[med-svn] [perlprimer] 06/12: New upstream version 1.2.3
Charles Plessy
plessy at moszumanska.debian.org
Sun Jan 22 14:25:56 UTC 2017
This is an automated email from the git hooks/post-receive script.
plessy pushed a commit to branch master
in repository perlprimer.
commit 00b28bafc03f1141f6abfb03883b47b301f09b48
Author: Charles Plessy <plessy at debian.org>
Date: Sun Jan 22 17:32:44 2017 +0900
New upstream version 1.2.3
---
Changelog | 12 ++
LICENSE | 339 ++++++++++++++++++++++++++++++++++++++++++++
README.md | 23 +++
ReadMe | 85 -----------
ReadMe.txt | 91 ------------
perlprimer.pl => perlprimer | 302 +++++++++++++++++++++++----------------
todo | 11 --
7 files changed, 554 insertions(+), 309 deletions(-)
diff --git a/Changelog b/Changelog
index 2f13c95..0434975 100644
--- a/Changelog
+++ b/Changelog
@@ -1,3 +1,15 @@
+22/1/17 (PerlPrimer-1.2.3)
+- Added the option of using Onodera and Melcher (2004) rules for the 3' GC clamp. This option is not currently enabled by default, but can be selected in the prefs "Exclusions" tab
+
+21/1/17 (PerlPrimer-1.2.2)
+- Added a "-1" button to the range options: use "Set from ORF" and then "-1" to only find primers that cover the entire ORF.
+
+20/1/17 (PerlPrimer-1.2.1)
+- Added a name column when copying primers (the name is built from the gene name, the coordinates of the primer and the direction).
+
+16/1/17 (PerlPrimer-1.2)
+- Re-wrote Ensembl routines to use the Ensembl REST API (a much more elegant solution -- thanks to Ensembl for developing this). Ensembl compatibility should never break again. (Some code tidying still needs to be done to remove some redundancies, but the code is fully functional. Please note that Ensembl REST sadly provides no fuzzy searching for genes: the correct gene name or Ensembl ID must be used.)
+
17/2/11 (PerlPrimer-1.1.20)
- Fixed Ensembl compatibility
- Fixed small error in Tm calculation code (thanks to Henning Lenz for pointing this out)
diff --git a/LICENSE b/LICENSE
new file mode 100644
index 0000000..23cb790
--- /dev/null
+++ b/LICENSE
@@ -0,0 +1,339 @@
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diff --git a/README.md b/README.md
new file mode 100644
index 0000000..8a30d30
--- /dev/null
+++ b/README.md
@@ -0,0 +1,23 @@
+# PerlPrimer
+
+PerlPrimer is a free, open-source GUI application written in Perl that designs primers for standard PCR, bisulphite PCR, real-time PCR (QPCR) and sequencing. It aims to automate and simplify the process of primer design.
+
+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 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 standard PCR conditions. The result is a highly accurate prediction of primer melting temperature, giving rise to a maximum yield of product when amplified.
+
+PerlPrimer is written in Perl and Perl/Tk. In addition, for QPCR functionality PerlPrimer uses the open-source Spidey executable from NCBI, and restriction enzyme data from the REBASE project is used when adding cloning sites. The program is designed to be cross-platform compatible and has been developed and tested on both Microsoft Windows and GNU/Linux-based operating systems. Users have also reported success using the program under Mac OS X.
+
+Please cite the reference below if this program is useful.
+
+ Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004 20(15):2471-2472 [Pubmed]
+
+
diff --git a/ReadMe b/ReadMe
deleted file mode 100644
index 8c17be7..0000000
--- a/ReadMe
+++ /dev/null
@@ -1,85 +0,0 @@
-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 the NCBI 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.
-
-
-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.
