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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>
                    </blockquote>
                    <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>
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      <br>
      <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>
    </blockquote>
    <br>
    <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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