampsize;
+ } else {
+ $seqbound3 = ($dnaseq_len-$$min_ampsize>$$min_range ? $$min_range : $dnaseq_len-$$min_ampsize);
+ }
+ } else {
+ $seqbound3 = ($$max_range ? $dnaseq_len-$$max_range+$$pri_win_max : $dnaseq_len)
+ }
+ }
+
+ return unless defined($seqbound5) && defined($seqbound3);
+
+ my $count;
+ for my $i ($seqbound5 .. $seqbound3) {
+ for my $primer_win ($$pri_win_min .. $$pri_win_max) {
+ $count++;
+ next if $i+$primer_win>$dnaseq_len;
+
+ my $currwindow=substr($primer_seq, $i, $primer_win);
+
+ next if check_degenerate($currwindow);
+
+ # Check for %C content
+ next if cc($currwindow) < $bisul_min_c;
+
+ #next if ga($currwindow) < 50;
+
+ # Check for exclude_cpg
+ # If exclude_cpg is set, no primers with CpG residues are include
+ # Otherwise, primers are allowed to have up to a maximum of 2 CpG residues
+ if ($exclude_cpg) {
+ next if $currwindow =~ /CG/
+ } else {
+ my $count_cpg = 0;
+ while ($currwindow =~ /CG/g) {
+ $count_cpg++
+ }
+ next if $count_cpg >2;
+ }
+
+ # Check for 3' C content
+ if ($exclude_3c) {
+ # It is important that primer specificity to the converted template is given
+ # by having C residues at the 3' end
+
+ # The following gives the most flexibility: a primer is accepted if
+ # it either ends in a C or has two of the last three bases as C's
+ next unless ((substr($currwindow, -1, 1) eq "C") or (cc(substr($currwindow, -3, 3)) > 50));
+ }
+
+ # now convert the primer ...
+ my $currwindowbs = bisulphite($currwindow);
+
+ # do we exclude repeats and runs before or after bisulphite conversion?
+ my $checkref = ($pre_bs ? \$currwindowbs : \$currwindow);
+
+ if ($exclude_rr_bs) {
+ # Run exclusion:
+ next if ($$checkref =~ /(C{$run_bs,}|A{$run_bs,}|G{$run_bs,}|T{$run_bs,})/);
+
+ # Repeat exclusion:
+ next if ($$checkref =~ /(.{2,})\1{$repeat_bs_real,}/);
+ }
+
+ # Need to complement if we're creating reverse primers,
+ # as the two converted strands of DNA are non-complementary
+ if ($reverse==1) {
+ $currwindowbs=complement($currwindowbs);
+ }
+
+ my ($tm)=tm($currwindowbs);
+
+ if (($$min_tm < $tm) && ($tm < $$max_tm)) {
+ # Add the original, unconverted sequence to the array as well:
+ # Note - this will not be complemented if reversed seq
+ push @primer_list, [$i, $primer_win, $currwindowbs, $tm, $currwindow];
+ }
+ }
+ }
+ return @primer_list;
+}
+
+
+#-----------------------------------------#
+# Calculation of amplicon lengths and #
+# compatible temperature matching primers #
+#-----------------------------------------#
+
+sub calc_amplicon {
+ my $dnaseq_r_len = shift;
+ my $count_amplicon=0;
+ @primer_pairs=();
+ my $count=0;
+
+ my ($min_tm, $max_tm, $max_diff, $pri_win_min, $pri_win_max, $min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_tm max_tm max_diff pri_win_win pri_win_max min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ # (the reverse sequence was reversed to make it 5'->3')
+ # So I either have to recalculate the revese numbers or
+ # not reverse the sequence and count backwards!
+ #
+ # Solution:
+ #
+ # realpos_start = total_length - calc_pos
+ # and closest_real_base = realpos_start - window
+ # thus = total_length - calc_pos - window
+
+ foreach my $key_f (@PF) {
+ return if ($cancel==1);
+
+ my ($pos_f, $len_f, $seq_f, $tm_f, $unconverted_f ) = @$key_f;
+
+ foreach my $key_r (@PR) {
+ return if ($cancel==1);
+ my ($pos_r, $len_r, $seq_r, $tm_r, $unconverted_r ) = @$key_r;
+
+ my $realpos_r = $dnaseq_r_len - $pos_r - 1;
+ my $amp_size=$realpos_r-$pos_f;
+
+ # Skip all pairs where forward > reverse ...
+ next unless ($realpos_r-$len_r>$pos_f+$len_f);
+
+ # Range check (messy):
+ # Now should be covered in windowing! but need for qpcr
+ if (($$min_range) && ($$max_range)) {
+ next unless ($pos_f < $$min_range && $$max_range < $realpos_r);
+ }
+
+ # Temperature check:
+ ### could be more efficient
+ next unless ($tm_r > ($tm_f-$$max_diff) && $tm_f > ($tm_r-$$max_diff));
+
+ # QPCR specific:
+ if ($qpcr_flag == 1) {
+ # Amplicon size check
+ next if ($amp_size < $$min_ampsize || $amp_size > $$max_ampsize);
+
+ #check amp range spans i/e boundary
+ my $qpcr_check=0;
+ if ($ie_span) {
+ foreach my $i (@intron_exon_bounds) {
+ $qpcr_check=1 if $pos_f<$i && $i<$realpos_r
+ }
+ next unless $qpcr_check==1;
+ }
+
+ #check at least one primer falls across i/e boundary
+ $qpcr_check=0;
+ if (($ie_overlap) && ($exclude_ie)) {
+ foreach my $i (@intron_exon_bounds) {
+ $qpcr_check=1 if $pos_f<($i-$exclude_ie) && ($i+$exclude_ie)<($pos_f+$len_f);
+ $qpcr_check=1 if ($realpos_r-$len_r)<($i-$exclude_ie) && ($i+$exclude_ie)<$realpos_r;
+ }
+ next unless $qpcr_check==1;
+ }
+ }
+
+ # all OK up to here, so let's check primer-dimers:
+ # NB: this is a big speed hit ...
+ # my (@pd_score, @pd_sorted)=();
+#
+ # push(@pd_score,primer_dimer($seq_f,$seq_f));
+ # push(@pd_score,primer_dimer($seq_f,$seq_r));
+ # push(@pd_score,primer_dimer($seq_r,$seq_r));
+ # @pd_sorted=sort{$a <=> $b} @pd_score;
+
+ # extensible
+ my $pd_score = primer_dimer($seq_f,$seq_f);
+
+ my $new_score = primer_dimer($seq_f,$seq_r);
+ $pd_score = $new_score if $new_score < $pd_score;
+
+ $new_score = primer_dimer($seq_r,$seq_r);
+ $pd_score = $new_score if $new_score < $pd_score;
+
+ # non_extensible
+ my $pd_score_full = primer_dimer($seq_f,$seq_f,1);
+
+ $new_score = primer_dimer($seq_f,$seq_r,1);
+ $pd_score_full = $new_score if $new_score < $pd_score_full;
+
+ $new_score = primer_dimer($seq_r,$seq_r,1);
+ $pd_score_full = $new_score if $new_score < $pd_score_full;
+
+ $tm_f = sprintf("%.2f", $tm_f);
+ $tm_r = sprintf("%.2f", $tm_r);
+
+ push @primer_pairs, [ $seq_f, $pos_f, $len_f, $tm_f,
+ $seq_r, $pos_r, $len_r, $tm_r, $realpos_r, $amp_size, $pd_score, ( $unconverted_f ? $unconverted_f : 0), ( $unconverted_r ? $unconverted_r : 0), $pd_score_full ];
+
+ }
+ $count++;
+ my $percent = sprintf("%.f", ($count/$pfkeys)*100);
+ sbarprint("\nCalculating amplicons ... $percent% completed ...");
+ }
+ sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+}
+
+
+#-----------------------------------------#
+# Calculation of amplicon lengths and #
+# compatible temperature matching primers #
+#-----------------------------------------#
+
+sub calc_seq_primers {
+ # scaled down calc_amplicon routine
+ my $count_amplicon=0;
+ @primer_pairs=();
+ my $count=0;
+
+ my $pdcheck=1;
+
+ my ($min_range, $max_range)
+ = get_variables(qw(min_range max_range));
+
+ my $last_seq;
+
+ # $pd_full = 1;
+ # $pd_extensible = 0;
+
+ foreach (@PF) {
+ return if ($cancel==1);
+
+ $count++;
+ my $percent = sprintf("%.f", ($count/$pfkeys)*100);
+ sbarprint("\nFinding primers ... $percent% completed ...");
+
+ my ($pos, $len, $seq, $tm_f ) = @$_;
+
+ # take the first one that fits if last_seq hasn't been set ...
+ unless (defined($last_seq)) {
+ next if $pos < $$min_range;
+ if ($pos > $seq_spacing_max+$$min_range) {
+ dialogue("Contiguous primers could not be found within the set parameters");
+ last;
+ }
+ } else {
+ next if $pos > $$max_range;
+ next if $pos < $last_seq + $seq_spacing_min;
+ if ($pos > $last_seq + $seq_spacing_max) {
+ dialogue("Contiguous primers could not be found within the set parameters");
+ last;
+ }
+ }
+
+ # calculate primer-dimers
+ my $pd = primer_dimer($seq,$seq,1);
+
+ # exclude all primer-dimers if asked ...
+ if ($exclude_pd_seq == 1) {
+ next if $pd < -$seq_pd_min;
+ }
+
+ $tm_f = sprintf("%.2f", $tm_f);
+
+ push @primer_pairs, [ $seq, $pos, $len, $tm_f, $pd ];
+ $last_seq = $pos;
+ }
+ # $pd_full = 0;
+
+ # $pd_extensible = 1;
+ sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+}
+
+# sub calc_seq_primers {
+ # # scaled down calc_amplicon routine
+ # my $count_amplicon=0;
+ # @primer_pairs=();
+ # my $count=0;
+#
+ # my $pdcheck=1;
+ #
+ # my ($min_range, $max_range)
+ # = get_variables(qw(min_range max_range));
+ #
+ # my $last_seq;
+ # my $seq_spacing_mid = int(($seq_spacing_max+$seq_spacing_min)/2);
+ #
+ # $pd_full = 1;
+ # # $pd_extensible = 0;
+ #
+ # # build hash table
+ # my $i;
+ # my $flag=0;
+ # my $skip;
+ # my $interval=1;
+ # my %seq_primers;
+ # foreach (@PF) {
+ # $i++;
+ # my ($pos) = @$_;
+ # $seq_primers{$pos} = $i;
+ # }
+ #
+ # my $j;
+ # for ($j=$$min_range; $j < $$max_range;) {
+ # my ($pos, $len, $seq, $tm_f);
+ # return if ($cancel==1);
+ #
+ # if ($skip) {
+ # $flag = 1-$flag;
+ # if ($flag) {
+ # $j+=$interval;
+ # } else {
+ # $j-=$interval;
+ # $interval++;
+ # }
+ # $skip=0;
+ # }
+ #
+ # $count++;
+ # # my $percent = sprintf("%.f", ($count/$pfkeys)*100);
+ # sbarprint("\nFinding primers ... $count primers checked ...");
+ #
+ #
+ # # take the first one that fits if last_seq hasn't been set ...
+ # unless (defined($last_seq)) {
+ # if ($seq_primers{$j}) {
+ # ($pos, $len, $seq, $tm_f ) = @{ $PF[$seq_primers{$j}] };
+ # } else {
+ # # we want the first primer to be as near to the start as possible
+ # print "$j\n";
+ # $j++;
+ # next;
+ # }
+ # print "$j: OK! pos was $pos\n";
+ #
+ # next if $pos < $$min_range;
+ # if ($pos > $seq_spacing_max+$$min_range) {
+ # dialogue("Contiguous primers could not be found within the set parameters");
+ # last;
+ # }
+ # } else {
+ # if ($seq_primers{$j}) {
+ # ($pos, $len, $seq, $tm_f ) = @{ $PF[$seq_primers{$j}] };
+ # } else {
+ # $skip=1;
+ # next;
+ # }
+ # if ($pos > $$max_range) {
+ # $skip=1;
+ # next;
+ # }
+ # if ($pos < $last_seq + $seq_spacing_min) {
+ # $skip=1;
+ # next;
+ # }
+ # if ($pos > $last_seq + $seq_spacing_max) {
+ # dialogue("Contiguous primers could not be found within the set parameters");
+ # last;
+ # }
+ # }
+ #
+ # # exclude all primer-dimers if asked ...
+ # my $pd;
+ # if ($exclude_pd_seq == 1) {
+ # if ($pd = primer_dimer($seq,$seq) < -$seq_pd_min) {
+ # $skip=1;
+ # next;
+ # }
+ # }
+ #
+ # $tm_f = sprintf("%.2f", $tm_f);
+ #
+ # push @primer_pairs, [ $seq, $pos, $len, $tm_f, $pd ];
+ # $interval = 1;
+ # $last_seq = $pos;
+ # $j = $pos + $seq_spacing_mid;
+ # }
+ #
+ # # $pd_extensible = 1;
+ # sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+# }
+
+#-------------------------#
+# calculate %base content #
+#-------------------------#
+
+# %GC
+sub gc {
+ $_ = $_[0];
+ my($gc,$countgc,$counttotal);
+ $gc=0;
+ $countgc=0;
+
+ $countgc = tr/GCgc/GCgc/;
+ $counttotal = length();
+
+ $gc = $countgc/$counttotal*100;
+ return $gc;
+}
+
+# %C
+sub cc {
+ $_ = $_[0];
+ my($cc,$countc,$counttotal);
+ $cc=0;
+ $countc=0;
+
+ $countc = tr/C/C/;
+ $counttotal = length();
+
+ $cc = $countc/$counttotal*100;
+ return $cc;
+}
+
+
+#----------------#
+# Clean sequence #
+#----------------#
+
+sub clean_seq {
+ $_ = shift;
+ my $nb_page = which_nb_page();
+
+ my ($fasta_name) = /^\s*>(.*\n)/;
+ s/^\s*>(.*\n)//g; #remove FASTA formatting if it exists
+ # print "name = $fasta_name\n";
+ if ($fasta_name && $open_file{$nb_page} eq "File not saved") {
+ # if FASTA details are present ...
+ my $name = format_file_name($1);
+
+ $open_file{$nb_page} = $name;
+ $top->configure(-title=>"PerlPrimer v$version - $name");
+ }
+
+ s/[\s\n\r]//g; #remove spaces/new lines
+ s/-//g; #remove gaps in sequence
+ s/\d//g; #remove digits
+
+ return $_;
+}
+
+
+#-------------------------------#
+# forward and reverse sequences #
+#-------------------------------#
+
+sub getseq {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ if (($$min_range) && ($$max_range)) {
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range - $$min_range if ($$max_range - $$min_range)>$$max_ampsize;
+ }
+
+ # forward
+ $_ = uc(shift);
+ $_ = clean_seq($_);
+
+ my $dnaseq_f = $_;
+
+ # reverse
+ my $dnaseq_r_top = reverse($dnaseq_f);
+ my $dnaseq_r = complement($dnaseq_r_top);
+
+ my $dnaseq_f_len = length($dnaseq_f);
+ my $dnaseq_r_len = length($dnaseq_r);
+
+ return ($dnaseq_f, $dnaseq_f_len, $dnaseq_r_top, $dnaseq_r, $dnaseq_r_len);
+}
+
+
+#-----
+# subroutine for calculating Tm as per SantaLucia(1998), cited above, with Mg++
+# (and K+, Tris++) concentration calculated via equations 7 and 8 from
+# von Ahsen, et al, 2001, Clinical Chemistry 47(11):1956-1961
+#-----
+
+sub tm {
+ my ($primer) = @_;
+ $primer = uc($primer); # if user enters primer directly as lower-case
+ my ($i, $nn, $initterm, $endterm);
+ my $primer_len = length($primer);
+ my ($deltaH, $deltaS);
+
+ #-----------------------------#
+ # calculate deltaH and deltaS #
+ #-----------------------------#
+
+ for ($i=0; $i<$primer_len-1; $i++) {
+ $nn = substr($primer, $i, 2);
+
+ $deltaH+= $oligo_dH{$nn};
+ $deltaS+= $oligo_dS{$nn};
+ }
+
+ #-------------------------#
+ # initial term correction #
+ #-------------------------#
+
+ $initterm="init" . substr($primer, 0, 1);
+ $deltaH+= $oligo_dH{$initterm};
+ $deltaS+= $oligo_dS{$initterm};
+
+ $endterm="init" . substr($primer, -1, 1);
+ $deltaH+= $oligo_dH{$endterm};
+ $deltaS+= $oligo_dS{$endterm};
+
+ # Tm at 1M NaCl
+ # $tm= ($deltaH * 1000) / ($deltaS + (1.987 * log($oligo_conc / 4))) - 273.15;
+
+ #------------------------------------------#
+ # correct for salt concentration on deltaS #
+ #------------------------------------------#
+
+ # Big problems if [dNTPs] > [Mg++] !! This is a quick fix ...
+ my $salt_correction;
+ if ($mg_conc > $dntp_conc) {
+ $salt_correction = sqrt($mg_conc - $dntp_conc);
+ } else {
+ $salt_correction = 0;
+ }
+
+ my $na_eq=($monovalent_cation_conc + 120 * $salt_correction)/1000;
+
+ # deltaS correction:
+ $deltaS += (0.368 * ($primer_len - 1) * log($na_eq));
+
+ my $oligo_conc_mols = $oligo_conc / 1000000000;
+
+ # Salt corrected Tm
+ # NB - for PCR I'm assuming for the moment that the [strand target] << [oligo]
+ # and that therefore the C(t) correction term approx equals [oligo]
+ my $corrected_tm=(($deltaH * 1000) / ($deltaS + (1.987 * log($oligo_conc_mols)))) - 273.15;
+ return ($corrected_tm, $deltaH, $deltaS);
+}
+
+
+#----------------------------------------------------------#
+# Recalculate the oligo_dG hash on current salt conditions #
+#----------------------------------------------------------#
+
+sub recalculate_dG {
+ # because dG = dH - TdS, and dS is dependent on the salt concentration ...
+ my $temperature = shift || $pd_temperature;
+
+ # Big problems if [dNTPs] > [Mg++] !! This is a quick fix ...
+ my $salt_correction;
+ if ($mg_conc > $dntp_conc) {
+ $salt_correction = sqrt($mg_conc - $dntp_conc);
+ } else {
+ $salt_correction = 0;
+ }
+
+ my $na_eq=($monovalent_cation_conc + 120 * $salt_correction)/1000;
+
+ # the length of each NN dimer is 2, therefore the modifier is 1
+ my $entropy_adjust = (0.368 * log($na_eq));
+
+ foreach my $key (keys(%oligo_dH_full)) {
+ next if $key =~ /init/; # the length of each monomer is 1, thus the modifier of dS is 0 and the values are precalulated
+
+ my $dS = $oligo_dS_full{$key} + $entropy_adjust;
+ my $dG = $oligo_dH_full{$key}-((273.15+$temperature)*($dS/1000));
+ $oligo_dG{$key} = $dG;
+ }
+}
+
+#--------------------------#
+# Primer-dimer calculation #
+#--------------------------#
+
+sub primer_dimer {
+ # This is my second attempt at implementing a primer-dimer system:
+ # The first attempt aligned each combination together explicitly; this attempt
+ # creates a binding matrix and then reads each primer combination from the
+ # matrix. It's not significantly faster, but it does have the advantage of
+ # extending this algorithm to allow for unequal loops (although this has not
+ # been implemented as yet) and providing the possiblity of reading all
+ # primer-dimers (not just 3') by adjusting a single variable (which is used when
+ # displaying primer information.
+
+ my ($primer_f, $primer_r, $pd_full) = @_;
+ return unless ($primer_f) && ($primer_r);
+
+ my ($k, $l);
+ @score=();
+ %primer_hash=();
+ @score_sort=();
+ @bind_string=();
+ %rating_hash=();
+
+ # $pl = greatest length
+ $pfl=length($primer_f);
+ $prl=length($primer_r);
+ $pl = ($pfl>$prl ? $pfl : $prl);
+
+ my $rcompr = reverse(complement($primer_r));
+ my $rcomprlc = lc($rcompr);
+ my $fprimer_r=lc(reverse($primer_f));
+ $rprimer_r=reverse($primer_r);
+
+ # Scan the primers against each other:
+ # The default is to only consider 3' primer-dimers, for speed concerns (5'
+ # pd's will reduce the primer population, but won't cause extendible dimers) -
+ # however, setting $pd_full to 1 will calculate all primer-dimers. This is
+ # currently used only when viewing individual primers, for both speed concerns
+ # and because it's 3' primer-dimers that are the real problem in PCR.
+
+ # create a binding array for each of the four bases
+ for $l (0 .. $pfl-1) {
+ my $mbase = substr($fprimer_r, $l, 1);
+ $primer_hash{$mbase}[$l]=1;
+ for $k qw(a g c t) {
+ $primer_hash{$k}[$l] ||=0;
+ }
+ }
+
+ # create the primer matrix
+ my @primer_comp;
+ for $k (0 .. $prl-1) {
+ $primer_comp[$k]=$primer_hash{substr($rcomprlc, $k, 1)};
+ }
+
+ # read each combination from the matrix, calculate dG for each dimer
+ my $pd_len = ($pd_full ? $pfl+$prl-1 : $pl-2);
+ for $k (0 .. $pd_len) {
+ $score[$k]=0;
+ my $bind;
+ my $score_p=0;
+
+ # extensible primer short-circuit - ignore all primers that will
+ # not create extensible (i.e. amplifiable) dimers
+ my $start = $k>$pfl-1 ? $pfl-1 : $k;
+ my $end = $k>$prl-1 ? $prl-1 : $k;
+ if ($pd_extensible && !$pd_full) {
+ next unless $primer_comp[0][$start] == 1;
+ next unless $primer_comp[$end][$start-$k] == 1;
+ }
+
+ # elsif ($pd_extensible) {
+ # # no point reconsidering them!
+ # next if $primer_comp[0][$start] == 1 && $primer_comp[$end][$start-$k] == 1;
+ # }
+
+ # read the binding data
+ for $l (0 .. $prl-1) {
+ if (($k-$l)<$pfl) {
+ $bind .= $primer_comp[$l][$k-$l] if ($k-$l)>=0;
+ } else {
+ # spacer
+ $bind .= "2";
+ }
+ }
+
+ # Single matched bases surrounded by mismatches are unstable,
+ # so we remove them with the regexp (look ahead is needed otherwise
+ # strings of consecutive match/mismatches are not caught)
+ $bind =~ s/01(?=[^1])/00/gx;
+
+ # Short circuit if there's nothing to bind
+ next unless $bind =~ /[1]/;
+
+ # Find start and end of similarity
+ my ($pb_init,$pb_end);
+ for $l (0 .. length($bind)-1) {
+ # at first I tried finding the initiating terminal bases with
+ # regexps, but that was much slower ...
+ if (substr($bind, $l, 1) eq "1") {
+ defined($pb_init) || ($pb_init = $l);
+ $pb_end=$l;
+ }
+ }
+
+ if (defined($pb_init)) {
+ # deltaG calculation
+ for $l ($pb_init .. $pb_end-1) {
+ next if substr($bind, $l, 2) eq "00";
+ next if substr($bind, $l, 1) eq "2";
+ $score_p+=$oligo_dG{substr($primer_f, $pfl-$k+$l-1, 2).substr($rprimer_r, $l, 2)};
+ }
+
+ # init term corrections
+ my $initterm="init" . substr($rprimer_r, $pb_init, 1);
+ $score_p+= $oligo_dG{$initterm};
+
+ my $endterm="init" . substr($rprimer_r, $pb_end, 1);
+ $score_p+= $oligo_dG{$endterm};
+
+ # add to the hash ...
+ $score[$k]=sprintf("%.2f",$score_p);
+ $bind_string[$k]=$bind;
+ $rating_hash{$score[$k]}=$k;
+ }
+ }
+
+ # sort the dimers to give the most stable:
+ @score_sort = sort { $a <=> $b } @score;
+
+ # Returns the most stable dimer
+ return $score_sort[0];
+}
+
+sub primer_dimer_new {
+ # This is my second attempt at implementing a primer-dimer system:
+ # The first attempt aligned each combination together explicitly; this attempt
+ # creates a binding matrix and then reads each primer combination from the
+ # matrix. It's not significantly faster, but it does have the advantage of
+ # extending this algorithm to allow for unequal loops (although this has not
+ # been implemented as yet) and providing the possiblity of reading all
+ # primer-dimers (not just 3') by adjusting a single variable (which is used when
+ # displaying primer information.
+
+ ### TODO:
+ # It is easily possible to calculate *all* primers when searching for primer
+ # pairs ... I really should add a preferences option to do this ...
+
+ my ($primer_f, $primer_r, $pd_full) = @_;
+ return unless ($primer_f) && ($primer_r);
+
+ my ($k, $l);
+ @score=();
+ %primer_hash=();
+ @score_sort=();
+ @bind_string=();
+ %rating_hash=();
+
+ # $pl = greatest length
+ $pfl=length($primer_f);
+ $prl=length($primer_r);
+ $pl = ($pfl>$prl ? $pfl : $prl);
+
+ my $rcompr = reverse(complement($primer_r));
+ my $rcomprlc = lc($rcompr);
+ my $fprimer_r=lc(reverse($primer_f));
+ $rprimer_r=reverse($primer_r);
+
+ # Scan the primers against each other:
+ # The default is to only consider 3' primer-dimers, for speed concerns (5'
+ # pd's will reduce the primer population, but won't cause extendible dimers) -
+ # however, setting $pd_full to 1 will calculate all primer-dimers. This is
+ # currently used only when viewing individual primers, for both speed concerns
+ # and because it's 3' primer-dimers that are the real problem in PCR.
+
+ # create a binding array for each of the four bases
+ for $l (0 .. $pfl-1) {
+ my $mbase = substr($fprimer_r, $l, 1);
+ $primer_hash{$mbase}[$l]=1;
+ for $k qw(a g c t) {
+ $primer_hash{$k}[$l] ||=0;
+ }
+ }
+
+ # create the primer matrix
+ my @primer_comp;
+ for $k (0 .. $prl-1) {
+ $primer_comp[$k]=$primer_hash{substr($rcomprlc, $k, 1)};
+ }
+
+ # print the matrix - for debugging
+ print "$primer_f vs. $primer_r - full pd = $pd_full\n";
+ print " \t";
+ for $l (0 .. $pfl-1) {
+ my $mbase = substr($fprimer_r, $l, 1);
+ print "$mbase ";
+ }
+ print "\n";
+ for $k (0 .. $prl-1) {
+ my $base = substr($rprimer_r, $k, 1);
+ print "$base:\t@{$primer_comp[$k]}\n";
+ }
+
+ my @binding_data;
+ # read each combination from the matrix, calculate dG for each dimer
+ my $pd_len = ($pd_full ? $pfl+$prl-1 : $pl-2);
+ for my $k (0 .. $pd_len) {
+ $score[$k]=0;
+ my $bind;
+ my $score_p=0;
+
+ # starting coords
+ my $pf_coord_start = ($k >= $pfl ? $pfl-1 : $k);
+ my $pr_coord_start = ($k - $pfl > 0 ? $k - $pfl : 0);
+ my ($pf_coord, $pr_coord, $first, $flag);
+
+ # read through each combination finding multiple matches
+ for ($pf_coord = $pf_coord_start, $pr_coord = $pr_coord_start;
+ $pf_coord>=0 && $pr_coord<$prl;
+ $pf_coord--,$pr_coord++) {
+ # read base
+ print "error: \[$pr_coord\] \[$pf_coord\]\n\n" unless defined($primer_comp[$pr_coord][$pf_coord]);
+ if ($primer_comp[$pr_coord][$pf_coord]==1) {
+ # binding bases
+ $bind++;
+ if ($flag) {
+ next;
+ } else {
+ $first=$pf_coord;
+ $flag=1;
+ }
+ } elsif ($flag) {
+ # end of a binding stretch
+ push @binding_data, [$k, $first, $bind] if $bind > 1;
+ $bind=0;
+ $flag=0;
+ }
+
+ # set up for next loop
+ # $pf_coord--;
+ # $pr_coord++;
+ # last if $pf_coord<0 || $pr_coord>$prl;
+ # redo;
+ }
+ }
+
+ # check for best binding for each possibility that yeilds two or more matched bases together
+ my $last;
+ my @unequal_loops;
+ foreach my $i (@binding_data) {
+ # skip multiple matches
+ # next if @$i[0] == $last;
+ #
+ # $last = @$i[0];
+ my $pf_coord = @$i[1];
+ my $length = @$i[2];
+
+ print "@$i\n";
+ }
+
+
+
+
+ # # extensible primer short-circuit - ignore all primers that will
+ # # not create extensible (i.e. amplifiable) dimers
+ # # my $start = $k>$pfl-1 ? $pfl-1 : $k;
+ # # my $end = $k>$prl-1 ? $prl-1 : $k;
+ # # if ($pd_extensible && !$pd_full) {
+ # # next unless $primer_comp[0][$start] == 1;
+ # # next unless $primer_comp[$end][$start-$k] == 1;
+ # # }
+ #
+ # # read the binding data
+ # for $l (0 .. $prl-1) {
+ # if (($k-$l)<$pfl) {
+ # $bind .= $primer_comp[$l][$k-$l] if ($k-$l)>=0;
+ # } else {
+ # # spacer
+ # $bind .= "2";
+ # }
+ # }
+ #
+ # # Single matched bases surrounded by mismatches are unstable,
+ # # so we remove them with the regexp (look ahead is needed otherwise
+ # # strings of consecutive match/mismatches are not caught)
+ # $bind =~ s/01(?=[^1])/00/gx;
+ #
+ # # Short circuit if there's nothing to bind
+ # next unless $bind =~ /[1]/;
+ #
+ # # Find start and end of similarity
+ # my ($pb_init,$pb_end);
+ # for $l (0 .. length($bind)-1) {
+ # # at first I tried finding the initiating terminal bases with
+ # # regexps, but that was much slower ...
+ # if (substr($bind, $l, 1) eq "1") {
+ # defined($pb_init) || ($pb_init = $l);
+ # $pb_end=$l;
+ # }
+ # }
+ #
+ # if (defined($pb_init)) {
+ # # deltaG calculation
+ # for $l ($pb_init .. $pb_end-1) {
+ # next if substr($bind, $l, 2) eq "00";
+ # next if substr($bind, $l, 1) eq "2";
+ # $score_p+=$oligo_dG{substr($primer_f, $pfl-$k+$l-1, 2).substr($rprimer_r, $l, 2)};
+ # }
+ #
+ # # init term corrections
+ # my $initterm="init" . substr($rprimer_r, $pb_init, 1);
+ # $score_p+= $oligo_dG{$initterm};
+ #
+ # my $endterm="init" . substr($rprimer_r, $pb_end, 1);
+ # $score_p+= $oligo_dG{$endterm};
+ #
+ # # add to the hash ...
+ # $score[$k]=sprintf("%.2f",$score_p);
+ # $bind_string[$k]=$bind;
+ # $rating_hash{$score[$k]}=$k;
+ # }
+ # }
+ #
+ # # sort the dimers to give the most stable:
+ # @score_sort = sort { $a <=> $b } @score;
+ #
+ # # Returns the most stable dimer
+ # return $score_sort[0];
+}
+
+
+#--------------------------------#
+# Rountine to draw primer-dimers #
+#--------------------------------#
+
+sub draw_dimer {
+ # This all seems a bit cumbersome!!
+ my ($primer_f, $primer_r, $pos, $FH) = @_;
+
+ my $rprimer_r=reverse($primer_r);
+ my $dimer_binding="";
+ my $pr_space="";
+ my $fspace="";
+ my $rspace="";
+
+ my $fspace_def = $pl-$pfl>0 ? $pl-$pfl : 0;
+ $fspace=" "x($fspace_def+($pos>$pl-1?$pos-$pl+1:0));
+
+ if ($pos+1>=$pfl) {
+ $rspace=" "x($pl-$pos-1);
+ } else {
+ $rspace=$fspace;
+ }
+
+ $pr_space=" "x($pfl-$pos-1);
+
+ for my $j (0 .. $pos) {
+ next unless $j < $prl;
+ if (substr($bind_string[$pos],$j,1)==1) {
+ $dimer_binding=$dimer_binding."|"
+ } elsif (substr($bind_string[$pos],$j,1)==0) {
+ $dimer_binding=$dimer_binding."."