-
-
-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.
diff --git a/ReadMe.txt b/ReadMe.txt
deleted file mode 100644
index f37ecc9..0000000
--- a/ReadMe.txt
+++ /dev/null
@@ -1,91 +0,0 @@
-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.
diff --git a/perlprimer.pl b/perlprimer
old mode 100644
new mode 100755
similarity index 98%
rename from perlprimer.pl
rename to perlprimer
index 83f7872..1efbee3
--- a/perlprimer.pl
+++ b/perlprimer
@@ -3,8 +3,7 @@
# PerlPrimer
# Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
-# version 1.1.21 (21 Nov 2011)
-# Copyright � 2003-2011, Owen Marshall
+# Copyright � 2003-2017, 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
@@ -29,7 +28,7 @@ use strict;
my ($version, $commandline, $win_exe);
BEGIN {
- $version = "1.1.21";
+ $version = "1.2.3";
$win_exe = 0;
($commandline) = @ARGV;
@@ -39,7 +38,7 @@ BEGIN {
PerlPrimer v$version
Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
-Copyright � 2003-2011 Owen Marshall\n
+Copyright � 2003-2017 Owen Marshall\n
Usage: perlprimer.pl [file.ppr]\n
EOT
exit 0;
@@ -49,7 +48,7 @@ EOT
if ($win_exe) {
print <<EOT;
PerlPrimer v$version
-Copyright � 2003-2011 Owen Marshall
+Copyright � 2003-2017 Owen Marshall
Designs primers for PCR, Bisulphite PCR, QPCR (Realtime), and Sequencing
This window is required for PerlPrimer to run -
@@ -113,6 +112,9 @@ use IO::Socket;
use Win32::GUI();
use File::Copy;
use File::Glob ':glob';
+use HTTP::Tiny;
+use JSON;
+use File::Basename;
require Tk::NoteBook;
require Tk::HList;
require Tk::ItemStyle;
@@ -321,6 +323,7 @@ my $bs=0;
my $qpcr_flag=0;
my $cancel=0;
my $defer_to_caps=0;
+my $onodera_clamp=0;
# benchmarking - for code optimisation
my $benchmark=0;
@@ -456,7 +459,7 @@ Schizosaccharomyces_pombe");
my @ensembl_types = split("\n",
"genomic
cdna
-coding
+cds
utr5
utr3");
@@ -771,6 +774,7 @@ my %pref_variables = (
repeats_bs => \$repeat_bs,
runs_bs => \$run_bs,
exclude_rr_bs => \$exclude_rr_bs,
+ onodera_clamp => \$onodera_clamp,
max_gc => \$max_gc,
min_gc => \$min_gc,
mg_conc => \$mg_conc,
@@ -920,6 +924,7 @@ read_prefs();
my %balloonmsg = (
'primer_getgene', "Find the longest ORF within the sequence and set the selected range",
'primer_reset', "Reset the range to the ORF boundaries",
+ 'primer_stepin_1', "Reduce the inner range by 1bp on each side",
'primer_stepin', "Reduce the inner range by 10bp on each side",
'primer_stepout', "Increase the outer range by 10bp on each side",
'exclude_gc', "Exclude primers with a GC content outside the range specified in the preferences\n(default is 40-60% GC)",
@@ -1466,7 +1471,8 @@ pack_gui('LabFrame', 'Amplified range', 'primer_range_l', \$page_primer_designf)
pack_gui('Label', 'bases');
nr('', 0);
pack_gui('Button', 'Set from ORF', 'primer_getgene', \&reset_bounds);
- pack_gui('Button', '-10', 'primer_stepin', \&step_in);
+ pack_gui('Button', '-1', 'primer_stepin_1', sub{step_in(1)});
+ pack_gui('Button', '-10', 'primer_stepin', sub{step_in(10)});
pack_gui('Button', '+10', 'primer_stepout', \&step_out);
pack_gui('LabFrame', 'Options', 'primer_options_l', \$page_primer_designf);
@@ -1632,7 +1638,8 @@ pack_gui('LabFrame', 'Amplified range', 'bisul_range_l', \$page_bisul_seqf);
pack_gui('Label', 'bases');
nr('', 0);
pack_gui('Button', 'Set from CpG island', 'bisul_getcpg', \&reset_bounds);
