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Hi Will,<br>
<br>
Just as a follow up, the problem was the old version of the bioperl
which is in the INSTALL.pl.<br>
Re-installing VEP with a link to pointing to a newer version of
bioperl did result to the same output with my custom cache asyou got
with your run.<br>
<br>
Thanks for all your help,<br>
Heidi<br>
<br>
<div class="moz-cite-prefix">27.3.2013 8:53, Heidi Viitaniemi
kirjoitti:<br>
</div>
<blockquote cite="mid:51529786.2000503@utu.fi" type="cite">
<meta http-equiv="Content-Type" content="text/html;
charset=ISO-8859-1">
Hi Will,<br>
<br>
And thank you very much for looking into this. <br>
<br>
The reason why I want to build my own cahce from gtf and fasta of
the Gasterosteus_aculeatus is that on groupXIX the two last (of
the three ) supercontigs are flipped in the Ensembl genome
(Ross&Peichel 2008). I also first thougt that reverse
complementing the fasta and gtf for groupXIX was the problem but
ENSGACG00000003129 is located on a reagion that I didn't touch in
the gtf or the fasta.<br>
<br>
I'll check the versions of Bioperl in our server and try using
only the fasta and gtf for ENSGACG00000003129.<br>
<br>
Thanks,<br>
Heidi<br>
<br>
<br>
<div class="moz-cite-prefix">26.3.2013 16:04, Will McLaren
kirjoitti:<br>
</div>
<blockquote
cite="mid:CAMVEDX01YHiQeoJmKLuNEy9MaLg-y_FJeKxTQaiwQxxWBsffPA@mail.gmail.com"
type="cite">
<div dir="ltr">Hi Heidi,
<div><br>
</div>
<div>Thanks for your patience, I've had a chance to look at
this now.</div>
<div><br>
</div>
<div style="">If I build a cache file from the following
files:</div>
<div style=""><br>
</div>
<div style=""> <a moz-do-not-send="true"
href="ftp://ftp.ensembl.org/pub/release-70/gtf/gasterosteus_aculeatus/Gasterosteus_aculeatus.BROADS1.70.gtf.gz">ftp://ftp.ensembl.org/pub/release-70/gtf/gasterosteus_aculeatus/Gasterosteus_aculeatus.BROADS1.70.gtf.gz</a><br>
</div>
<div style=""><br>
</div>
<div style="">and</div>
<div style=""><br>
</div>
<div style=""><a moz-do-not-send="true"
href="ftp://ftp.ensembl.org/pub/release-70/fasta/gasterosteus_aculeatus/dna/Gasterosteus_aculeatus.BROADS1.70.dna.toplevel.fa.gz">ftp://ftp.ensembl.org/pub/release-70/fasta/gasterosteus_aculeatus/dna/Gasterosteus_aculeatus.BROADS1.70.dna.toplevel.fa.gz</a><br>
</div>
<div style=""><br>
</div>
<div style="">I get (I think!) the correct output from the
VEP:</div>
<div style=""><br>
</div>
<div style="">perl <a moz-do-not-send="true"
href="http://gtf2vep.pl">gtf2vep.pl</a> -i
Gasterosteus_aculeatus.BROADS1.70.gtf.gz -fasta
Gasterosteus_aculeatus.BROADS1.70.dna.toplevel.fa -species
gasterosteus_aculeatus -dir test/ -db 70<br>
</div>
<div style="">perl <a moz-do-not-send="true"
href="http://variant_effect_predictor.pl">variant_effect_predictor.pl</a>
-i gastero_in.txt -species gasterosteus_aculeatus -force
-off -dir test/ -db 70<br>
</div>
<div style="">grep -v # variant_effect_output.txt</div>
<div style=""><br>
</div>
<div style="">
<div>groupXIX_2822477_C/T groupXIX:2822477 T
ENSGACG00000003129 ENSGACT00000004109 Transcript
missense_variant 67 49 17 A/T
Gcg/Acg -</div>
<div>groupXIX_2822500_T/C groupXIX:2822500 C
ENSGACG00000003129 ENSGACT00000004109 Transcript
missense_variant 44 26 9 D/G
gAc/gGc -</div>
<div>groupXIX_2822523_C/T groupXIX:2822523 T