+ } else {
+ $dimer_binding=$dimer_binding." "
+ }
+ }
+
+ print $FH "$fspace"."5' "."$primer_f"." 3'\n".
+ "$rspace"." "."$pr_space"."$dimer_binding\n".
+ "$rspace"."$pr_space"."3' "."$rprimer_r"." 5'\n\n";
+}
+
+
+#----------------------#
+# Program exit/restart #
+#----------------------#
+
+sub end_prog {
+ # This sends the program to the pref writing routine after Mainloop() ...
+ $top->destroy;
+}
+
+
+sub restart {
+ exec "$0";
+}
+
+
+
+#---------------------#
+# GUI button routines #
+#---------------------#
+
+sub get_tm {
+ my ($report) = @_;
+ # Lower case bug fix:
+ $fprimer = uc($fprimer);
+ $rprimer = uc($rprimer);
+
+ my ($deltaG, $deltaH, $deltaS);
+ my $oligo_conc_mols = $oligo_conc / 1000000000;
+
+ # my $check = check_degenerate($fprimer, 1);
+ # print "fprimer was $fprimer; check was $check\n";
+ if ($fprimer && !check_degenerate($fprimer, 1)) {
+ ($fprimer_tm, $deltaH, $deltaS) = tm($fprimer);
+ $fprimer_gc = int(gc($fprimer));
+ $fprimer_tm = sprintf("%.2f", $fprimer_tm);
+
+ # since dG = dH - TdS, we don't need to calculate based on NN's ...
+ $deltaG = $deltaH-((273.15+$pd_temperature)*($deltaS/1000));
+
+ $fprimer_ds = sprintf("%.2f", $deltaS);
+ $fprimer_dh = sprintf("%.2f", $deltaH);
+ $fprimer_dg = sprintf("%.2f", $deltaG);
+ $fprimer_len = length($fprimer);
+ }
+
+ if ($rprimer && !check_degenerate($rprimer, 1)) {
+ ($rprimer_tm, $deltaH, $deltaS) = tm($rprimer);
+ $rprimer_gc = int(gc($rprimer));
+ $rprimer_tm = sprintf("%.2f", $rprimer_tm);
+
+ $deltaG = $deltaH-((273.15+$pd_temperature)*($deltaS/1000));
+
+ $rprimer_ds = sprintf("%.2f", $deltaS);
+ $rprimer_dh = sprintf("%.2f", $deltaH);
+ $rprimer_dg = sprintf("%.2f", $deltaG);
+ $rprimer_len = length($rprimer);
+ }
+
+ my $repeat_real = $repeat-1;
+ $packed_widgets{dim}->delete(0.1,"end");
+ $packed_widgets{dim}->insert('end', "Warning: forward primer run found\n", 'red') if ($fprimer =~ /(C{$run,}|A{$run,}|G{$run,}|T{$run,})/);
+ $packed_widgets{dim}->insert('end', "Warning: forward primer repeat found : $1\n", 'red') if ($fprimer =~ /(.{2,})\1{$repeat_real,}/);
+
+ $packed_widgets{dim}->insert('end', "Warning: reverse primer run found\n", 'red') if ($rprimer =~ /(C{$run,}|A{$run,}|G{$run,}|T{$run,})/);
+ $packed_widgets{dim}->insert('end', "Warning: reverse primer repeat found\n", 'red') if ($rprimer =~ /(.{2,})\1{$repeat_real,}/);
+
+ $packed_widgets{dim}->insert('end', "Most stable 3' extensible primer-dimers (at $pd_temperature°C), if any\n\n", 'blue');
+
+ my ($pd1, $pd2, $pd3);
+ if ($fprimer && !check_degenerate($fprimer)) {
+ $pd1 = primer_dimer($fprimer,$fprimer);
+ $pos=$rating_hash{$score_sort[0]};
+
+ unless ($score_sort[0]==0) {
+ $packed_widgets{dim}->insert('end', "Forward vs. Forward: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($fprimer, $fprimer, $pos, \ *DIMER);
+ }
+
+ if ($rprimer && !check_degenerate($rprimer)) {
+ $pd2 = primer_dimer($fprimer,$rprimer);
+ $pos=$rating_hash{$score_sort[0]};
+ unless ($score_sort[0]==0) {
+ $packed_widgets{dim}->insert('end', "Forward vs. Reverse: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($fprimer, $rprimer, $pos, \ *DIMER);
+ }
+ }
+ }
+
+ if ($rprimer && !check_degenerate($rprimer)) {
+ $pd3 = primer_dimer($rprimer,$rprimer);
+ $pos=$rating_hash{$score_sort[0]};
+
+ unless ($score_sort[0]==0) {
+ $packed_widgets{dim}->insert('end', "Reverse vs. Reverse: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($rprimer, $rprimer, $pos, \ *DIMER) unless ($score_sort[0]==0);
+ }
+ }
+
+ # $pd_full = 1;
+ $packed_widgets{dim}->insert('end', "\nMore stable non-extensible primer-dimers (at $pd_temperature°C), if any\n\n", 'blue');
+
+ if ($fprimer && !check_degenerate($fprimer)) {
+ primer_dimer($fprimer,$fprimer,1);
+ $pos=$rating_hash{$score_sort[0]};
+
+ if ($score_sort[0]<$pd1) {
+ $packed_widgets{dim}->insert('end', "Forward vs. Forward: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($fprimer, $fprimer, $pos, \ *DIMER);
+ }
+
+ if ($rprimer && !check_degenerate($rprimer)) {
+ primer_dimer($fprimer,$rprimer,1);
+ $pos=$rating_hash{$score_sort[0]};
+ if ($score_sort[0]<$pd2) {
+ $packed_widgets{dim}->insert('end', "Forward vs. Reverse: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($fprimer, $rprimer, $pos, \ *DIMER);
+ }
+ }
+ }
+ if ($rprimer && !check_degenerate($rprimer)) {
+ primer_dimer($rprimer,$rprimer,1);
+ $pos=$rating_hash{$score_sort[0]};
+
+ if ($score_sort[0]<$pd3) {
+ $packed_widgets{dim}->insert('end', "Reverse vs. Reverse: $score_sort[0] kcal/mol\n\n", 'black');
+ draw_dimer($rprimer, $rprimer, $pos, \ *DIMER);
+ }
+ }
+
+ # $pd_full = 0;
+
+ # again ... messy tie in for report generating routine ...
+ # Yes, I know this is a really, really, ugly way to do things!!
+ if ($report) {
+ my $dimer_text = $packed_widgets{dim}->get('0.1','end');
+ my $primer_text = <<EOT;
+Forward primer: $fprimer
+
+Tm: $fprimer_tm°C\t\tLength: $fprimer_len bases
+dS°: $fprimer_ds eu\t\tdH°: $fprimer_dh kcal/mol
+dG°$pd_temperature: $fprimer_dg kcal/mol
+
+
+Reverse primer: $rprimer
+
+Tm: $rprimer_tm°C\t\tLength: $rprimer_len bases
+dS°: $rprimer_ds eu\t\tdH°: $rprimer_dh kcal/mol
+dG°$pd_temperature: $rprimer_dg kcal/mol
+
+
+$dimer_text\n
+EOT
+ return $primer_text;
+ }
+
+ $packed_widgets{dim}->see(0.1);
+}
+
+
+sub blast_primers {
+ return if check_packages("HTTP::Request", "LWP::UserAgent");
+
+ ### Todo: find matching sequence names from f and r primer blast results
+ ### - easily check possible amp problems
+
+ # I've tried to make this the way blast *should* be, with colour output
+ # and the ability to limit the results ...
+ if (($fprimer eq "") && ($rprimer eq "")) {
+ dialogue("Please enter primers to BLAST");
+ return;
+ }
+ my ($blast_out_f,$blast_out_r)=("Waiting on Blast Server for forward primer results ...","Waiting on Blast Server for reverse primer results ...");
+ my ($blast_header_f, @blast_summary_f, @blast_results_f1, @blast_results_f2, @blast_results_f3);
+ my ($blast_header_r, @blast_summary_r, @blast_results_r1, @blast_results_r2, @blast_results_r3);
+ my $display='f';
+ my $blast_search_string;
+ $blast_status = "";
+
+ my $blast_display = sub {
+ # I don't know whether this is messy or beautiful ...
+ # But I really love the fact that I can do it!!
+ my ($blast_header_ref, $blast_summary_ref, $blast_results_1_ref, $blast_results_2_ref, $blast_results_3_ref, $search_string);
+ if ($display eq 'f') {
+ $blast_header_ref = \$blast_header_f;
+ $blast_summary_ref = \@blast_summary_f;
+ $blast_results_1_ref = \@blast_results_f1;
+ $blast_results_2_ref = \@blast_results_f2;
+ $blast_results_3_ref = \@blast_results_f3;
+ } else {
+ $blast_header_ref = \$blast_header_r;
+ $blast_summary_ref = \@blast_summary_r;
+ $blast_results_1_ref = \@blast_results_r1;
+ $blast_results_2_ref = \@blast_results_r2;
+ $blast_results_3_ref = \@blast_results_r3;
+ }
+
+ $packed_widgets{'blast_text'}->delete('0.1', 'end');
+ unless ($$blast_header_ref) {
+ return
+ }
+ $packed_widgets{'blast_text'}->insert('0.1', $$blast_header_ref, 'black');
+
+ ### TODO: hyperlinks??
+
+ # Users might get confused about "."'s in the string box ...
+ ($search_string = $blast_search_string) ||=".";
+ for my $i (0 .. $#$blast_summary_ref) {
+ $_ = $$blast_summary_ref[$i];
+ next unless /$search_string/;
+ if (m/^(\w*\|[\w\|\.\d]+)(.*?)([\d\.]+\s+[\d\.\w\-]+\s*$)/) {
+ $packed_widgets{'blast_text'}->insert('end', $1);
+ $packed_widgets{'blast_text'}->insert('end', $2, 'blue');
+ $packed_widgets{'blast_text'}->insert('end', $3."\n");
+ } else {
+ ## Debug only
+ # print "WARNING: failed parsing $_ from BLAST results\n";
+ }
+
+ }
+ for my $i (0 .. $#$blast_results_1_ref) {
+ next unless $$blast_results_1_ref[$i] =~ /$search_string/;
+ $packed_widgets{'blast_text'}->insert('end', "\n\n".$$blast_results_1_ref[$i]."\n\n ", 'blue');
+ $packed_widgets{'blast_text'}->insert('end', $$blast_results_2_ref[$i]."\n\n", 'black');
+ $packed_widgets{'blast_text'}->insert('end', $$blast_results_3_ref[$i]."\n");
+ }
+ };
+
+ # GUI code
+ # destroy old window if one exists (and cancel last blast search ...)
+ if (Exists($blast_d)) {
+ # $top->afterCancel($rptid);
+ $rptid->cancel;
+ $blast_d->destroy;
+ }
+
+ $blast_d = $top->Toplevel(-title=>'BLAST Search');
+ $blast_d->withdraw();
+ my $blast_d_f = $blast_d->Frame()->pack(-expand=>1, -side=>'top', -fill=>'both');
+ my $blast_d_fb = $blast_d->Frame()->pack(-side=>'left', -anchor=>'sw');
+ my $blast_d_fs = $blast_d->Frame()->pack(-side=>'right', -anchor=>'se');
+ $blast_d->Icon(-image => $pixmap);
+
+ nr(\$blast_d_f, $frame_pady, 1);
+ pack_gui('ROText', '', 'blast_text', 95, 30, -scrollbars=>'osoe');
+
+ # buttons
+ nr(\$blast_d_fb);
+ pack_gui('Button', "OK", "blast_full", sub{
+ $rptid->cancel if $rptid;
+ $blast_d->destroy;
+ }, "active");
+
+ pack_gui('Button', "Forward", "blast_f", sub {
+ $display='f';
+ &$blast_display;
+ }, 'disabled');
+
+ pack_gui('Button', "Reverse", "blast_r", sub {
+ $display='r';
+ &$blast_display;
+ }, 'disabled');
+
+ # Status line
+ pack_gui('Label', 'Status: ', 'blast_status_l');
+ pack_gui('Label', \$blast_status, 'blast_status');
+
+ # text search button
+ nr(\$blast_d_fs);
+ pack_gui('Label', 'String:');
+ pack_gui('Entry', \$blast_search_string, 'blast_search_string', 20);
+ pack_gui('Button', 'Search', "blast_search", sub {
+ &$blast_display;
+ }, 'disabled');
+
+ $packed_widgets{blast_text}->configure(-fg=>'grey30');
+ $packed_widgets{blast_text}->tagConfigure('blue',
+ -foreground => 'midnightblue');
+ $packed_widgets{blast_text}->tagConfigure('black',
+ -foreground => 'black');
+
+ $blast_status = "Sending BLAST request ...";
+ $blast_d->update;
+ $blast_d->deiconify();
+ $top->update;
+
+ my $blast_summary;
+
+ if ($fprimer) {
+ # Blast forward primer
+ $blast_status="Blasting forward primer ...";
+
+ ($blast_header_f, $blast_summary) = blast($fprimer);
+ if ($blast_summary) {
+ @blast_summary_f = split("\n",$blast_summary);
+ @blast_results_f1 = @blast_results_1;
+ @blast_results_f2 = @blast_results_2;
+ @blast_results_f3 = @blast_results_3;
+
+ $display='f';
+ &$blast_display;
+
+ $packed_widgets{blast_f}->configure(-state=>'normal');
+ $packed_widgets{blast_search}->configure(-state=>'normal');
+ } else {
+ $blast_status="Unable to connect to server";
+ }
+ }
+
+ if ($rprimer) {
+ #Blast reverse primer
+ $blast_status="Blasting reverse primer ...";
+
+ ($blast_header_r, $blast_summary) = blast($rprimer);
+ if ($blast_summary) {
+ @blast_summary_r = split("\n",$blast_summary);
+ @blast_results_r1 = @blast_results_1;
+ @blast_results_r2 = @blast_results_2;
+ @blast_results_r3 = @blast_results_3;
+
+ $packed_widgets{blast_r}->configure(-state=>'normal');
+ $packed_widgets{blast_search}->configure(-state=>'normal');
+
+ $blast_status="Blast search complete";
+ } else {
+ $blast_status="Unable to connect to server";
+ }
+ }
+}
+
+sub get_primers {
+ my ($max_ampsize, $max_range_5p, $max_range_3p)
+ = get_variables(qw(max_ampsize max_range_5p max_range_3p));
+
+ $cancel=0;
+ $bs=0;
+ my $seq = get_seq();
+ check_range();
+
+ # set the max_ampsize in case the user has modified
+ # either max_range_5p or max_range_3p by hand ...
+ if (($$max_range_5p)&&($$max_range_3p)) {
+ $$max_ampsize = $$max_range_3p-$$max_range_5p
+ }
+
+ @PF=@PR=();
+ my ($gene_5p, $gene_3p)=find_gene($seq);
+
+ sbarprint("\nMoving forward and reverse into arrays ...");
+ my ($dnaseq_f, $dnaseq_f_len, $dnaseq_r_top, $dnaseq_r, $dnaseq_r_len) = getseq($seq);
+ return if ($cancel==1);
+
+ # Check that there was actually a DNA sequence found!
+ if (length($dnaseq_f)==0) {
+ sbarprint("\nNo DNA sequence");
+ $cancel = 1;
+ return;
+ }
+
+ read_windows(\&primer_window, $dnaseq_f, $dnaseq_f_len, $dnaseq_r);
+ return if ($cancel==1);
+
+ sbarprint("\nCalculating amplicons ...");
+ calc_amplicon($dnaseq_r_len);
+
+ @primer_pairs_pr_s = @primer_pairs;
+ sort_primers('13',1);
+ sort_primers('10');
+
+ sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+ # draw_dna(\$packed_widgets{primer_canvas},\$packed_widgets{seq});
+ draw_dna();
+}
+
+
+sub get_seq_primers {
+ my ($min_range, $max_range, $slist) = get_variables(qw(min_range max_range primers));
+
+ $cancel=0;
+ $bs=0;
+ my $seq = get_seq();
+ # check_range();
+
+ # unless boundaries are set, set them to sequence limits
+ unless (($$max_range)&&($$max_range)) {
+ $$min_range = 0;
+ $$max_range = length($seq);
+ }
+
+ $$max_range = length($seq) if $$max_range > length($seq);
+
+ @PF=@PR=();
+ my ($gene_5p, $gene_3p)=find_gene($seq);
+
+ sbarprint("\nMoving forward and reverse into arrays ...");
+ my ($dnaseq_f, $dnaseq_f_len) = getseq($seq);
+ return if ($cancel==1);
+
+ # Check that there was actually a DNA sequence found!
+ if (length($dnaseq_f)==0) {
+ sbarprint("\nNo DNA sequence");
+ return;
+ }
+
+ read_windows(\&primer_window, $dnaseq_f, $dnaseq_f_len);
+ return if ($cancel==1);
+
+ sbarprint("\nFinding primers ...");
+ calc_seq_primers();
+
+ @$slist = @primer_pairs;
+ sort_primers('1');
+
+ sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+ # draw_dna(\$packed_widgets{primer_canvas},\$packed_widgets{seq});
+ draw_dna();
+}
+
+
+sub get_qprimers {
+ $cancel=0;
+ $bs=0;
+
+ my ($spidey_out, $mrna_seq) = run_spidey(1);
+ return if $cancel == 1;
+
+ @PF=@PR=();
+ my ($gene_5p, $gene_3p)=find_gene($mrna_seq);
+
+ sbarprint("\nMoving forward and reverse into arrays ...");
+ my ($dnaseq_f, $dnaseq_f_len, $dnaseq_r_top, $dnaseq_r, $dnaseq_r_len) = getseq($mrna_seq);
+ return if ($cancel==1);
+
+ # Check that there was actually a DNA sequence found!
+ if (length($dnaseq_f)==0) {
+ sbarprint("\nNo DNA sequence");
+ return;
+ }
+
+ read_windows(\&primer_window, $dnaseq_f, $dnaseq_f_len, $dnaseq_r);
+ return if ($cancel==1);
+
+ sbarprint("\nCalculating amplicons ...");
+ # set qpcr flag for amplicon and draw_dna subroutines:
+ $qpcr_flag=1;
+ calc_amplicon($dnaseq_r_len);
+
+ @primer_pairs_q_s = @primer_pairs;
+ sort_primers('13',1);
+ sort_primers('10');
+
+ sbarprint("\nFinished ... found ".($#primer_pairs+1)." primer pairs");
+ draw_dna();
+
+ # unset flag
+ $qpcr_flag=0;
+}
+
+
+sub find_re_sites {
+ my $page = which_nb_page();
+ unless ($page eq "pd") {
+ dialogue("This feature is only relevant for the Standard PCR page");
+ return;
+ }
+
+ my $seq = get_seq();
+ my ($max_range_5p, $max_range_3p)
+ = get_variables(qw(max_range_5p max_range_3p));
+ $seq = substr($seq, $$max_range_5p, $$max_range_3p-$$max_range_5p) if $seq;
+
+ # find the rebase file (different versions are released constantly)
+ my @gcg_paths = glob("$HOME"."gcg.*");
+ print @gcg_paths;
+ unless (@gcg_paths) {
+ # search for the file in the program directory
+ @gcg_paths = glob("$program_directory"."gcg.*");
+ }
+
+ my $gcg_path;
+ foreach (@gcg_paths) {
+ if (/.*gcg\.\d*$/) {
+ $gcg_path = $_;
+ }
+ }
+
+ unless ($gcg_path) {
+ dialogue("Error: Cannot find the Restriction Enzyme database.\n\nYou need the GCG database from REBASE (http://rebase.neb.com/rebase/rebase.html) for this function to work. The file 'gcg.###' (where ### is the version) should be included in the PerlPrimer distribution - please place that file either in the directory where the program is located, or in the directory $HOME, and try again.\n\nAlternatively, you can download the latest version of the database from the REBASE ftp site.");
+ return;
+ }
+
+ # open rebase
+ unless (open (REBASE, "<$gcg_path")) {
+ dialogue("Error: Cannot open the Restriction Enzyme database: $!");
+ return;
+ }
+ my @re_data = <REBASE>;
+ close REBASE;
+
+ # parse data
+ my $i;
+ my %enzymes;
+ my %enzyme_sites;
+ my %enzymes_full;
+ foreach (@re_data) {
+ s/[\n\r]//g;
+ my ($enzyme, $re_site) = /^\;*([A-Z|a-z|0-9|\;]+)\s+\d+\s+([A-z\'_]+)\s+/;
+
+ next unless $enzyme && $re_site;
+ my $original_site = $re_site;
+ $re_site =~ s/\'//g;
+ $re_site =~ s/_//g;
+
+ if ($enzymes_full{$re_site}) {
+ $enzymes_full{$re_site}.=" | $enzyme";
+ } else {
+ $enzymes_full{$re_site}=$enzyme;
+ }
+
+ $enzyme_sites{$enzyme}=$original_site;
+
+ if ($simple_sites) {
+ next if $re_site =~ /[rymkswbdhvn]/i;
+ next if length($re_site) > 6;
+ next if length($re_site) < 6;
+ } else {
+ $re_site =~ s/r/[ga]/ig;
+ $re_site =~ s/y/[ct]/ig;
+ $re_site =~ s/m/[ac]/ig;
+ $re_site =~ s/k/[gt]/ig;
+ $re_site =~ s/s/[gc]/ig;
+ $re_site =~ s/w/[at]/ig;
+ $re_site =~ s/b/[cgt]/ig;
+ $re_site =~ s/d/[agt]/ig;
+ $re_site =~ s/h/[act]/ig;
+ $re_site =~ s/v/[acg]/ig;
+ $re_site =~ s/n/[acgt]/ig;
+ }
+
+ $enzymes{$enzyme}=$re_site;
+ }
+
+ my @no_matches;
+ if ($exclude_found_sites) {
+ foreach my $key (keys(%enzymes)) {
+ if ($seq !~ /$enzymes{$key}/i) {
+ push @no_matches, "$key - $enzyme_sites{$key}";
+ }
+ }
+ } else {
+ foreach my $key (keys(%enzymes)) {
+ push @no_matches, "$key - $enzyme_sites{$key}";
+ }
+ }
+
+ unless (Exists($cloning_d)) {
+ $cloning_d = $top->Toplevel(-title=>'Cloning site configuration');
+ my $cloning_f = $cloning_d->Frame()->pack(-fill=>'both', -pady=>7);
+ my $cloning_fb = $cloning_d->Frame()->pack(-side=>'bottom', -fill=>'none');
+
+ my $re_enzymes = [sort @no_matches];
+
+ # deconstruct current info if it exists
+ my ($tmp_site, $enzyme, $key);
+ if ($primer_seq_5f) {
+ $_ = uc($primer_seq_5f);
+ ($cloning_anchor, $tmp_site) = /([A-Z]*)_([A-Z]*)/;
+ if ($enzymes_full{$tmp_site}) {
+ $enzyme = $enzymes_full{$tmp_site};
+ ($key) = ($enzyme =~ /([A-z0-9]*)/);
+ $forward_re_site = "$enzyme - $enzyme_sites{$key}";
+ } else {
+ $forward_re_site = $tmp_site;
+ }
+ }
+
+ if ($primer_seq_5r) {
+ $_ = uc($primer_seq_5r);
+ my $tmp_site;
+ ($cloning_anchor, $tmp_site) = /([A-Z]*)_([A-Z]*)/;
+ if ($enzymes_full{$tmp_site}) {
+ $enzyme = $enzymes_full{$tmp_site};
+ ($key) = ($enzyme =~ /([A-z0-9]*)/);
+ $reverse_re_site = "$enzyme - $enzyme_sites{$key}";
+ } else {
+ $reverse_re_site = $tmp_site;
+ }
+ }
+
+ nr(\$cloning_f);
+ pack_gui('Label', 'Forward restriction enzyme site: ', 'cloning_re_f');
+ pack_gui('BrowseEntry', \$forward_re_site, 'cloning_re_f', $re_enzymes);
+ nr();
+ pack_gui('Label', 'Reverse restriction enzyme site: ', 'cloning_re_r');
+ pack_gui('BrowseEntry', \$reverse_re_site, 'cloning_re_r', $re_enzymes);
+ nr('', 5);
+ pack_gui('Label', "5' anchor sequence", "cloning_anchor");
+ pack_gui('Entry', \$cloning_anchor, "cloning_anchor", 15);
+
+ nr(\$cloning_fb);
+ pack_gui('Button', 'OK', 'cloning_ok', sub {
+ my ($enzyme) = ($forward_re_site =~ /([A-z0-9]*)/);
+ $primer_seq_5f = lc("$cloning_anchor\_$enzymes{$enzyme}");
+ ($enzyme) = ($reverse_re_site =~ /([A-z0-9]*)/);
+ $primer_seq_5r = lc("$cloning_anchor\_$enzymes{$enzyme}");
+ $cloning_d->destroy;
+ return;
+ }, "active");
+ pack_gui('Button', 'Cancel', 'cloning_cancel', sub {$cloning_d->destroy;});
+
+ $cloning_d->Icon(-image => $pixmap);
+ } else {
+ $cloning_d->deiconify;
+ $cloning_d->raise;
+ }
+
+}
+
+
+sub run_spidey {
+ my ($print_alignment) = @_;
+ $print_alignment ||= 0;
+
+ # Find the spidey executable
+ my @spidey_files = glob("$spidey_path*pidey.*");
+ @spidey_files = glob("$spidey_path*pidey*") unless @spidey_files;
+
+ unless (@spidey_files) {
+ dialogue("Error: cannot find the Spidey executable in $spidey_path\n\nIf this is not the directory where Spidey is located, please specify the correct location in the Preferences");
+ $cancel=1;
+ return;
+ }
+
+ my %sizes_names;
+ foreach (@spidey_files) {
+ my $size = (stat($_))[7];
+ $sizes_names{$size}=$_;
+ }
+ my $largest = (sort {$b <=> $a} keys %sizes_names)[0];
+ my $spidey_exec = $sizes_names{$largest};
+
+ my $spidey_command = "\"$spidey_exec\" -i \"$tmp.dna_tmp\" -m \"$tmp.mrna_tmp\" -p $print_alignment";
+
+ my $mrna_seq = $packed_widgets{"qmrna_seq"}->get(0.1,"end");
+ unless (length(clean_seq($mrna_seq))) {
+ dialogue("Error: Please enter an mRNA sequence\n");
+ $cancel=1;
+ return;
+ }
+
+ my $dna_seq = $packed_widgets{"qdna_seq"}->get(0.1,"end");
+ unless (length(clean_seq($dna_seq))) {
+ dialogue("Error: Please enter a DNA sequence\n");
+ $cancel=1;
+ return;
+ }
+
+ # Spidey will only accept input as files, so we need to move the two
+ # sequences to temporary files
+ unless (open (MRNA, ">".$tmp.".mrna_tmp")) {
+ dialogue("Error: could not write mRNA temp file: $!\n");
+ $cancel=1;
+ return;
+ }
+ print MRNA $mrna_seq;
+ close (MRNA);
+
+ unless (open (DNA, ">".$tmp.".dna_tmp")) {
+ dialogue("Error: could not write DNA temp file: $!\n");
+ $cancel=1;
+ return;
+ }
+ print DNA $dna_seq;
+ close (DNA);
+
+ # run spidey ...
+ sbarprint("\nRunning spidey ...");
+ $_ = `$spidey_command`;
+
+ # open(SPIDEY, "$spidey_command |");
+ # $_ = join("",<SPIDEY>);
+ # close SPIDEY;
+
+ # abort and complain if we don't see the --SPIDEY signature
+ unless (/--SPIDEY/) {
+ print "$_\n";
+ if (m/no valid bioseqs(.*)/i) {
+ dialogue("Error: No valid bioseqs$1\n\n(If all sequences are present, this may mean that the temp directory is incorrectly set in the preferences - please make sure that this directory is writable)");
+ sbarprint("\nCancelled - No valid bioseqs$1");
+ } else {
+ dialogue("Error: Cannot run Spidey executable correctly\n(Please check the console for error messages)");
+ sbarprint("\nCancelled - Spidey executable not found");
+ }
+ $cancel=1;
+ return;
+ }
+
+ # Warn if problems occur ...
+ print "\n\nWarning: incomplete sequence data or large intron present\n" unless /overall percent identity: 100\.0%/;
+ print "\n\nWarning: mRNA coverage incomplete\n" unless /mRNA coverage: 100%/;
+
+ # ... and try using the large intron option if they have
+ unless (/overall percent identity: 100\.0%/ && /mRNA coverage: 100%/) {
+ print "Trying spidey again using large intron option ...\n";
+ $_ = `$spidey_command -X`;
+
+ # if things are still crap, display a dialogue with spidey's output,
+ # so the user can work out what's gone wrong
+ dialogue("Alignment error:\nSequence data is incomplete\n\n$_") unless /overall percent identity: 100\.0%/;
+ dialogue("Alignment error:\nmRNA coverage is incomplete\n\n$_") unless /mRNA coverage: 100%/;
+ }
+
+ # delete those tmp files
+ unlink("$tmp.mrna_tmp") || dialogue("Error: could not delete temporary file $spidey_path.mrna_tmp");
+ unlink("$tmp.dna_tmp") || dialogue("Error: could not delete temporary file $spidey_path.dna_tmp");
+
+ sbarprint("\n");
+
+ return ($_, $mrna_seq) unless $print_alignment;
+
+ # isolate intron/exon boundaries
+ @intron_exon_bounds = ();
+ while (/(\d+)-(\d+) \(mRNA\)/g) {
+ push (@intron_exon_bounds, $2);
+ }
+
+ # last boundary is end of mRNA sequence: remove
+ pop @intron_exon_bounds;
+
+ return ($_, $mrna_seq);
+}
+
+
+sub get_bisulphite {
+ # Criteria:
+ # Nested or heminested PCR
+ # Primer amplicon not greater than 450bp
+ # Selection for bisulphite-converted templates
+ # Even distribution of bases prior to conversion
+ # As high GA content as posible [currently not used]
+ # Lack of CpG residues or degeneracy
+ # Long (25-30mer) primer
+ $cancel=0;
+
+ # old benchmarking code - used for optimising routines
+ my $t_old = new Benchmark if $benchmark;
+ $bs = 1;
+ check_range();
+
+ my ($max_ampsize, $max_range_5p, $max_range_3p)
+ = get_variables(qw(max_ampsize max_range_5p max_range_3p));
+
+ # set the max_ampsize in case the user has modified
+ # either max_range_5p or max_range_3p by hand ...
+ if (($$max_range_5p)&&($$max_range_3p)) {
+ $$max_ampsize = $$max_range_3p-$$max_range_5p
+ }
+
+ my $seq = $packed_widgets{"bisul_seq"}->get(0.1,"end");
+ my ($dnaseq_f, $dnaseq_f_len, $dnaseq_r_top, $dnaseq_r, $dnaseq_r_len) = getseq($seq);
+
+ # Check that there was actually a DNA sequence found!
