<br><br><div id="WISESTAMP_SIG_8122"></div><br><br><div class="gmail_quote">---------- Forwarded message ----------<br>From: <b class="gmail_sendername">ketan padiya</b> <span dir="ltr"><<a href="mailto:ketanmicro@gmail.com">ketanmicro@gmail.com</a>></span><br>
Date: Tue, Feb 22, 2011 at 11:42 AM<br>Subject: Re: [ensembl-dev] problem in variant effect predictor stand alone<br>To: Will McLaren <<a href="mailto:wm2@ebi.ac.uk">wm2@ebi.ac.uk</a>><br><br><br><br>Hi Will,<br><br>
Still the new perl script giving errors,<br><br>[orf@localhost new]$ perl <a href="http://variant_effect_predictor.pl" target="_blank">variant_effect_predictor.pl</a> -i ../../samtools-0.1.12a/samfiles/q20/vcf/GKUNU9Q04_chr28_q20_sort.vcf -o chr28.txt -s cow -w -b 1000<br>
<span style="color: rgb(255, 0, 0);">WARNING: Start 7355 or end . coordinate invalid on line 4</span><br style="color: rgb(255, 0, 0);"><span style="color: rgb(255, 0, 0);">WARNING: Start 27062066 or end . coordinate invalid on line 356</span><br style="color: rgb(255, 0, 0);">
<span style="color: rgb(255, 0, 0);">WARNING: Start 28411255 or end . coordinate invalid on line 492</span><br style="color: rgb(255, 0, 0);"><span style="color: rgb(255, 0, 0);">WARNING: Start 40292556 or end . coordinate invalid on line 596</span><br>
<br>
Analyzing chromosome 28 at <a href="http://variant_effect_predictor.pl" target="_blank">variant_effect_predictor.pl</a> line 622, <GEN0> line 688.<br><br> - fetched 382 transcripts at <a href="http://variant_effect_predictor.pl" target="_blank">variant_effect_predictor.pl</a> line 632, <GEN0> line 688.<br>
<br>Could not connect to database bos_taurus_core_61_4j as user anonymous using [DBI:mysql:database=bos_taurus_core_61_4j;host=<a href="http://ensembldb.ensembl.org" target="_blank">ensembldb.ensembl.org</a>;port=5306] as a locator:<br>
Unknown MySQL server host '<a href="http://ensembldb.ensembl.org" target="_blank">ensembldb.ensembl.org</a>' (2) at /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/DBConnection.pm line 290, <GEN0> line 688.<br>
<br>-------------------- EXCEPTION --------------------<br>MSG: Could not connect to database bos_taurus_core_61_4j as user anonymous using [DBI:mysql:database=bos_taurus_core_61_4j;host=<a href="http://ensembldb.ensembl.org" target="_blank">ensembldb.ensembl.org</a>;port=5306] as a locator:<br>
Unknown MySQL server host '<a href="http://ensembldb.ensembl.org" target="_blank">ensembldb.ensembl.org</a>' (2)<br>STACK Bio::EnsEMBL::DBSQL::DBConnection::connect /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/DBConnection.pm:299<br>
STACK Bio::EnsEMBL::DBSQL::DBConnection::db_handle /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/DBConnection.pm:618<br>STACK Bio::EnsEMBL::DBSQL::DBConnection::prepare /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/DBConnection.pm:647<br>
STACK Bio::EnsEMBL::DBSQL::BaseAdaptor::prepare /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseAdaptor.pm:164<br>STACK Bio::EnsEMBL::DBSQL::AttributeAdaptor::fetch_all_by_ /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/AttributeAdaptor.pm:282<br>
STACK Bio::EnsEMBL::DBSQL::AttributeAdaptor::AUTOLOAD /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/AttributeAdaptor.pm:100<br>STACK Bio::EnsEMBL::Slice::get_all_Attributes /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Slice.pm:1237<br>
STACK Bio::EnsEMBL::Slice::is_circular /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Slice.pm:536<br>STACK Bio::EnsEMBL::Slice::project /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Slice.pm:881<br>STACK Bio::EnsEMBL::DBSQL::SequenceAdaptor::fetch_by_Slice_start_end_strand /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/SequenceAdaptor.pm:220<br>
STACK Bio::EnsEMBL::Slice::subseq /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Slice.pm:642<br>STACK Bio::EnsEMBL::Exon::seq /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Exon.pm:1467<br>STACK Bio::EnsEMBL::Transcript::spliced_seq /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Transcript.pm:768<br>
