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<div>Hi Will,</div>
<div><font face="tahoma"></font> </div>
<div><font face="tahoma">Thanks for your help with this. Yes, I am aware of the Ensembl<a></a> rhesus gene annotations, but the RhesusBase<a></a> annotations I am using are newer and include RNA-Seq<a></a> data, so they are supposed to be better than the Ensembl<a></a>
predictions.</font></div>
<div><font face="tahoma"></font> </div>
<div><font face="tahoma">Your modified version of gtf2vep.pl<a></a> does indeed make the cache files, but I am now running into issues running vep<a></a> using the cache files. I am getting the following errors. I took your approach to stopping the errors by
checking for undefined values and dealing with them. Each successive error occurs after fixing an error.</font></div>
<div><font face="tahoma"></font> </div>
<div>Can't call method "strand" on an undefined value at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Transcript.pm<a></a> line 1141, <GEN0<a></a>> line 5035.</div>
<div><font face="tahoma"></font> </div>
<div>Can't call method "strand" on an undefined value at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Transcript.pm<a></a> line 1095, <GEN0<a></a>> line 5035.</div>
<div dir="ltr"><font color="#000000" size="2" face="Tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Can't call method "phase" on an undefined value at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Transcript.pm<a></a> line 930, <GEN0<a></a>> line 45035.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr">Can't call method "strand" on an undefined value at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Translation.pm<a></a> line 399, <GEN0<a></a>> line 45035</div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr">Can't call method "strand" on an undefined value at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Translation.pm<a></a> line 428, <GEN0<a></a>> line 45035.</div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">With each successive fix, vep<a></a> runs a bit farther, but
</font><font face="tahoma">I get a lot of the following warnings:</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr">Use of uninitialized value in addition (+) at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Variation/Utils<a></a>/VariationEffect.pm<a></a> line 536, <GEN0<a></a>> line 5035.<br>
Use of uninitialized value in subtraction (-) at /home/rharris1<a></a>/work/ensembl<a></a>/vep<a></a>/variant_effect_predictor<a></a>//Bio/EnsEMBL<a></a>/Variation/Utils<a></a>/VariationEffect.pm<a></a> line 519, <GEN0<a></a>> line 5035.</div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">The vep<a></a> results I do get look to be correct for intergenic<a></a>, upstream, downstream, and intronic<a></a> variants, but variants in exons<a></a> are sometimes not being reported correctly. In several cases, a variant
that should be identified as synonymous or missense<a></a> is identified as "5_prime_UTR_variant,3_prime_UTR_variant<a></a>". </font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">I just noticed that in the Ensembl<a></a> gtf<a></a> file (which I ran through gtf2vep.pl<a></a> and vep<a></a> and it worked) the negative strand genes are in reverse chromosome order. I changed my gtf<a></a> to that order
and it ran through the original gtf2vep.pl<a></a> (before your fixes) without throwing an error, but I still get errors using vep<a></a>. Should the negative strand genes be in reverse chromosome order? </font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Please let me know if you have any ideas about this.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Thanks,</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Alan</font></div>
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<hr tabindex="-1">
<font color="#000000" size="2" face="Tahoma"><b>From:</b> dev-bounces@ensembl.org [dev-bounces@ensembl.org] On Behalf Of Will McLaren [wm2@ebi.ac.uk]<br>
<b>Sent:</b> Wednesday, January 08, 2014 7:23 AM<br>
<b>To:</b> Ensembl developers list<br>
<b>Subject:</b> Re: [ensembl-dev] Problem with gtf2vep.pl<br>
</font><br>
</div>
<div></div>
<div>
<div dir="ltr">Hello Alan,
<div><br>
</div>
<div>Thanks for the detailed report. There's an odd bug happening here which I can't get to the bottom of at the moment.</div>
<div><br>
</div>
<div>I've added a fix for now which stops the error happening, and the cache builds fine for me from your input.</div>
<div><br>
</div>
<div>Since we're in the process of switching our code hosting to Git, for now I have only pushed the fix to our GitHub - you can get the fixed script here:</div>
<div><br>
</div>
<div><a href="https://github.com/Ensembl/ensembl-tools/blob/release/74/scripts/variant_effect_predictor/gtf2vep.pl" target="_blank">https://github.com/Ensembl/ensembl-tools/blob/release/74/scripts/variant_effect_predictor/gtf2vep.pl</a></div>
<div><br>
</div>
<div>Let me know if this isn't convenient and I can get the fix pushed to our CVS tree too.</div>
<div><br>
</div>
<div>PS I assume you are aware we build a cache file for macaque already? <a href="ftp://ftp.ensembl.org/pub/release-74/variation/VEP/" target="_blank">ftp://ftp.ensembl.org/pub/release-74/variation/VEP/</a></div>
<div><br>
</div>
<div>Thanks again</div>
<div><br>
</div>
<div>Will McLaren</div>
<div>Ensembl Variation</div>
</div>
<div class="gmail_extra"><br>
<br>
<div class="gmail_quote">On 8 January 2014 05:38, Harris, Ronald Alan <span dir="ltr">
<<a href="mailto:rharris1@bcm.edu">rharris1@bcm.edu</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="PADDING-LEFT: 1ex; MARGIN: 0px 0px 0px 0.8ex; BORDER-LEFT: #ccc 1px solid">
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<div dir="ltr"><font color="#000000" face="Tahoma">Hi,</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">I have been trying to use <a href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a><a></a> to generate a cache file based on RhesusBase<a></a> (<a href="http://www.rhesusbase.org/" target="_blank">http://www.rhesusbase.org/</a>)
gene annotations on the UCSC<a></a> rheMac2<a></a>/Ensembl<a></a> MMUL_1<a></a> assembly. I downloaded their rb2<a></a> gene predictions as a gtf<a></a> file through their UCSC<a></a> mirror, changed the source column to "protein_coding<a></a>", added "exon_number<a></a>"
and the appropriate number in the description field, and sorted the annotations by chromosome position. The gtf<a></a> file can be downloaded from here:</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><a href="https://bigfile.bcm.edu/download.php?claimID=tnwUAesf9rRRH3u5&claimPasscode=B8mm8RNVZG4Ub6Xy&fid=52811&emailAddr=rharris1@bcm.edu" target="_blank">https://bigfile.bcm.edu/download.php?claimID=tnwUAesf9rRRH3u5&claimPasscode=B8mm8RNVZG4Ub6Xy&fid=52811&emailAddr=rharris1@bcm.edu</a></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">When I run <a href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a><a></a> I get this error:</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Can't call method "start" on an undefined value at <a href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a><a></a> line 376.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">This error occurs after generating some of the cache files in the .vep<a></a> directory. I tried to run <a href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a><a></a> using gtf<a></a> files with only a single chromosome
and it looks like the error consistently occurs when trying to generate the 1-1000000 cache file. Oddly, if I just run <a href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a><a></a> on the annotations from 1-1000000 on a single chromosome I do not get this
error.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">I don't think this is due to chr<a></a> in chromosome names because the fasta<a></a> file I am using has chr<a></a> in the chromosome names.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">I would appreciate any help you could give me with this.</font></div>
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Thanks,</font></div>
<span class="HOEnZb"><font color="#888888">
<div dir="ltr"><font face="tahoma"></font> </div>
<div dir="ltr"><font face="tahoma">Alan</font></div>
</font></span></div>
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