+ if (length($dnaseq_f)==0) {
+ sbarprint("\nNo DNA sequence");
+ return;
+ }
+
+ @PF=@PR=();
+ return if ($cancel==1);
+
+ # Only use top strand for primer design (because conversion makes
+ # the two templates non-complementary), so use $dnaseq_r_top
+ read_windows(\&bisul_window, $dnaseq_f, $dnaseq_f_len, $dnaseq_r_top);
+ return if ($cancel==1);
+
+ sbarprint("\nCalculating amplicons ...");
+ calc_amplicon($dnaseq_r_len);
+
+ @primer_pairs_bs_s = @primer_pairs;
+ sort_primers('13',1);
+ sort_primers('10');
+
+ $bs = 0;
+
+ # benchmarking code
+ if ($benchmark) {
+ my $t_new = new Benchmark;
+ my $diff = timediff ($t_new, $t_old);
+ my $str = timestr ($diff, "all", "5.3f");
+ print "Time for bisulphite was $str\n\n";
+ }
+}
+
+sub check_range {
+ my ($max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ $$max_range_5p = undef if defined($$max_range_5p) && $$max_range_5p eq "";
+ $$max_range_3p = undef if defined($$max_range_3p) && $$max_range_3p eq "";
+ unless (defined($$max_range_5p)&&defined($$max_range_3p)) {
+ return unless defined($$min_range)&&defined($$max_range);
+ $$max_range_5p = $$min_range unless $$max_range_5p;
+ $$max_range_3p = $$max_range unless $$max_range_3p;
+ $$max_ampsize = $$max_range_3p - $$max_range_5p;
+ }
+}
+
+sub copy_selected_primers {
+ # Get variable refs
+ my ($ref, $slist) = get_variables(qw(hlist primers));
+ my $page = which_nb_page();
+
+ # get selected primers
+ my @sel = $$ref->selectionGet;
+
+ # Clipboard copying routine
+ my $clip = "Forward Primer\tPos\tLen\tTm\tReverse Primer\tPos\tLen\tTm\tAmp\tdG\n";
+
+ my (@gene_array, $gene_frame, $seq);
+ if ($page eq "pd" && (($primer_seq_5f) || ($primer_seq_5r))) {
+ $seq = get_seq();
+ @gene_array=find_gene($seq);
+ $gene_frame=$gene_array[0][0]%3;
+ }
+
+ foreach (@sel) {
+ my $sel = $_;
+ for my $j ( 0 .. 4, 8, 6, 7, 9, 10 ) {
+ if ($page eq "bis") {
+ # Primer redundancy for CpG residues:
+ # Replaces T with Y (pyrimidine) for forward
+ # Replaces A with R (purine) for reverse
+
+ my $temp_seq;
+ if ($j == 0) {
+ $temp_seq = $$slist[$sel][0];
+ my $f_original = $$slist[$sel][11];
+ for my $i (0 .. length($f_original)) {
+ if (substr($f_original, $i, 2) eq "CG") {
+ substr($temp_seq, $i, 1) = 'Y';
+ }
+ }
+ $clip .= "$temp_seq\t";
+ } elsif ($j == 4) {
+ $temp_seq = $$slist[$sel][4];
+ my $r_original = $$slist[$sel][12];
+ for my $i (0 .. length($r_original)) {
+ if (substr($r_original, $i, 2) eq "CG") {
+ substr($temp_seq, $i, 1) = 'R';
+ }
+ }
+ $clip .= "$temp_seq\t";
+ } else {
+ $clip .= "$$slist[$sel][$j]\t";
+ }
+ } elsif ($page eq "pd") {
+ if ($j == 0 && $primer_seq_5f) {
+ my $fprimer = $$slist[$sel][$j];
+ my ($primer_seq_5f_real, $insert_f) = add_cloning($seq, $fprimer, $$slist[$sel][1]);
+ $clip .= uc("$primer_seq_5f_real$insert_f\_$fprimer\t");
+ } elsif ($j == 4 && $primer_seq_5r) {
+ my $rprimer = $$slist[$sel][$j];
+ my ($primer_seq_5r_real, $insert_r) = (add_cloning($seq, '', '', '', $rprimer, $$slist[$sel][8]))[5,6];
+ # print
+ $clip .= uc("$primer_seq_5r_real$insert_r\_$rprimer\t");
+ } else {
+ $clip .= "$$slist[$sel][$j]\t";
+ }
+ } else {
+ $clip .= "$$slist[$sel][$j]\t";
+ }
+ }
+ $clip .= "\n";
+ }
+
+ $top->clipboardClear;
+ $top->clipboardAppend($clip);
+}
+
+sub select_all_primers {
+ my ($ref, $slist) = get_variables(qw(hlist primers));
+ $$ref->selectionSet(0,$#$slist);
+}
+
+sub primer_take_range {
+ # Get variable refs
+ my ($max_range_5p, $min_range, $max_range, $max_range_3p, $hlist, $slist)
+ = get_variables(qw(max_range_5p min_range max_range max_range_3p hlist primers));
+
+ # get selected primer
+ my @sel = $$hlist->selectionGet;
+ my $primer = shift @sel;
+
+ $$max_range_5p = $$min_range = $$slist[$primer][1]+$$slist[$primer][2];
+ $$max_range_3p = $$max_range = $$slist[$primer][8]-$$slist[$primer][6];
+
+ draw_dna();
+ browse_primer($primer);
+}
+
+sub menu_popup {
+ my ($popup_ref) = get_variables(qw(popup));
+ $$popup_ref->Popup(
+ -popanchor => 'nw',
+ -popover => 'cursor');
+}
+
+sub menu_text {
+ $text_widget_ref = $_[0];
+ $popup_text->Popup(
+ -popanchor => 'nw',
+ -popover => 'cursor');
+}
+
+sub sort_primers {
+ my ($sort_criteria, $quiet) = @_;
+ my ($list, $slist) = get_variables(qw(hlist primers));
+ my @old_slist = @$slist;
+
+ $$list->delete('all');
+
+ # Re-sort forwards or backwards
+ # allow the ability to simply redisplay the list (when opening files)
+ if ($sort_criteria) {
+ $sort_criteria = $sort_prev if $sort_criteria eq "r";
+
+ # We want dG to be sorted from highest to lowest by default:
+ # this is really inelegant!
+ my $sort_order = $sort_reverse;
+ $sort_order = 1-$sort_reverse if ($sort_criteria eq '10' || $sort_criteria eq '13');
+
+ @$slist = sort {@$a[$sort_criteria] <=> @$b[$sort_criteria]} @old_slist if $sort_order==0;
+ @$slist = sort {@$b[$sort_criteria] <=> @$a[$sort_criteria]} @old_slist if $sort_order==1;
+ }
+
+ return if $quiet;
+
+ my $num_elements = @{ @$slist[0] } if @$slist;
+ $num_elements ||=0;
+
+ # Re-draw
+ if ($num_elements == 5) { # sequencing quick fix!!
+ for my $i ( 0 .. $#$slist ) {
+ $$list->add($i);
+ $$list->itemCreate($i, 0, -text=>"$$slist[$i][0]", -style=> $style_primer);
+ $$list->itemCreate($i, 1, -text=>"$$slist[$i][1]");
+ $$list->itemCreate($i, 2, -text=>"$$slist[$i][2]");
+ $$list->itemCreate($i, 3, -text=>"$$slist[$i][3]", -style=> $style_tm);
+
+ $$list->itemCreate($i, 4, -text=>"$$slist[$i][4]");
+ }
+ } else {
+ for my $i ( 0 .. $#$slist ) {
+ $$list->add($i);
+ $$list->itemCreate($i, 0, -text=>"$$slist[$i][0]", -style=> $style_primer);
+ $$list->itemCreate($i, 1, -text=>"$$slist[$i][1]");
+ $$list->itemCreate($i, 2, -text=>"$$slist[$i][2]");
+ $$list->itemCreate($i, 3, -text=>"$$slist[$i][3]", -style=> $style_tm);
+
+ $$list->itemCreate($i, 4, -text=>"$$slist[$i][4]", -style=> $style_primer);
+ $$list->itemCreate($i, 5, -text=>"$$slist[$i][8]");
+ $$list->itemCreate($i, 6, -text=>"$$slist[$i][6]");
+ $$list->itemCreate($i, 7, -text=>"$$slist[$i][7]", -style=> $style_tm);
+
+ $$list->itemCreate($i, 8, -text=>"$$slist[$i][9]");
+ $$list->itemCreate($i, 9, -text=>"$$slist[$i][10]");
+ $$list->itemCreate($i, 10, -text=>"$$slist[$i][13]") if defined($$slist[$i][13]);
+ }
+ }
+
+ # save last value so that "reverse" can be applied if neccessary
+ $sort_prev = $sort_criteria;
+}
+
+sub cancel {
+ # Set cancel flag
+ # (this stops the primer-pairs calculation,
+ # which can go on for some time ...)
+ $cancel = 1;
+ sbarprint("\nOperation cancelled");
+}
+
+sub get_primers_auto_in {
+ my ($sub_ref, $primers_ref) = get_variables(qw(primer_sub primers));
+ &$sub_ref();
+ while ($#$primers_ref < 0) {
+ step_in();
+ return if $cancel==1;
+ &$sub_ref();
+ }
+}
+
+sub get_primers_auto_out {
+ my ($sub_ref, $primers_ref) = get_variables(qw(primer_sub primers));
+ &$sub_ref();
+ while ($#$primers_ref < 0) {
+ step_out();
+ return if $cancel==1;
+ &$sub_ref();
+ }
+}
+
+sub get_primers_cloning {
+ my $page = which_nb_page();
+ unless ($page eq 'pd') {
+ dialogue("This feature is only relevant for the Standard PCR page");
+ return;
+ }
+ my $seq = get_seq();
+ unless ($seq) {
+ dialogue("No DNA sequence found!");
+ return;
+ }
+
+ my ($sub_ref, $primers_ref) = get_variables(qw(primer_sub primers));
+ &$sub_ref();
+ while ($#$primers_ref < 0) {
+ step_cloning();
+ return if $cancel==1;
+ &$sub_ref();
+ }
+}
+
+
+#----------#
+# New File #
+#----------#
+
+sub new_file {
+ my ($widget_ref) = @_;
+ my $page = which_nb_page();
+ if ($page eq "primer") {
+ dialogue("You can't open a new file from the primer information page - please switch to the relevant project page first");
+ return;
+ }
+
+ my ($seq_ref, $hlist) = get_variables(qw(seq hlist));
+ $seq_ref = $widget_ref if ref($widget_ref);
+
+ $_ = $$seq_ref->get(0.1,"end");
+ my ($seq_check) = /atcg/i;
+
+ # print "seq was $_\n\n\$seq was $seq\n";
+ # my $seq = $$seq_ref->search(-regexp => "[a|g|t|c|A|T|G|C]", "0.1");
+
+ # If there's a sequence present, prompt to make sure the user wants to
+ # lose all changes ...
+ if (defined($seq_check) && $file_data_overwrite) {
+ my $answer = dialogue("Save before closing?", 'Yes', 'No', 'Cancel');
+ if ($answer =~ /cancel/i) {
+ $cancel = 1;
+ return;
+ } elsif ($answer =~ /yes/i) {
+ pp_file_save();
+ }
+ }
+
+ if ($page eq "qpcr") {
+ # only for qpcr - quick and dodgy fix
+ $packed_widgets{qdna_seq}->delete(0.1,"end");
+ $packed_widgets{qmrna_seq}->delete(0.1,"end");
+ }
+
+ # Clear variables
+ foreach my $key (keys %{ $variables{$page}}) {
+ my $pointer = $variables{$page}{$key};
+ if (eval {defined($$pointer)}) {
+ undef($$pointer);
+ if ($default_variables{$page}{$pointer}) {
+ $$pointer = $default_variables{$page}{$pointer};
+ }
+ }
+ }
+
+ # Clear arrays
+ foreach my $key (keys %{ $arrays{$page}}) {
+ my $pointer = $arrays{$page}{$key};
+ if (eval {@$pointer}) {
+ undef(@$pointer);
+ }
+ }
+
+ # Clear sequence and hlist displays
+ $$seq_ref->delete(0.1,"end");
+ $$hlist->delete('all');
+ draw_dna();
+
+ # Clear title
+ $top->configure(-title=>"PerlPrimer v$version");
+ $open_file{$page} = 'File not saved';
+}
+
+
+#-----------------------------------------------#
+# Saving/loading/deleting user-defined defaults #
+#-----------------------------------------------#
+
+sub save_defaults {
+ # Save user-defined default values
+ my $page = which_nb_page();
+ my $file_defaults = "$HOME.perlprimer.$page";
+ my $defaults;
+ # get list of variables and values
+ foreach my $key (keys %{ $variables{$page}}) {
+ my $pointer = $variables{$page}{$key};
+ if (eval {defined($$pointer)}) {
+ if (defined($default_variables{$page}{$pointer})) {
+ $default_variables{$page}{$pointer} = $$pointer;
+ $defaults .= "$key = $$pointer\n";
+ }
+ }
+ }
+ unless (open (DEFAULTS, ">$file_defaults")) {
+ dialogue("Error: could not open $file_defaults for writing: $!");
+ return;
+ }
+ print DEFAULTS $defaults;
+ close DEFAULTS;
+}
+
+sub restore_defaults {
+ # Remove user-specified preferences file if exists
+ my $page = which_nb_page();
+ my $file_defaults = "$HOME.perlprimer.$page";
+
+ if (-e $file_defaults) {
+ my $answer = dialogue("Warning: This will permanently remove the user-defined default values for this page and restore the built-in values (requires program restart)",'OkCancel');
+ return if $answer =~ /cancel/i;
+ unlink($file_defaults) || dialogue("Error: cannot delete file $file_defaults: $1");
+ dialogue("Built-in values restored. Please restart the program for changes to take effect");
+ } else {
+ dialogue("Error - User-defined defaults have not been saved for this page.\n\nIf you are trying to reset the parameters for this tab and remove all sequence data, use the \"New File\" command from the File menu");
+ }
+}
+
+sub load_defaults {
+ # load user-defined default values (same idea as save defaults above, but in reverse)
+ foreach my $key (keys (%nb_page_ref)) {
+ my $page = $nb_page_ref{$key};
+ my $file_defaults = "$HOME.perlprimer.$nb_page_ref{$key}";
+ if (-e $file_defaults) {
+ print "Loading user-defined defaults file: $file_defaults\n";
+ unless (open (DEFAULTS, "<$file_defaults")) {
+ dialogue("Error: could not open $file_defaults for reading: $!");
+ return;
+ }
+ while (<DEFAULTS>) {
+ chomp;
+ my ($variable, $value) = split / = /;
+ my $pointer = $variables{$page}{$variable};
+ if (defined($default_variables{$page}{$pointer})) {
+ $$pointer = $value;
+ $default_variables{$page}{$pointer} = $value;
+ }
+ }
+ close DEFAULTS;
+ }
+ }
+}
+
+#----------------#
+# Blast routines #
+#----------------#
+
+sub blast {
+ # for more info on using BLAST via http requests, see:
+ # http://www.ncbi.nlm.nih.gov/BLAST/Doc/
+ # which details the request syntax
+
+ unless (Exists($blast_d)) {
+ # this routine should not continue running if the user has destroyed
+ # the blast search dialogue. Hopefully this will stop it!
+ # $top->afterCancel($rptid);
+ $rptid->cancel;
+ return;
+ }
+
+ return unless (my $query=shift);
+
+ unless ($local_blast) {
+ # Standard NCBI server blast over http
+ my $blast_put = "http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?QUERY=$query&DATABASE=$blast_database&ENTREZ_QUERY=($blast_entrez_query)&EXPECT=$blast_expect&WORD_SIZE=$blast_word_size&FORMAT_TYPE=Text&PROGRAM=blastn&SERVICE=plain&CMD=Put";
+
+ # send off the query
+ $_ = http_get($blast_put);
+
+ # abort if unsuccessfull
+ return if $_ eq " ";
+
+ # find the RID string
+ /QBlastInfoBegin(.*)QBlastInfoEnd/sm;
+ $_ = $1;
+ /RID = ([\d\w\-.]*)/;
+
+ $rid_get = "http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?RID=$1&FORMAT_TYPE=Text&CMD=Get";
+
+ # Hah! How about this for a simple way to unblock your GUI ... the only way I
+ # know of to use non-blocking, sleeping subroutines without forks - which we
+ # can't use with ActivePerl for Win32 :(
+ $flag=undef;
+ $blast_count = time();
+ $rptid = $top->repeat(15000, \&blast_wait);
+ $blast_d->waitVariable(\$flag);
+ # $blast_d->afterCancel($rptid);
+ $rptid->cancel;
+
+ # Having asked for text output, blast still provides some HTML formatting!
+ s/\<!\-\-.*\-\-\>//sg;
+ s/\<[\w\\]*\>//g;
+ s/<a href=".*?ALT="New"> //g;
+ } else {
+ # local BLAST server
+ # need to save the primer sequence to a temp file for input to local BLAST
+ unless (open (PRIMER, ">$tmp.primer_tmp")) {
+ dialogue("Error: could not write BLAST temp file: $!\n");
+ return;
+ }
+ print PRIMER $query;
+ close (PRIMER);
+
+ my $local_blast_command = $local_blast_directory."blastall -p blastn -d $local_blast_database -W $blast_word_size -i $tmp.primer_tmp";
+ $_ = `$local_blast_command`;
+
+ # delete tmp file
+ unlink "$tmp.primer_tmp";
+ }
+
+ # OK, let's try something really nifty ... let's pull each entry out:
+ my $blast_out = $_;
+ undef(@blast_results_1);
+ undef(@blast_results_2);
+ undef(@blast_results_3);
+
+ # s/\r//g;
+
+ /(BLASTN.*?Value.*?)(\w+.*?)\>/s;
+ my ($blast_header, $blast_summary) = ($1,$2);
+
+ # search for each sequence identifier and find corresponding blast entry
+ while (m/^(\w*\|[\w\|\.\d]+)/mg) {
+ my $found_result = $1;
+ if ($blast_out =~ /\n\>(\Q$found_result\E.*?Length\s*?=\s*?\d*)[\n\s]*(Score\s*?=.*?Strand\s*?=\s*?\w*\s*?\/\s*?\w*)[\n\s]*(Query.*?Sbjct.*?)\n/s) {
+ push @blast_results_1, $1;
+ push @blast_results_2, $2;
+ push @blast_results_3, $3;
+ }
+
+ }
+
+ return ($blast_header, $blast_summary);
+}
+
+sub blast_wait {
+ #print "Waiting for server response ...\n";
+ my $time_now = time();
+ my $time = convert_time($time_now-$blast_count);
+
+ $blast_status = "Waiting for server response ... $time elapsed";
+ $top->update;
+
+ # print "status: $blast_status\n";
+ $_ = http_get($rid_get);
+ unless (/Status=WAITING/) {
+ $flag=0;
+ } else {
+ #### debugging ... really need to use a timeout with repeat above ...
+ # print "output was $_\n";
+ }
+}
+
+sub convert_time {
+ my ($time) = @_;
+
+ my $seconds = $time % 60;
+ my $minutes_total = ($time-$seconds)/60;
+ # my $minutes = $minutes_total % 60;
+ # my $hours = ($minutes_total-$minutes)/60;
+
+ my $time_readable = sprintf("%02d:%02d", $minutes_total, $seconds);
+
+ return $time_readable;
+}
+
+
+
+#------------------#
+# Ensembl routines #
+#------------------#
+
+sub get_ensembl {
+ return if check_packages("HTTP::Request", "LWP::UserAgent");
+ # retrieve a gene sequence (or a gene and genomic sequence) from ensembl.org ...
+
+ # Why do we use Ensembl? Simply because it happens to provide easy sequence
+ # retrieval, including the genomic sequence of a gene (i.e. not just the cDNA
+ # sequence). However, unlike NCBI, Ensembl provides no documentation on their
+ # cgi scripts; this was all worked out from going through the page sources.
+ my $nb_page = which_nb_page();
+ if ($nb_page eq "primer") {
+ dialogue("Please switch to the project page that you wish to enter the Ensembl data into");
+ return;
+ }
+
+ unless (Exists($ensembl)) {
+ $ensembl = $top->Toplevel(-title=>'Retrieve gene from Ensembl');
+ my $ensembl_f = $ensembl->Frame()->pack(-fill=>'both', -pady=>7);
+ my $ensembl_fb = $ensembl->Frame()->pack(-side=>'bottom', -fill=>'none');
+
+ nr(\$ensembl_f);
+ pack_gui('Label', 'Gene name ', "ensembl_gene");
+ pack_gui('Entry', \$ensembl_gene, "ensembl_gene", 15);
+ nr();
+ pack_gui('Label', 'Organism', 'ensembl_organsim');
+ pack_gui('BrowseEntry', \$ensembl_organism, 'ensembl_organsim', \@ensembl_species, 20);
+ nr();
+ pack_gui('Label', 'Retrieve', 'ensembl_type');
+ pack_gui('BrowseEntry', \$ensembl_type, 'ensembl_type', \@ensembl_types, 10);
+
+ nr(\$ensembl_fb);
+ pack_gui('Button', 'OK', 'ensembl_ok', \&fetch_ensembl, "active");
+ pack_gui('Button', 'Cancel', 'ensembl_cancel', sub {$ensembl->destroy;});
+
+ $ensembl->Icon(-image => $pixmap);
+ } else {
+ $ensembl->deiconify;
+ $ensembl->raise;
+ }
+}
+
+sub fetch_ensembl {
+ my $page = which_nb_page();
+ unless (($ensembl_gene) && ($ensembl_organism) && ($ensembl_type)) {
+ dialogue("Please make sure gene name, organism and retrieval type are filled in");
+ return;
+ }
+
+ # Search for the gene:
+
+ # As of 03/2006, Ensembl now uses "searchview" rather than "textview", and the
+ # species delimiter is important; searchview does not seem to have a species argument
+
+ # As of 04/2006, Ensembl uses "textview" again. Various unprintable expletives come
+ # mind at this point, all directed towards the developers of the Ensembl web structure ...
+
+ # ... and as of 06/2006, Ensembl is back to "searchview"! Worse, textview works (thereby removing
+ # my supposedly failsafe error message) but returns no matches. Sheesh ...
+ $_ = http_get("http://www.ensembl.org/$ensembl_organism/searchview?species=$ensembl_organism&idx=Gene&q=$ensembl_gene");
+
+ s/<\/*span.*?>//g; # rip out highlight spans
+ s/<\/*font.*?>//g; # rip out font spans
+
+ # find the Ensembl gene ID, and count the number - if there's more than one
+ # we'll have to ask the user to be more specific
+ my $gene_id;
+ my @gene_names;
+ my $name;
+ my %ids;
+
+ # as of 07/2005, we're actually looking for the transcript ID, not the gene ID ...
+ # Here, we scrape both genes and associated transcripts from the server:
+ while (m/Ensembl gene ([\w\d]+) .*?:(.*?)<br \/>(.*?)<br \/>/mg) {
+ my ($gene_id, $transcripts, $name) = ($1, $2, $3);
+ $name ||= "$gene_id: no description available";
+ my @enst;
+ while ($transcripts =~ m/(ENS[A-Z]*T\d+)/g) {
+ push @enst, $1;
+ }
+ # A hack - Drosophila uses different transcript definitions ...
+ while ($transcripts =~ m/(CG\d+-RA)/g) {
+ push @enst, $1;
+ }
+ $name =~ s/\<.*?\>//g;
+ push @gene_names, $name;
+ $ids{$name}=[$gene_id, @enst];
+ }
+
+ @gene_names = sort(@gene_names);
+
+ if (@gene_names) {
+ # Ask user to confirm gene identity or pick the gene of interest if multiple matches
+ my $ensembl_mm = $top->Toplevel(-title=>"Select gene of interest ...");
+ my $ensembl_mm_f = $ensembl_mm->Frame()->pack(-fill=>'both', -pady=>7);
+ my $ensembl_mm_fb = $ensembl_mm->Frame()->pack(-side=>'bottom', -fill=>'none');
+ nr(\$ensembl_mm_f);
+ pack_gui('Label', "Found ".($#gene_names+1)." matching gene".($#gene_names > 0 ? 's' : '')." ...", "ensemble_mm_d_note");
+ nr();
+ $name = $gene_names[0];
+ pack_gui('BrowseEntry', \$name, 'ensembl_mm_d_genes', \@gene_names, 40);
+
+ my $cancel=1;
+ nr(\$ensembl_mm_fb);
+ pack_gui('Button', 'OK', 'ensembl_ok', sub {
+ $cancel=undef;
+ $ensembl_mm->destroy;
+ $gene_id = $ids{$name}[0];
+ }, "active");
+ pack_gui('Button', 'Cancel', 'ensembl_cancel', sub {
+ $ensembl_mm->destroy;
+ });
+
+ $ensembl_mm->Icon(-image => $pixmap);
+
+ # (we need it to freeze execution at this point, since the user may
+ # wish to cancel and refine their choice)
+ $ensembl_mm->waitWindow;
+ return if $cancel;
+ }
+
+ my $transcript;
+ if (%ids) {
+ $transcript = $ids{$name}[1];
+ if ($#{ $ids{$name}} > 1) {
+ # multiple transcripts: ask user to select transcript ID
+ my @transcripts = @{ $ids{$name} }[1 .. $#{$ids{$name}}];
+ my $ensembl_mt = $top->Toplevel(-title=>"Please select transcipt ...");
+ my $ensembl_mt_f = $ensembl_mt->Frame()->pack(-fill=>'both', -pady=>7);
+ my $ensembl_mt_fb = $ensembl_mt->Frame()->pack(-side=>'bottom', -fill=>'none');
+ nr(\$ensembl_mt_f);
+ pack_gui('Label', "Ensemble gene $ids{$name}[0] has ".($#transcripts+1)." transcript".($#transcripts > 0 ? 's' : '')." ...", "ensemble_mt_d_note");
+ nr();
+ pack_gui('BrowseEntry', \$transcript, 'ensembl_mt_d_genes', \@transcripts, 20);
+
+ my $cancel=1;
+ nr(\$ensembl_mt_fb);
+ pack_gui('Button', 'OK', 'ensembl_ok', sub {
+ $cancel=undef;
+ $ensembl_mt->destroy;
+ }, "active");
+ pack_gui('Button', 'Cancel', 'ensembl_cancel', sub {
+ $ensembl_mt->destroy;
+ });
+ pack_gui('Button', 'View transcripts', 'ensembl_view_transcripts', sub {
+ my $command = "\"$browser\" http://www.ensembl.org/$ensembl_organism/geneview?gene=$gene_id";
+ if ($os eq 'win') {
+ system "start $command";
+ } else {
+ system "$command &";
+ }
+ });
+
+
+ $ensembl_mt->Icon(-image => $pixmap);
+
+ # (we need it to freeze execution at this point, since the user may
+ # wish to cancel and refine their choice)
+ $ensembl_mt->waitWindow;
+ return if $cancel;
+ }
+ }
+
+ unless ($gene_id) {
+ # no matches
+ if (/Your query found no matches/si) {
+ dialogue("Your query matched no entries in the search database");
+ } elsif ($_ eq " ") {
+ # returned if response->is_error below
+ return;
+ } else {
+ dialogue("Error: Unable to find gene_id in response from server.\n\nThis probably means that the Ensembl server has changed formats - please report this in an email to owenjm\@users.sf.net or submit a bug report at http://perlprimer.sf.net ...\n\nThanks!");
+ # print "output was\n$_\n";
+ }
+
+ return;
+ }
+
+ new_file();
+
+ if ($page eq 'qpcr') {
+ # retrieve both gene and transcript sequences - retrieval type is ignored
+ $_ = http_get(convert_ensembl($ensembl_organism,$transcript,'genomic'));
+ $packed_widgets{qdna_seq}->delete(0.1,"end");
+ $packed_widgets{qdna_seq}->insert(0.1,$_);
+
+ $_ = http_get(convert_ensembl($ensembl_organism,$transcript,'cdna'));
+ $packed_widgets{qmrna_seq}->delete(0.1,"end");
+ $packed_widgets{qmrna_seq}->insert(0.1,$_);
+
+ # Run spidey automatically to show the user the intron/exon boundaries
+ run_spidey(1);
+ } else {
+ # retrieve requested sequence
+ my ($seq_ref) = get_variables('seq');
+ $_ = http_get(convert_ensembl($ensembl_organism,$transcript,$ensembl_type));
+ $$seq_ref->delete(0.1,"end");
+ $$seq_ref->insert(0.1,$_);
+ }
+
+ # update title
+ my $file_name = "$ensembl_gene"."_($ensembl_organism)";
+ $file_name = format_file_name($file_name);
+ $top->configure(-title=>"PerlPrimer v$version - $file_name");
+ $open_file{$page} = $file_name;
+ sbarprint("\n$ensembl_gene ($ensembl_organism) retrieved sucessfully");
+
+ # destroy dialogue, draw the sequence
+ $ensembl->destroy;
+ draw_dna();
+ reset_bounds();
+ return;
+}
+
+sub convert_ensembl {
+ # argument to http address converter
+ my ($ensembl_organism,$transcript,$ensembl_type) = @_;
+ my $address = "http://www.ensembl.org/$ensembl_organism/exportview?seq_region_name=&type1=transcript&anchor1=$transcript&type2=bp&anchor2=&downstream=&upstream=&format=fasta&action=export&_format=Text&options=$ensembl_type&output=txt";
+ return $address;
+}
+
+
+#------------------------#
+# http retrieval routine #
+#------------------------#
+
+sub http_get {
+ return if check_packages("HTTP::Request", "LWP::UserAgent");
+ # simple html retrieval ... used by BLAST and Ensembl routines
+ # I'd love to fork this and stop the GUI blocking here - but I cannot find
+ # an effective way to do this ... :(
+ my ($address, $method) = @_;
+ # print "$address\n";
+ my $ua = LWP::UserAgent->new();
+
+ # timeout - default value of 180 seconds is too long
+ $ua->timeout(60);
+
+ # proxy server
+ if ($use_proxy) {
+ # allow for users using "http://" in the proxy address
+ $http_proxy =~ /(http:\/\/)*(.*)/;
+ my $proxy = $2;
+ $ua->proxy('http', "http://$proxy:$http_port");
+ } else {
+ # still allow env_proxy override
+ $ua->env_proxy();
+ }
+
+ my $req = HTTP::Request->new(GET => "$address");
+
+ my $response = $ua->request($req);
+ if ($response->is_error) {
+ my $code = $response->code;
+ my $message = $response->message;
+
+ dialogue("Error: $code $message");
+ return " ";
+ }
+
+ $top->update;
+ my $http = $response->content;
+ # print "http:$http\n";
+
+ return $http;
+}
+
+
+#---------------------#
+# Status bar updating #
+#---------------------#
+
+sub sbarprint {
+ print SBAR $_[0];
+ $top->update;
+}
+
+
+#------------#
+# HList subs #
+#------------#
+
+sub header_create {
+ my ($widget_r, @headers) = @_;
+ my $i=0;
+ foreach (@headers) {
+ $$widget_r->header('create', $i, -text => $_);
+ $i++;
+ }
+
+ $style_primer = $$widget_r->ItemStyle("text",
+ -background => '#eeeeee',
+ -foreground => 'red');
+ $style_tm = $$widget_r->ItemStyle("text",
+ -background => '#eeeeee',
+ -foreground => 'blue');
+}
+
+
+sub browse_bisulphite {
+ # my $seq = get_seq();
+ # Believe it or not, when a primer-pair is double-clicked to send the user
+ # to the hlist-command sub, the browse-command sub is still executed! In
+ # our case, that's pretty bad since we're now requesting a sequence on a
+ # page that doesn't have one. So there's a failsafe here:
+ # return if $seq eq "1";
+
+ my ($hlist_sel) = @_;
+
+ # This routine colours original C residues red and original CpG C's blue
+ # if ($primer_pairs_bs_s[$hlist_sel][10]) {
+ # brute force!!