- pack_gui('Button', '-10', 'primer_stepin', \&step_in);
+ pack_gui('Button', '-1', 'primer_stepin_1', sub{step_in(1)});
+ pack_gui('Button', '-10', 'primer_stepin', sub{step_in(10)});
pack_gui('Button', '+10', 'primer_stepout', \&step_out);
pack_gui('LabFrame', 'Options', 'bisul_options_l', \$page_bisul_seqf);
@@ -2421,9 +2428,14 @@ sub primer_window {
}
if ($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 $gc_clamp > 50;
+ if ($onodera_clamp) {
+ # Onodera and Melcher exclusion
+ next unless onodera(substr($currwindow, -3, 3));
+ } else {
+ # 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) {
@@ -2897,6 +2909,11 @@ sub cc {
return $cc;
}
+# Onodera and Melcher exclusion
+sub onodera {
+ $_ = shift;
+ return 1 if (m/[G|C][A|T][G|C]/ || m/[A|T][G|C][G|C]/ && !(m/[A|T]CG/));
+}
#----------------#
# Clean sequence #
@@ -3093,23 +3110,24 @@ sub primer_dimer {
# 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) {
+ foreach $l (0 .. $pfl-1) {
my $mbase = substr($fprimer_r, $l, 1);
$primer_hash{$mbase}[$l]=1;
- for $k qw(a g c t) {
+ my @nucleotides = qw(a g c t);
+ foreach $k (@nucleotides) {
$primer_hash{$k}[$l] ||=0;
}
}
# create the primer matrix
my @primer_comp;
- for $k (0 .. $prl-1) {
+ foreach $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) {
+ foreach $k (0 .. $pd_len) {
$score[$k]=0;
my $bind;
my $score_p=0;
@@ -3129,7 +3147,7 @@ sub primer_dimer {
# }
# read the binding data
- for $l (0 .. $prl-1) {
+ foreach $l (0 .. $prl-1) {
if (($k-$l)<$pfl) {
$bind .= $primer_comp[$l][$k-$l] if ($k-$l)>=0;
} else {
@@ -3148,7 +3166,7 @@ sub primer_dimer {
# Find start and end of similarity
my ($pb_init,$pb_end);
- for $l (0 .. length($bind)-1) {
+ foreach $l (0 .. length($bind)-1) {
# at first I tried finding the initiating terminal bases with
# regexps, but that was much slower ...
if (substr($bind, $l, 1) eq "1") {
@@ -3159,7 +3177,7 @@ sub primer_dimer {
if (defined($pb_init)) {
# deltaG calculation
- for $l ($pb_init .. $pb_end-1) {
+ foreach $l ($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)};
@@ -3228,44 +3246,44 @@ sub primer_dimer_new {
# 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) {
+ foreach $l (0 .. $pfl-1) {
my $mbase = substr($fprimer_r, $l, 1);
$primer_hash{$mbase}[$l]=1;
- for $k qw(a g c t) {
+ foreach $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)};
+ foreach my $kk (0 .. $prl-1) {
+ $primer_comp[$kk]=$primer_hash{substr($rcomprlc, $kk, 1)};
}
# print the matrix - for debugging
print "$primer_f vs. $primer_r - full pd = $pd_full\n";
print " \t";
- for $l (0 .. $pfl-1) {
+ foreach $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";
+ foreach my $kk (0 .. $prl-1) {
+ my $base = substr($rprimer_r, $kk, 1);
+ print "$base:\t@{$primer_comp[$kk]}\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;
+ foreach my $kk (0 .. $pd_len) {
+ $score[$kk]=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 $pf_coord_start = ($kk >= $pfl ? $pfl-1 : $kk);
+ my $pr_coord_start = ($kk - $pfl > 0 ? $kk - $pfl : 0);
my ($pf_coord, $pr_coord, $first, $flag);
# read through each combination finding multiple matches
@@ -3285,7 +3303,7 @@ sub primer_dimer_new {
}
} elsif ($flag) {
# end of a binding stretch
- push @binding_data, [$k, $first, $bind] if $bind > 1;
+ push @binding_data, [$kk, $first, $bind] if $bind > 1;
$bind=0;
$flag=0;