ENSGACG00000003129 ENSGACT00000004109 Transcript
initiator_codon_variant 21 3 1
M/I atG/atA -</div>
<div>groupXIX_2822541_T/A groupXIX:2822541 A
ENSGACG00000003129 ENSGACT00000004109 Transcript
5_prime_UTR_variant</div>
<div> 3 - - - - -</div>
</div>
<div style=""> <br>
</div>
<div style="">This works the same if I use the version 67
files as it appears you have.</div>
<div style=""><br>
</div>
<div style="">So I suspect there is something different about
your FASTA file - you could check that the sequence of the
groupXIX file matches that in the file I link to above (do
an md5sum or some such thing).</div>
<div style=""><br>
</div>
<div style="">It is also possible that an issue with older
versions of BioPerl is to blame - there was a known bug in
the way BioPerl indexes large FASTA file. Normally for
Ensembl we recommend using BioPerl 1.2.3 (which contains the
bug), but VEP works fine with the latest version. I'd try
updating your BioPerl install to the latest version, remove
the *.fa.index file that is generated next to your .fa file,
and try re-running <a moz-do-not-send="true"
href="http://gtf2vep.pl">gtf2vep.pl</a></div>
<div style=""><br>
</div>
<div style="">Beyond this it's hard to say what's happening
without seeing the contents of your GTF and FASTA files. If
the problem persists, perhaps you could just pull out the
lines in the GTF for ENSGACT00000004109 and the sequence for
groupXIX and if that still gives you the same problem, send
them to me so I can debug.</div>
<div style=""><br>
</div>
<div style="">Hope this helps!</div>
<div style=""><br>
</div>
<div style="">Will</div>
</div>
<div class="gmail_extra"><br>
<br>
<div class="gmail_quote">On 19 March 2013 12:07, Heidi
Viitaniemi <span dir="ltr"><<a moz-do-not-send="true"
href="mailto:hmviit@utu.fi" target="_blank">hmviit@utu.fi</a>></span>
wrote:<br>
<blockquote class="gmail_quote" style="margin:0 0 0
.8ex;border-left:1px #ccc solid;padding-left:1ex">
<div text="#000000" bgcolor="#FFFFFF"> Hi Will,<br>
<br>
And thank you for your response. I'll wait for the
solution. I like the idea that you can incorporate your
own data to run VEP.<br>
<br>
Thanks,<br>
Heidi Viitaniemi<br>
<br>
<br>
<br>
<div>19.3.2013 13:42, Will McLaren kirjoitti:<br>
</div>
<div>
<div class="h5">
<blockquote type="cite">
<div dir="ltr">Hello Heidi,
<div><br>
</div>
<div>Thanks for finding this - the causes of
this bug are I believe somewhat complex so may
take a while to get to the bottom of it.</div>
<div><br>
</div>
<div>Just wanted to let you know that your mail
is not being ignored!</div>
<div><br>
</div>
<div>Regards</div>
<div><br>
</div>
<div>Will McLaren</div>
<div>Ensembl Variation</div>
</div>
<div class="gmail_extra"><br>
<br>
<div class="gmail_quote">On 18 March 2013 13:48,
Heidi Viitaniemi <span dir="ltr"><<a
moz-do-not-send="true"
href="mailto:hmviit@utu.fi"
target="_blank">hmviit@utu.fi</a>></span>
wrote:<br>
<blockquote class="gmail_quote"
style="margin:0 0 0 .8ex;border-left:1px
#ccc solid;padding-left:1ex">
<div text="#000000" bgcolor="#FFFFFF"> Hi,<br>
<br>
I'm running version 2.7 on a unix server.
I want to create a custom cache using my
own gtf and fasta with <a
moz-do-not-send="true"
href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a>.