+ my $f_original = $primer_pairs_bs_s[$hlist_sel][11];
+ my $f_converted = $primer_pairs_bs_s[$hlist_sel][0];
+
+ my $r_original = $primer_pairs_bs_s[$hlist_sel][12];
+ my $r_comp_original = complement($r_original);
+ my $r_converted = $primer_pairs_bs_s[$hlist_sel][4];
+
+ $status_bar->insert('end',"\nF: 5' ", 'grey');
+
+ for my $i (0 .. length($f_original)) {
+ if (substr($f_original, $i, 2) eq "CG") {
+ $status_bar->insert('end',substr($f_converted, $i, 1), 'blue');
+ } elsif (compare_bs($f_original, $f_converted, $i)) {
+ $status_bar->insert('end',substr($f_converted, $i, 1), 'red')
+ } else {
+ $status_bar->insert('end',substr($f_converted, $i, 1))
+ }
+ }
+
+ $status_bar->insert('end'," 3' R: 5' ", 'grey');
+
+ for my $i (0 .. length($r_comp_original)) {
+ if (substr($r_comp_original, $i, 2) eq "GC") {
+ $status_bar->insert('end',substr($r_converted, $i, 1), 'blue');
+ } elsif (compare_bs($r_comp_original, $r_converted, $i)) {
+ $status_bar->insert('end',substr($r_converted, $i, 1), 'red')
+ } else {
+ $status_bar->insert('end',substr($r_converted, $i, 1))
+ }
+ }
+
+ $status_bar->insert('end'," 3'", 'grey');
+ $status_bar->see('end');
+
+ browse_primer($hlist_sel);
+ # }
+}
+
+sub browse_primer {
+ my ($hlist, $slist, $canv) = get_variables(qw(hlist primers canvas));
+ my $seq = get_seq();
+ return if $seq eq "1";
+
+ my $hlist_sel;
+
+ if (defined($_[0])) {
+ $hlist_sel = $_[0];
+ } else {
+ my @sel = $$hlist->selectionGet;
+ $hlist_sel = shift @sel;
+ }
+
+ my $width = $$canv->width;
+ my $height= $$canv->height;
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+ $$canv->delete('primerf','primerr');
+
+ my $fprimerpos = $$slist[$hlist_sel][1];
+ my $rprimerpos = $$slist[$hlist_sel][8];
+
+ my $fprimerposl = $$slist[$hlist_sel][2];
+ my $rprimerposl = $$slist[$hlist_sel][6];
+
+ my ($fprimer_x1,$fprimer_x2,$rprimer_x1,$rprimer_x2);
+ if (defined($fprimerpos)) {
+ $fprimer_x1 = $fprimerpos*$dna_canvas_size + $dna_canvas_offset;
+ $fprimer_x2 = ($fprimerpos+$fprimerposl)*$dna_canvas_size + $dna_canvas_offset;
+ }
+
+ if (defined($rprimerpos)) {
+ $rprimer_x1 = $rprimerpos*$dna_canvas_size + $dna_canvas_offset;
+ $rprimer_x2 = ($rprimerpos-$rprimerposl)*$dna_canvas_size + $dna_canvas_offset;
+ }
+
+ my $line_arrow_h = 4;
+ my $line_arrow_w = 4;
+
+ $$canv->createLine($fprimer_x1,$dc_sel_y1,$fprimer_x2,$dc_sel_y1,$fprimer_x2-$line_arrow_w,$dc_sel_y1-$line_arrow_h, -fill=>'red', -width=>2, -tag=>'primerf') if $fprimerpos;
+ $$canv->createLine($rprimer_x1,$dc_sel_y2,$rprimer_x2,$dc_sel_y2,$rprimer_x2+$line_arrow_w,$dc_sel_y2+$line_arrow_h, -fill=>'red', -width=>2, -tag=>'primerr') if $rprimerpos;
+}
+
+
+sub compare_bs {
+ return 1 if substr($_[0], $_[2], 1) ne substr($_[1], $_[2], 1);
+}
+
+
+sub jump_to_tm {
+ # an arrow key shortcut ... jumps from the primer listings to detailed info
+ # including primer-dimers
+ my $nb_page = which_nb_page();
+ return if $nb_page eq "primer";
+ $stored_page = $nb_page;
+
+ my ($hlist, $slist) = get_variables(qw(hlist primers));
+ my @sel = $$hlist->selectionGet;
+ my $hlist_sel = $sel[0];
+
+ return unless @$slist;
+
+ $fprimer = $$slist[$hlist_sel][0];
+ $rprimer = $$slist[$hlist_sel][4] if $$slist[$hlist_sel][5];
+
+ get_tm();
+
+ $nb->raise('primer');
+ $packed_widgets{dim}->focus;
+}
+
+
+sub jump_back {
+ # jumps back from the primer information page to the project page
+ my $nb_page = which_nb_page();
+ return unless $nb_page eq "primer";
+ return unless $stored_page;
+
+ $nb->raise($stored_page);
+ my ($hlist) = get_variables(qw(hlist));
+ $$hlist->focus;
+}
+
+
+sub hlist_command {
+ my $hlist_sel = $_[0];
+ my ($hlist_ref, $slist) = get_variables(qw(hlist primers));
+
+ my $nb_page = which_nb_page();
+ return unless ref($slist) eq 'ARRAY';
+ $stored_page = $nb_page;
+
+ $fprimer = $$slist[$hlist_sel][0];
+ if ($$slist[$hlist_sel][5]) {
+ $rprimer = $$slist[$hlist_sel][4];
+ } else {
+ $rprimer = "";
+ }
+
+ get_tm();
+
+ $nb->raise('primer');
+}
+
+
+#----------------------------#
+# Generate Report subroutine #
+#----------------------------#
+
+sub generate_report {
+ my ($hlist_ref, $slist) = get_variables(qw(hlist primers));
+ my $nb_page = which_nb_page();
+ if ($nb_page eq "primer") {
+ dialogue("You can't generate a report from the primer information page - please switch to the project page first");
+ return;
+ }
+
+ my ($hlist_sel) = $$hlist_ref->selectionGet;
+ unless (defined(@$slist) && defined($hlist_sel)) {
+ dialogue("The Generate Report function saves the statistics and alignment of a particular primer pair - please select a primer pair first");
+ return;
+ }
+
+ my $fprimer_mod = $fprimer = $$slist[$hlist_sel][0];
+ my $rprimer_mod = $rprimer = $$slist[$hlist_sel][4] unless $nb_page eq 'seq';
+
+ my $fprimerpos = $$slist[$hlist_sel][1];
+ my $rprimerpos = $$slist[$hlist_sel][8] unless $nb_page eq 'seq';
+ my $amplicon_size = $$slist[$hlist_sel][9] unless $nb_page eq 'seq';
+
+ # Generate modified primers:
+ my (@gene_array, $gene_frame, $seq);
+ if ($nb_page eq "pd" && (($primer_seq_5f) || ($primer_seq_5r))) {
+ $seq = get_seq();
+ @gene_array=find_gene($seq);
+ $gene_frame=$gene_array[0][0]%3;
+ }
+
+ for my $primer ($fprimer, $rprimer) {
+ if ($nb_page eq "bis") {
+ # Primer redundancy for CpG residues:
+ # Replaces T with Y (pyrimidine) for forward
+ # Replaces A with R (purine) for reverse
+
+ my $f_original = $$slist[$hlist_sel][11];
+ for my $i (0 .. length($f_original)) {
+ if (substr($f_original, $i, 2) eq "CG") {
+ substr($fprimer_mod, $i, 1) = 'Y';
+ }
+ }
+ my $r_original = $$slist[$hlist_sel][12];
+ for my $i (0 .. length($r_original)) {
+ if (substr($r_original, $i, 2) eq "CG") {
+ substr($rprimer_mod, $i, 1) = 'R';
+ }
+ }
+ } elsif ($nb_page eq "pd") {
+ if ($primer_seq_5f) {
+ my ($primer_seq_5f_real, $insert_f) = add_cloning($seq, $fprimer, $fprimerpos);
+ $fprimer_mod = uc("$primer_seq_5f_real$insert_f\_$fprimer");
+ }
+
+ if ($primer_seq_5r) {
+ my ($primer_seq_5r_real, $insert_r) = (add_cloning($seq, '', '', '', $rprimer, $rprimerpos))[5,6];
+ $rprimer_mod = uc("$primer_seq_5r_real$insert_r\_$rprimer");
+ }
+ }
+ }
+
+ my $tm_text = get_tm(1);
+ my $text = dna_magnify('',1);
+
+ my $time = localtime;
+ my $amplicon = "$amplicon_size bases ($fprimerpos - $rprimerpos)" unless $nb_page eq 'seq';
+ $amplicon ||= "";
+
+ my $output = <<EOT;
+[$open_file{$nb_page} - Report generated at $time]
+
+PRIMERS
+-------
+
+Forward: 5' $fprimer_mod 3'
+Reverse: 5' $rprimer_mod 3'
+
+$amplicon
+
+PRIMER DETAILS
+--------------
+
+$tm_text
+
+SEQUENCE MAP
+------------
+
+$text
+EOT
+ my $file;
+
+ unless ($open_file{$nb_page} eq 'File not saved') {
+ my $filename = $open_file{$nb_page};
+ $filename =~ s/\.ppr//g;
+ $filename .= "_report";
+ $file = $top->getSaveFile(-defaultextension=>'.txt', -initialfile=>$filename, -filetypes=>$file_types_text);
+ } else {
+ $file = $top->getSaveFile(-defaultextension=>'.txt', -filetypes=>$file_types_text);
+ }
+
+ if (defined($file)) {
+ open (REPORT, ">$file") || dialogue("Error: Could not open file: $!");
+ print REPORT $output;
+ close (REPORT);
+ sbarprint("\n$file report generated");
+ }
+}
+
+
+#---------------#
+# Gui dialogues #
+#---------------#
+
+sub dialogue {
+ my ($message, @buttons) = @_;
+ @buttons = ('OK') unless @buttons;
+
+ my ($title, $text) = $message =~ /(\w+?)\:\s*/;
+ $title ||= "Warning";
+ $text ||= $message;
+
+ my $icon = 'info';
+ $icon = 'error' if $title =~ /error/i;
+ $icon = 'question' if $#buttons > 1;
+
+ ### Neither messageBox or Dialog are really suitable here - clunky, ugly,
+ ### different appearances under different OSes. Need to write our own dialogue
+ ### handling code sometime ...
+
+ # messageBox code
+ my $type = join("", @buttons);
+ my $selected = $top->messageBox(
+ -title=>$title,
+ -message=> $text,
+ -type => $type,
+ -icon=> $icon,
+ );
+
+ # dialog code (currently not used)
+ # my $dialogue = $top->Dialog(
+ # -title => $title,
+ # -text => $text,
+ # -bitmap => $pixmap,
+ # -default_button => $buttons[0],
+ # -buttons => \@buttons,
+ # );
+ #
+ # my $selected = $dialogue->Show();
+
+ return $selected;
+}
+
+
+sub info {
+ if (Exists($info_d)) {
+ $info_d->deiconify();
+ $info_d->raise();
+ return;
+ }
+
+ my $text = <<EOT;
+PerlPrimer v$version
+Copyright © 2003-2006 Owen Marshall\n
+EOT
+ my $text2 = <<EOT;
+An application to design primers for PCR, Bisulphite PCR, Real-time PCR and Sequencing.
+
+This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.\n
+EOT
+ my $address = <<EOT;
+http://perlprimer.sourceforge.net
+EOT
+ $info_d = $top->Toplevel(-title=>'About PerlPrimer ...');
+ my $info_d_f = $info_d->Frame()->pack(-padx=>4, -pady=>4, -expand=>1, -fill=>'both');
+ my $info_icon = $info_d_f->Label(-image => $pixmap)->pack(-side=>'left', -anchor=>'n');
+
+ # Because of a few tweaks and the custom layout, this is the one window that
+ # doesn't use pack_gui()
+
+ # Want the text widget to have the same background colour as the window
+ # (so the user can't tell it's a text widget!) We're using a text widget
+ # rather than a frame to get some formatting options (i.e. bold text)
+ my $colour = $info_d->cget(-bg);
+ my $info_text = $info_d_f->ROText(
+ -relief => 'flat',
+ -wrap => 'word',
+ -bg => $colour,
+ -width => 50,
+ -height => 12,
+ -font => $gui_font,
+ )->pack( -expand=>0, -fill=>'both', -anchor=>'nw');
+ $info_text->tagConfigure('bold',
+ -font => "$gui_font bold");
+ $info_text->tagConfigure('center',
+ -justify => 'center');
+ $info_text->insert('end', $text, 'bold');
+ $info_text->insert('end', $text2);
+ $info_text->insert('end', $address);
+ my $info_OK = $info_d->Button(
+ -text => 'OK',
+ -font => $gui_font,
+ -padx=>4,
+ -pady=>$button_pady,
+ -command => sub {$info_d->destroy},
+ )->pack(-pady=>4, -side=>'bottom');
+ $info_d->Icon(-image => $pixmap);
+}
+
+
+sub canvas_info {
+ if (Exists($canvas_info_d)) {
+ $canvas_info_d->deiconify();
+ $canvas_info_d->raise();
+ return;
+ }
+
+ $canvas_info_d = $top->Toplevel(-title=>'PerlPrimer Help');
+ my $canvas_info_d_f = $canvas_info_d->Frame()->pack(-expand=>1, -fill=>'both');
+ my $canvas_info_d_fb = $canvas_info_d->Frame()->pack(-side=>'bottom', -fill=>'none');
+ nr(\$canvas_info_d_f, $frame_pady, 1);
+ pack_gui('ROText', '', 'canvas_info_text', 80, 35, -scrollbars=>'oe', -wrap=>'word');
+ nr(\$canvas_info_d_fb);
+ pack_gui('Button', "OK", "canvas_info_OK", sub {$canvas_info_d->destroy}, "active");
+ $packed_widgets{canvas_info_text}->configure(-font=>"$gui_font");
+ $packed_widgets{canvas_info_text}->tagConfigure('bold',
+ -font => "$gui_font bold");
+ $packed_widgets{canvas_info_text}->tagConfigure('center',
+ -justify => 'center');
+
+ my $info_text_dna=<<EOT;
+\nThe grey line represents the DNA sequence, with genes [Standard PCR and Real-time PCR tabs] or CpG islands [Bisulphite PCR tab] marked in dark blue. Intron/exon boundaries are displayed in the Real-time PCR tab.
+EOT
+
+ my $info_text_ranges=<<EOT;
+\nThe light blue rectangle [Standard PCR and Bisulphite PCR tabs only] marks the minimum amplified area. It can be automatically set to the gene boundaries by clicking the "Set from ORF" or "Set from CpG Islands" buttons, or can be resized using the left mouse button. Alternatively, you can set it precisely in the "Amplified range" section.
+
+The orange rectangle marks the maximum amplified area - no primers will be taken from outside of this range. You can resize it by using the middle mouse button or Ctrl-left mouse button.
+EOT
+
+ my $info_text_primers=<<EOT;
+\nClicking on a primer pair in the Results list displays the primers in red, drawn to scale.
+EOT
+
+ my $info_text_other=<<EOT;
+\nRight-clicking on the DNA will open a detailed view aligning the primers, DNA, translated ORF [Standard PCR and Real-time PCR tabs] or CpG island boundaries [Bisulphite PCR tab]. Intron/exon boundaries [Real-time PCR tab] or CpG residues [Bisulphite PCR tab] are also highlighted. The "Copy printable" button in this dialogue window will copy the entire layout to the clipboard wrapped at 80 characters, which can then be pasted into a word processor/text editor and printed.
+EOT
+
+ $packed_widgets{canvas_info_text}->insert('1.0', " ", 'center');
+ $packed_widgets{canvas_info_text}->imageCreate('end', -image=>$top->Pixmap(-data => $dna_canvas_pixmap));
+
+ $packed_widgets{canvas_info_text}->insert('end', "\n\n\nDNA sequence\n", 'bold');
+ $packed_widgets{canvas_info_text}->insert('end', $info_text_dna);
+ $packed_widgets{canvas_info_text}->insert('end', "\n\nAmplified ranges\n", 'bold');
+ $packed_widgets{canvas_info_text}->insert('end', $info_text_ranges);
+ $packed_widgets{canvas_info_text}->insert('end', "\n\nPrimers\n", 'bold');
+ $packed_widgets{canvas_info_text}->insert('end', $info_text_primers);
+ $packed_widgets{canvas_info_text}->insert('end', "\n\nOther features\n", 'bold');
+ $packed_widgets{canvas_info_text}->insert('end', $info_text_other);
+}
+
+
+sub acknowledgements {
+ # just raise the dialogue if it already exists ....
+ if (Exists($ack_d)) {
+ $ack_d->deiconify();
+ $ack_d->raise();
+ return;
+ }
+
+ $ack_d = $top->Toplevel(-title=>'Acknowledgements');
+ my $ack_d_f = $ack_d->Frame()->pack(-expand=>1, -fill=>'both');
+ my $ack_d_fb = $ack_d->Frame()->pack(-side=>'bottom', -fill=>'none');
+ nr(\$ack_d_f, $frame_pady, 1);
+ pack_gui('ROText', '', 'ack_text', 70, 35, -scrollbars=>'oe', -wrap=>'word');
+ nr(\$ack_d_fb);
+ pack_gui('Button', "OK", "ack_OK", sub {$ack_d->destroy}, "active");
+ $packed_widgets{ack_text}->configure(-font=>"$gui_font");
+ $packed_widgets{ack_text}->tagConfigure('bold',
+ -font =>"$gui_font bold");
+ my $text_rebase=<<EOT;
+Restriction enzyme data are provided by the REBASE project (http://rebase.neb.com)
+
+Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE - restriction enzymes and methylases. Nucleic Acids Res. 2003; 31:418-20.
+EOT
+
+
+ my $text_thermo=<<EOT;
+Thermodynamic parameters are based on the following papers:
+
+Allawi HT, SantaLucia J Jr. Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry. 1997 Aug 26;36(34):10581-94
+
+SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5.
+
+Allawi HT, SantaLucia J Jr. Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA. Biochemistry. 1998 Feb 24;37(8):2170-9.
+
+Allawi HT, SantaLucia J Jr. Thermodynamics of internal C.T mismatches in DNA. Nucleic Acids Res. 1998 Jun 1;26(11):2694-701.
+
+Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects. Biochemistry. 1998 Jun 30;37(26):9435-44.
+
+Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry. 1999 Mar 23;38(12):3468-77.
+EOT
+
+ my $text_entropy=<<EOT;
+Entropy corrections for PCR salt conditions are based on:
+
+von Ahsen N, Wittwer CT, Schütz E. Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas. Clinical Chemistry. 2001; 47(11):1956-1961
+EOT
+
+ my $text_cpg=<<EOT;
+Parameters for Bisulphate PCR primer design are based upon:
+
+Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ. Identification and resolution of artifacts in bisulfite sequencing. Methods. 2002 Jun;27(2):101-7.
+EOT
+
+ my $text_thanks=<<EOT;
+Alf Eaton
+Alexander Kozik
+Chris Vega
+Katrina Bell
+Richard Saffery
+Nick Wong
+Karl Billeter
+Steffen Moeller
+EOT
+
+ $packed_widgets{ack_text}->insert('0.1', "Restriction enzyme data\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_rebase);
+
+ $packed_widgets{ack_text}->insert('end', "\n\nThermodynamic parameters\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_thermo);
+
+ $packed_widgets{ack_text}->insert('end', "\n\nEntropy corrections\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_entropy);
+
+ $packed_widgets{ack_text}->insert('end', "\n\nBisulphate PCR primer design\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_cpg);
+
+ $packed_widgets{ack_text}->insert('end', "\n\nSpecial thanks for comments, testing and suggestions\n(in no particular order)\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_thanks);
+
+ # icon
+ $ack_d->Icon(-image => $pixmap);
+}
+
+
+sub view_spidey_out {
+ my ($spidey_out) = run_spidey(1);
+
+ my $spidey_d = $top->Toplevel(-title=>'Spidey output');
+ my $spidey_d_f = $spidey_d->Frame()->pack(-expand=>1, -fill=>'both');
+ my $spidey_d_fb = $spidey_d->Frame()->pack(-side=>'bottom', -fill=>'x');
+ $spidey_d->Icon(-image => $pixmap);
+
+ nr(\$spidey_d_f, $frame_pady, 1);
+ pack_gui('ROText', '', 'spidey_text', 100, 25, -scrollbars=>'osoe');
+
+ nr(\$spidey_d_fb);
+ pack_gui('Button', "OK", "spidey_OK", sub {$spidey_d->destroy}, "active");
+ pack_gui('Button', "Full output", "spidey_full", sub {
+ ($spidey_out) = run_spidey(0);
+ $packed_widgets{'spidey_text'}->delete('0.1', 'end');
+ $packed_widgets{'spidey_text'}->insert('0.1', $spidey_out)
+ });
+
+ $packed_widgets{spidey_text}->insert('0.1', $spidey_out);
+}
+
+#-------------#
+# Preferences #
+#-------------#
+
+sub prefs {
+ # Preferences dialogue
+
+ # Preference file is $pref_file; all variables saved/restored are given in
+ # the hash %pref_variables. It should be fairly clear that this makes the
+ # preference system easily extensible and yet also extremely simple ...
+
+ # just raise the dialogue if it already exists ....
+ if (Exists($prefs)) {
+ $prefs->deiconify();
+ $prefs->raise();
+ return;
+ }
+
+ $prefs = $top->Toplevel(-title=>'Preferences');
+ my $prefs_nb = $prefs->NoteBook(
+ -inactivebackground=>"#$nb_colour",
+ -relief => 'raised',
+ -bd => 1,
+ )->pack(
+ -expand=>1,
+ -padx=>4,
+ -pady=>4,
+ -fill=>'both',
+ );
+
+ my $prefs_page_general = $prefs_nb->add('files', -label=>'General', -anchor=>'nw');
+ my $prefs_page_repeats = $prefs_nb->add('repeats', -label=>'Exclusions', -anchor=>'nw');
+ my $prefs_page_blast = $prefs_nb->add('blast', -label=>'BLAST', -anchor=>'nw');
+ my $prefs_page_cloning = $prefs_nb->add('cloning', -label=>'Cloning', -anchor=>'nw');
+ my $prefs_page_orfcpg = $prefs_nb->add('orfcpg', -label=>'CpG Islands', -anchor=>'nw');
+ my $prefs_page_dimers = $prefs_nb->add('dimers', -label=>'Dimers', -anchor=>'nw');
+ my $prefs_page_connection = $prefs_nb->add('connection', -label=>'Network', -anchor=>'nw');
+ my $prefs_page_gui = $prefs_nb->add('gui', -label=>'GUI', -anchor=>'nw');
+
+ my $prefs_page_general_f = $prefs_page_general->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_repeats_f = $prefs_page_repeats->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_blast_f = $prefs_page_blast->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_orfcpg_f = $prefs_page_orfcpg->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_cloning_f = $prefs_page_cloning->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_dimers_f = $prefs_page_dimers->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_connection_f = $prefs_page_connection->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+ my $prefs_page_gui_f = $prefs_page_gui->Frame()->pack(-anchor=>'nw', -expand=>0, -fill=>'none');
+
+ my $prefs_fb = $prefs->Frame()->pack(-side=>'bottom', -padx=>2, -fill=>'none');
+
+ # new Tk choose directory code:
+ my $browse_directory = sub {
+ my ($dir_ref, $title) = @_;
+ my $dir = $prefs->chooseDirectory();
+ return unless defined($dir);
+
+ if ($os eq 'win') {
+ # directory separator correction if running through cygwin
+ $dir =~ s/\//\\/g;
+ }
+
+
+ $dir = $dir.$dir_sep unless ($dir =~ /$dir_sep$/);
+ $$dir_ref = $dir;
+ };
+
+ my $browse_file = sub {
+ my ($file_ref, $title) = @_;
+ my $file = $top->getOpenFile(-initialfile=>$$file_ref);
+ return unless defined($file);
+
+ if ($os eq 'win') {
+ # directory separator correction if running through cygwin
+ $file =~ s/\//\\/g;
+ }
+ $$file_ref = $file;
+ };
+
+ nr(\$prefs_page_general_f, 2);
+ nr();
+ pack_gui('Label', "Directories", "prefs_directories_l", -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', "Home directory", "prefs_files_home");
+ pack_gui('Label', \$HOME, "prefs_files_home");
+ # my $open_home = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_directory, (\$HOME, "home")])->pack(-side=>'left');
+
+ nr('',0);
+ pack_gui('Label', "Temp directory", "prefs_files_tmp");
+ pack_gui('Entry', \$tmp, "prefs_files_tmp", 20);
+ my $open_tmp = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_directory, (\$tmp, "tmp")])->pack(-side=>'left');
+ nr('',0);
+ pack_gui('Label', "Path to Spidey executable", "prefs_files_spidey");
+ pack_gui('Entry', \$spidey_path, "prefs_files_spidey", 20);
+ my $open_spidey = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_directory, (\$spidey_path, "Spidey")])->pack(-side=>'left');
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "Opening files", "prefs_files_l", -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Prompt to save existing project before opening new data', "prefs_files_overwrite", \$file_data_overwrite);
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "PCR component concentrations", "prefs_oligo", -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', "Mg++: ");
+ pack_gui('Entry', \$mg_conc, "prefs_mg_conc", 5);
+ pack_gui('Label', "mM");
+
+ nr();
+ pack_gui('Label', "Oligos: ");
+ pack_gui('Entry', \$oligo_conc, "prefs_oligo_conc", 5);
+ pack_gui('Label', "nM");
+ nr();
+ pack_gui('Label', "dNTPs: ");
+ pack_gui('Entry', \$dntp_conc, "prefs_oligo_conc", 5);
+ pack_gui('Label', "mM");
+ nr();
+ pack_gui('Label', "Monovalent cations: ");
+ pack_gui('Entry', \$monovalent_cation_conc, "prefs_monocat_conc", 5,);
+ pack_gui('Label', "mM");
+
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "ORF / CpG island finding behaviour", '', -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Defer to capitalised regions', "prefs_defer", \$defer_to_caps);
+ nr('',2);
+
+
+
+ nr(\$prefs_page_repeats_f, 2);
+ nr();
+ pack_gui('Label', "PCR excluded repeats / runs", '', -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Exclude primers containing more than', "prefs_exclude_rr", \$exclude_rr);
+ nr();
+ pack_gui('Entry', \$repeat, "prefs_rr_repeats", 3);
+ pack_gui('Label', 'Repeats or');
+ pack_gui('Entry', \$run, "prefs_rr_runs", 3);
+ pack_gui('Label', 'Runs');
+
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "Bisulphite PCR excluded repeats / runs", '', -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Exclude primers containing more than', "prefs_exclude_rr_bs", \$exclude_rr_bs);
+ nr();
+ pack_gui('Entry', \$repeat, "prefs_rrbs_repeats", 3);
+ pack_gui('Label', 'Repeats or');
+ pack_gui('Entry', \$run, "prefs_rrbs_repeats", 3);
+ pack_gui('Label', 'Runs');
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', 'Exclude %GC content', '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', "Only consider primers with ", "prefs_gc_max");
+ pack_gui('Entry', \$min_gc, "prefs_gc_min", 3);
+ pack_gui('Label', '-');
+ pack_gui('Entry', \$max_gc, "prefs_gc_max", 3);
+ pack_gui('Label', '% GC content');
+ nr();
+ pack_gui('Label', '(Used when "Exclude %GC" is checked on a project page)');
+
+ nr('',2);
+
+ nr(\$prefs_page_blast_f, 2);
+ nr();
+ pack_gui('Label', "BLAST search parameters", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Expect value: ', "prefs_blast_expect");
+ pack_gui('Entry', \$blast_expect, "prefs_blast_expect", 4);
+ nr();
+ pack_gui('Label', 'Word size: ', "prefs_blast_wordsize");
+ pack_gui('Entry', \$blast_word_size, "prefs_blast_wordsize", 4);
+ nr();
+ pack_gui('Radiobutton', "Use remote BLAST server", 'remote_blast_r', -variable=>\$local_blast, -value=>0);
+ pack_gui('Radiobutton', "Use local BLAST server", 'remote_blast_r', -variable=>\$local_blast, -value=>1);
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "Remote BLAST (NCBI server)", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Database: ', 'prefs_blast_database');
+ pack_gui('BrowseEntry', \$blast_database, 'prefs_blast_database', \@blast_database_array, 10);
+ nr();
+ pack_gui('Label', 'Limit to organism (Entrez query): ', 'prefs_blast_entrez');
+ pack_gui('BrowseEntry', \$blast_entrez_query, 'prefs_blast_entrez', \@blast_entrez_array, 20);
+ nr('',7);
+ nr();
+ pack_gui('Label', "Local BLAST", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'BLAST server location', "prefs_blast_expect");
+ pack_gui('Entry', \$local_blast_directory, "prefs_blast_expect", 30);
+ my $open_blast_srv = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_directory, (\$local_blast_directory, "BLAST directory")])->pack(-side=>'left');
+ nr('',0);
+ pack_gui('Label', 'Database: ', "prefs_blast_expect");
+ pack_gui('Entry', \$local_blast_database, "prefs_blast_expect", 30);
+ my $open_blast_database = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_file, (\$local_blast_database, "BLAST database")])->pack(-side=>'left');
+ nr('',0);
+
+ nr(\$prefs_page_orfcpg_f, 2);
+ nr();
+ pack_gui('Label', "CpG island prediction", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', "Window size: ", "prefs_bioinf_window");
+ pack_gui('Entry', \$cpg_window, "prefs_bioinf_window", 5);
+ pack_gui('Label', 'bases');
+ nr();
+ pack_gui('Label', "Minimum island size: ", "prefs_bioinf_island");
+ pack_gui('Entry', \$min_cpg_island, "prefs_bioinf_island", 5);
+ pack_gui('Label', 'bases');
+ nr();
+ pack_gui('Label', "Minimum obs/exp: ", "prefs_bioinf_oe");
+ pack_gui('Entry', \$cpg_oe, "prefs_bioinf_oe", 5);
+ nr();
+ pack_gui('Label', "Minimum GC content: ", "prefs_bioinf_gc");
+ pack_gui('Entry', \$cpg_gc, "prefs_bioinf_gc", 5);
+ pack_gui('Label', '%');
+ nr('',0);
+ pack_gui('Checkbutton', 'Emulate cpgplot', "prefs_cpgplot_method", \$cpgplot_method);
+ nr('', 7);
+
+
+
+
+ nr(\$prefs_page_cloning_f, 2);
+ nr();
+ pack_gui('Label', "Restriction enzyme cloning sequences", '', -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Use 6-base cutting enzymes only', "prefs_simple_sites", \$simple_sites);
+ nr('',0);
+ pack_gui('Checkbutton', 'Only list enzymes that do not cut sequence', "prefs_exclude_found_sites", \$exclude_found_sites);
+ nr('',7);
+
+ nr(\$prefs_page_dimers_f, 2);
+ nr();
+ pack_gui('Label', "Primer-dimer parameters", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Calculate primer-dimer dG at ', 'prefs_dimer_temp');
+ pack_gui('Entry', \$pd_temperature, 'prefs_dimer_temp', 3);
+ pack_gui('Label', '°C');
+
+ nr(\$prefs_page_connection_f, 2);
+ nr();
+ pack_gui('Label', "Web browser", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Browser: ', 'prefs_connection_browser');
+ pack_gui('Entry', \$browser, 'prefs_connection_browser', 30);
+ my $open_browser = pack_button($row_counter[-1], $top->Pixmap(-data => $icon_open_small), [$browse_file, (\$browser, "Browser")])->pack(-side=>'left');
+
+ nr('', 7);
+ nr();
+ pack_gui('Label', "Proxy server", '', -font=>$gui_font_bold);
+ nr('',0);
+ pack_gui('Checkbutton', 'Use http proxy server', "prefs_connection_proxy", \$use_proxy);
+ nr();
+ pack_gui('Label', 'Address: ', 'prefs_connection_address');
+ pack_gui('Entry', \$http_proxy, 'prefs_connection_address', 30);
+ pack_gui('Label', 'Port', 'prefs_connection_port');
+ pack_gui('Entry', \$http_port, 'prefs_connection_port', 5);
+ nr('',7);
+ nr();
+ pack_gui('Label', "Interaction with external applications", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Listen to port ',"prefs_contigviewer_port");
+ pack_gui('Entry', \$tcp_port, "prefs_contigviewer_port", 6);
+ nr('',0);
+ pack_gui('Checkbutton', 'Automatically find primers upon receiving data', "prefs_contigviewer_autofind", \$ipc_autofind);
+
+
+ nr(\$prefs_page_gui_f, 3);
+ nr();
+ pack_gui('Label', "Fonts", '', -font=>$gui_font_bold);
+ nr();
+ my @font_families = sort $top->fontFamilies;
+ pack_gui('Label', 'Main font:', 'prefs_gui_family');
+ pack_gui('BrowseEntry', \$gui_font_face, 'prefs_gui_family', \@font_families);
+ pack_gui('Label', ' Size:', 'prefs_gui_size');
+ pack_gui('BrowseEntry', \$gui_font_size, 'prefs_gui_size', [(3 .. 32)], 3);
+ nr();
+ pack_gui('Label', 'List font:', 'prefs_gui_list_family');
+ pack_gui('BrowseEntry', \$list_font_face, 'prefs_gui_list_family', \@font_families);
+ pack_gui('Label', ' Size:', 'prefs_gui_list_size');
+ pack_gui('BrowseEntry', \$list_font_size, 'prefs_gui_list_size', [(3 .. 32)], 3);
+ nr();
+ pack_gui('Label', 'Fixed font:', 'prefs_gui_text_family');
+ pack_gui('BrowseEntry', \$text_font_face, 'prefs_gui_text_family', \@font_families);
+ pack_gui('Label', ' Size:', 'prefs_gui_text_size');
+ pack_gui('BrowseEntry', \$text_font_size, 'prefs_gui_text_size', [(3 .. 32)], 3);
+ nr();
+
+ pack_gui('Checkbutton', 'Use OS font defaults', 'prefs_gui_override', \$font_override);
+ nr('',7);
+
+ nr();
+ pack_gui('Label', "Previously opened files", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'List the last ', "prefs_gui_mru_number");
+ pack_gui('Entry', \$mru_number, "prefs_gui_mru_number", 3);
+ pack_gui('Label', 'files', "prefs_gui_mru_number");
+ nr('',7);
+
+ nr();
+ pack_gui('Label', "Mouse Wheel", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Label', 'Mouse wheel scrolls ', "prefs_gui_wheel");
+ pack_gui('Entry', \$scroll_factor, "prefs_gui_wheel", 3);
+ pack_gui('Label', 'lines');
+ nr('',2);
+
+ nr(\$prefs_fb);
+ pack_gui('Button', 'OK', 'prefs_ok', sub {
+ # check if [dNTPs] > [Mg++] ...