}
@@ -3325,7 +3343,7 @@ sub primer_dimer_new {
# # }
#
# # read the binding data
- # for $l (0 .. $prl-1) {
+ # foreach $l (0 .. $prl-1) {
# if (($k-$l)<$pfl) {
# $bind .= $primer_comp[$l][$k-$l] if ($k-$l)>=0;
# } else {
@@ -3344,7 +3362,7 @@ sub primer_dimer_new {
#
# # Find start and end of similarity
# my ($pb_init,$pb_end);
- # for $l (0 .. length($bind)-1) {
+ # foreach $l (0 .. length($bind)-1) {
# # at first I tried finding the initiating terminal bases with
# # regexps, but that was much slower ...
# if (substr($bind, $l, 1) eq "1") {
@@ -3355,7 +3373,7 @@ sub primer_dimer_new {
#
# if (defined($pb_init)) {
# # deltaG calculation
- # for $l ($pb_init .. $pb_end-1) {
+ # foreach $l ($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)};
@@ -4248,7 +4266,9 @@ sub copy_selected_primers {
my @sel = $$ref->selectionGet;
# Clipboard copying routine
- my $clip = "Forward Primer\tPos\tLen\tTm\tReverse Primer\tPos\tLen\tTm\tAmp\tdG\n";
+ my $clip = ($page eq "seq") ?
+ "Name\tForward Primer\tPos\tLen\tTm\n" :
+ "Name\tForward Primer\tPos\tLen\tTm\tName\tReverse Primer\tPos\tLen\tTm\tAmp\tdG\n";
my (@gene_array, $gene_frame, $seq);
if ($page eq "pd" && (($primer_seq_5f) || ($primer_seq_5r))) {
@@ -4257,10 +4277,28 @@ sub copy_selected_primers {
$gene_frame=$gene_array[0][0]%3;
}
- foreach (@sel) {
- my $sel = $_;
- for my $j ( 0 .. 4, 8, 6, 7, 9, 10 ) {
- if ($page eq "bis") {
+ my ($file_name,$dir,$ext) = fileparse($open_file{$page}, qr/\.[^.]*/);
+ $file_name =~ s/_.*//g;
+
+ foreach my $sel (@sel) {
+ my $nb_page = which_nb_page();
+
+ my @details;
+ if ($page eq "seq") {
+ @details = (0 .. 3);
+ } else {
+ @details = ( 0 .. 4, 8, 6, 7, 9, 10 );
+ }
+
+ for my $j (@details) {
+ # Names
+ if ($j == 0) {
+ $clip .= $file_name."_".$$slist[$sel][1]."F\t";
+ } elsif ($j == 4) {
+ $clip .= $file_name."_".$$slist[$sel][8]."R\t";
+ }
+
+ if ($page eq "bis") {
# Primer redundancy for CpG residues:
# Replaces T with Y (pyrimidine) for forward
# Replaces A with R (purine) for reverse
@@ -4725,10 +4763,9 @@ 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.
+ # Now uses Ensembl's very nice REST API to retrieve all details -- should never break again!
+ # (a very quick kludge for now ... will tidy up code later ...)