STACK Bio::EnsEMBL::Transcript::translateable_seq /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Transcript.pm:824<br>STACK Bio::EnsEMBL::Transcript::translate /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Transcript.pm:1655<br>
STACK Bio::EnsEMBL::Utils::TranscriptAlleles::type_variation /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Utils/TranscriptAlleles.pm:289<br>STACK Bio::EnsEMBL::Variation::DBSQL::TranscriptVariationAdaptor::_calc_consequences /home/orf/EnsEMBL/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/DBSQL/TranscriptVariationAdaptor.pm:586<br>
STACK main::whole_genome_fetch <a href="http://variant_effect_predictor.pl:659" target="_blank">variant_effect_predictor.pl:659</a><br>STACK toplevel <a href="http://variant_effect_predictor.pl:372" target="_blank">variant_effect_predictor.pl:372</a><br>
Ensembl API version = 61<br>---------------------------------------------------<br><br>Please help me to rum script successfully<br><br>Thanks<div><div></div><div class="h5"><br><div></div><br><br><div class="gmail_quote">
On Mon, Feb 21, 2011 at 4:04 PM, Will McLaren <span dir="ltr"><<a href="mailto:wm2@ebi.ac.uk" target="_blank">wm2@ebi.ac.uk</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">Hi Ketan,<div><br>I apologise, there were a couple of bugs which where giving you your problems.</div>
<div><br></div><div>They are now fixed, I have attached the patched file here.</div><div><br></div><div>You should rerun this code on your data files, even if they worked OK before, as there was a bug which would have affected all insertions of 1bp.</div>
<div><br></div><div>Thanks</div><div><br></div><div><font color="#888888">Will</font><div><div></div><div><br><br><div class="gmail_quote">On 16 February 2011 04:59, ketan padiya <span dir="ltr"><<a href="mailto:ketanmicro@gmail.com" target="_blank">ketanmicro@gmail.com</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">Thanks for quick reply,<br><br>Sending the input lines causing problems<br><br>chr1 5959615 . tatggtccaatggtccaa tatggtccaa 44.2 . INDEL;DP=2;AF1=1;CI95=0.5,1;DP4=0,0,0,2;MQ=60 PL:GT:GQ 83,6,0:1/1:49<br>
chr1 108473380 . taaaa taaa 3.66 . INDEL;DP=2;AF1=1;CI95=0.5,1;DP4=0,0,2,0;MQ=60 PL:GT:GQ 40,6,0:1/1:49<br>chr1 127416494 . acc ac 3.66 . INDEL;DP=3;AF1=1;CI95=0.5,1;DP4=0,0,0,2;MQ=60 PL:GT:GQ 40,6,0:1/1:49<br>
<br>and there go'se another problem,<br><br>When applying following command, it did well with chr1 and gave results, all vcf extracted using samtools<br><br>[orf@localhost snp_effect_predictor]$ perl <a href="http://variant_effect_predictor.pl" target="_blank">variant_effect_predictor.pl</a> -i ../samtools-0.1.12a/samfiles/q20/vcf/GKUNU9Q04_chr3_q20_sort.vcf -o chr3.txt -format vcf -s cow -w -b 5000<br>
<br>-------------------- EXCEPTION --------------------<br>MSG: Start must be less than or equal to end+1<br>STACK Bio::EnsEMBL::Feature::new /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/Feature.pm:139<br>STACK Bio::EnsEMBL::Variation::VariationFeature::new /home/orf/EnsEMBL/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/VariationFeature.pm:177<br>
STACK toplevel <a href="http://variant_effect_predictor.pl:304" target="_blank">variant_effect_predictor.pl:304</a><br>Ensembl API version = 61<br>---------------------------------------------------<br>I am also attaching vcf file for this chr 3.<div>
<div></div><div><br>
<br><br><div></div><br><br><div class="gmail_quote">On Wed, Feb 16, 2011 at 10:14 AM, Will McLaren <span dir="ltr"><<a href="mailto:wm2@ebi.ac.uk" target="_blank">wm2@ebi.ac.uk</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">
Dear Ketyan,<div><br></div><div>Are you able to send me the input lines that are causing these errors? It is difficult for me to diagnose the problem without seeing the data.</div><div><br>Thanks</div><div><br></div><div>
<font color="#888888">Will</font><div><div></div><div><br>
<br><div class="gmail_quote">On 16 February 2011 13:08, ketan padiya <span dir="ltr"><<a href="mailto:ketanmicro@gmail.com" target="_blank">ketanmicro@gmail.com</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">