+ if ($dntp_conc > $mg_conc) {
+ my $answer = dialogue("Setting [dNTPs] > [Mg++] will have unexpected results - Tm calculations may be inaccurate", 'OK', 'Cancel');
+ return if $answer eq 'Cancel';
+ }
+
+ # if the salt concentration has been changed, we need to recalculate %oligo_dG
+ recalculate_dG();
+
+ # write the data to the pref_file ...
+ my $file_data = "";
+ foreach my $i (keys %pref_variables) {
+ my $pointer = $pref_variables{$i};
+ $file_data .= "$i = $$pointer\n";
+ }
+ foreach my $i (keys %pref_arrays) {
+ my $pointer = $pref_arrays{$i};
+ $file_data .= "$i = [".join(",",@$pointer)."]\n";
+ }
+ open (PREFS, ">$pref_file") || dialogue("Error: Could not open prefs file for writing: $!");
+ print PREFS $file_data;
+ close (PREFS);
+
+ # You really don't want to know why we have to read the prefs file
+ # just after writing it ...
+
+ # (hint: if we don't, and we open and OK the prefs window twice,
+ # perl starts saying that 1.5-0.2 = 1 ... I don't know why ... )
+
+ read_prefs();
+ $prefs->destroy;
+ }, 'active');
+
+ pack_gui('Button', 'Revert', 'prefs_cancel', sub {
+ # This is perhaps a bit inefficient, re-reading the pref_file each time
+ # the dialogue is cancelled. However, it does not seem to cause a
+ # noticable time-lag and it's certainly the simplest way to do things
+ # (otherwise we'd have to save each value to a temporary hash and update
+ # each variable on prefs_OK ... to make this extensible we'd have to make
+ # a hash with the same keys as %pref_variables ...
+ # it's definitely possible, but a bit of a fuss!!
+ read_prefs();
+ $prefs->destroy;
+ });
+
+ $prefs->Icon(-image => $pixmap);
+}
+
+
+sub read_prefs {
+ return unless -e $pref_file;
+ open (PREFS, "<$pref_file") || dialogue("Error: Could not open prefs file for reading: $!");
+ my $pointer;
+ while (<PREFS>) {
+ s/[\n\r]//g;
+ next if /^$/;
+ my ($key, $value) = split / = /;
+ if ($value =~ /\[(.+)\]/) {
+ # an array
+ $pointer = $pref_arrays{$key};
+ @$pointer = split(",",$1);
+ } else {
+ $pointer = $pref_variables{$key};
+ $$pointer = $value;
+ }
+ }
+ close (PREFS);
+}
+
+
+#------------------------#
+# DNA graphical routines #
+#------------------------#
+
+sub find_gene {
+ # Find genes (or CpG islands, or whatever else you might fancy) based on
+ # case (upper-case marks genes) (can have more than one ORF in the same
+ # sequence, which the ORF-finding sub doesn't allow; also speeds things
+ # up a bit by not having to ORF/CpG-find each time)
+ $_ = shift;
+ return if length($_) == 0;
+ my ($subroutine) = get_variables(qw(find_sub));
+
+ # s/\>.*\n//g; #remove FASTA formatting if it exists
+ $_ = clean_seq($_);
+
+ my @array = ();
+ my $prev = 0;
+ my $nb_page = which_nb_page();
+
+ # if the user wishes to defer to capitalised regions, do so ...
+ # i.e. a sequence with lowercase and capitalised regions will be assumed to
+ # use caps to mark the gene/cpg island
+ # NB - this is no longer the default behaviour
+ if ($defer_to_caps) {
+ my $i;
+ while (/([a-z]*)([A-Z]*)(?=[a-z]*)/xg) {
+ $i++;
+ push @array, [$prev+length($1), $prev+length($1)+length($2)] if (length($2)>0 && length($1)>0);
+ $prev += length($1)+length($2);
+ }
+
+ return @array if @array;
+ }
+
+ # if we didn't find any marked regions, let's hand it over to our built in,
+ # trusty orf and cpg finding routines ...
+ return &$subroutine($_);
+}
+
+
+sub find_orf {
+ # no gene marked - try to find ORF since user has requested it
+ $_ = lc(shift);
+ $_ = clean_seq($_);
+
+ my ($seq_ref) = get_variables('seq');
+
+ my @orf=();
+ my $seq = $_;
+ for my $i (0 .. 2) {
+ $orf[$i][0] = 0;
+ my $init;
+ my $met_init;
+ my $orf_count = 0;
+ my $orf_start = -1;
+ my $j;
+ for ($j = $i; $j<(length($seq)-3); $j+=3) {
+ my $codon = uc(substr($seq, $j, 3));
+ my $aa = $genetic_code{$codon};
+ $aa ||= "X";
+ unless ($aa eq "*") {
+ # only take ORF from initiating Met codon or rare alternatives
+ unless ($init) {
+ if ($codon =~ m/[ATGC]TG/) {
+ $init=1;
+ $orf_start = $j;
+ } else {
+ next;
+ }
+ }
+
+ # prefer initiation from downstream ATG if it exists ...
+ if (!$met_init && ($aa eq 'M')) {
+ $met_init=1;
+ $orf_start = $j;
+ $orf_count = 0;
+ }
+
+ $orf_count++;
+ next;
+ }
+ # stop codon
+ if ($orf_count > $orf[$i][0]) {
+ $orf[$i][0]=$orf_count+1;
+ $orf[$i][1]=$orf_start;
+ # end orf
+ $orf[$i][2]=$j+3;
+ # frame
+ $orf[$i][3]=$i;
+ }
+ $orf_count=$met_init=$init=0;
+ $orf_start=-1;
+ }
+ if ($orf_count > $orf[$i][0]) {
+ $orf[$i][0]=$orf_count+1;
+ $orf[$i][1]=$orf_start;
+ # end orf
+ $orf[$i][2]=$j+3;
+ # frame
+ $orf[$i][3]=$i;
+ }
+ $orf_start=$orf_count=$met_init=0;
+ }
+
+ # sort by orf length:
+ @orf = sort {@$b[0] <=> @$a[0]} @orf;
+
+ return unless $orf[0][0] > 1;
+
+ # Recreate DNA sequence based on ORF
+ my $dna3 ="";
+ my $dna1 = substr($seq, 0, $orf[0][1]);
+ my $dna2 = uc(substr($seq, $orf[0][1], $orf[0][0]*3));
+ $dna3 = substr($seq, $orf[0][0]*3+$orf[0][1], ) unless $orf[0][0]*3+$orf[0][1]>=length($seq);
+
+ push my @orf_return, [$orf[0][1], $orf[0][2]];
+
+ # Re-enter DNA sequence using capitalised ORF, lowercase upstream and downstream regions
+ $$seq_ref->delete(0.1,"end");
+ $$seq_ref->insert(0.1,$dna1.$dna2.$dna3);
+
+ return @orf_return;
+ # return $orf[0][1], $orf[0][2];
+}
+
+sub find_cpg {
+ # no CpG marked - try to find CpG islands from sequence
+ # two methods: correct and cpgplot emulation
+ $_ = lc(shift);
+ $_ = clean_seq($_);
+
+ my $seq = $_;
+ my $seq_len = length($seq);
+ my @cpg_island =();
+
+ my $cpg_flag = 0;
+ my $cpg_start = 0;
+ my $cpg_count = 0;
+ my $island_max = 0;
+ my $last_cpg_pos = 0;
+ my $win_count =0;
+
+ # when cpgplot method is 1, we're emulating cpgplot behaviour (see comment
+ # below)
+ my $cpg_av = ($cpgplot_method ? 10 : 9);
+
+ # CpG finding routine
+ #
+ # This is based on the method first described in Gardiner-Garden and Frommer
+ # (1987) and used in many other programs since. The most common utility is
+ # cpgplot, but cpgplot does not give the same results as this routine!
+ # Analysing the source, it's apparent that cpgplot is buggy and in fact does
+ # not use the parameters it claims to, but slightly an underestimate.
+ #
+ # The worst bug of all in cpgplot is that the avg readings for each window are
+ # inflated: they are not an average at all; rather, 11 frames are read and the
+ # totals divided by 10!! The second bug comes in calculating the number of CpG
+ # residues in a window - cpgplot actually adds in an extra base when
+ # performing this calculation, but does not add in an extra base when counting
+ # the c's and g's in a sequence, or the %GC content!!
+ #
+ # PerlPrimer has the option to deliberately allow for this and return the
+ # same results as cpgplot, simply because cpgplot has been around for so long
+ # seems to be the de facto standard for CpG island prediction. However, by
+ # default PerlPrimer uses my algorithmically correct approach.
+
+ my (@cpg_oe, @cpg_pgc)=();
+
+ # We use an average of 10 (or 11 in cpgplot's case) window values at each
+ # point, so to save recalculating the same value 10 times we save it in an
+ # array ...
+ for my $i (0 .. $seq_len-$cpg_window) {
+ $_ = substr($seq, $i, $cpg_window);
+ # calculate O/E
+ my $g = tr/Gg/Gg/;
+ my $c = tr/Cc/Cc/;
+ my $exp_gc = ($c*$g)/(length($_)-1);
+ if ($cpgplot_method) {
+ $_ = substr($seq, $i, $cpg_window+1);
+ }
+ my $cg = s/cg/cg/ig;
+
+ # save data
+ $cpg_pgc[$i] = gc($_);
+ if ($exp_gc == 0) {
+ $cpg_oe[$i] = 0;
+ } else {
+ $cpg_oe[$i] = $cg/$exp_gc;
+ }
+ }
+
+ # Calculate the averages and find the islands
+ for my $i (0 .. $seq_len-$cpg_window-10) {
+ # the real position is in the middle of the window...
+ my $cpg_real_pos = int($i+($cpg_window/2));
+
+ # of course, this is (10 or 11)/2 bases upstream from the real mid-point,
+ # since we're averaging 10 frames from here (another bug in cpgplot!)
+ $cpg_real_pos += int($cpg_av/2) unless $cpgplot_method;
+
+ my $sum_cg = 0;
+ my $sum_obs_exp = 0;
+
+ # Cpgplot uses an 11 window "average" (it thinks it's only using 10 ...)
+ for my $j ($i .. $i+$cpg_av) {
+ $sum_cg += $cpg_pgc[$j];
+ $sum_obs_exp += $cpg_oe[$j];
+ }
+
+ my $av_obs_exp = $sum_obs_exp/10;
+ my $av_cg = $sum_cg/10;
+
+ if (($av_cg > $cpg_gc) && ($av_obs_exp>$cpg_oe)) {
+ # A possible island: set the start point if it's the first
+ $cpg_start=$cpg_real_pos if $cpg_flag==0;
+ $cpg_flag=1;
+ next;
+ }
+ unless ($cpg_flag==0) {
+ if ($cpg_real_pos-$cpg_start < $min_cpg_island) {
+ # don't save the island if it's less than the minimum size
+ $cpg_flag=0;
+ $cpg_count=0;
+ $cpg_start=0;
+ next;
+ }
+
+ # Save the island
+ # This should have an end point of $cpg_real_pos-1, as we're
+ # now a base ahead of the last positive, but cpgplot has it
+ # as $cpg_real_pos+1 (?!?)
+ my $cpg_island_end = ($cpgplot_method ? $cpg_real_pos-1 : $cpg_real_pos+1);
+ push @cpg_island, [$cpg_start, $cpg_island_end];
+
+ # reset flags
+ $cpg_flag=0;
+ $cpg_count=0;
+ $cpg_start=0;
+ }
+ }
+
+ $packed_widgets{"bisul_seq"}->delete(0.1,"end");
+ my $cpg_pos = 0;
+
+ for my $i (0 .. $#cpg_island) {
+ print "Island No. $i: start $cpg_island[$i][0], end $cpg_island[$i][1], length ",$cpg_island[$i][1]-$cpg_island[$i][0],"\n";
+
+ # Recreate DNA sequence based on ORF
+ my $dna1 = substr($seq, $cpg_pos, $cpg_island[$i][0]-$cpg_pos);
+ # print "pos, len = $cpg_pos, $cpg_island[$i][0]\n";
+ my $dna2 = uc(substr($seq, $cpg_island[$i][0], $cpg_island[$i][1]-$cpg_island[$i][0]));
+ # print "dna2 pos, len = $cpg_island[$i][0], $cpg_island[$i][1]\n";
+ $cpg_pos = $cpg_island[$i][1];
+ # print "pos = $cpg_pos\n";
+
+ # Re-enter DNA sequence using capitalised ORF, lowercase 5' and 3' regions
+ $packed_widgets{"bisul_seq"}->insert("1.end",$dna1.$dna2);
+ }
+
+ # Last part of the reconstructed sequence:
+ my $dna3 = substr($seq, $cpg_pos);
+ $packed_widgets{"bisul_seq"}->insert("1.end",$dna3);
+
+ return (-1) unless @cpg_island;
+ return @cpg_island;
+ # return the start of the first island and the end of the last island
+ return $cpg_island[0][0], $cpg_island[$#cpg_island][0]+$cpg_island[$#cpg_island][1];
+}
+
+sub draw_dna {
+ my $old_defer = $defer_to_caps; # save state
+ $defer_to_caps = 1 if @_ && $_[0] == 1;
+
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p, $hlist, $slist, $canvas)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p hlist primers canvas));
+
+ my $seq = get_seq();
+ return unless $seq =~ /[a|t|c|g]/i;
+
+ # delete old canvas elements
+ $$canvas->delete('dna', 'amp_range', 'gene_range', 'dna_range','primerf','primerr', 'direction', 'ie_boundary');
+
+
+ # get dimensions of canvas
+ my $width = $$canvas->width;
+ my $height= $$canvas->height;
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+ my $canv = which_nb_page();
+
+ # Amplicon size
+ if (($$max_ampsize)&&($$min_ampsize)&&($$max_range)) {
+ $min_amp_canvas{$canv} = $$max_range_5p * $dna_canvas_size + $dna_canvas_offset if defined($$max_range_5p);
+ $max_amp_canvas{$canv} = $$max_range_3p * $dna_canvas_size + $dna_canvas_offset if defined($$max_range_3p);
+ $$canvas->createRectangle($min_amp_canvas{$canv},$dc_sel_y1-$dc_sel_offset2,$max_amp_canvas{$canv},$dc_sel_y2+$dc_sel_offset2, -fill=>'orange', -outline=>'orange4', -width=>1, -tag=>'amp_range');
+ }
+
+ # range
+ if (defined($$max_range)&&defined($$min_range)) {
+ $min_range_canvas{$canv} = $$min_range * $dna_canvas_size + $dna_canvas_offset;
+ $max_range_canvas{$canv} = $$max_range * $dna_canvas_size + $dna_canvas_offset;
+ $$canvas->createRectangle($min_range_canvas{$canv},$dc_sel_y1,$max_range_canvas{$canv},$dc_sel_y2, -fill=>'dodgerblue', -outline=>'dodgerblue4', -tag=>'dna_range');
+ }
+
+ # max_range_5p and 3p variables set
+ check_range();
+
+ # draw DNA
+ $$canvas->createRectangle($dna_canvas_offset,$dc_dna_y1,$width-($dna_canvas_offset+1),$dc_dna_y2, -fill=>'grey50', -tag=>'dna');
+ $$canvas->createText(5,$dna_canvas_middle, -text=>"5'", -justify=>'right', -tag=>'direction');
+ $$canvas->createText($width-6,$dna_canvas_middle, -text=>"3'", -justify=>'right', -tag=>'direction');
+
+ # gene (from capitals, if present)
+ my @gene_array = find_gene($seq);
+
+ for my $i (0 ... $#gene_array) {
+ my($gene_5p,$gene_3p)=($gene_array[$i][0], $gene_array[$i][1]);
+ if (defined($gene_5p)&&defined($gene_3p)) {
+ $min_range_canvas{$canv} = $gene_5p * $dna_canvas_size + $dna_canvas_offset;
+ $max_range_canvas{$canv} = $gene_3p * $dna_canvas_size + $dna_canvas_offset;
+ $$canvas->createRectangle($min_range_canvas{$canv},$dc_dna_y1,$max_range_canvas{$canv},$dc_dna_y2, -fill=>'midnightblue', -tag=>'gene_range');
+ }
+ }
+
+ # QPCR specific:
+ if ($canv eq 'qpcr') {
+ foreach my $i (@intron_exon_bounds) {
+ my $iex = $i * $dna_canvas_size + $dna_canvas_offset;
+ $$canvas->createLine($iex, $dc_dna_y1, $iex, $dc_dna_y2, -fill=>'white', -tag=>'ie_boundary');
+ }
+ }
+
+ my @sel = $$hlist->selectionGet;
+ my $hlist_sel = shift @sel;
+
+ if (defined($hlist_sel)) {
+
+ # my $width = $$canv->width;
+ # my $height= $$canv->height;
+ # my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+ $$canvas->delete('primerf','primerr');
+
+ my $fprimerpos = $$slist[$hlist_sel][1];
+ my $rprimerpos = $$slist[$hlist_sel][8];
+
+ my $fprimerposl = $$slist[$hlist_sel][2];
+ my $rprimerposl = $$slist[$hlist_sel][6];
+
+ my ($fprimer_x1,$fprimer_x2,$rprimer_x1,$rprimer_x2);
+ if (defined($fprimerpos)) {
+ $fprimer_x1 = $fprimerpos*$dna_canvas_size + $dna_canvas_offset;
+ $fprimer_x2 = ($fprimerpos+$fprimerposl)*$dna_canvas_size + $dna_canvas_offset;
+ }
+
+ if (defined($rprimerpos)) {
+ $rprimer_x1 = $rprimerpos*$dna_canvas_size + $dna_canvas_offset;
+ $rprimer_x2 = ($rprimerpos-$rprimerposl)*$dna_canvas_size + $dna_canvas_offset;
+ }
+
+ my $line_arrow_h = 4;
+ my $line_arrow_w = 4;
+
+ $$canvas->createLine($fprimer_x1,$dc_sel_y1,$fprimer_x2,$dc_sel_y1,$fprimer_x2-$line_arrow_w,$dc_sel_y1-$line_arrow_h, -fill=>'red', -width=>2, -tag=>'primerf') if $fprimerpos;
+ $$canvas->createLine($rprimer_x1,$dc_sel_y2,$rprimer_x2,$dc_sel_y2,$rprimer_x2+$line_arrow_w,$dc_sel_y2+$line_arrow_h, -fill=>'red', -width=>2, -tag=>'primerr') if $rprimerpos;
+ }
+
+ $defer_to_caps = $old_defer; # restore state
+}
+
+
+sub view_intron_exon_structure {
+ # Draws a typical genomic DNA diagram showing exons and introns
+ if (Exists($view_ie)) {
+ $view_ie->destroy;
+ }
+
+ $cancel=0;
+
+ # Create dialogue
+ $view_ie = $top->Toplevel(-title=>'Intron/Exon Genomic Structure');
+ my $view_ie_f = $view_ie->Frame()->pack(-expand=>1, -fill=>'both');
+ my $view_ie_fb = $view_ie->Frame()->pack(-side=>'bottom', -fill=>'none');
+
+ nr(\$view_ie_f);
+ pack_gui('Canvas', '', 'view_ie_canvas', 0, 0, -width=>600, -height=>$dna_canvas_height+35);
+
+ nr(\$view_ie_fb);
+ pack_gui('Button', 'OK', 'view_ie_ok', sub {$view_ie->destroy}, 'active');
+
+ $view_ie->Icon(-image => $pixmap);
+
+ my $canvas = \$packed_widgets{view_ie_canvas};
+
+ # get spidey output
+ ($_) = run_spidey(1);
+
+ if ($cancel==1) {
+ # cannot find spidey
+ $cancel=0;
+ $view_ie->destroy;
+ return;
+ }
+
+ # isolate genomic structure
+ my @exon_structure;
+ while (m/Exon \d+[\(\-\)]*: (\d+)-(\d+) \(gen\)/g) {
+ push @exon_structure, [$1, $2];
+ }
+
+ # Get strand orientation - will need to reverse array if minus strand
+ my ($strand) = m/Strand: (\w+)/;
+ @exon_structure = reverse(@exon_structure) if $strand eq "minus";
+
+ # length of genomic sequence
+ my $gen_length = $exon_structure[-1][1];
+
+ # get dimensions of canvas
+ my $width = $$canvas->width;
+ my $height= $$canvas->height;
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/$gen_length;
+
+ my $intron_y1 = $dc_dna_y1+2;
+ my $intron_y2 = $dc_dna_y2-2;
+
+ my $text_y = $dna_canvas_height+5;
+
+ my $previous_canvas_j;
+ my $count;
+ foreach my $a (@exon_structure) {
+ $count++;
+ my ($i, $j) = @$a;
+ my ($canvas_i, $canvas_j) = ($i * $dna_canvas_size + $dna_canvas_offset, $j * $dna_canvas_size + $dna_canvas_offset);
+ if ($previous_canvas_j) {
+ # draw intron
+ $$canvas->createRectangle($previous_canvas_j, $intron_y1, $canvas_i, $intron_y2, -fill=>'grey');
+ }
+
+ # draw exon
+ $$canvas->createRectangle($canvas_i, $dc_dna_y1, $canvas_j, $dc_dna_y2, -fill=>'midnightblue');
+
+ my $canvas_text = ($canvas_i + $canvas_j)/2;
+ # label exon
+ $$canvas->createText($canvas_text, $text_y, -fill=>'midnightblue', -text=>$count);
+
+ $previous_canvas_j = $canvas_j;
+ }
+
+ # draw sequence length info
+ for my $i (0 .. 5) {
+ my $base_real = $i * $gen_length / 5;
+ my $base = int($base_real/1000) * 1000;
+ my $canvas_text = $base * $dna_canvas_size + $dna_canvas_offset;
+ $$canvas->createText($canvas_text, $text_y+20, -justify=>'left', -fill=>'grey30', -text=>int($base/1000) . "kb");
+ }
+}
+
+
+sub items_drag {
+ my ($min_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_range_5p min_range max_range max_range_3p));
+
+ my $canv = which_nb_page();
+
+ my ($x,$ref) = @_;
+
+ my $width = $$ref->width;
+ my $dna_max = $width-$dna_canvas_offset;
+ my $dna_min = $dna_canvas_offset;
+
+ my $seq = get_seq();
+ return unless $seq =~ /[a|t|c|g]/i;
+
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+ $x = sprintf("%.0f", $$ref->canvasx($x));
+ $x = $dna_min if $x < $dna_min;
+ $x = $dna_max if $x > $dna_max;
+
+ if ($canv eq 'qpcr') {
+ # get DNA base
+ $x = ($x-$dna_canvas_offset) / $dna_canvas_size;
+
+ # exon selection
+ my $last_ie = 0;
+ my $exon_count;
+ foreach (@intron_exon_bounds) {
+ $exon_count++;
+ if ($x>$last_ie && $x<$_) {
+ select_exon($exon_count, 5);
+ return;
+ }
+ }
+ $exon_count++;
+ select_exon($exon_count, 5);
+ return;
+ }
+
+ if (($$max_range)&&($$min_range)) {
+ $min_range_canvas{$canv} = $$min_range * $dna_canvas_size + $dna_canvas_offset;
+ $max_range_canvas{$canv} = $$max_range * $dna_canvas_size + $dna_canvas_offset;
+ }
+
+ if (($min_amp_canvas{$canv}) && ($max_amp_canvas{$canv})) {
+ $x = $min_amp_canvas{$canv} if $x < $min_amp_canvas{$canv};
+ $x = $max_amp_canvas{$canv} if $x > $max_amp_canvas{$canv};
+ }
+
+ if ($x-$min_range_canvas{$canv}<$max_range_canvas{$canv}-$x) {
+ $$ref->coords('dna_range', $x, $dc_sel_y1, $max_range_canvas{$canv},$dc_sel_y2);
+ $$min_range = sprintf("%.0f", ($x-$dna_canvas_offset) / $dna_canvas_size);
+ $$min_range = $$max_range_5p if $$max_range_5p && $min_range<$$max_range_5p;
+
+ } else {
+ $$ref->coords('dna_range', $min_range_canvas{$canv}, $dc_sel_y1, $x, $dc_sel_y2);
+ $$max_range = sprintf("%.0f", ($x-$dna_canvas_offset) / $dna_canvas_size);
+ $$max_range = $$max_range_3p if $$max_range_3p && $$max_range>$$max_range_3p;
+ }
+
+ $$min_ampsize = $$max_range - $$min_range;
+}
+
+sub select_exon {
+ my ($exon, $end) = @_;
+ $ie_limit ||= 1;
+ $ie_limit_5p = $exon if $end == 5;
+ $ie_limit_3p = $exon if $end == 3;
+}
+
+sub amplicon_drag {
+ my ($x,$widget_ref) = @_;
+
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ my $canv = which_nb_page();
+
+
+ my $width = $$widget_ref->width;
+ my $dna_max = $width-$dna_canvas_offset;
+ my $dna_min = $dna_canvas_offset;
+
+ my $seq = get_seq();
+ return unless $seq =~ /[a|t|c|g]/i;
+
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+
+ $x = sprintf("%.0f", $$widget_ref->canvasx($x));
+ $x = $dna_min if $x < $dna_min;
+ $x = $dna_max if $x > $dna_max;
+
+ if ($canv eq 'qpcr') {
+ # get DNA base
+ $x = ($x-$dna_canvas_offset) / $dna_canvas_size;
+
+ # exon selection
+ my $last_ie = 0;
+ my $exon_count;
+ foreach (@intron_exon_bounds) {
+ $exon_count++;
+ if ($x>$last_ie && $x<$_) {
+ select_exon($exon_count, 3);
+ return;
+ }
+ }
+ $exon_count++;
+ select_exon($exon_count, 3);
+ return;
+ }
+
+
+ $seq = clean_seq($seq);
+ my $seq_len = length($seq)-1;
+
+ my $dna_max2 = $$max_range*$dna_canvas_size + $dna_canvas_offset;
+ my $dna_min2 = $$min_range*$dna_canvas_size + $dna_canvas_offset;
+
+ # grab default values if not assigned
+ if (($min_ampsize)&&($max_ampsize)) {
+ $min_amp_canvas{$canv} = $$min_range * $dna_canvas_size + $dna_canvas_offset unless $min_amp_canvas{$canv};
+ $max_amp_canvas{$canv} = ($$max_ampsize+$$min_range) * $dna_canvas_size + $dna_canvas_offset unless $max_amp_canvas{$canv} ;
+ }
+
+
+
+ if ($x-$min_amp_canvas{$canv}<$max_amp_canvas{$canv}-$x) {
+ # Changing amp_size at 5' end
+ $x = $dna_min2 if $x > $dna_min2;
+ $$widget_ref->coords('amp_range', $x, $dc_sel_y1-$dc_sel_offset2, $max_amp_canvas{$canv},$dc_sel_y2+$dc_sel_offset2);
+ $min_amp_canvas{$canv}=$x;
+ $$max_range_5p = sprintf("%.0f", ($x-$dna_canvas_offset) / $dna_canvas_size);
+ } else {
+ # Changing amp_size at 3' end
+ $x = $dna_max2 if $x < $dna_max2;
+ $$widget_ref->coords('amp_range', $min_amp_canvas{$canv}, $dc_sel_y1-$dc_sel_offset2, $x, $dc_sel_y2+$dc_sel_offset2);
+ $max_amp_canvas{$canv}=$x;
+ $$max_range_3p = sprintf("%.0f", ($x-$dna_canvas_offset) / $dna_canvas_size);
+ $$max_range_3p = $seq_len if $$max_range_3p>$seq_len;
+
+ }
+ $$max_ampsize = sprintf("%.0f", ($max_amp_canvas{$canv}-$min_amp_canvas{$canv}) / $dna_canvas_size);
+}
+
+sub get_gene {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+ my $seq = get_seq();
+ my @gene_array = find_gene($seq);
+
+ return unless @gene_array;
+ ($$min_range, $$max_range)=($gene_array[0][0], $gene_array[$#gene_array][1]-1);
+
+
+ $$max_range_5p ||= $$min_range;
+ $$max_range_3p ||= $$max_range;
+ $$max_range_5p = $$min_range if $$min_range < $$max_range_5p;
+ $$max_range_3p = $$max_range if $$max_range > $$max_range_3p;
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range - $$min_range; # if ($max_range - $min_range)>$max_ampsize;
+
+ draw_dna(1);
+}
+
+sub get_cpg {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+ my $seq = get_seq();
+ my @gene_array = find_gene($seq);
+ if ($gene_array[0] == -1) {
+ sbarprint("\nNo CpG Island found!");
+ return;
+ }
+
+ ($$min_range, $$max_range)=($gene_array[0][0], $gene_array[$#gene_array][1]-1);
+
+ $$max_range_5p ||= $$min_range;
+ $$max_range_3p ||= $$max_range;
+ $$max_range_5p = $$min_range if $$min_range < $$max_range_5p;
+ $$max_range_3p = $$max_range if $$max_range > $$max_range_3p;
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range - $$min_range; # if ($max_range - $min_range)>$max_ampsize;
+
+ draw_dna(1);
+}
+
+sub reset_bounds {
+ my $page = which_nb_page();
+ return if $page eq "qpcr";
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p, $sub_ref)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p subroutine));
+ $$max_range_5p = $$max_range_3p = $$min_range = $$max_range = $$min_ampsize = $$max_ampsize = undef;
+ &$sub_ref();
+}
+
+sub step_in {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p, $sub_ref, $canv_ref, $seq_ref)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p subroutine canvas seq));
+
+ &$sub_ref() unless defined($$max_range_5p) && defined($$max_range_3p);
+ $$min_range += 10;
+ $$max_range -= 10;
+ if ($$min_range >= $$max_range) {
+ sbarprint("\nNo primers found");
+ $cancel=1;
+ return;
+ }
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range_3p - $$max_range_5p;
+
+ draw_dna(1);
+}
+
+sub step_out {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p, $sub_ref, $canv_ref, $seq_ref)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p subroutine canvas seq));
+ my $seq_len = length(get_seq());
+ &$sub_ref() unless defined($$max_range_5p) && defined($$max_range_3p);
+
+ $cancel=1 if ($$max_range_5p==0) && ($$max_range_3p==$seq_len);
+
+ $$max_range_5p -= 10;
+ $$max_range_3p += 10;
+ $$max_range_5p = 0 if $$max_range_5p < 0;
+ $$max_range_3p = $seq_len if $$max_range_3p>$seq_len;
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range_3p - $$max_range_5p;
+
+ draw_dna(1);
+}
+
+sub step_cloning {
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p, $sub_ref, $canv_ref, $seq_ref)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p subroutine canvas seq));
+ my $seq_len = length(get_seq());
+ &$sub_ref() unless defined($$max_range_5p) && defined($$max_range_3p);
+
+ $cancel=1 if ($$max_range_5p==0) && ($$max_range_3p==$seq_len);
+
+ $$max_range_5p -= 10;
+ $$max_range -= 10;
+ $$max_range_5p = 0 if $$max_range_5p < 0;
+ $$max_range = $$min_range if $$max_range < $$min_range;
+
+ return if $$max_range_5p == 0 && $$max_range == $$min_range;
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range_3p - $$max_range_5p;
+
+ draw_dna(1);
+}
+
+sub add_cloning {
+ my ($seq, $fprimer, $fprimerpos, $fprimerposl, $rprimer, $rprimerpos, $rprimerposl) = @_;
+
+ # Adding an extra sequence is all very well.