+
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");
@@ -4768,21 +4805,49 @@ sub fetch_ensembl {
return;
}
- # Search for the gene:
- # New search method as of v1.1.18 (modified in 1.1.20, and again in 1.1.21 thanks to Nick Kennedy)
- $_ = http_get("http://www.ensembl.org/$ensembl_organism/Search/Details?species=$ensembl_organism;idx=Gene;end=2;q=$ensembl_gene");
- #$_ = http_get("http://www.ensembl.org/$ensembl_organism/Search/Details?species=$ensembl_organism;idx=Gene;q=$ensembl_gene");
- #print "http://www.ensembl.org/$ensembl_organism/Search/Details?species=$ensembl_organism;idx=Gene;q=$ensembl_gene\n";
+ print STDERR "Gene was $ensembl_gene ...\n";
+
+ my $http = HTTP::Tiny->new();
+
+ my $server = 'http://rest.ensembl.org';
+ my $ext = "/xrefs/symbol/$ensembl_organism/$ensembl_gene?content-type=application/json";
+ my $response = $http->get($server.$ext, {
+ headers => { 'Content-type' => 'application/json' }
+ });
+
+ die "Failed!\n" unless $response->{success};
+
+
+ my $gene_id;
+
+ if(length $response->{content}) {
+ my $gene = decode_json($response->{content});
+
+ unless (@{$gene}[0]) {
+ # no matches
+ dialogue("Sorry, a matching Ensembl ID for that gene name was not found. Gene names need to be typed exactly (both Ensembl names and Ensembl IDs can be used).\n\nPlease try again.");
+ # print "output was\n$_\n";
+ return;
+ }
+
+
+ #print Dumper @{$gene};
+ $gene_id = @{$gene}[0]->{id};
- s/<\/*span.*?>//g; # rip out highlight spans
- s/<\/*font.*?>//g; # rip out font spans
+ printf("Gene: %s\n",$gene_id);
+ }
+
- # Print the HTML output for debugging:
- # print "$_\n\n";
-
+ $ext = "/lookup/id/$gene_id?expand=1";
+ $response = $http->get($server.$ext, {
+ headers => { 'Content-type' => 'application/json' }
+ });
+
+ die "Failed!\n" unless $response->{success};
+
+
# 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;
@@ -4790,34 +4855,13 @@ sub fetch_ensembl {
my %trans;
my @enst_readable;
- # As of 03/2010, Ensembl now returns the matching genes first, without listing transcripts
- # So we need a new http call to get the page we really want, and we'll need to prompt the
- # if there's more than one match here ...
-
- #~ # Find genes and gene_ids
- #~ while (m/\<a href="(.*?)"\>[\w_]+ ([\w_]+) Gene: ([\w\d\.\-]+) \(.*?: ([\w\d\.\-]+)\)/mg) {
- #~ my ($href, $gene_type, $gene_id, $name) = ($1, $2, $3, $4);
- #~ #print "($href, $gene_type, $gene_id, $name)\n\n";
+ if(length $response->{content}) {
+ my $gene = decode_json($response->{content});
+
+ push @gene_names, $gene->{description};
- #~ $name ||= "$gene_id: no description available";
- #~ push @gene_names, $name;
- #~ $ids{$name}=[$gene_id, $href];
- #~ }
-
- # As of 02/2011, Ensembl has changed formats again, leading to a slightly different parsing ... and again for version 1.1.21
-
- # Find genes and gene_ids
- while (m/\<div class=\"hit\"\>.*?href=\"(.*?)\".*?strong\>(.*?)\W*\<.*?\[(.*?): (.*?) ].*?Description.*?dd\>\W*(.*?)\W*\</sg) {
- my ($href, $name, $gene_type, $gene_id, $gene_des) = ($1, $2, $3, $4, $5);
- #print "($href, $gene_type, $gene_id, $name, $gene_des)\n\n";
- $name .= " -- $gene_des";
- $name ||= "$gene_id: no description available";
- push @gene_names, $name;
- $ids{$name}=[$gene_id, $href];
}
- # No need to sort -- genes are listed in order of relevance
- #@gene_names = sort(@gene_names);
if (@gene_names) {
# Ask user to confirm gene identity or pick the gene of interest if multiple matches
@@ -4849,36 +4893,34 @@ sub fetch_ensembl {
return if $cancel;
}
- # Having selected the gene, we can now get the transcripts ... (parsing changed for 1.1.21)
- if ($name && ($ids{$name}[1])) {
- $_ = http_get("http://www.ensembl.org/$ids{$name}[1]");
- #print "$_\n";
- my ($transcripts) = m/id="transcripts"(.*?)\/table/sg;
- while ($transcripts =~ m/\/Summary\?.*?t=([\w\d\.]+).*?<td.*?>.*?<td.*?>.*?<td.*?>([\d\-]+).*?<td.*?>.*?>([\w\s]+)</sg) {
- #print "$1 $2 $3\n";
- push @enst, $1;
- if ($2 eq '-') {
- # Processed transcript, not protein coding
- push @enst_readable, "$1 $3";
+ # Having selected the gene, we can now get the transcripts ...