This works without problem and also
running VEP seems to go fine. The problem
is that, in the output it seems that the
cDNA_position, CDS_position and
Protein_position are correct given my
input gtf file but the calls for
Amino_acids and Codons seem completely
random. If I run against the cache
retrieved from ensembl these are all
correct. The version of the genome didn't
have an effect on the output, the gtf's
haven't changed. The gtf and the fasta
that I'm using for the custom originate
from the ensembl reference so I don't see
any reason why the custom cache shouldn't
perform the same way as the reference from
ensembl cache. Could there be bug that
somehow messes up the link between the
custom gtf and fasta in my run? Below are
the commands I ran and a snippet of the
output's I got.<br>
<br>
Thanks,<br>
Heidi Viitaniemi<br>
<br>
For custom cache I'm running (wrong output
for Amino_acids and Codons)<br>
perl <a moz-do-not-send="true"
href="http://gtf2vep.pl" target="_blank">gtf2vep.pl</a>
-i GasAcu1.67_group_xixflip.gtf -f
gasAcu_group_withoutbac_inv7.fa -d 67 -s
Gasterosteus_aculeatus_XIXflipped_18032013<br>
perl <a moz-do-not-send="true"
href="http://variant_effect_predictor.pl"
target="_blank">variant_effect_predictor.pl</a>
-offline 1 -dir $HOME/.vep -i
ens_realigned_AK_F.var.vcf -format vcf
-fork 4 -db_version 67 -species
Gasterosteus_aculeatus_XIXflipped_18032013
-numbers -per_gene -buffer_size 10000 -o
VEP_18032013_exon_pergene_AK_F.var.vcf.txt<br>
<br>
<table style="border-collapse:collapse"
width="1587" border="0" cellpadding="0"
cellspacing="0" height="89">
<colgroup><col style="width:125pt"
width="167"> <col
style="width:119pt" width="158"> <col
style="width:48pt" width="64"> <col
style="width:136pt" width="181"> <col
style="width:118pt" width="157"> <col
style="width:68pt" width="90"> <col
style="width:136pt" width="181"> <col
style="width:85pt" span="2"
width="113"> <col
style="width:48pt" span="6"
width="64"> <col
style="width:136pt" width="181"> </colgroup><tbody>
<tr style="min-height:15.0pt"
height="20">
<td
style="min-height:15.0pt;width:125pt"
width="167" height="20">groupXIX_2822477_C/T</td>
<td style="width:119pt" width="158">groupXIX:2822477</td>
<td style="width:48pt" width="64">T</td>
<td style="width:136pt" width="181">ENSGACG00000003129</td>
<td style="width:118pt" width="157">ENSGACT00000004109</td>
<td style="width:68pt" width="90">Transcript</td>
<td style="width:136pt" width="181">missense_variant</td>
<td style="width:48pt" width="64"
align="right">67</td>
<td style="width:48pt" width="64"
align="right">49</td>
<td style="width:48pt" width="64"
align="right">17</td>
<td style="width:48pt" width="64">G/R</td>
<td style="width:48pt" width="64">Gga/Aga</td>
<td style="width:48pt" width="64">-</td>
<td style="width:136pt" width="181">EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822500_T/C</td>
<td>groupXIX:2822500</td>
<td>C</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>missense_variant</td>
<td align="right">44</td>
<td align="right">26</td>
<td align="right">9</td>
<td>Y/C</td>
<td>tAt/tGt</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822523_C/T</td>
<td>groupXIX:2822523</td>
<td>T</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>synonymous_variant</td>
<td align="right">21</td>
<td align="right">3</td>
<td align="right">1</td>
<td>R</td>
<td>cgG/cgA</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822541_T/A</td>
<td>groupXIX:2822541</td>
<td>A</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>5_prime_UTR_variant</td>
<td align="right">3</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
</tbody>
</table>
<br>
<br>
For ensembl cache I'm running (correct
output for Amino_acids and Codons)<br>
perl <a moz-do-not-send="true"
href="http://variant_effect_predictor.pl"
target="_blank">variant_effect_predictor.pl</a>