+ # But we want to automagically keep it in frame when creating
+ # fusion constructs ...
+
+ # Warning!! This is clumsy!
+
+ my @gene_array=find_gene($seq);
+ my $gene_frame=$gene_array[0][0]%3;
+
+ my $insert_f = "";
+ my $insert_r = "";
+ my ($primer_seq_5f_real, $rprimer_seq_5r_real, $primer_seq_5r_real);
+
+ if ($primer_seq_5f && $fprimer) {
+ # calculate frame position for forward primer + insert
+ $_ = $primer_seq_5f;
+
+ my ($fspl1, $fspl2)=split('_');
+ my $renz_offset_f = 3-length($fspl2);
+
+ s/[\|_]//g;
+ $primer_seq_5f_real = $_;
+
+ if (defined($primer_seq_5f_frame) && ($primer_seq_5f_frame ne "")) {
+ my $fjoinframe = 3-$primer_seq_5f_frame;
+ my $primer_frame_f= (($fprimerpos - $gene_frame)%3 + $fjoinframe + $renz_offset_f)%3;
+
+ # Add spacers if needed between primer sequences and the inserts,
+ # and adjust the spacing of the primerpos and primerposl variables
+ # so that the primers are still displayed in the correct positions
+ $insert_f = "A" x $primer_frame_f;
+ }
+
+ $fprimer = "$primer_seq_5f_real$insert_f$fprimer";
+ $fprimerpos -= length($insert_f)+length($primer_seq_5f_real);
+ $fprimerposl += length($insert_f)+length($primer_seq_5f);
+ }
+
+ if ($primer_seq_5r && $rprimer) {
+ # calculate frame position for reverse primer + insert
+ $_ = $primer_seq_5r;
+
+ my ($rspl1, $rspl2)=split('_');
+ # my $renz_offset_r = 3-length($rspl2);
+
+ s/[\|_]//g;
+ $primer_seq_5r_real = $_;
+ $rprimer_seq_5r_real = reverse($primer_seq_5r_real);
+
+ if (defined($primer_seq_5r_frame) && ($primer_seq_5r_frame ne "")) {
+ my $rjoinframe = $primer_seq_5r_frame;
+ my $primer_frame_r= (2-($rprimerpos - $gene_frame)%3 + $rjoinframe)%3;
+ $insert_r = "A" x $primer_frame_r;
+ }
+
+ $rprimer = "$rprimer$insert_r$rprimer_seq_5r_real";
+ $rprimerpos += length($insert_r)+length($primer_seq_5r_real);
+ $rprimerposl += length($insert_r)+length($primer_seq_5r_real);
+ }
+
+ return ($primer_seq_5f_real, $insert_f, $fprimer, $fprimerpos, $fprimerposl, $primer_seq_5r_real, $insert_r, $rprimer, $rprimerpos, $rprimerposl);
+}
+
+sub dna_magnify {
+ # This is one long, convoluted subroutine that attempts
+ # to please all of the people all of the time :)
+ # A bit ungainly, but it just kept on growing ...
+ # Design could still do with a bit of tweaking ...
+ my ($x, $report) = @_;
+ my $seq = get_seq();
+ return unless $seq =~ /[a|t|c|g]/i;
+
+ my $page = which_nb_page();
+ my ($max_range_5p, $min_range, $max_range, $max_range_3p, $primer_array, $list_ref, $ref)
+ = get_variables(qw(max_range_5p min_range max_range max_range_3p primers hlist canvas));
+ my ($fprimer, $rprimer, $fprimerpos, $rprimerpos, $fprimerposl, $rprimerposl, $primer_seq_5f_real, $insert_f, $primer_seq_5r_real, $insert_r);
+
+ # get dimensions of canvas
+ my $width = $$ref->width;
+ my $dna_canvas_size=($width-($dna_canvas_offset*2))/length($seq);
+
+ # This sub used to jump to the exact position right clicked; however, all I ever
+ # want to do is look at the primers, so I feel the routine would be better jumping
+ # straight to the forward primer ...
+ # my $base_pos = sprintf("%.0f",($x-$dna_canvas_offset)/$dna_canvas_size);
+
+ my $text_ref = \$packed_widgets{view_base_text};
+
+ # Local subroutines:
+ my $copy_report = sub {
+ # Copies the content into 80-column wrapped text
+ my ($clip, $last_i);
+ my $length = 80;
+ for (my $i=$length; $i<=length($seq)+$length; $i+=$length) {
+ $last_i ||= 0;
+ for my $j (0 .. 4) {
+ $clip .= $$text_ref->get("$j.$last_i","$j.$i");
+ $clip .= "\n";
+ }
+ $clip .= "\n\n";
+ $last_i=$i;
+ }
+ return $clip;
+ };
+
+ my $copy_printable = sub {
+ # copy report for the clipboard ...
+ $top->clipboardClear;
+ my $clip = &$copy_report();
+ $top->clipboardAppend($clip);
+ };
+
+ my $view_fprimer = sub {
+ $$text_ref->see("1.".($fprimerpos+10));
+ };
+
+ my $view_rprimer = sub {
+ $$text_ref->see("1.".($rprimerpos-10));
+ };
+
+ ### In progress!!
+ # my $multiline_pos = sub {
+ # my ($x,$y) = @_;
+ # # if ($multiline) {
+ # # # single lines to multiple lines
+ # #
+ # # }
+ # };
+
+
+ # if previous window exists we destroy it first, to avoid confusion
+ # (trying to simply erase the contents in the text box and
+ # re-configuring the buttons has about the same effect with Perl/Tk,
+ # so there's not much point in doing so ...)
+ if (Exists($view_base)) {
+ $view_base->destroy;
+ }
+
+ $view_base = $top->Toplevel(-title=>'Details ...');
+ my $view_basef = $view_base->Frame()->pack(-expand=>1, -fill=>'both');
+ my $view_basefb = $view_base->Frame()->pack(-side=>'left');
+ my $view_basefbr = $view_base->Frame()->pack(-side=>'right');
+
+ $view_base->withdraw if $report;
+
+ nr(\$view_basef);
+ pack_gui('ROText', '', 'view_base_text', 80, 4, -scrollbars=>'os', -wrap=>'none');
+
+ nr(\$view_basefb);
+ pack_gui('Button', 'OK', 'view_base_ok', sub {$view_base->destroy}, 'active');
+ pack_gui('Button', 'Copy printable', 'view_base_copy', \&$copy_printable);
+
+ nr(\$view_basefbr);
+ pack_gui('Button', 'Forward', 'view_base_pf', \&$view_fprimer, 'disabled' );
+ pack_gui('Button', 'Reverse', 'view_base_pr', \&$view_rprimer, 'disabled' );
+
+ $$text_ref->tagConfigure('red',
+ -foreground => 'red');
+ $$text_ref->tagConfigure('blue',
+ -foreground => 'midnightblue',
+ -background => '#cee3ee');
+ $$text_ref->tagConfigure('blue_utr',
+ -foreground => '#459ecc',
+ -background => '#dde8ee');
+ $$text_ref->tagConfigure('blue_cpg',
+ -foreground => 'royalblue',
+ -background => 'royalblue');
+ $$text_ref->tagConfigure('numbers',
+ -foreground => 'grey75',
+ -background => 'grey35');
+ $$text_ref->tagConfigure('grey60',
+ -foreground => 'grey50');
+ $$text_ref->tagConfigure('grey40',
+ -foreground => 'grey40');
+ $$text_ref->tagConfigure('black',
+ -foreground => 'black',
+ -background => 'grey89');
+ $$text_ref->tagConfigure('codon',
+ -foreground => 'black',
+ -background => 'grey81');
+ $$text_ref->tagConfigure('codon_utr',
+ -foreground => 'black',
+ -background => 'grey84');
+ $$text_ref->tagConfigure('codonbg',
+ -foreground => 'black',
+ -background => 'grey93');
+ $$text_ref->tagConfigure('orange',
+ -foreground => 'orange');
+ $$text_ref->tagConfigure('dodgerblue',
+ -foreground => 'dodgerblue');
+ $$text_ref->tagConfigure('ie',
+ -foreground => 'white',
+ -background => 'red');
+
+ for my $i (1 .. 3) {
+ $$text_ref->insert("$i.0","\n")
+ }
+
+ # my $list_ref = which_hlist();
+ my @sel= $$list_ref->selectionGet;
+ my $sel=shift(@sel);
+
+ # Selected primers, if any ...
+ if (defined($sel)) {
+ $fprimer = $$primer_array[$sel][0];
+ $rprimer = reverse($$primer_array[$sel][4]) if $$primer_array[$sel][5];
+
+ $fprimerpos = $$primer_array[$sel][1];
+ $rprimerpos = $$primer_array[$sel][8];
+
+ $fprimerposl = $$primer_array[$sel][2];
+ $rprimerposl = $$primer_array[$sel][6];
+
+ # need to do this before adding sequences ...
+ my $spacerrp = " "x($rprimerpos-$rprimerposl-$fprimerpos-$fprimerposl-5) if $rprimer;
+
+ # Added sequences?
+ if ($page eq 'pd' && (($primer_seq_5f) || ($primer_seq_5r))) {
+ ($primer_seq_5f_real, $insert_f, $fprimer, $fprimerpos, $fprimerposl, $primer_seq_5r_real, $insert_r, $rprimer, $rprimerpos, $rprimerposl)
+ = add_cloning($seq, $fprimer, $fprimerpos, $fprimerposl, $rprimer, $rprimerpos, $rprimerposl);
+ }
+
+ my $spacerfp = " "x($fprimerpos-3);
+ # print "($rprimerpos-$rprimerposl-$fprimerpos-$fprimerposl-5) = ".($rprimerpos-$rprimerposl-$fprimerpos-$fprimerposl-5);
+ $$text_ref->insert('2.0',$spacerfp."5' ", 'grey60');
+ $$text_ref->insert('2.end',$fprimer, 'red');
+
+ if ($rprimer) {
+ $$text_ref->insert('2.end'," 3'".$spacerrp."3' ", 'grey60');
+ $$text_ref->insert('2.end',$rprimer, 'red');
+ $$text_ref->insert('2.end'," 5'", 'grey60');
+ } else {
+ $$text_ref->insert('2.end'," 3'", 'grey60');
+ }
+
+ # Make buttons active or inactive:
+ $packed_widgets{view_base_pf}->configure(-state=>($fprimer ? 'active' : 'disabled'));
+ $packed_widgets{view_base_pr}->configure(-state=>($rprimer ? 'active' : 'disabled'));
+ }
+
+ # Numbering along the top (starts at 0)
+ my $numbers="0.........";
+ for my $i(1 .. length($seq)/10) {
+ $numbers.=($i*10)."."x(9-length($i));
+ }
+
+ # insert the numbers and the sequence
+ $$text_ref->insert('1.0',$numbers,'numbers');
+ $$text_ref->insert('3.0',$seq, 'grey40');
+
+ # translation of ORFs
+ # (or if Bisulphite sequencing highlights CpG island)
+ my @gene_array=find_gene($seq);
+ my $prev_end = 0;
+ for my $i (0 .. $#gene_array) {
+ my $peptide="";
+ my ($start, $end)=($gene_array[$i][0],$gene_array[$i][1]);
+
+ # if f_primer starts before ORF, translate from there up
+ my ($new_start, $new_peptide);
+ if ($fprimerpos && $fprimerpos < $start) {
+ my $start_mod = $start%3;
+ my $fprimer_mod = $fprimerpos%3;
+ $new_start = $fprimerpos + ($start_mod-$fprimer_mod);
+ # $start = $new_start;
+ }
+
+
+ $$text_ref->tagAdd('black', "3.$start", "3.$end");
+ $$text_ref->tagAdd('black', "3.$new_start", "3.$end") if $new_start;
+ if ($page eq 'bis') {
+ $peptide = "-"x($end-$start);
+ } else {
+ my ($j,$k)=(0,0);
+ if ($new_start) {
+ for ($j=$new_start; $j<$start; $j+=3) {
+ my $codon = uc(substr($seq, $j, 3));
+ my $aa = $genetic_code{$codon};
+ $aa ||= "X";
+ $new_peptide .= $aa." ";
+ if ($k == 1) {
+ my $tend = $j+3;
+ $$text_ref->tagAdd('codon_utr', "3.$j", "3.$tend");
+ }
+ $k = 1 - $k;
+ }
+ }
+
+ for ($j=$start; $j<$end; $j+=3) {
+ my $codon = uc(substr($seq, $j, 3));
+ my $aa = $genetic_code{$codon};
+ $aa ||= "X";
+ $peptide .= $aa." ";
+ if ($k == 1) {
+ my $tend = $j+3;
+ $$text_ref->tagAdd('codon', "3.$j", "3.$tend");
+ }
+ $k = 1 - $k;
+ }
+ }
+
+ $start = $new_start if $new_start;
+ my $spacer=" "x($start-$prev_end);
+ $prev_end=$end;
+ $$text_ref->insert("4.0",$spacer);
+
+ unless ($page eq 'bis') {
+ $$text_ref->insert("4.end",$new_peptide,'blue_utr') if $new_peptide;
+ $$text_ref->insert("4.end",$peptide,'blue');
+ } else {
+ $$text_ref->insert("4.end",$peptide,'blue_cpg');
+ }
+ }
+
+ # highlight intron-exon boundaries for QPCR
+ # or ranges otherwise
+ if ($page eq 'qpcr') {
+ foreach my $i (@intron_exon_bounds) {
+ my $tag_start=$i-1;
+ my $tag_end=$i+1;
+ $$text_ref->tagAdd('ie', "3.$tag_start", "3.$tag_end");
+ }
+ } else {
+ my $sel_range=$$max_range+1;
+ my $sel_range_3p=$$max_range_3p+1 if $$max_range_3p;
+ $$text_ref->tagAdd('orange', "3.$$max_range_5p", "3.$sel_range_3p") if $$max_range_3p;
+ $$text_ref->tagAdd('dodgerblue', "3.$$min_range", "3.$sel_range");
+ }
+
+ if ($page eq 'bis') {
+ # CpG labeling
+ for my $i (0 .. length($seq)) {
+ if (substr($seq, $i, 2) =~ /CG|cg/ ) {
+ my $tagcpg = $i+2;
+ $$text_ref->tagAdd('ie', "3.$i", "3.$tagcpg");
+ }
+ }
+
+ }
+
+ # this is very inefficient and messy, but gets the job done ...
+ if ($report) {
+ my $sequence_report = &$copy_report;
+ $view_base->destroy;
+ return $sequence_report;
+ }
+
+ # icon
+ $view_base->Icon(-image => $pixmap);
+
+ # Show the position where the user clicked
+ # my $center_base_pos = $base_pos-30;
+ # $$text_ref->see("1.$center_base_pos");
+
+ # Show the forward primer if it exists
+ if ($fprimerpos) {
+ my $center_base_pos = $fprimerpos-30;
+ $$text_ref->see("1.$center_base_pos");
+ }
+}
+
+
+#---------------#
+# File commands #
+#---------------#
+
+sub pp_file_open {
+ # NB - there's several reasons why we're not using XML as a file format here.
+ # The first is simplicity, both for the programmer and for the end-user (who
+ # would have to download and install a CPAN module otherwise - which might be
+ # daunting to many). The second is that I can't abide XML.
+
+ # The system that follows is both simple and highly extensible (adding
+ # support for another varible simple entails a single line in either the
+ # %variables or %arrays hashes - everything else is automatic ... )
+
+ my ($file, $widget_ref, @file_data) = @_;
+ $file ||= $top->getOpenFile(-filetypes=>$file_types);
+ return unless defined($file);
+
+ unless (@file_data) {
+ unless (open (SEQ, "<$file")) {
+ dialogue("Could not open file $file: $!");
+ return;
+ }
+ sbarprint("\nOpening $file ...");
+ @file_data = <SEQ>;
+ close SEQ;
+ } else {
+ sbarprint("\nReading data from port $tcp_port ...");
+ }
+
+ # clear previous selections:
+ # new_file();
+
+ my ($nb_page, $name) = open_file_type($file, $widget_ref, @file_data);
+ unless ($nb_page) {
+ $cancel = 0;
+ return;
+ }
+
+ # redraw dna
+ draw_dna();
+
+ # reset qpcr_flag
+ $qpcr_flag = 0;
+ sbarprint("\n$file opened successfully");
+ $file =~ s/\//\\/g if $os eq 'win'; # path bug in Win32 Perl/Tk
+
+ # since we're taking the name from FASTA files directly ...
+ my $title_filename = ($name ? $name : $file);
+
+ # my $full_path = $file;
+ # $file =~ s/.*[\/\\]//g;
+ $top->configure(-title=>"PerlPrimer v$version - $title_filename");
+ $open_file{$nb_page} = $title_filename;
+
+ recently_used_files($file);
+}
+
+sub open_file_type {
+ my ($file, $widget_ref, @file_data) = @_;
+ my ($page, $name);
+ foreach (@file_data) {
+ # This loop is in case the first line is blank.
+ # It's probably unnecessary ..
+ next if /^$/;
+ if (/nb = .+/) {
+ # file appears to be a perlprimer file
+ return ($page, $name) = open_ppr($file, @file_data);
+ } elsif (/^\>.+/) {
+ # file appears to be FASTA format
+ return ($page, $name) = open_fasta($file, $widget_ref, @file_data);
+ # } elsif (/[efijlopqz\d]+/i) {
+ # # file is unknown format
+ # dialogue("File does not appear to be a PerlPrimer file or in FASTA format.\nIf you are trying to open a DNA sequence file, please use the open icon next to the sequence entry field");
+ # last;
+ # } else {
+ # return ($page, $name) = open_fasta($file, @file_data);
+ }
+ }
+}
+
+sub open_ppr {
+ my $file = shift;
+ my @file_data = @_;
+
+ my $nb_page;
+ my $array_flag=0;
+ @save_selection=();
+ $save_seq=$save_seq2="";
+ my %flag=();
+
+ my $pointer;
+ my ($key, $value, $lines, $open_percent);
+ my $total_lines = @file_data + 1;
+ foreach (@file_data) {
+ # we're supporting both *nix and win32 ...
+ s/[\n\r]//g;
+
+ $open_percent = sprintf("%.f", $lines++/$total_lines*100);
+ sbarprint("\nOpening $file ... $open_percent\%");
+
+ # get nb_page entry (NB: must come before all data entry)
+ # or look for [array] section
+ if (/nb = (.*)/) {
+ $nb->raise($1);
+ $top->update;
+ new_file();
+ if ($cancel) {
+ sbarprint("\nFile open cancelled");
+ return;
+ }
+ $nb_page = $1;
+ next;
+ } elsif (/\[arrays\]/) {
+ $array_flag=1;
+ next;
+ }
+
+ # ignore blank lines
+ next if /^$/;
+
+ # grab values
+ # my ($key, $value) = split / = /;
+ if (/ = /) {
+ ($key, $value) = split / = /;
+ } elsif ($array_flag==0) {
+ # allow multi-line variables - eg for genomic DNA
+ $$pointer .= "\n$_";
+ next;
+ }
+
+ # set variables
+ unless ($array_flag==1) {
+ $pointer = $variables{$nb_page}{$key};
+ $$pointer = $value;
+ next;
+ }
+
+ # array parsing
+ $pointer = $arrays{$nb_page}{$key};
+ unless ($flag{$key}) {
+ # clear the array
+ @$pointer=();
+ $flag{$key}=1;
+ }
+ # this is so easy!
+ my @temp = split(/ /,$value);
+ if ($#temp == 0) {
+ push (@$pointer, $temp[0]);
+ } else {
+ push (@$pointer, [@temp]);
+ }
+ }
+
+ # restore sequence(s)
+ my ($seq_ref, $hlist) = get_variables(qw(seq hlist));
+ $$seq_ref->delete(0.1,"end");
+ $$seq_ref->insert(0.1,$save_seq);
+ if ($save_seq2) {
+ # only for qpcr
+ $packed_widgets{qdna_seq}->delete(0.1,"end");
+ $packed_widgets{qdna_seq}->insert(0.1,$save_seq2);
+ }
+
+ # restore results
+ sort_primers();
+
+ # restore intron-exon boundaries if qpcr
+ $qpcr_flag = 1 if $nb_page eq 'qpcr';
+
+ # restore selection
+ for my $i (0 .. $#save_selection) {
+ $$hlist->selectionSet("$save_selection[$i]");
+ }
+ if (@save_selection) {
+ browse_primer($save_selection[$#save_selection]);
+ $$hlist->see($save_selection[$#save_selection]);
+ }
+
+ return $nb_page;
+}
+
+sub open_fasta {
+ my ($file, $widget_ref, @file_data) = @_;
+ my ($flag, $dna, $name, $range_5a, $range_5b, $range_3a, $range_3b, $page);
+ my $nb_page = which_nb_page();
+ my ($key, $value, $lines, $open_percent);
+ my $total_lines = @file_data + 1;
+ foreach (@file_data) {
+ $open_percent = sprintf("%.f", $lines++/$total_lines*100);
+ sbarprint("\nOpening $file ... $open_percent\%");
+
+ next if /^$/;
+ # if ($flag) {
+ # $dna .= "$_";
+ # next;
+ # } elsif (/^\>/) {
+ if (/^\>/) {
+ next if $flag;
+ ($name, $range_5a, $range_5b, $range_3a, $range_3b, $page) = /^\>\s*(.+?)\s*(?:5prime_region\[(\d+)-(\d+)\])?\s*(?:3prime_region\[(\d+)-(\d+)\])?\s*(?:page\[(\d+)\])?\s*$/;
+
+ # process name (remove whitespace, other illegal filename chars)
+ $name = format_file_name($name);
+
+ # page defaults to 1 if there's other info that suggests this is a socket file
+ $page ||= 1 if ($range_5a || $range_5b || $range_3a || $range_3b);
+ $nb_page = $nb_page_ref{$page} if $page;
+ $nb->raise($nb_page);
+
+ $top->update;
+ if ($cancel) {
+ sbarprint("\nFile open cancelled");
+ return;
+ }
+ $flag = 1;
+ } else {
+ $dna .= "$_";
+ }
+ }
+
+ # insert dna sequence if present
+ if ($dna && $dna =~ /[agct]/i) {
+ ($widget_ref) = get_variables(qw(seq)) unless ref($widget_ref);
+ $_ = $$widget_ref->get(0.1,"end");
+
+ # New file if we're overwriting a previous sequence
+ my ($seq_check) = /atcg/i;
+ new_file($widget_ref) if $seq_check;
+
+ $$widget_ref->delete(0.1,"end");
+ $$widget_ref->insert(0.1,$dna);
+ }
+
+ # set ranges if specified
+ my ($min_ampsize, $max_ampsize, $max_range_5p, $min_range, $max_range, $max_range_3p)
+ = get_variables(qw(min_ampsize max_ampsize max_range_5p min_range max_range max_range_3p));
+
+ if ($range_5a && $range_5b && $range_3a && $range_3b) {
+ $$max_range_5p = $range_5a;
+ $$min_range = $range_5b;
+ $$max_range = $range_3a;
+ $$max_range_3p = $range_3b;
+
+ $$min_ampsize=$$max_range - $$min_range;
+ $$max_ampsize=$$max_range_3p - $$max_range_5p;
+ }
+
+ # set range to ORF if not
+ reset_bounds() unless $$min_range && $$max_range;
+
+ return ($nb_page, $name);
+}
+
+sub format_file_name {
+ my ($name) = @_;
+ $name =~ s/\s+/_/g; # replace spaces with underscores
+ $name =~ s/[\(\)\,\.\;]//g; # remove brackets and punctuation
+ $name =~ s/\|/-/g; # replace pipes with dashes
+ $name =~ s/_$//g; # remove final, terminating underscore
+ $name =~ s/-_/-/g; # fix -_ problems
+ return $name;
+}
+
+sub pp_file_save {
+ my $nb_page = which_nb_page();
+ if ($nb_page eq "primer") {
+ dialogue("You can't save a file from the primer information page - please save from the project page instead");
+ return;
+ }
+
+ # Either prompt for file name if file not saved or "Save as ..." was
+ # called, or save without prompting if file has been saved before
+ # (It's amazing how complicated "simple" behaviour can be ...)
+ my ($flag) = @_ || 0;
+ my $file;
+ if ($open_file{$nb_page} eq 'File not saved') {
+ $file = $top->getSaveFile(-filetypes=>$file_types, -defaultextension=>'.ppr');
+ } elsif (($flag == 1) || ($open_file{$nb_page} !~ /\.ppr/)) {
+ $file = $top->getSaveFile(-filetypes=>$file_types, -defaultextension=>'.ppr', -initialfile=>$open_file{$nb_page});
+ } else {
+ $file = $open_file{$nb_page};
+ }
+
+ return unless defined($file);
+ $file .= '.ppr' unless $file =~ /\.ppr/;
+
+ sbarprint("\nSaving $file ...");
+
+ my $total_keys = keys(%{ $variables{$nb_page} }) + keys(%{ $arrays{$nb_page} }) + 1;
+ my ($keys_saved, $saved_percent);
+
+ # Now the guts of the saving routine - see the pp_file_open routine for general
+ # comments ... this is all straightforward stuff
+
+ # save sequence data
+ $save_seq = get_seq();
+ $save_seq2 = $packed_widgets{qdna_seq}->get(0.1,"end") if $nb_page eq "qpcr";
+
+ # save selection data
+ my ($hlist) = get_variables('hlist');;
+ @save_selection = $$hlist->selectionGet;
+
+ # save variables
+ my $file_data = "nb = $nb_page\n";
+ my $pointer;
+ foreach my $i (keys %{ $variables{$nb_page} }) {
+ $pointer = $variables{$nb_page}{$i};
+ $file_data .= "$i = $$pointer\n" if defined($$pointer);
+ $saved_percent = sprintf("%.f", $keys_saved++/$total_keys*100);
+ sbarprint("\nSaving $file ... $saved_percent\%");
+ }
+
+ # save arrays
+ $file_data .= "[arrays]\n";
+ foreach my $i (keys %{ $arrays{$nb_page} }) {
+ $pointer = $arrays{$nb_page}{$i};
+ for my $j (0 .. $#$pointer) {
+ unless (ref($$pointer[$j])) {
+ $file_data .= "$i = $$pointer[$j]\n";
+ } else {
+ $file_data .= "$i = @{$$pointer[$j]}\n";
+ }
+ }
+ $saved_percent = sprintf("%.f", $keys_saved++/$total_keys*100);
+ sbarprint("\nSaving $file ... $saved_percent\%");
+ }
+
+ # write file
+ open (SEQ, ">$file") || dialogue("Could not open file for saving: $!\n");
+ print SEQ $file_data;
+ close (SEQ);
+
+ sbarprint("\n$file saved successfully");
+
+ # Set the program title to reflect the new file name
+ # $file =~ s/.*\/.*\///g;
+ $file =~ s/\//\\/g if $os eq 'win'; # path bug in Win32 Perl/Tk
+ $top->configure(-title=>"PerlPrimer v$version - $file");
+ $open_file{$nb_page} = $file;
+
+ recently_used_files($file);
+}
+
+sub recently_used_files {
+ # recently used files
+ my ($file) = @_;
+ if ($file) {
+ for my $i (0 .. $#mru) {
+ $mru[$i] = "" if $mru[$i] eq $file;
+ }
+ push(@mru, $file);
+ }
+
+ # the subroutine gets called when the program loads, so we need an escape
+ return if @mru == 0;
+
+ # clean up the space from before if it exists
+ my @new_mru;
+ for my $i (0 .. $#mru) {
+ push(@new_mru, $mru[$i]) if ($mru[$i]);
+ }
+
+ # take only the last requested files so the list doesn't grow too big
+ my $start = $#new_mru-($mru_number-1)<0 ? 0 : $#new_mru-($mru_number-1);
+ my $end = $#new_mru;
+ @mru = @new_mru[$start .. $end];
+
+ # clear the old menu
+ $menu_mru->delete(1,'end');
+
+ # ... and insert the new list
+ for my $i (0 .. $#mru) {
+ $menu_mru->insert(0,'command', -label=>"$mru[$i]", -command =>[sub {pp_file_open($mru[$i])}] );
+ }
+}
+
+sub open_seq {
+ # opens file and writes contents to text widget
+ my ($widget_ref) = @_;
+ my $file = $top->getOpenFile(-filetypes=>$file_types_dna);
+ if (defined($file)) {
+ pp_file_open($file, $widget_ref);
+ }
+}
+
+sub save_seq {
+ # saves sequence in text widget to file
+ my ($widget_ref) = @_;
+ my $file = $top->getSaveFile(-filetypes=>$file_types_dna, -defaultextension=>'.fasta');
+ my $seq = $$widget_ref->get(0.1, 'end');
+ my $nb_page = which_nb_page();
+ if (defined($file)) {
+ my $out;
+ if ($file =~ /\.fasta$/) {
+ my $description = $open_file{$nb_page};
+ $description = $file if $description eq 'File not saved';
+ $description =~ s/.*[\\\/]//g;
+
+ # open description window
+ my $descript_w = $top->Toplevel(-title=>'Enter FASTA description line');
+ nr(\$descript_w);
+ pack_gui('Entry',\$description, 'description_text', 35);
+ pack_gui('Button', 'OK', 'description_ok', sub {$descript_w->destroy}, 'active');
+ $descript_w->Icon(-image => $pixmap);
+ $descript_w->grab;
+ $descript_w->waitWindow;
+
+ my $fasta = ">$description\n$seq";
+ $out = $fasta;
+ } else {
+ $out = $seq;
+ }
+
+ open (SEQ, ">$file") || dialogue("Could not open file: $!");
+ print SEQ $out;
+ close (SEQ);
+ sbarprint("\n$file saved successfully");
+ }
+}
+
+
+#------------------#
+# Text subroutines #
+#------------------#
+
+sub clear_text {
+ my $ref = $_[0];
+ $$ref->delete(0.1,'end');
+}
+
+sub select_all_text {
+ my $ref = $_[0];
+ $$ref->selectAll;
+}
+
+sub lc_text {
+ my $ref = $_[0];
+ my $text = $$ref->get(0.1,"end");
+ my $text_lc = lc($text);
+ $$ref->delete(0.1,'end');
+ $$ref->insert(0.1,$text_lc);
+}
+
+sub reverse_complement {
+ my $ref = $_[0];
+ my $text = $$ref->get(0.1,"end");
+ $text =~ s/[\n\s]//g;
+ my $text_comp_rev = reverse(complement($text));
+ $$ref->delete(0.1,'end');
+ $$ref->insert(0.1,$text_comp_rev);
+}
+
+
+#----------------#
+# Reference subs #
+#----------------#
+
+sub get_seq {
+ my ($seq_ref) = get_variables('seq');
+ return 1 unless $$seq_ref;
+ $_ = $$seq_ref->get(0.1,"end");
+
+ $_ = clean_seq($_);
+ # s/(\>.*\n)//g; #remove FASTA formatting if it exists
+ # if ($1 && !$open_file{$nb_page}) {
+ # # if FASTA details are present ...
+ # my $name = format_file_name($1);
+ #
+ # $open_file{$nb_page} = $name;
+ # $top->configure(-title=>"PerlPrimer v$version - $name");
+ # }
+ #
+ # s/[\n\r]//g; # remove line breaks
+ return $_;
+}
+
+sub which_nb_page {
+ return $nb->raised;
+}
+
+sub get_variables {
+ my $note_page = which_nb_page();
+ $null = undef;
+ my @return_args;
+ foreach my $var (@_) {
+ if ($page_specific_vars{$note_page}{$var}) {
+ push @return_args, $page_specific_vars{$note_page}{$var};
+ } else {
+ push @return_args, \undef;
+ }
+ }
+ return @return_args;
+}
+
+sub check_packages {
+ my $failed;
+ foreach (@_) {
+ if ($failed_packages =~ / $_ /) {
+ dialogue("PerlPrimer requires the package $_ for this feature to work\n\nPlease download and install $_ from CPAN (http://cpan.org)");
+ $failed = 1;
+ }
+ }
+ return 1 if $failed;
+}
+
+sub check_packages_no_warn {
+ my $failed;
+ foreach (@_) {
+ if ($failed_packages =~ / $_ /) {
+ $failed = 1;
+ }
+ }
+ return 1 if $failed;
+}
+
+
+#----------------------#
+# Balloon state toggle #
+#----------------------#
+
+sub balloon_toggle {
+ $Balloon->configure(-state => 'none') if $balloon_help == 0;
+ $Balloon->configure(-state => 'balloon') if $balloon_help == 1;
+}
+
+
+sub check_path {
+ $_ = shift;
+ my $regexp_sep = "\\"."$dir_sep";
+ return $_.$dir_sep unless m/$regexp_sep$/;
+ return $_;
+}
+
+
+sub load_data {
+ #-----
+ #
+ # NN thermodynamics hashes (AA = 5' AA 3'/3' TT 5') derived from ...