+
+
+ if(length $response->{content}) {
+ my $gene = decode_json($response->{content});
+ $gene_id = $gene->{id};
+ foreach my $v (@{ $gene->{Transcript}}) {
+ if ($v->{Translation}) {
+ push @enst_readable, "$v->{id} $v->{display_name} (size: $v->{Translation}->{length} aa)";
} else {
- # Protein coding
- push @enst_readable, "$1 $3 (size: $2 aa)";
+ push @enst_readable, "$v->{id} $v->{display_name}"
}
- $ids{$1} = [$2, $3];
- }
+
+ push @enst, $v->{id};
+ }
}
-
+
my $transcript;
if (@enst) {
- $transcript = $enst[0];
- if ($#enst > 1) {
+ $transcript = $enst_readable[0];
+ if ($#enst > 0) {
# multiple transcripts: ask user to select transcript ID
my @transcripts = @enst;
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");
+ pack_gui('Label', "Ensemble gene $name has ".($#transcripts+1)." transcript".($#transcripts > 0 ? 's' : '')." ...", "ensemble_mt_d_note");
nr();
pack_gui('BrowseEntry', \$transcript, 'ensembl_mt_d_genes', \@enst_readable, 50);
@@ -4930,11 +4972,11 @@ sub fetch_ensembl {
if ($page eq 'qpcr') {
# retrieve both gene and transcript sequences - retrieval type is ignored
- $_ = http_get(convert_ensembl($ensembl_organism,$transcript,'genomic','Location'));
+ $_ = convert_ensembl($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'));
+ $_ = convert_ensembl($ensembl_organism,$transcript,'cdna');
$packed_widgets{qmrna_seq}->delete(0.1,"end");
$packed_widgets{qmrna_seq}->insert(0.1,$_);
@@ -4943,7 +4985,7 @@ sub fetch_ensembl {
} else {
# retrieve requested sequence
my ($seq_ref) = get_variables('seq');
- $_ = http_get(convert_ensembl($ensembl_organism,$transcript,$ensembl_type));
+ $_ = convert_ensembl($ensembl_organism,$transcript,$ensembl_type);
$$seq_ref->delete(0.1,"end");
$$seq_ref->insert(0.1,$_);
}
@@ -4965,21 +5007,18 @@ sub fetch_ensembl {
sub convert_ensembl {
# argument to http address converter (addresses changed for 1.1.21)
my ($ensembl_organism,$transcript,$ensembl_type,$export_type) = @_;
- $export_type||='Transcript';
+ my $http = HTTP::Tiny->new();
+
+ my $server = 'http://rest.ensembl.org';
+ my $ext = "/sequence/id/$transcript?content-type=text/plain;type=$ensembl_type";
+ my $response_cds = $http->get($server."/sequence/id/$transcript?content-type=text/plain;type=$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";
- # my $address = "http://www.ensembl.org/$ensembl_organism/$export_type/Export/fasta?db=core;t=$transcript;st=$ensembl_type;_format=Text";
- # return $address;
+ #print $server."/sequence/id/$transcript?content-type=text/plain;type=$ensembl_type"."\n";
+
+ if(length $response_cds->{content}) {
+ return $response_cds->{content};
+ }
- # Thanks to Karl Kashofer for the following fix ...