Thanks for reply, It worked well.<br><br>One more question for INDEL, variant_effect_predictor perl script don't recognize INDEL string and gives warning like,<br><br>WARNING: Invalid allele string atggtccaatggtccaa/atggtccaa on line 92<br>
WARNING: Invalid allele string aaaa/aaa on line 1249<br>WARNING: Invalid allele string cc/c on line 1378<div><div></div><div><br><br><br><div class="gmail_quote">On Tue, Feb 15, 2011 at 6:37 PM, Will McLaren <span dir="ltr"><<a href="mailto:wm2@ebi.ac.uk" target="_blank">wm2@ebi.ac.uk</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">Dear Ketyan,<div><br><div class="gmail_quote"><div>On 15 February 2011 20:13, ketan padiya <span dir="ltr"><<a href="mailto:ketanmicro@gmail.com" target="_blank">ketanmicro@gmail.com</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">
I have downloaded variant effect predictor and EnsEMBL API for that my problems are,<br><br>1) After every reboot of system i have to give the PERL5LIB path, Why?<br></blockquote><div><br></div></div><div>You can configure your system to load this PERL5LIB automatically every time it starts. How you do this depends on what type of system you are using. For example, if you are using the CSH shell system, you can edit the file named ".cshrc" in your home directory, then add lines like:</div>
<div><br></div><div><div>setenv PERL5LIB ${PERL5LIB}:${HOME}/src/ensembl/modules</div><div>setenv PERL5LIB ${PERL5LIB}:${HOME}/srcl/ensembl-compara/modules</div><div>setenv PERL5LIB ${PERL5LIB}:${HOME}/src/ensembl-functgenomics/modules</div>
<div>setenv PERL5LIB ${PERL5LIB}:${HOME}/src/ensembl-variation/modules</div></div><div><br></div><div>Or, if you use Bash, you can add the lines to ".bashrc", also in your home directory:</div><div><br></div><div>
<span style="color: rgb(85, 85, 85); font-family: 'Luxi Sans',Helvetica,Arial,Geneva,sans-serif; font-size: 13px;"><pre style="font-family: 'Courier New',Courier,monospace; font-size: 1em; margin-left: 1em;">
PERL5LIB=${PERL5LIB}:${HOME}/src/bioperl-live
PERL5LIB=${PERL5LIB}:${HOME}/src/ensembl/modules
PERL5LIB=${PERL5LIB}:${HOME}/src/ensembl-compara/modules
PERL5LIB=${PERL5LIB}:${HOME}/src/ensembl-variation/modules
PERL5LIB=${PERL5LIB}:${HOME}/src/ensembl-functgenomics/modules
export PERL5LIB</pre></span></div><div><div> </div><blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">2) Variant effect predictor is taking too long to process given vcf file (~2000 line of SNP/INDELs)<br>
</blockquote><div><br></div></div><div>For this and point 3), I recommend you try adding the following flags to your command:</div><div><br></div><div>perl <a href="http://variant_effect_predictor.pl/" target="_blank">variant_effect_predictor.pl</a> -i ../samtools-0.1.12a/samfiles/q20/vcf/GKUNU9Q04_chr1_q20_sort.vcf -o chr1.txt -format vcf -s cow -w -b 5000</div>
<div><br></div><div>Using -format forces the program to read your file as a VCF.</div><div><br></div><div>Using -w means the script runs in "whole-genome" mode, which is better suited to large data sets that cover, for example, one chromosome. You should make sure when you use this that the VCF input file is sorted by chromosome and then position.</div>
<div><br></div><div>Setting a larger buffer size with -b helps whole genome mode work faster.</div><div><br></div><div>Please note that the -w option is only available from version 61 of Ensembl.</div><div><br></div><div>
Thanks and good luck!</div><div><br></div><div>Will McLaren</div><div>Ensembl Variation</div><div><br></div><div> </div><blockquote class="gmail_quote" style="margin: 0pt 0pt 0pt 0.8ex; border-left: 1px solid rgb(204, 204, 204); padding-left: 1ex;">
<div><div></div><div>
3) In the end it gives error, <br><br>[orf@localhost variant_effect_predictor]$ perl <a href="http://variant_effect_predictor.pl" target="_blank">variant_effect_predictor.pl</a> -i ../samtools-0.1.12a/samfiles/q20/vcf/GKUNU9Q04_chr1_q20_sort.vcf -o chr1.txt -s cow<br>
