<span id="mailbox-conversation"><div>Hi all,</div>
<div><br></div>
<div>Developer of LOFTEE here - I've seen this kind of thing before (Issue #2). The issue is actually with the FASTA index file created by VEP/BioPerl. When you're in online mode, it's getting the right sequence of the splice site, but when offline with a malformed index, it always returns NN resulting in many NON_CAN_SPLICE and NON_CAN_SPLICE_SURR annotations.</div>
<div><br></div>
<div>I suggest deleting the Homo_sapiens.GRCh37.75.dna.primary_assembly.fa.index file and recreating it: to do this, just run VEP on a small test file. Important note: you must let VEP run to completion, even though Checking/Creating FASTA Index is near the beginning and it starts writing one at that time, it can be a corrupt index if you cancel it at that point. I typically just annotate a single variant so it finishes quickly. Don't ask how I figured all this out...</div>
<div><br></div>
<div>Hope that helps!</div></span><div class="mailbox_signature">
<br>-Konrad</div>
<br><br><div class="gmail_quote"><p>On Fri, Oct 9, 2015 at 4:40 AM, Will McLaren <span dir="ltr"><<a href="mailto:wm2@ebi.ac.uk" target="_blank">wm2@ebi.ac.uk</a>></span> wrote:<br></p><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex;"><div>
<div dir="ltr">Hi Alex,<div><br></div>
<div>Regarding issue 1, have you considered using VCF output instead of the default tab-delimited output? <a href="http://www.ensembl.org/info/docs/tools/vep/vep_formats.html#vcfout">http://www.ensembl.org/info/docs/tools/vep/vep_formats.html#vcfout</a>
</div>
<div><br></div>
<div>Have you tried contacting the VAX authors? Michael Yourshaw is usually very responsive when I have communicated with him in the past.</div>
<div><br></div>
<div>I'm sure you can appreciate we have to prioritise debugging and fixing our own code, but please do get back to us if you still have any outstanding issues.</div>
<div><br></div>
<div>You may also like to try another available LoF plugin, LOFTEE from Daniel MacArthur's lab: <a href="https://github.com/konradjk/loftee">https://github.com/konradjk/loftee</a>
</div>
<div><br></div>
<div>Regards</div>
<div><br></div>
<div>Will McLaren</div>
<div>Ensembl Variation</div>
</div>
<div class="gmail_extra">
<br><div class="gmail_quote">On 9 October 2015 at 03:22, Alex Beesley <span dir="ltr"><<a href="mailto:Alex.Beesley@telethonkids.org.au">Alex.Beesley@telethonkids.org.au</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
<div style="word-wrap:break-word;color:rgb(0,0,0);font-size:14px;font-family:Calibri,sans-serif">
<div>
<div>
<div>Dear Team</div>
</div>
</div>
<span>
<div style="word-wrap:break-word">
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
<br></div>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
I am experiencing significant difficulties with both the LoF.pm and VCFCols.pm plugins with VEP (FYI I am using a GRCh37 cache downloaded using the installer script and default settings (ensembl-tools release-82)).</div>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
<br></div>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
# Issue 1</div>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
I want to use VCFCols.pm in order to obtain the original REF and ALT alleles from the VCF (to aid with interpretation of complex variants). However it seems that the only way to run VCFCols.pm plugin is in the online mode – if one tries to run it in offline
mode (see first code example below), VEP returns an error relating to "$config->{ga}->fetch_by_transcript_stable_id($transcript_id)”. However, when running online (see second code example), it is extremely slow. This is incredibly frustrating because I do
not wish to use any of the VAX functionality or its related databases, I simply wish to grab the original REF, ALT and other VCF column headers (including the genotypes and FORMAT fields) in my VEP output. Is there another way to grab the original VCF columns
in the VEP output other than using VCFCols.pm? Or a way to modify the plugin such that it can work offline?</div>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
<br></div>
<div>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