- # for genomic retrieval we need to append "genomic=unmasked"
- if ($ensembl_type eq 'genomic') {
- my $address = "http://www.ensembl.org/$ensembl_organism/Export/Output/Transcript?output=fasta;db=core;t=$transcript;param=$ensembl_type;genomic=unmasked;_format=Text";
- return $address;
- } else {
- my $address = "http://www.ensembl.org/$ensembl_organism/Export/Output/Transcript?output=fasta;db=core;t=$transcript;param=$ensembl_type;_format=Text";
- return $address;
- };
}
@@ -5225,7 +5264,7 @@ sub generate_report {
}
my ($hlist_sel) = $$hlist_ref->selectionGet;
- unless (defined(@$slist) && defined($hlist_sel)) {
+ unless (@$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;
}
@@ -5377,7 +5416,7 @@ sub info {
my $text = <<EOT;
PerlPrimer v$version
-Copyright � 2003-2011 Owen Marshall\n
+Copyright � 2003-2017 Owen Marshall\n
EOT
my $text2 = <<EOT;
An application to design primers for PCR, Bisulphite PCR, Real-time PCR and Sequencing.
@@ -5385,7 +5424,7 @@ An application to design primers for PCR, Bisulphite PCR, Real-time PCR and Sequ
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
+https://github.com/owenjm/perlprimer
EOT
$info_d = $top->Toplevel(-title=>'About PerlPrimer ...');
my $info_d_f = $info_d->Frame()->pack(-padx=>4, -pady=>4, -expand=>1, -fill=>'both');
@@ -5500,11 +5539,16 @@ Restriction enzyme data are provided by the REBASE project (http://rebase.neb.co
Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE - restriction enzymes and methylases. Nucleic Acids Res. 2003; 31:418-20.
EOT
-
+ my $text_gc_clamp=<<EOT;
+Strict GC clamp rules (need to be enabled in preferences) are based on:
+
+Onodera K, Melcher U. Selection for 3' end triplets for polymerase chain reaction primers. Mol Cell Probes. 2004 Dec;18(6):369-72.
+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
+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.
@@ -5552,6 +5596,9 @@ EOT
$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\nGC clamp parameters\n\n", 'bold');
+ $packed_widgets{ack_text}->insert('end', $text_gc_clamp);
+
$packed_widgets{ack_text}->insert('end', "\n\nBisulphate PCR primer design\n\n", 'bold');
$packed_widgets{ack_text}->insert('end', $text_cpg);
@@ -5751,6 +5798,14 @@ sub prefs {
pack_gui('Label', '% GC content');
nr();
pack_gui('Label', '(Used when "Exclude %GC" is checked on a project page)');
+ nr('', 7);
+
+ nr();
+ pack_gui('Label', "3' GC clamp", '', -font=>$gui_font_bold);
+ nr();
+ pack_gui('Checkbutton', 'Use Onodera and Melcher (2004) rules', "prefs_onodera", \$onodera_clamp);
+ nr();
+ pack_gui('Label', '(Selects for WSS or SWS but not WCG at 3\' end)');
nr('',2);
@@ -6630,12 +6685,15 @@ sub reset_bounds {
}
sub step_in {
+ my $step_length=shift;
+ $step_length ||= 10;
+
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;
- $$max_range -= 10;
+ $$min_range += $step_length;
+ $$max_range -= $step_length;
if ($$min_range >= $$max_range) {
sbarprint("\nNo primers found");
$cancel=1;
@@ -7307,7 +7365,7 @@ sub open_fasta {
}
sub format_file_name {
- my ($name) = @_;
+ my $name = shift;
$name =~ s/\s+/_/g; # replace spaces with underscores
$name =~ s/[\(\)\,\.\;]//g; # remove brackets and punctuation
$name =~ s/\|/-/g; # replace pipes with dashes
diff --git a/todo b/todo
deleted file mode 100644
index 22ac09b..0000000
--- a/todo
+++ /dev/null
@@ -1,11 +0,0 @@
-For v1.2
-
-* Use an in vitro PCR server rather than straight BLAST for checking primers. MFEPrimer (http://biocompute.bmi.ac.cn/MFEprimer/) looks a good candidate.
-
-* 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 unequal primer-dimer internal loops
-
-* More emphasis on RealTime PCR design? Show primers on intron/exon map, for example?
-
-* Ability to designate regions with starting/ending coordinates or starting/ending sequence strings, and assign colours and PCR ranges to such regions
--
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