+ #
+ # Allawi HT, SantaLucia J Jr. Thermodynamics and NMR of internal G.T mismatches in DNA.
+ # Biochemistry. 1997 Aug 26;36(34):10581-94
+ #
+ # SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics.
+ # Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5.
+ #
+ # ... with mismatch dG data (AGTG = 5' AG 3'/3' TG 5') derived from ...
+ #
+ # Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches.
+ # Biochemistry. 1999 Mar 23;38(12):3468-77.
+ #
+ # Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects.
+ # Biochemistry. 1998 Jun 30;37(26):9435-44.
+ #
+ # Allawi HT, SantaLucia J Jr. Thermodynamics of internal C.T mismatches in DNA.
+ # Nucleic Acids Res. 1998 Jun 1;26(11):2694-701.
+ #
+ # Allawi HT, SantaLucia J Jr. Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA.
+ # Biochemistry. 1998 Feb 24;37(8):2170-9.
+ #
+ # Allawi HT, SantaLucia J Jr. Thermodynamics and NMR of internal G.T mismatches in DNA.
+ # Biochemistry. 1997 Aug 26;36(34):10581-94
+ #
+ #-----
+
+ #-------------------#
+ # deltaH (kcal/mol) #
+ #-------------------#
+
+ %oligo_dH=qw(
+ AA -7.9 TT -7.9
+ AT -7.2 TA -7.2
+ CA -8.5 TG -8.5
+ GT -8.4 AC -8.4
+ CT -7.8 AG -7.8
+ GA -8.2 TC -8.2
+ CG -10.6 GC -9.8
+ GG -8.0 CC -8.0
+ initC 0.1 initG 0.1
+ initA 2.3 initT 2.3
+ );
+
+ %oligo_dH_full=(
+ qw(AATT -7.9 TTAA -7.9
+ ATTA -7.2 TAAT -7.2
+ CAGT -8.5 TGAC -8.5
+ GTCA -8.4 ACTG -8.4
+ CTGA -7.8 AGTC -7.8
+ GACT -8.2 TCAG -8.2
+ CGGC -10.6 GCCG -9.8
+ GGCC -8.0 CCGG -8.0
+
+ initC 0.1 initG 0.1
+ initA 2.3 initT 2.3),
+
+ # Like pair mismatches
+
+ qw(AATA 1.2 ATAA 1.2
+ CAGA -0.9 AGAC -0.9
+ GACA -2.9 ACAG -2.9
+ TAAA 4.7 AAAT 4.7
+
+ ACTC 0.0 CTCA 0.0
+ CCGC -1.5 CGCC -1.5
+ GCCC 3.6 CCCG 3.6
+ TCAC 6.1 CACT 6.1
+
+ AGTG -3.1 GTGA -3.1
+ CGGG -4.9 GGGC -4.9
+ GGCG -6.0 GCGG -6.0
+ TGAG 1.6 GAGT 1.6
+
+ ATTT -2.7 TTTA -2.7
+ CTGT -5.0 TGTC -5.0
+ GTCT -2.2 TCTG -2.2
+ TTAT 0.2 TATT 0.2 ),
+
+ # G.T mismatches
+
+ qw(AGTT 1.0 TTGA 1.0
+ ATTG -2.5 GTTA -2.5
+ CGGT -4.1 TGGC -4.1
+ CTGG -2.8 GGTC -2.8
+ GGCT 3.3 TCGG 3.3
+ GGTT 5.8 TTGG 5.8
+ GTCG -4.4 GCTG -4.4
+ GTTG 4.1 GTTG 4.1
+ TGAT -0.1 TAGT -0.1
+ TGGT -1.4 TGGT -1.4
+ TTAG -1.3 GATT -1.3),
+
+ # G.A mismatches
+
+ qw(AATG -0.6 GTAA -0.6
+ AGTA -0.7 ATGA -0.7
+ CAGG -0.7 GGAC -0.7
+ CGGA -4.0 AGGC -4.0
+ GACG -0.6 GCAG -0.6
+ GGCA 0.5 ACGG 0.5
+ TAAG 0.7 GAAT 0.7
+ TGAA 3.0 AAGT 3.0),
+
+ # C.T mismatches
+
+ qw(ACTT 0.7 TTCA 0.7
+ ATTC -1.2 CTTA -1.2
+ CCGT -0.8 TGCC -0.8
+ CTGC -1.5 CGTC -1.5
+ GCCT 2.3 TCCG 2.3
+ GTCC 5.2 CCTG 5.2
+ TCAT 1.2 TACT 1.2
+ TTAC 1.0 CATT 1.0),
+
+ # A.C mismatches
+
+ qw(AATC 2.3 CTAA 2.3
+ ACTA 5.3 ATCA 5.3
+ CAGC 1.9 CGAC 1.9
+ CCGA 0.6 AGCC 0.6
+ GACC 5.2 CCAG 5.2
+ GCCA -0.7 ACCG -0.7
+ TAAC 3.4 CAAT 3.4
+ TCAA 7.6 AACT 7.6),
+
+ );
+
+ #--------------------#
+ # deltaS (cal/K.mol) #
+ #--------------------#
+
+ %oligo_dS=qw(
+ AA -22.2 TT -22.2
+ AT -20.4 TA -21.3
+ CA -22.7 TG -22.7
+ GT -22.4 AC -22.4
+ CT -21.0 AG -21.0
+ GA -22.2 TC -22.2
+ CG -27.2 GC -24.4
+ GG -19.9 CC -19.9
+ initC -2.8 initG -2.8
+ initA 4.1 initT 4.1
+ sym -1.4
+ );
+
+ %oligo_dS_full=(
+ qw(AATT -22.2 TTAA -22.2
+ ATTA -20.4 TAAT -21.3
+ CAGT -22.7 TGAC -22.7
+ GTCA -22.4 ACTG -22.4
+ CTGA -21.0 AGTC -21.0
+ GACT -22.2 TCAG -22.2
+ CGGC -27.2 GCCG -24.4
+ GGCC -19.9 CCGG -19.9
+
+ initC -2.8 initG -2.8
+ initA 4.1 initT 4.1
+ sym -1.4),
+
+ # Like pair mismatches
+
+ qw(AATA 1.7 ATAA 1.7
+ CAGA -4.2 AGAC -4.2
+ GACA -9.8 ACAG -9.8
+ TAAA 12.9 AAAT 12.9
+
+ ACTC -4.4 CTCA -4.4
+ CCGC -7.2 CGCC -7.2
+ GCCC 8.9 CCCG 8.9
+ TCAC 16.4 CACT 16.4
+
+ AGTG -9.5 GTGA -9.5
+ CGGG -15.3 GGGC -15.3
+ GGCG -15.8 GCGG -15.8
+ TGAG 3.6 GAGT 3.6
+
+ ATTT -10.8 TTTA -10.8
+ CTGT -15.8 TGTC -15.8
+ GTCT -8.4 TCTG -8.4
+ TTAT -1.5 TATT -1.5),
+
+ # G.T mismatches
+
+ qw(AGTT 0.9 TTGA 0.9
+ ATTG -8.3 GTTA -8.3
+ CGGT -11.7 TGGC -11.7
+ CTGG -8.0 GGTC -8.0
+ GGCT 10.4 TCGG 10.4
+ GGTT 16.3 TTGG 16.3
+ GTCG -12.3 GCTG -12.3
+ GTTG 9.5 GTTG 9.5
+ TGAT -1.7 TAGT -1.7
+ TGGT -6.2 TGGT -6.2
+ TTAG -5.3 GATT -5.3),
+
+ # G.A mismatches
+
+ qw(AATG -2.3 GTAA -2.3
+ AGTA -2.3 ATGA -2.3
+ CAGG -2.3 GGAC -2.3
+ CGGA -13.2 AGGC -13.2
+ GACG -1.0 GCAG -1.0
+ GGCA 3.2 ACGG 3.2
+ TAAG 0.7 GAAT 0.7
+ TGAA 7.4 AAGT 7.4),
+
+ # C.T mismatches
+
+ qw(ACTT 0.2 TTCA 0.2
+ ATTC -6.2 CTTA -6.2
+ CCGT -4.5 TGCC -4.5
+ CTGC -6.1 CGTC -6.1
+ GCCT 5.4 TCCG 5.4
+ GTCC 13.5 CCTG 13.5
+ TCAT 0.7 TACT 0.7
+ TTAC 0.7 CATT 0.7),
+
+ # A.C mismatches
+
+ qw(AATC 4.6 CTAA 4.6
+ ACTA 14.6 ATCA 14.6
+ CAGC 3.7 CGAC 3.7
+ CCGA -0.6 AGCC -0.6
+ GACC 14.2 CCAG 14.2
+ GCCA -3.8 ACCG -3.8
+ TAAC 8.0 CAAT 8.0
+ TCAA 20.2 AACT 20.2),
+
+ );
+
+
+ # Genetic code hash
+ %genetic_code=qw(
+ TTT F TTC F TTA L TTG L
+ CTT L CTC L CTA L CTG L
+ ATT I ATC I ATA I ATG M
+ GTT V GTC V GTA V GTG V
+ TCT S TCC S TCA S TCG S
+ CCT P CCC P CCA P CCG P
+ ACT T ACC T ACA T ACG T
+ GCT A GCC A GCA A GCG A
+ TAT Y TAC Y TAA * TAG *
+ CAT H CAC H CAA Q CAG Q
+ AAT N AAC N AAA K AAG K
+ GAT D GAC D GAA E GAG E
+ TGT C TGC C TGA * TGG W
+ CGT R CGC R CGA R CGG R
+ AGT S AGC S AGA R AGG R
+ GGT G GGC G GGA G GGG G
+ );
+}
+
+#----------------------#
+# Icon and pixmap data #
+#----------------------#
+
+# ... just a little bit of bloat :)
+
+sub load_icon_data {
+
+$perlprimer_icon = <<'end_of_pixmap';
+/* XPM */
+static char * dna_icon2_xpm[] = {
+"32 32 25 1",
+" c #FFFFFF",
+". c #402E33",
+"+ c #A0302D",
+"@ c #918FC4",
+"# c #B89590",
+"$ c #CAC7EE",
+"% c #E4B82A",
+"& c #969096",
+"* c #E3382B",
+"= c #924E32",
+"- c #E76029",
+"; c #EFD4C7",
+"> c #69647F",
+", c #F9FBF8",
+"' c #DD6C69",
+") c #80696A",
+"! c #C0817E",
+"~ c #9F6160",
+"{ c #E08B27",
+"] c #F7B6B3",
+"^ c #D2D1CF",
+"/ c #B3AEB2",
+"( c #F92C2B",
+"_ c #AEACF1",
+": c #E7A19D",
+" *(;,,;:>@_>##!((;=((+ ",
+" -;,,/>@_@/^,;(-;)=. ",
+" +*~&#/__&,,,-(({ ",
+" .!,::#.>*',;*(** ",
+" ),;~&,'--,,~=++ ",
+" &/&,!(*%:^) ",
+" +#,'*({(#& ",
+" .(:,^+=**/^. ",
+" +(%%],,#&&&)++ ",
+" +({*(!&>>....~ ",
+" +#+{(()$@@@>)!^^ ",
+" +%;^/))/$____::,,!(+ ",
+" ({(*;/&&&@)>)!#,,/*+ ",
+" +-((~^^,__@:],@$^,;= ",
+" .=~&,,,___:]___,#+*+ ",
+" =*#/&&&&>.!]$_$^!(*** ",
+" +**+#__#:@@/^//,,]'%* ",
+" ++./__::__,,,^+'#/{ ",
+" &/@#!@_$,^'*(-(+ ",
+" !,##>&&^/,]({{*+ ",
+" &,:@_@~'/^,,{{+ ",
+" /~>@@**-(~,:+ ",
+" ).&(%-(*,) ",
+" )#^^,;%{*~/&) ",
+" (-*+,,'(~))&/. ",
+" {((*;,;+))!))+ ",
+" %-(',,,>)!&^^'= ",
+" =*=!](*!,/>>)~#,,/*+ ",
+" **(;,''~.>>>&:];':(* ",
+" *(*,,,^>>@@_$/^'**++ ",
+" +=*{,,/)~@__>^,,,-* ",
+" *(''*!)#~.>@$]];,;((+ "};
+end_of_pixmap
+
+$icon_open = <<'end_of_pixmap';
+/* XPM */
+static char * open_20_xpm[] = {
+"20 20 27 1",
+" c None",
+". c #020501",
+"+ c #37372E",
+"@ c #5B5D5A",
+"# c #8E8F7E",
+"$ c #BABCAC",
+"% c #B8BB98",
+"& c #ACB0A3",
+"* c #1A1B18",
+"= c #43433B",
+"- c #CDD0AD",
+"; c #737561",
+"> c #A1A68A",
+", c #898F73",
+"' c #D7D9C6",
+") c #525548",
+"! c #AEB293",
+"~ c #C4C7A4",
+"{ c #242521",
+"] c #61624E",
+"^ c #808673",
+"/ c #C2C5B4",
+"( c #959B7E",
+"_ c #D5D8B6",
+": c #B1B39D",
+"< c #999B8E",
+"[ c #6C6D59",
+" ",
+" ",
+" ..... ",
+" +#<<<^= ",
+" @____~[{ ",
+"=$_~~~-(+ ",
+"=/%!!%%>;======+ ",
+"+->(>>>>>>:::::#. ",
+"{/,)+++++++++++{.. ",
+"{:]<$$$///$////$$$@ ",
+"{$=&__---_-----~~!) ",
+"{<<'_---------~%!,+ ",
+"{<<'-~--~~~~%~%:>]* ",
+"*&/~%%%%%%%!!>>>^+ ",
+"*$/!!>>>>>>>((,,]. ",
+"{&,^;;;;[[[]]]))+ ",
+" ............... ",
+" ",
+" ",
+" "};
+end_of_pixmap
+
+$icon_open_small = <<'end_of_pixmap';
+/* XPM */
+static char * open_xpm[] = {
+"18 18 64 1",
+" c None",
+". c #000000",
+"+ c #E4E5DF",
+"@ c #D5D6CB",
+"# c #D6D7CA",
+"$ c #A3A39D",
+"% c #F5F6F0",
+"& c #8D907B",
+"* c #92957E",
+"= c #90937D",
+"- c #979B84",
+"; c #6D705F",
+"> c #EAECDB",
+", c #8A8C7D",
+"' c #8E917B",
+") c #91947F",
+"! c #8B8E7A",
+"~ c #999B87",
+"{ c #919480",
+"] c #989B86",
+"^ c #B1B4A2",
+"/ c #A2A394",
+"( c #F7F7F7",
+"_ c #878A75",
+": c #666858",
+"< c #4B4D3F",
+"[ c #4D4F40",
+"} c #404135",
+"| c #424337",
+"1 c #434437",
+"2 c #404236",
+"3 c #3C3D32",
+"4 c #48493C",
+"5 c #1A1A16",
+"6 c #C6C6BE",
+"7 c #848672",
+"8 c #25261F",
+"9 c #F1F2E9",
+"0 c #DDE0C7",
+"a c #D6DABB",
+"b c #CDD2AC",
+"c c #C7CCA7",
+"d c #989C80",
+"e c #C6C7BE",
+"f c #5F6152",
+"g c #888980",
+"h c #A7AB8C",
+"i c #878A70",
+"j c #9FA19A",
+"k c #EFF0E5",
+"l c #9EA284",
+"m c #80817B",
+"n c #96968D",
+"o c #E3E5D1",
+"p c #83866D",
+"q c #97998D",
+"r c #EDEFE2",
+"s c #A2A688",
+"t c #767671",
+"u c #E7E9DA",
+"v c #D1D3BD",
+"w c #BBBF9D",
+"x c #989B80",
+"y c #6E715C",
+" ",
+" ",
+" ",
+" .... ",
+" .+@#$. ",
+" .%&*=-;..... ",
+" .>,')!~{]{^/. ",
+" .(_:<[}||12345 ",
+" .67890abbbbbcd. ",
+" .efg0bbbbbbbhi. ",
+" .j8kabbbbbbbl. ",
+" .mnobbbbbbbbp. ",
+" .qrbbbbbbbbs. ",
+" .tuvwwwwwwxy. ",
+" ........... ",
+" ",
+" ",
+" "};
+end_of_pixmap
+
+$icon_save = <<'end_of_pixmap';
+/* XPM */
+static char * save_20_xpm[] = {
+"20 20 27 1",
+" c None",
+". c #1B2226",
+"+ c #728BA0",
+"@ c #ADC9E0",
+"# c #424B54",
+"$ c #CA766B",
+"% c #5F7688",
+"& c #E3E5E1",
+"* c #8DAAC4",
+"= c #2A3138",
+"- c #AEAEAC",
+"; c #878A89",
+"> c #656764",
+", c #DF9A8E",
+"' c #4E3F3B",
+") c #55534E",
+"! c #DCDDDA",
+"~ c #7D8690",
+"{ c #3B424A",
+"] c #F8FAF7",
+"^ c #C7C6C1",
+"/ c #6E757D",
+"( c #4A5E6D",
+"_ c #7F98AD",
+": c #757879",
+"< c #9BBAD0",
+"[ c #F5E8E8",
+" ",
+" ",
+" .{='''''''''''={. ",
+" #@+,,,,,,,,,,,/*# ",
+" {<%,$$$$$$$$$$/_= ",
+" #<~![[[[[[[[[[/+= ",
+" {<~!]]]]][&[[[:_= ",
+" {<~^!&!!&!&&!!/+= ",
+" {</[]]]]]]]]]]~+= ",
+" {<~^!!!!&!&!!!/+= ",
+" {</!]]]]]]]]]]:+= ",
+" {<_~//////////~+. ",
+" {*__++++++_+++_+. ",
+" {*_%:;----;;((+_. ",
+" {*+#^^;:^^^-:(+_. ",
+" #*+#[^))^^^^;(%_. ",
+" {*+#&-)'-^^[/%%_. ",
+" .%%#^->:-!&&:(%~. ",
+" .==))>>>::>=={(. ",
+" "};
+end_of_pixmap
+
+$icon_clear = <<'end_of_pixmap';
+/* XPM */
+static char * clear_20_xpm[] = {
+"20 20 27 1",
+" c None",
+". c #000100",
+"+ c #504B3D",
+"@ c #989981",
+"# c #332B22",
+"$ c #6D7163",
+"% c #ADB8A6",
+"& c #1E1E1B",
+"* c #CEBF94",
+"= c #8D745E",
+"- c #554537",
+"; c #A6AA95",
+"> c #847B60",
+", c #3D3E32",
+"' c #5A5C4F",
+") c #BDA793",
+"! c #B5AB88",
+"~ c #E3D3A4",
+"{ c #CFBAAA",
+"] c #48382B",
+"^ c #282017",
+"/ c #6A634C",
+"( c #797E6C",
+"_ c #6D5644",
+": c #A38E79",
+"< c #C9BE9F",
+"[ c #11100E",
+" ",
+" ",
+" ^-& ",
+" -)_. ",
+" ]))^ ",
+" ^={_. ",
+" #=:^ ",
+" #=_^ .. ",
+" ]_- .['& ",
+" ]_[(@%, . ",
+" -;%%$+# .",
+" &$%%$>*' . . ",
+" &(%%$>~<!&.. ",
+" [%%'>~~>@>.. .",
+" [$'>~<~!'(/.. ",
+" .'*~)@;@^',.. ",
+" #>!!/]'+. ",
+" [,+,&.. ",
+" ",
+" "};
+end_of_pixmap
+
+$icon_info = <<'end_of_pixmap';
+/* XPM */
+static char * info_20_xpm[] = {
+"20 20 147 2",
+" c None",
+". c #ABABB1",
+"+ c #D6D6DE",
+"@ c #E5E5EF",
+"# c #E5E5F2",
+"$ c #D8D8E6",
+"% c #BBBBC9",
+"& c #6F7078",
+"* c #CECED4",
+"= c #F4F4F9",
+"- c #F8F8FB",
+"; c #F0F0F7",
+"> c #E9E9F4",
+", c #E4E4F2",
+"' c #DEDFF0",
+") c #D7D8EA",
+"! c #8B8B97",
+"~ c #C4C4CA",
+"{ c #F6F6FB",
+"] c #FCFCFD",
+"^ c #F9F9FC",
+"/ c #F1F1F8",
+"( c #ECECF6",
+"_ c #E6E6F3",
+": c #E0E1F1",
+"< c #DADDEF",
+"[ c #D6D9EB",
+"} c #6C6D77",
+"| c #EEEEF3",
+"1 c #FBFBFD",
+"2 c #F3F3F9",
+"3 c #EEEEF7",
+"4 c #E8E8F5",
+"5 c #E2E2F2",
+"6 c #D7DBED",
+"7 c #C5C8DA",
+"8 c #BFBFC4",
+"9 c #FAFAFC",
+"0 c #F6F6FA",
+"a c #F2F3F9",
+"b c #E7E8F5",
+"c c #DCE0EF",
+"d c #D6DCEC",
+"e c #D5DBED",
+"f c #55575E",
+"g c #CBCBD2",
+"h c #F2F2F9",
+"i c #F7F7FB",
+"j c #F9F9FB",
+"k c #F7F8FB",
+"l c #F4F4FA",
+"m c #E0E4F1",
+"n c #D6DFEC",
+"o c #D4DCED",
+"p c #6A6C77",
+"q c #C6C6CD",
+"r c #EFEFF7",
+"s c #F6F6F8",
+"t c #F9F9FA",
+"u c #F6F7FA",
+"v c #ECECF2",
+"w c #E1E6F1",
+"x c #D6E3EB",
+"y c #D4DEEC",
+"z c #676973",
+"A c #A8A8AF",
+"B c #EAEAF4",
+"C c #EDEEF5",
+"D c #F1F1F7",
+"E c #E7E7E9",
+"F c #F2F2F8",
+"G c #EBECF3",
+"H c #E3E6ED",
+"I c #DCE5EC",
+"J c #D5E8EA",
+"K c #D3E0EC",
+"L c #404248",
+"M c #DADBE6",
+"N c #E3EAEF",
+"O c #E5EBF1",
+"P c #E0E3E6",
+"Q c #E5EAF2",
+"R c #E3E8F2",
+"S c #D8DDE6",
+"T c #CED6DC",
+"U c #D6E9E9",
+"V c #D3E9E9",
+"W c #B2BBC8",
+"X c #8D8F96",
+"Y c #DEE3ED",
+"Z c #DCE7EB",
+"` c #C6D0D0",
+" . c #D8E2E9",
+".. c #D7E0E9",
+"+. c #CED5D4",
+"@. c #D4E4E6",
+"#. c #D3EBE9",
+"$. c #C8D6DF",
+"%. c #43454B",
+"&. c #8D8E98",
+"*. c #D5D8E6",
+"=. c #CBCFD2",
+"-. c #C5CBC5",
+";. c #CFD5CE",
+">. c #C1C9C2",
+",. c #D1DDDE",
+"'. c #C4D0DA",
+"). c #55585F",
+"!. c #847F75",
+"~. c #C9C3B4",
+"{. c #C7C5C0",
+"]. c #CDC9BE",
+"^. c #D0CAB5",
+"/. c #C1B99B",
+"(. c #2E2E2C",
+"_. c #F0E4BA",
+":. c #EBE0B7",
+"<. c #E1D29A",
+"[. c #D5C58C",
+"}. c #AE9E66",
+"|. c #13110A",
+"1. c #F1E5BC",
+"2. c #F1E7C5",
+"3. c #E6D69E",
+"4. c #D8C890",
+"5. c #AFA06C",
+"6. c #15130C",
+"7. c #EFE4BF",
+"8. c #E7DEBE",
+"9. c #DACD9C",
+"0. c #CDBE8A",
+"a. c #B0A16D",
+"b. c #14120B",
+"c. c #E7DDB6",
+"d. c #E2D8B4",
+"e. c #D1C28C",
+"f. c #C3B47D",
+"g. c #9A8D5F",
+"h. c #0F0D08",
+"i. c #898060",
+"j. c #BCB18A",
+"k. c #AD9F6C",
+"l. c #82764C",
+"m. c #312C1A",
+"n. c #2D2D2D",
+"o. c #404040",
+"p. c #060606",
+" ",
+" . + @ # $ % & ",
+" * = - ; > , ' ) ! ",
+" ~ { ] ^ / ( _ : < [ } ",
+" | ^ 1 - 2 3 4 5 < 6 7 ",
+" 8 = ^ ] 9 0 a 3 b c d e f ",
+" g h i j j ^ k l 3 m n o p ",
+" q r 2 s t 9 9 u v w x y z ",
+" A B C D E F ; G H I J K L ",
+" M N O P Q R S T U V W ",
+" X Y Z ` ...+. at .#.$.%. ",
+" &.*.=.-.;.>.,.'.). ",
+" !.~.{.].^./.(. ",
+" _.:.<.[.}.|. ",
+" 1.2.3.4.5.6. ",
+" 7.8.9.0.a.b. ",
+" c.d.e.f.g.h. ",
+" i.j.k.l.m. ",
+" n.o.p. ",
+" "};
+end_of_pixmap
+
+$icon_magnify = <<'end_of_pixmap';
+/* XPM */
+static char * stock_zoom_in_20_xpm[] = {
+"20 20 108 2",
+" c None",
+". c #3E3E3E",
+"+ c #535353",
+"@ c #585858",
+"# c #565656",
+"$ c #525252",
+"% c #3F3F3F",
+"& c #484848",
+"* c #797979",
+"= c #A7A7A7",
+"- c #BDBDBD",
+"; c #C1C1C1",
+"> c #B5B5B5",
+", c #8C8C8C",
+"' c #2B2B2B",
+") c #4D4D4D",
+"! c #9A9A9A",
+"~ c #CBCBCB",
+"{ c #D9D9D9",
+"] c #E2E2E2",
+"^ c #E6E6E6",
+"/ c #E4E4E4",
+"( c #D5D5D5",
+"_ c #6E6E6E",
+": c #272727",
+"< c #D1D1D1",
+"[ c #F3F3F3",
+"} c #F8F8F8",
+"| c #F9F9F9",
+"1 c #F7F7F7",
+"2 c #F1F1F1",
+"3 c #C6C6C6",
+"4 c #626262",
+"5 c #1A1A1A",
+"6 c #757575",
+"7 c #CFCFCF",
+"8 c #FAFAFA",
+"9 c #FCFCFC",
+"0 c #A1A1A1",
+"a c #454545",
+"b c #DDDDDD",
+"c c #F0F0F0",
+"d c #E8E8E8",
+"e c #C3C3C3",
+"f c #343434",
+"g c #434343",
+"h c #AAAAAA",
+"i c #E0E0E0",
+"j c #FDFDFD",
+"k c #A2A2A2",
+"l c #F2F2F2",
+"m c #EAEAEA",
+"n c #DFDFDF",
+"o c #808080",
+"p c #080808",
+"q c #4C4C4C",
+"r c #C9C9C9",
+"s c #F4F4F4",
+"t c #9F9F9F",
+"u c #737373",
+"v c #909090",
+"w c #9D9D9D",
+"x c #B7B7B7",
+"y c #060606",
+"z c #D4D4D4",
+"A c #E9E9E9",
+"B c #444444",
+"C c #424242",
+"D c #7A7A7A",
+"E c #DBDBDB",
+"F c #C0C0C0",
+"G c #050505",
+"H c #EDEDED",
+"I c #D7D7D7",
+"J c #8E8E8E",
+"K c #D2D2D2",
+"L c #A3A3A3",
+"M c #030303",
+"N c #929292",
+"O c #979797",
+"P c #414141",
+"Q c #D6D6D6",
+"R c #C8C8C8",
+"S c #BCBCBC",
+"T c #D8D8D8",
+"U c #DEDEDE",
+"V c #CDCDCD",
+"W c #949494",
+"X c #171717",
+"Y c #181818",
+"Z c #6A6A6A",
+"` c #CECECE",
+" . c #D3D3D3",
+".. c #3B3B3B",
+"+. c #ACACAC",
+"@. c #C2C2C2",
+"#. c #898989",
+"$. c #393939",
+"%. c #000000",
+"&. c #0A0A0A",
+"*. c #262626",
+"=. c #656565",
+"-. c #A0A0A0",
+";. c #878787",
+">. c #474747",
+",. c #141414",
+"'. c #101010",
+"). c #313131",
+" ",
+" . + @ # $ % ",
+" & * = - ; > , # ' ",
+" ) ! ~ { ] ^ / ( > _ : ",
+" % ! < / [ } | 1 2 ^ 3 4 5 ",
+" ' 6 7 ^ 8 9 0 a b 1 c d e f ",
+" g h i [ 8 j k a b } l m n o p ",
+" q r d s t 0 u a v w x m n > y ",
+" ) z A l g B B B B C D ^ E F G ",
+" a 3 ^ H z I J g - K I i ( L M ",
+" ' N E / m H O P 7 ^ i Q R # ",
+" ) S T U ] h u V E z ~ W X ",
+" Y Z F ` .z .K ` 3 0 ..y ",
+" Y # +.; @.; ; x #.$.p %. ",
+" &.*.=.N -.;.>.,.y %.%.%.%. ",
+" %.%.%.%.%. 5 *.y %. ",
+" %.>.$.&.%. ",
+" '.B ).G ",
+" ,.X G ",
+" "};
+end_of_pixmap
+
+$icon_new = <<'end_of_pixmap';
+/* XPM */
+static char * new_20_xpm[] = {
+"20 20 160 2",
+" c None",
+". c #2D2D2D",
+"+ c #4E4E4E",
+"@ c #545454",
+"# c #555555",
+"$ c #505050",
+"% c #3D3D3D",
+"& c #727272",
+"* c #F3F3F3",
+"= c #FEFEFE",
+"- c #FFFFFF",
+"; c #EEEEEE",
+"> c #B2B2B2",
+", c #838383",
+"' c #7E7E7E",
+") c #EAEAEA",
+"! c #CFCFCF",
+"~ c #E3E3E3",
+"{ c #7D7D7D",
+"] c #808080",
+"^ c #FDFDFC",
+"/ c #FDFDFD",
+"( c #FCFCFB",
+"_ c #CDCDCC",
+": c #F6F6F6",
+"< c #DADADA",
+"[ c #666666",
+"} c #FEFEFD",
+"| c #FCFCFC",
+"1 c #FBFBFB",
+"2 c #FAFAF9",
+"3 c #FBFBFA",
+"4 c #FAFAF8",
+"5 c #DCDCDB",
+"6 c #B6B6B5",
+"7 c #D0D0D0",
+"8 c #CDCDCD",
+"9 c #A6A6A6",
+"0 c #626262",
+"a c #F9F9F8",
+"b c #F8F8F7",
+"c c #F9F8F7",
+"d c #F8F7F6",
+"e c #E3E3E2",
+"f c #828282",
+"g c #484747",
+"h c #3F3E3E",
+"i c #323232",
+"j c #242423",
+"k c #141412",
+"l c #FAFAFA",
+"m c #F7F7F6",
+"n c #F6F6F4",
+"o c #F7F6F5",
+"p c #F6F5F4",
+"q c #F4F4F2",
+"r c #EAEAE8",
+"s c #DAD9D7",
+"t c #CACAC8",
+"u c #B0AFAD",
+"v c #878582",
+"w c #3B3A35",
+"x c #F5F4F3",
+"y c #F4F3F2",
+"z c #F2F2F0",
+"A c #EDEDEB",
+"B c #E8E7E5",
+"C c #E4E3E1",
+"D c #CFCECC",
+"E c #ADABA7",
+"F c #464440",
+"G c #F3F3F1",
+"H c #F1F1EF",
+"I c #F0EFED",
+"J c #EDECEA",
+"K c #EBEAE8",
+"L c #E6E5E3",
+"M c #DFDEDB",
+"N c #BAB7B2",
+"O c #47443F",
+"P c #F3F2F1",
+"Q c #F0F0EE",
+"R c #F1F0EE",
+"S c #EEEDEB",
+"T c #EAE9E7",
+"U c #E6E5E2",
+"V c #E4E3E0",
+"W c #E2E1DE",
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+" "};
+end_of_pixmap
+
+$icon_separator = <<'end_of_pixmap';
+/* XPM */
+static char * sep_20_xpm[] = {
+"4 20 3 1",
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+" .+ ",
+" .+ ",
+" .+ ",
+" ",
+" ",
+" ",
+" "};
+end_of_pixmap
+
+$icon_ensembl = <<'end_of_pixmap';
+/* XPM */
+static char * ensembl_20_xpm[] = {
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+end_of_pixmap
+
+$icon_dna_save = <<'end_of_pixmap';
+/* XPM */
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+end_of_pixmap
+
+$icon_dna_open = <<'end_of_pixmap';
+/* XPM */
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+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + @ # # # $ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . # & . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * = - ; > , . . . . . . . . . . ' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ) ! ~ { . ] ^ * . . . . . . . . / ( . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ : . . . . < [ . . . . . . . . % + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . } | . . . + % . . . . & 1 ! - 2 3 . . + 4 ; + 5 . 6 7 8 . 9 0 a b # c d . . + 4 ; < e # f + % . . . . + f # g h . . + 4 ; i + % . + j k l m $ c n . o p @ - q r . + 4 ; i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ; , . . . . s t r . + % . . + % @ + b o p @ - q r . + 4 ; i . . . + 4 ; u b # c d . . + v k w x . s y # z + % o p @ - q r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A B C | . . + @ # # # D 4 E F F G H . . + I . J q . K L M N ; D O P . Q + R . + I . S T . U + % . . . . + V . W e . . + I . { + % . + : . X + Y s + R H Z . 8 ` .. + I . { . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..+.. . . . @.+ 3 . + % . . + % % + . H Z . 8 ` .. + I . { . . . + I . 9 P . Q + R . + #.. $...%.&.*.. U + % H Z . 8 ` .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . =.-.;.>.. . . + % . . . . ,.'.. . '.).. . + !.. , ~.. {.].^./.(.. r _.u :.+ <.. + !.. [.s . }.+ % . . . . + ) . R + 8 . + !.. . + % . + s . % + F . + % [.@ # # |.H . + !.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ; 1.. . . . 2.7 r . + % . . + % % + . [.@ # # |.H . + !.. . . . . + !.. r _.u :.+ <.. + 3.. N + <.[.s . / + % [.@ # # |.H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . } 4.;.-.=.. . . . + % . . . . ,.'.. . 5.6.. . + % . . ( 7.8.* g V < . ` :.] 9.+ % . + % . [.0.. 8 + % . . . . + s . / + a.. + % . . + % . + % . % + . . + % ).9.. . b.8 . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c.d.. . . . .` . . + e.. %.+ % % + . ).9.. . b.8 . + % . . . . . + % . ` :.] 9.+ % . + % . . + % ).s . R + % ).9.. . b.8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A f.g.f.A . . . . . + % . . . . p E F F G 4 . . + % . . h.i.j.. # @ s . t k.. l.+ 3.. + % . m.n.. o.+ % . . . . + u . 0 p.. . + % . . + % . + % . % + . . + % q.f { r.s.U . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . } t.. !.- u.N . v.^ a . . w.x.. y.+ % & + @.q.f { r.s.U . + % . . . . . + % . t k.. l.+ 3.. + % . . + % = u . z.+ <.q.f { r.s.U . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . =.-.;.4.} . . . . . . + % . . . . <.H ! # 2 o . . + % . . A.+ h . 9 B.. . ].c C.d ~.D.. + % . 2.E.# O + % . . . . + _ F.e G.. . + % . . + % . + % . % + . . + % % m.! L n { . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H.f.I.t.. 5.= ! - > , . . . J.; k u + % N s.C.% m.! L n { . + % . . . . . + % . ].c C.d ~.D.. + % . . + % s e @ :.+ r.% m.! L n { . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.;.-.=.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A K.C 4.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L.M.. 5 @ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t.g.f.A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N.-.C O.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l.P.F.Q.R.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.I.. . . . t.} . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N.S.C =.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.I.I.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . =.C C N.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.T.T.T.I.I.I.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.U.C V.W.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.X.X.I.I.I.I.I.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.Y.Z.I.`. +T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.X.X.I.I.I.I.I.X.X.X.X.X.X.X..+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+X.X.X.X.X.X.X.X.Y.++I. at +#+X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . + + + + + . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X..+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+.+X.X.X.X.X.X.X.X.%+&+++*+X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . + . . . . . ",
+". . . . . . + . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X..+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+.+X.X.X.X.X.X.X.X.X.%+Y.X.X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + . . + . . . . . ",
+". . . . . . + . . . . . . + . . . . . . + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + . . . . . . . . . + . . + . . . . . ",
+". . . . . . + + + + . . . . . . . . . . + T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=++ T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + . . . . . . . + + . . . . . . . . . ",
+". . . . . . . . . . + . . . . . . . . . + T T T T T T T T T T T T T T T T T T T T T T T T T T T T T -+T T T T T T T T T T T T T T T T T T T T T + =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+;+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=++ T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + . . . . . . . . . + . . . . . . . . ",
+". . . . . . . . . . + . . . . . . . . . + T T T T T T T T T T T T T T T T T T T T T T T T T T T T >+,+-+T T T T T T T T T T T T T T T T T T T T + =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+'+)+;+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=++ T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + . . . . . . . . . + . . . . . . . . ",
+". . . . . . + . . . + . . . . . . . . . + T T T T T T T T T T T T T T T T T T T T T T T T T T T !+~+{+]+T T T T T T T T T T T T T T T T T T T T + =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+'+)+'+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=++ T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + . . . . . + . . . + . . . . . . . . ",
+". . . . . . + . . . + . . . . . . . . . + T T T T T T T T T T T T T T T T T T T T T T T T T T ^+/+/+^+T T T T T T T T T T T T T T T T T T T T T + =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+'+)+'+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=++ T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T + . . . . . + . . . + . . . . . . . . ",