-offline -dir $HOME/.vep -i
ens_realigned_AK_F.var.vcf -format vcf
-fork 4 -db_version 69 -species
gasterosteus_aculeatus -numbers -per_gene
-buffer_size 10000 -o
ensVEP_18032013_exon_pergene_AK_F.var.vcf.txt<br>
<br>
<span><font color="#888888"> </font></span><span><font
color="#888888"> </font></span><span><font
color="#888888"> </font></span>
<table
style="border-collapse:collapse;width:1101pt"
width="1468" border="0" cellpadding="0"
cellspacing="0">
<colgroup><col style="width:122pt"
width="163"> <col
style="width:92pt" width="122"> <col
style="width:48pt" width="64"> <col
style="width:109pt" width="145"> <col
style="width:107pt" width="143"> <col
style="width:117pt" width="156"> <col
style="width:170pt" width="227"> <col
style="width:48pt" span="7"
width="64"> </colgroup><tbody>
<tr style="min-height:15.0pt"
height="20">
<td
style="min-height:15.0pt;width:122pt"
width="163" height="20">groupXIX_2822477_C/T</td>
<td style="width:92pt" width="122">groupXIX:2822477</td>
<td style="width:48pt" width="64">T</td>
<td style="width:109pt" width="145">ENSGACG00000003129</td>
<td style="width:107pt" width="143">ENSGACT00000004109</td>
<td style="width:117pt" width="156">Transcript</td>
<td style="width:170pt" width="227">missense_variant</td>
<td style="width:48pt" width="64"
align="right">67</td>
<td style="width:48pt" width="64"
align="right">49</td>
<td style="width:48pt" width="64"
align="right">17</td>
<td style="width:48pt" width="64">A/T</td>
<td style="width:48pt" width="64">Gcg/Acg</td>
<td style="width:48pt" width="64">-</td>
<td style="width:48pt" width="64">EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822500_T/C</td>
<td>groupXIX:2822500</td>
<td>C</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>missense_variant</td>
<td align="right">44</td>
<td align="right">26</td>
<td align="right">9</td>
<td>D/G</td>
<td>gAc/gGc</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822523_C/T</td>
<td>groupXIX:2822523</td>
<td>T</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>initiator_codon_variant</td>
<td align="right">21</td>
<td align="right">3</td>
<td align="right">1</td>
<td>M/I</td>
<td>atG/atA</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
<tr style="min-height:15.0pt"
height="20">
<td style="min-height:15.0pt"
height="20">groupXIX_2822541_T/A</td>
<td>groupXIX:2822541</td>
<td>A</td>
<td>ENSGACG00000003129</td>
<td>ENSGACT00000004109</td>
<td>Transcript</td>
<td>5_prime_UTR_variant</td>
<td align="right">3</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>EXON=1/2</td>
</tr>
</tbody>
</table>
<span><font color="#888888"> <br>
<br>
<pre cols="72">--
______________________________________________
Heidi Viitaniemi
PhD student
Division of Genetics and Physiology
Department of Biology
Itäinen Pitkäkatu 4A, 7th floor (Pharmacity)
University of Turku
20520 Turku
FINLAND </pre>
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<pre cols="72">--
______________________________________________
Heidi Viitaniemi
PhD student
Division of Genetics and Physiology
Department of Biology
Itäinen Pitkäkatu 4A, 7th floor (Pharmacity)
University of Turku
20520 Turku
FINLAND </pre>
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<pre class="moz-signature" cols="72">--
______________________________________________
Heidi Viitaniemi
PhD student
Division of Genetics and Physiology
Department of Biology
Itäinen Pitkäkatu 4A, 7th floor (Pharmacity)
University of Turku
20520 Turku
FINLAND </pre>
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<pre class="moz-signature" cols="72">--
______________________________________________
Heidi Viitaniemi
PhD student
Division of Genetics and Physiology
Department of Biology
Itäinen Pitkäkatu 4A, 7th floor (Pharmacity)
University of Turku
20520 Turku
FINLAND </pre>
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