WARNING: Start 5959615 or end . coordinate invalid on line 92 <span style="color: rgb(255, 0, 0);">/ INDEL</span><br>WARNING: Start 30571012 or end . coordinate invalid on line 572 <span style="color: rgb(255, 0, 0);">/ INDEL</span><br>
WARNING: Start 64306203 or end . coordinate invalid on line 819 <span style="color: rgb(255, 0, 0);">/ INDEL</span><br>WARNING: Start 76575493 or end . coordinate invalid on line 895<span style="color: rgb(255, 0, 0);"> / INDEL</span><br>
DBD::mysql::st execute failed: Lost connection to MySQL server during query at /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseAdaptor.pm line 521, <GEN0> line 1006.<br><br>-------------------- EXCEPTION --------------------<br>
MSG: Detected an error whilst executing SQL 'SELECT vf.variation_feature_id, vf.seq_region_id, vf.seq_region_start, vf.seq_region_end, vf.seq_region_strand, vf.variation_id, vf.allele_string, vf.variation_name, vf.map_weight, <a href="http://s.name" target="_blank">s.name</a>, s.somatic, vf.validation_status, vf.consequence_type, vf.class_so_id<br>
FROM ( (variation_feature vf, source s) <br> LEFT JOIN failed_variation fv ON fv.variation_id = vf.variation_id ) <br> WHERE s.somatic = 0 AND <br> (<br> fv.variation_id IS NULL OR<br> fv.subsnp_id IS NOT NULL<br>
)<br> AND vf.seq_region_id = 142972 AND vf.seq_region_start <= 23402307 AND vf.seq_region_end >= 23402307 AND vf.seq_region_start >= 23401807 AND<br> vf.source_id = s.source_id <br>': DBD::mysql::st execute failed: Lost connection to MySQL server during query at /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseAdaptor.pm line 521, <GEN0> line 1006.<br>
<br>STACK Bio::EnsEMBL::DBSQL::BaseAdaptor::generic_fetch /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseAdaptor.pm:522<br>STACK Bio::EnsEMBL::DBSQL::BaseFeatureAdaptor::_slice_fetch /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseFeatureAdaptor.pm:495<br>
STACK Bio::EnsEMBL::DBSQL::BaseFeatureAdaptor::fetch_all_by_Slice_constraint /home/orf/EnsEMBL/src/ensembl/modules/Bio/EnsEMBL/DBSQL/BaseFeatureAdaptor.pm:316<br>STACK Bio::EnsEMBL::Variation::DBSQL::VariationFeatureAdaptor::fetch_all_by_Slice_constraint /home/orf/EnsEMBL/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/DBSQL/VariationFeatureAdaptor.pm:121<br>
STACK Bio::EnsEMBL::Variation::DBSQL::VariationFeatureAdaptor::fetch_all_by_Slice /home/orf/EnsEMBL/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/DBSQL/VariationFeatureAdaptor.pm:175<br>STACK main::print_consequences <a href="http://variant_effect_predictor.pl:318" target="_blank">variant_effect_predictor.pl:318</a><br>
STACK toplevel <a href="http://variant_effect_predictor.pl:289" target="_blank">variant_effect_predictor.pl:289</a><br>Ensembl API version = 61<br>---------------------------------------------------<br><font color="#888888"><br clear="all">
<br>-- <br>Ketan Padiya<div>
Research Fellow<br>Anand Veterinary College<br>Gujarat<br>India.<br>+91 9428969448</div><div style="display: inline;"></div><br>
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<br></blockquote></div><br></div>
</blockquote></div><br><br clear="all"><br>-- <br>Ketan Padiya<div>Research Fellow<br>Anand Veterinary College<br>Gujarat<br>India.<br>+91 9428969448</div><div style="display: inline;"></div><br>
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<br></blockquote></div><br></div></div></div>
</blockquote></div><br><br clear="all"><br>-- <br>Ketan Padiya<div>Research Fellow<br>Anand Veterinary College<br>Gujarat<br>India.<br>+91 9428969448</div><div style="display: inline;"></div><br>
</div></div></blockquote></div><br></div></div></div>
</blockquote></div><br><br clear="all"><br>-- <br>Ketan Padiya<div>Research Fellow<br>Anand Veterinary College<br>Gujarat<br>India.<br>+91 9428969448</div><div style="display: inline;"></div><br>
</div></div></div><br><br clear="all"><br>-- <br>Ketan Padiya<div>Research Fellow<br>Anand Veterinary College<br>Gujarat<br>India.<br>+91 9428969448</div><div style="display: inline;"></div><br>