perl ${VEP}/<a href="http://variant_effect_predictor.pl">variant_effect_predictor.pl</a> -i ${INPUT_VCF} -o ${INPUT_VCF%*.vcf}.vep --cache --assembly GRCh37 --offline \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--force_overwrite --check_existing --fork 24 \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--everything --flag_pick \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin CADD,${CADD_SNV},${CADD_INDEL} \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin ExAC,${EXAC} \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<span style="white-space:pre-wrap"></span>—-plugin VCFCols \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin LoF,human_ancestor_fa:/home/san/alex/.vep/Plugins/loftee-master/human_ancestor.fa.gz \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--fields Uploaded_variation,Location,REF,ALT,INFO,FORMAT,LoF,LoF_filter,LoF_flags,CADD_RAW,CADD_PHRED,ExAC_AF</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
perl ${VEP}/<a href="http://variant_effect_predictor.pl">variant_effect_predictor.pl</a> -i ${INPUT_VCF} -o ${INPUT_VCF%*.vcf}.ONLINE.vep --cache --assembly GRCh37 --port 3337 \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--force_overwrite --check_existing --fork 24 \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--everything --flag_pick \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin CADD,${CADD_SNV},${CADD_INDEL} \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin ExAC,${EXAC} \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<span style="white-space:pre-wrap"></span>—-plugin VCFCols \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--plugin LoF,human_ancestor_fa:/home/san/alex/.vep/Plugins/loftee-master/human_ancestor.fa.gz \</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
--fields Uploaded_variation,Location,REF,ALT,INFO,FORMAT,LoF,LoF_filter,LoF_flags,CADD_RAW,CADD_PHRED,ExAC_AF</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<span style="font-family:Calibri,sans-serif;font-size:14px"># Issue 2</span></p>
<div style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14px">
When running VEP in either of the two modes shown above, I obtain different confidence calls from the LoF.pm in regards to frameshift mutations. Specifically, for the example shown below, the LoF.pm plugin will call the variant HC (high confidence) in ONLINE
mode, but LC (low confidence) when running offline. The particular flag thrown up for the LC call relates to non-canonical intron splice sites, however I have checked this particular variant on UCSC and the splice appear to be canonical, thus the ONLINE vep
output is correct, and the offline appears to be incorrect. Since I am using a local cache (and I have also tried using a local fasta file), I am at a loss to explain why I would get completely different results by these two approaches for a LoF call. As mentioned
above, my cache was downloaded using the installer script and default settings (ensembl-tools release-82).</div>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
# Running Offline</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
#Uploaded_variation Consequence IMPACT LoF</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
10_126691951_C/- - 10:126691951 - frameshift_variant HIGH LC NON_CAN_SPLICE_SURR </p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
10_126692023_G/- - 10:126692023 - frameshift_variant HIGH LC NON_CAN_SPLICE_SURR</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
# Running Online</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
#Uploaded_variation Consequence IMPACT LoF</p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
10_126691951_C/- - 10:126691951 - frameshift_variant HIGH HC </p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
10_126692023_G/- - 10:126692023 - frameshift_variant HIGH HC </p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
<br></p>
<p style="margin:0px"><font face="Calibri,sans-serif">I</font><font face="Calibri,sans-serif"> appreciate that neither VCFCols.pm nor LoF.pm were developed by your team, but I would be very grateful if you could help me on these issues as I have been struggling
to get VEP customised for my needs for some time now. In regards to issue 1, I believe a lot of your users would benefit from a tool that could grab the original VCF headers in the VEP output, and in regards to the second issue, there must be something strange
going on in regards to compatibility with the downloaded caches and the online databases but I am at a loss to explain it.</font></p>
<p style="margin:0px"><font face="Calibri,sans-serif"><br></font></p>
<p style="margin:0px"><font face="Calibri,sans-serif">Many thanks in advance</font></p>
<p style="margin:0px"><font face="Calibri,sans-serif">Alex Beesley</font></p>
<p style="color:rgb(0,0,0);font-family:Menlo;font-size:11px;margin:0px">
Telethon Kids Institute</p>
</div>
</div>
</span>
<div>Perth, Western Australia</div>
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