+". . . . . . . + + + . . . . . . . . . . + + + + + + + + + + + + + + + + + + + + + + + + + + (+_+:+<++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + [+}+[++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + (++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + . . . . . . + + + . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |+1+4.. . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X..+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+2+3+2+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+4+5+6+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+.+X.X.X.X.X.X.X.X.X.X.X.X.X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N.S.7+8+. . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X..+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+2+3+2+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+9+0+a+b+c+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+$+I.I.I.I.I.X.X.X.X.X.X.X.X.X.X.X.X.X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+7+S.N.. . . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X..+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+d+e+d+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+f+g+h+g+i+j+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+.+I.I.I.I.I.I.X.X.X.X.X.X.X.X.X.X.X.X.X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1+|+. . . . . . . . . . . . . . . . . . . . . . T.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.k+l+k+X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.&+m+n+o+n+m+&+X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.I.I.I.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.X.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . =.;.f.} . . . . . . . . . . . . . . . . . . . . . . T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.p+q+p+T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.r+s+t+u+t+s+r+T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.I.I.I.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v+w+-.-.A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . } | g.| . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+x+7+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.I.I.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v+A y+z+=.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . } B g.| . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+x+7+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A+B+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A+C+D+E+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.F+>.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+x+7+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A+C+G+H+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A+B+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+x+7+|+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v+8+I+J+x+7+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+1+t.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v+A+8+|+7+x+7+|+8+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + @ # # ~.K+. . . + n.. . . % + . . . { - [ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * = - ; > , . . . + @ # F.L y a . . + @ # # # $ . . . . . 1 L+. . . . 5.M+@ # k u { . . . . . . . . . . 5.M+@ # k u { . . . . . # & . . . . . . . . + % . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+t.. . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+x+7+|+. . . + @ # F.L y a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . # & . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . 5.N+v.. . + O+P+. . % + . . . Y Q+w 3.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ) ! ~ { . ] ^ * . . + % . . N *.R+. . + % . . . . . . . . { S+N . . . s ; T+F . s U+*.. . . . . . . . . s ; T+F . s U+*.. . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+|+7+1+t.. . + % . . N *.R+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . ] U+. . + V+g ' . % + . . . 2 U G : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ : . . . . < [ . . + % . . . s O+. . + % . . . . . . . . M.2 . . . . O V . . . . 1.0 . . . . . . . . . O V . . . . 1.0 . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8+t.. . . + % . . . s O+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . & ..r . + W+~.v . % + . . }.X+e.Y+N+N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ; , . . . . s t r . + % . . { Z+B.. . + % . . . . . . . . d.`+. . . . ! @. . . . . . . . + f # g h . . ! @. . . . . . . . . . . + % . . at +@@ @@k.. . + % a b # c d . . + v k w x . 9.e # f + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + v k w x . . + v k w x . o p @ - q r . + 4 ; i . . . + 4 ; u b # c d . . + v k w x . s y # z + % o p @ - q r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . { Z+B.. o p @ - q r #@#.. . l.H o p @ - q r . + 4 ; i . at +@@ @@k.. o p @ - q r . . . . + f # g h . . + 4 ; i + % . + j k l m $ c n . o p @ - q r . + 4 ; i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . r + b.. + % $@t $@% + . . P [ . . + at D.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..+.. . . . @.+ 3 . + @ # # w ` %@. . + @ # # # D . . . . E.&@. . . . *@3.. . =@# # # . . + V . W e . . *@3.. . =@# # # . . . . . + % . 6.- at . `+: . . + % O P . Q + R . + #.. $...%.` T . U + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + #.. $...%.. + #.. $...%.H Z . 8 ` .. + I . { . . . + I . 9 P . Q + R . + #.. $...%.&.*.. U + % H Z . 8 ` .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + @ # # w ` %@. H Z . 8 ` .X ;@. . B.5 H Z . 8 ` .. + I . { 6.- at . `+: . H Z . 8 ` .. . . . + V . W e . . + I . { + % . + : . X + Y s + R H Z . 8 ` .. + I . { . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . &@O+r . + % . v ~.W++ . . >@X+# # ,.#@. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ; 1.. . . . 2.7 r . + % N $.! m . . . + % . . . . . . . r., at . . . . . c L+. . . . % + . . + ) . R + 8 . c L+. . . . % + . . . . . + % . :.# G '@%.. . + % r _.u :.+ <.. + 3.. N + <.[.s . }.+ % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + 3.. N + <.. + 3.. N + <.[.@ # # |.H . + !.. . . . . + !.. r _.u :.+ <.. + 3.. N + <.[.s . / + % [.@ # # |.H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % N $.! m . . [.@ # # |.H N ( $@1.P.N [.@ # # |.H . + !.. . :.# G '@%.. [.@ # # |.H . . . . + ) . R + 8 . + !.. . + % . + s . % + F . + % [.@ # # |.H . + !.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . )@E.. . + % . ' g !@+ . !.X+a.. . s ~@& . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c.d.. . . . .` . . + % . . {@]@^@. . + % . . . . . . . /@(@. . . . . _ =@. . . . % + . . + s . / + a.. _ =@. . . . % + . . . . . + % . . _ at l.:@y . . + % ` :.] 9.+ % . + % . . + % [.0.. 8 + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + % . . + % . + % . . + % ).9.. . b.8 . + % . . . . . + % . ` :.] 9.+ % . + % . . + % ).s . R + % ).9.. . b.8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . {@]@^@. ).9.. . b.8 . u <@^.d.. ).9.. . b.8 . + % . . . _ at l.:@y . ).9.. . b.8 . . . . + s . / + a.. + % . . + % . + % . % + . . + % ).9.. . b.8 . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . _ at N+_.. . + % . . L+]@+ . u H . . . . y m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !.- u.N . v.^ a . . + % . . . f @@{ . + % . . . . . . . f X . . . . . s ! l.a.. ) p [@. . + u . 0 p.. . s ! l.a.. ) p [@. . . . . + % / |. @. A.w.. . + % t k.. l.+ 3.. + % . . + % m.n.. o.+ % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + % . . + % . + % . . + % q.f { r.s.U . + % . . . . . + % . t k.. l.+ 3.. + % . . + % = u . z.+ <.q.f { r.s.U . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . f @@{ q.f { r.s.U . D # # 2.. q.f { r.s.U . + % . / |. @. A.w.. q.f { r.s.U . . . . + u . 0 p.. . + % . . + % . + % . % + . . + % q.f { r.s.U . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + @ # @ }@u.. . . + % . . . n.+ . ~.6 . . . . |@g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.= ! - > , . . . + % . . . 3 w z.. + % . . . . . . . g ' . . . . . . s !@# - P.<@s . . + _ F.e G.. . . s !@# - P.<@s . . . . . + % . `+F.@ ~.1 at . . + % ].c C.d ~.D.. + % . . + % 2.E.# O + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . + % . . + % . + % . . + % % m.! L n { . + % . . . . . + % . ].c C.d ~.D.. + % . . + % s e @ :.+ r.% m.! L n { . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . 3 w z.% m.! L n { . . q.[ . . % m.! L n { . + % . . `+F.@ ~.1 at . % m.! L n { . . . . + _ F.e G.. . + % . . + % . + % . % + . . + % % m.! L n { . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L.M.. 5 @ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l.P.F.Q.R.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + % . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ",
+". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . "};
+
+end_of_pixmap
+}
Property changes on: trunk/packages/perlprimer/branches/upstream/current/perlprimer.pl
___________________________________________________________________
Name: svn:executable
+
Added: trunk/packages/perlprimer/branches/upstream/current/todo
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/todo 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/todo 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,14 @@
+For v1.2
+
+* Blast search dialogue should include a list of sequences that match both the forward and reverse primers
+
+* More intelligent sequencing primer design - search for primers around the midpoint between minimum and maximum distances, and then gradually move out
+
+* More complex primer-dimer routines - including detection of hair-pins and unequal primer-dimer internal loops
+
+* Ability to designate regions with starting/ending coordinates or starting/ending sequence strings, and assign colours and PCR ranges to such regions
+
+
+Post v1.2
+
+* Methylation specific PCR?
Added: trunk/packages/perlprimer/branches/upstream/current/tutorial.html
===================================================================
--- trunk/packages/perlprimer/branches/upstream/current/tutorial.html 2006-08-07 01:25:59 UTC (rev 86)
+++ trunk/packages/perlprimer/branches/upstream/current/tutorial.html 2006-08-07 13:20:50 UTC (rev 87)
@@ -0,0 +1,424 @@
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+
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+
+<title>PerlPrimer</title><link rel="shortcut icon" href="http://perlprimer.sourceforge.net/favicon.ico" type="image/x-icon"></head>
+
+
+<body><div class="header"><br></div>
+<div class="shadow1"></div>
+
+<div class="shadow2"></div>
+<div class="top"><br>
+<table border="0" cellpadding="0" cellspacing="0"><tbody><tr>
+<td width="100%">
+<table border="0" cellpadding="0" cellspacing="0"><tbody><tr>
+<td>
+<h2><b>PerlPrimer - open-source PCR primer design</b></h2>
+<a href="http://perlprimer.sourceforge.net/index.html">About</a> | <a href="http://perlprimer.sourceforge.net/news.html">News</a> | <a href="http://perlprimer.sourceforge.net/download.html">Download</a> | Tutorial | <a href="http://perlprimer.sourceforge.net/methodology.html">Methodology</a> | <a href="http://perlprimer.sourceforge.net/links.html">Links</a> | <a href="http://perlprimer.sourceforge.net/screenshots.html">Screenshots</a> | <a href="http://perlprimer.sourceforge.net/contact.html">Contact</a> </td></tr></tbody></table>
+</td><td halign="right" valign="middle">
+<a href="http://sourceforge.net/"> <img src="tutorial_files/sflogo.png" alt="SourceForge.net Logo" align="middle" border="0" height="62" width="210"></a>
+</td></tr></tbody></table>
+</div>
+<div class="content">
+
+<h3>Contents</h3>
+<a href="#introduction">Introduction (and sample files)</a>
+<p><a href="#standard">Standard PCR</a>
+<br><span class="toc"><a href="#standard_restriction">Adding restriction enzyme cloning sequences</a></span>
+<br><span class="toc"><a href="#standard_saving">Saving a printable summary of the selected primer pair</a></span>
+<br><span class="toc"><a href="#standard_exporting">Exporting selected primers to a spreadsheet</a></span>
+</p><p><a href="#realtime">Real-time PCR</a>
+</p><p><a href="#sequencing">Sequencing Primers</a>
+</p><p><a href="#advanced">Advanced Features</a>
+<br><span class="toc"><a href="#advanced_finding">Finding primers</a></span>
+<br><span class="toc"><a href="#advanced_amplifying">Amplifying user-defined regions</a></span>
+<br><span class="toc"><a href="#advanced_specifying">Specifying PCR component concentrations</a></span>
+<br><span class="toc"><a href="#advanced_blast">Advanced BLAST searching</a></span>
+<br><span class="toc"><a href="#advanced_blast_local">Using a local BLAST server</a></span>
+<br><span class="toc"><a href="#advanced_ipc">Using other programs to communicate with PerlPrimer</a></span>
+<a name="introduction"><h3>Introduction</h3></a>
+</p><p>This tutorial covers most of the commonly used features of
+PerlPrimer, for standard PCR, real-time PCR and sequencing, using the
+human gene <i>PGR</i> (encoding the Progesterone Receptor protein) as
+an example. Example files for each section may be downloaded below
+(right-click on the links and choose either "Save Target As ..." or
+"Save Link to Disk ...", depending on your browser).
+</p><p><a href="http://perlprimer.sourceforge.net/hs_pgr_cDNA.txt">Human <i>PGR</i> cDNA sequence</a>
+<br><a href="http://perlprimer.sourceforge.net/hs_pgr_genomic.txt">Human <i>PGR</i> genomic sequence</a>
+</p><p><a href="http://perlprimer.sourceforge.net/hs_pgr_cloning.ppr">standard PCR perlprimer file</a>
+<br><a href="http://perlprimer.sourceforge.net/hs_pgr_qpcr.ppr">real-time PCR perlprimer file</a>
+<br><a href="http://perlprimer.sourceforge.net/hs_pgr_sequencing.ppr">sequencing perlprimer file</a>
+</p><p>The tutorial is based upon PerlPrimer 1.1 - users of older
+versions may find some features unavailable and are advised to upgrade
+to the latest version. All screenshots were made using the program
+running under Linux, however, the program should behave identically
+under Windows and MacOS X.
+</p><p>In addition to this tutorial, PerlPrimer also comes with
+extensive "Balloon Help", enabled by default (and which can be turned
+on or off in the Help menu). Simply hold the mouse pointer over a
+button or feature and a explanatory message should be displayed.
+</p><p><a name="standard"><h3>Standard PCR</h3></a>
+</p><p>While a DNA sequence may be opened from the sequence input area (click on the <img src="tutorial_files/open_dna_20.png">
+icon), the simplest means of retrieving a sequence is to use the
+Ensembl database tool. Click on the "Retrieve gene from Ensembl" icon <img src="tutorial_files/ensembl_20.png">
+or select it from the File menu. Enter "pgr" as the gene name and
+select "Homo_sapiens" as the organism. Make sure that "cdna" is set as
+the retrieval type, and click OK.
+</p><p>You should see a screen similar to that shown below (note that
+on earlier versions the amplification range will not be set
+automatically - click the "Set from ORF" button to do so) The range
+will now be set by default to the largest Open Reading Frame (or ORF)
+present in the sequence. The range is displayed by two boxes around the
+DNA sequence - one orange (the outer range) and one blue (the inner
+range). All primers will be found within the orange box and outside of
+the blue box.
+</p><p><img src="tutorial_files/img1.png">
+</p><p>(The following example considers cloning a gene by PCR
+amplification into an expression plasmid. Note, however, that primers
+may be designed around other regions of interest as well as genes or
+that amplification boundaries may be entered manually (see the section
+"Advanced uses" below))
+</p><p>To clone the cDNA ORF into an expression plasmid, we will
+generally want a forward primer before or encompassing the initiating
+ATG, and a reverse primer before the terminal stop codon. The simplest
+way to do this to use the "Find primers for cloning" command from the
+Tools menu - this will automatically decrease the 5' outer range and 3'
+inner range until primers pairs are found. (There is another way to do
+this - see the "Advanced Features" section below). Using the standard
+default parameters, this should find four primer pairs, the first one
+amplifying from bases 1443 -> 4226. (see screen shot below)
+</p><p><img src="tutorial_files/img2.png">
+</p><p>Double-clicking on this primer pair (or pressing the right arrow
+key on the keyboard) will switch to the Primers tab, showing primer
+information and stable primer-dimers. The most stable extensible
+primer-dimer is fairly weak (dG°37 of -2.00 kcal/mol), and although the
+non-extensible Forward vs. Forward dimer may slightly reduce the primer
+population, it should not cause problems for the purpose of cloning the
+gene. (screen shot)
+</p><p><img src="tutorial_files/img3.png">
+</p><p>There is also a button on this page to BLAST primers using the
+NCBI BLAST server. Click on this and wait for the BLAST results to come
+up (this may take a minute or two - the server can be quite slow at
+times) Limit the results to human only by typing "Homo" into the search
+box and clicking the Search button. (screen shot) The forward primer is
+fairly unique, and although the reverse primer is less so it should be
+OK to amplify from. Return to the Standard PCR page and select this
+primer pair.
+</p><p><img src="tutorial_files/img4.png">
+</p><p><a name="standard_restriction"><h4>Adding restriction enzyme cloning sequences</h4></a>
+</p><p>The example provided here will clone the <i>PGR</i> ORF into
+GFP-fusion constructs (Clontech plasmids EGFP-C1 and EGFP-C2), creating
+both an N-terminal and a C-terminal fusion protein when translated.
+Select "Add cloning sequences" from the Tools menu. This will bring up
+a dialogue box with the ability to select restriction enzyme sites to
+add to the 5' end of each primer. By default, only those 6-base cutting
+enzymes that will not cut the DNA sequence within the outer range are
+listed. Looking at the restriction sites present in the plasmid MCS and
+comparing to the list of non-cutting enzymes, we can see that BglII and
+EcoRI can be used. Select these (see below) and click OK. Finally, we
+need to add the frame that the sites lie in, relevant to the EGFP gene
+(this is obviously only necessary when creating fusion proteins).
+Looking at the plasmid MCS, it should be clear that both BglII for
+EGFP-C1 and EcoRI for EGFP-N2 are in frame 0 (where the frame can be
+one of 0, 1 or 2 and 0 represents a sequence in frame). Enter "0" into
+each frame box.
+</p><p>EGFP-C1 MCS:
+</p><p><img src="tutorial_files/egfp_c1_mcs.png">
+</p><p>EGFP-N2 MCS:
+</p><p><img src="tutorial_files/egfp_n2_mcs.png">
+</p><p><img src="tutorial_files/img5.png">
+</p><p>Clicking on the <img src="tutorial_files/magnify.png"> icon next
+to the DNA graphic (also by right-clicking on the DNA graphic) will
+bring up an alignment of the DNA sequence and primers with cloning
+sequences attached. The ORF is translated and codons are highlighted
+(see below). Note the inserted Adenine base between the forward primer
+and the cloning site.
+</p><p><img src="tutorial_files/img6.png">
+</p><p><a name="standard_saving"><h4>Saving a printable summary of the selected primer pair</h4></a>
+</p><p>Clicking on the <img src="tutorial_files/report_20.png"> icon
+(or selecting "Generate Report" from the Tools menu) will save a text
+file listing the selected primer pair, statistics and primer-dimers of
+each primer and an alignment of the primers against the DNA sequence.
+</p><p>
+<a name="standard_exporting"><h4>Exporting selected primers to a spreadsheet</h4></a>
+</p><p>If satisfied with a primer pair or a group of primer pairs,
+right-click on the primer list and choose "Copy", or simply press
+Ctrl-C. The primers are copied in a tab-delimited format that can be
+pasted into any spreadsheet application.
+</p><p><a name="realtime"><h3>Real-time PCR</h3></a>
+</p><p>Designing real-time PCR primers with PerlPrimer is a simple and straightforward process. This tutorial uses the Human <i>PGR</i> gene as an example.
+</p><p>Click on the "Retrieve gene from Ensembl" icon (picture) or
+select it from the File menu, enter "pgr" as the gene name and select
+"Homo_sapiens" as the organism. Click OK. PerlPrimer will automatically
+download the genomic and cDNA sequences for the gene. You should have a
+screen similar to that below. </p><p><img src="tutorial_files/img7.png">
+</p><p>Click on the "Find Primers" button. PerlPrimer runs the
+application Spidey, which finds intron/exon boundaries (displayed as
+white lines across the DNA graphic). It then selects primer pairs that
+will produce an amplicon within the bounds specified, which span at
+least one intron/exon boundary and in which at least one primer pair
+lies across an intron/exon boundary. (Thus the primers should only
+amplify cDNA and will not amplify genomic DNA) Primers are sorted by
+extensible primer-dimer stability. In this case, no primers are found
+on the first run - probably because the regions around the intron/exon
+boundaries have an abnormally high or low GC content. To test this, try
+changing the cutoffs for GC% from 40%-60% to 35%-65% (you can do this
+in the Preferences, under the "Exclusions" tab).
+</p><p>Searching for primers again now yields over 400 primer pairs (see below).
+</p><p><img src="tutorial_files/img8.png">
+</p><p>Of these, the first primer pair should be fine - BLAST searching
+as detailed above suggests that both primers are specific. Clicking on
+the <img src="tutorial_files/magnify.png"> icon now displays the intron/exon boundaries with the primers aligned (see below).
+</p><p><img src="tutorial_files/img9.png">
+</p><p><a name="sequencing"><h3>Sequencing Primers</h3></a>
+</p><p>Finding sequencing primers for any sequence is also extremely simple. Using the example above of the human <i>PGR</i>
+cDNA sequence, open the sequence (or retrieve it from Ensembl), check
+that the default "Primers every ... bases" is appropriate for your
+average sequence read-length, and click the "Find Primers" button.
+Suitable primers will be listed, or if a gap is found where no primers
+match the required parameters, a dialogue will displayed informing the
+user that the search parameters will need to be relaxed. By default,
+primers are only listed if all primer-dimers have a dG°37 less than -5
+kcal/mol - this can be varied if required. In this case, we need to
+increase the maximum read length to 800bp to find primers (screenshot).
+</p><p><img src="tutorial_files/img10.png">
+</p><p><a name="advanced"><h3>Advanced Features</h3></a>
+</p><p><a name="advanced_finding"><h4>Finding primers</h4></a>
+</p><p>Two other buttons in the "Standard PCR" and "Bisulphite PCR"
+pages that have not been mentioned above are the "Find Inwards" and
+"Find Outwards" buttons - useful for amplifying regions of interest.
+The "Find Inwards" button reduces the inner amplified range by 10 base
+increments until suitable primers are found; the "Find Outwards" does
+the opposite - increasing the outer amplified range by 10 base
+increments until primers are found.
+</p><p>The inner and outer ranges can also be adjusted by using the
+mouse - dragging with the left mouse button affects the inner range;
+dragging with the middle mouse button (or the left button with the Ctrl
+key held down) affects the outer range.
+</p><p><a name="advanced_amplifying"><h4>Amplifying user-defined regions</h4></a>
+</p><p>While PerlPrimer will by default automatically find the largest
+ORF (or CpG islands) in a sequence, it is also possible to use a
+user-defined region, or number of regions. This can be achieved by
+capitalising the region of interest (most word processing/text editor
+programs should be able to do this) before opening the sequence in
+PerlPrimer, and selecting "Defer to capitalised regions" in the
+Preferences under the "General" tab. (click on the <img src="tutorial_files/preferences_20.png"> icon or select "Preferences" from the Tools menu). The internal ORF and CpG island detection algorithms are now bypassed.
+</p><p>It is also possible to manually specify the base positions to
+amplify from by typing in the numbers into the Amplified Range section.
+</p><p><a name="advanced_specifying"><h4>Specifying PCR component concentrations</h4></a>
+</p><p>PCR component concentrations are set by default to the standard PCR conditions of 1.5mM Mg<sup>++</sup>, 50mM Monovalent cations (i.e. Na<sup>+</sup>, K<sup>+</sup>, etc), 0.2 mM dNTPs and a final concentration of each primer as 200mM.
+</p><p>These concentrations can be changed in the Preferences under the "General" tab (click on the <img src="tutorial_files/preferences_20.png">
+icon or select "Preferences" from the Tools menu). Please note that
+changing any of these values (including the primer concentration) will
+affect the <i>T<sub>m</sub></i>.
+</p><p><a name="advanced_blast"><h4>Advanced BLAST searching</h4></a>
+</p><p>Most parameters of BLAST searches (including the Expect value,
+the database and the ability to limit searches to an organism) can be
+set in the Preferences, under the "Bioinformatics" tab.
+</p><p><a name="advanced_blast_local"><h4>Using a local BLAST server</h4></a>
+</p><p>As of version 1.1.5, users may install and use a local BLAST server and data files. <a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST-BLAST/">Binaries</a> and <a href="ftp://ftp.ncbi.nih.gov/blast/db/">databases</a>
+are available from the NCBI ftp site - please see the README file for
+stand-alone BLAST included with the BLAST distribution for details on
+setting up the server and database files.
+</p><p>Once a local BLAST server has been setup, select "Use local
+BLAST server" from the "BLAST" tab in the Preferences dialogue, select
+the directory where BLAST server executables are located and select the
+relevant database file.
+</p><p><a name="advanced_ipc"><h4>Using other programs to communicate with PerlPrimer</h4></a>
+</p><p>As of version 1.1.3, PerlPrimer now establishes a listening
+socket on local port 2500 which other applications can use to send data
+directly to PerlPrimer (see <a href="http://www.atgc.org/Py_ContigViewer/">Contig Viewer</a>
+for an example of an application that uses this feature). The data
+format uses a modified FASTA format with optional PCR amplification
+boundaries set in the FASTA header; the syntax for the modified
+description line is
+</p><p><tt>>Name of DNA sequence 5prime_region[?-?] 3prime_region[?-?] page[?]</tt>
+</p><p>where the optional page argument represents the PCR tab (thus 1
+= Standard PCR, 2 = Bisulphite PCR, 3 = Real-time PCR, etc). An example
+is:
+</p><p><tt>>Contig2942 test file 5prime_region[53-185] 3prime_region[324-464]
+GGTTCCAATGGAGTTGTGATAAACGAAGAGCAGCACAAGCTCCCAATACCAAATTTAGTACTACTGACCAATTATAAAGAGTAAATATAGAAGATT
+AGGGTTTTAAGATCTCTAACAAAATTGCACTGGGAAGAATCTGGTTCTTCAATTTTCTGGGATTTATCAGATCTGAAGAAACTGAAGAGTGAAGTG
+GTAATCGTTGATAAGTGTATGTTCAAGGGAGGATTGTTTGGATTTACTGAACTGTTGTTTGATGTGGTTTTGGAGAAGATAAGATCTGTGATTTGT
+AAAACCCAAAACGGATTCTTTTTGTACTTGATTGATAGATCTTCAGTTTTTTTGGGAGTTGAATCAGTTATTGTTTAATTTAGAAATGGAGACACT
+TGTTGTTGTTTCTCAGCATAAGAATCACTACTATGATAGAACTAGGGGTCAAGCTCCTATTCGATTTGGATCCTTTGGGTCACCTCCTTCTGTAGG
+GTTCAAAGAGATAAATTGCCGGAATTTTGAATCAAGTGCCGGTATACTTCCTACCCCGTTAAAGGCCTATTCCACACCAGTCACTAAAAAGGGTTA
+TTCATCCTCGTTTCGTTCCAAAACACCTTCACCCCCATCATCTATCCAGGGTAAATCTCACTCTGAAAGTCAAAAGAAGTCG</tt>
+</p><p>To change the PCR amplification boundaries without resending the
+DNA sequence, simply send the FASTA description line only. The modified
+FASTA above can also be opened manually using the <img src="tutorial_files/open_dna_20.png"> button in the sequence boxes.
+</p></div>
+<p></p><div class="footer">© 2003-2005 Owen Marshall ... Last updated Fri Feb 4 16:46:40 2005</div>
+</body></html>
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