<html><head><meta http-equiv="Content-Type" content="text/html charset=iso-8859-1"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Hi Marc,<br><div><br><div><div>On 29 Aug 2013, at 08:09, Marc Hoeppner <<a href="mailto:mphoeppner@gmail.com">mphoeppner@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite">
  
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    <div class="moz-cite-prefix">Hi Thibaut et al, <br>
      <br>
      sorry to bother you guys again - last question(s), I promise. <br>
      <br>
      1) Using WU-Blast<br>
      <br>
      I have been trying to switch to wublast to remove a bit of
      overhead from setting up the pipeline with NCBI. However, when
      following the documentation, I get this as a command line call:<br>
      <br>
      wublastp /references/databases/uniprot_swissprot/uniprot.fasta
      /tmp/seq.9741.42380.fa -gi -cpus=1<br>
      <br>
      The thing is that my version of Wu-blast (BLASTP 2.0a19MP-WashU
      [14-Jul-1998] [Build linux-x86 18:51:39 30-Jul-1998]) does not
      have a '-cpus' flag and I didn't set it. Hence, it fails.<br>
      <br>
      The flag is set within within the Blast module(s) automatically,
      like in Analysis::Runnable::Blast.pm:<br>
      <br>
      $self->options('-cpus=1') if(!$self->options);<br></div></div></blockquote>This is probably a bug, You can simply delete/comment this line. I will have a look.<br><blockquote type="cite"><div bgcolor="#FFFFFF" text="#000000"><div class="moz-cite-prefix">
      <br>
      So I am guessing you guys must be using some other version of
      Wu-Blast? I can't find any other than the one I got, since the
      original code from <a class="moz-txt-link-freetext" href="http://blast.wustl.edu/">http://blast.wustl.edu/</a> is now hosted elsewhere
      and the only legacy version of their blast suite (Wu-blast) is the
      one I downloaded. <br>
      <br>
      Any idea where I can get the version used by EnsEMBL? Or is it the
      same and the error lies elsewhere?<br>
      <br>
      2) Having to specify '-p blastn' as a parameter for DNA-Peptide
      blasts when running the NCBI suite.<br>
      (This is from a conversation with Thibaut directly - the pipeline
      fails to determine the right blastall program to use when running
      DNA/Peptide blasts)<br></div></div></blockquote>I don't see it as problem, when you prepare your analysis you just need to specify it in the config file.</div><div><br></div><div><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">[unigene] </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">db=unigene </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">db_file=/data2/projects/annotation/EnsEMBL/chicken/refseqs/unigene.fa </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">program=blastall </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">program_file=blastall </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">parameters=-p blastn -A 40 -a 1</span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">module=BlastGenscanDNA </span><br style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">input_id_type=CONTIG </span></div><div><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); "><br></span></div><div><span style="font-family: -moz-fixed; font-size: 12px; background-color: rgb(255, 255, 255); ">The line parameters contains program specific parameters. I'm not sure of the -A 40 but it seems to me to be similar to -hitdist 40</span></div><div><font face="-moz-fixed"><br></font></div><div><font face="-moz-fixed">I suppose that we were using NCBI blast for the protein alignment and WU blast for the dna alignment but I cannot say for sure.</font></div><div><font face="-moz-fixed"><br></font></div><div><font face="-moz-fixed">You can modify our code to suit your needs.</font></div><div><font face="-moz-fixed"><br></font></div><div><font face="-moz-fixed">I will look into it later.</font></div><div><font face="-moz-fixed"><br></font></div><div><font face="-moz-fixed">Regards</font></div><div><font face="-moz-fixed">Thibaut</font></div><div><font face="-moz-fixed"><br></font><blockquote type="cite"><div bgcolor="#FFFFFF" text="#000000"><div class="moz-cite-prefix">
      <br>
      So I had a look at the code, since I still didn't understand why
      BlastGenscanPep.pm would automatically figure out which blastall
      programm (blastall -p blastp ) to use and BlastGenscanDna.pm
      wouldn't . Seems like this descision process is coded into the
      BlastGenscanPep.pm module, but was left out of BlastGenscanDna.pm.
      I wonder why that is? Makes using NCBI for the pipeline a little
      awkward (which I wouldn't mind if I could get the stupid Wu-Blast
      to work ;))<br>
      <br>
      From BlastGenscanPep:<br>
      <br>
      if ( $blast{-type}=~m/ncbi/ ) {<br>
          if ( $options{-query_type}=~m/pep/ ) {<br>
            if ( $options{-database_type}=~m/pep/ ) {<br>
                 $options_string = '-p blastp' ;  <br>
            } elsif ( $options{-database_type}=~m/dna/ ) {<br>
               $options_string = '-p tblastn' ;<br>
            }<br>
          }<br>
      <br>
          if ( $options{-query_type}=~m/dna/ ) {<br>
            if ( $options{-database_type}=~m/dna/ ) {<br>
                 $options_string = '-p blastn' ;<br>
            }elsif ( $options{-database_type}=~m/pep/ ) {<br>
                 $options_string = '-p blastx' ;<br>
            }<br>
          }<br>
        }<br>
      <br>
      <br>
      <br>
      Cheers,<br>
      <br>
      Marc<br>
    </div>
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      Hi Marc,
      <div><br>
        <div>
          <div>On 28 Aug 2013, at 14:35, Marc Hoeppner <<a moz-do-not-send="true" href="mailto:mphoeppner@gmail.com">mphoeppner@gmail.com</a>>
            wrote:</div>
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              <div class="moz-cite-prefix">Hi Thibaut,<br>
                <br>
                thanks for the help, been able to fix my problems. <br>
                <br>
                Turns out that yes, the coordinate system rank seems to
                matter - but I think the problem was that Pmatch was
                configured to write to the GENEWISE_DB, which existed
                and had tables and everything, but wasn't loaded with
                any data (such as a coordinate system). <br>
              </div>
            </div>
          </blockquote>
          Whenever you write in a database using the Genebuild pipeline
          you need to check that some tables are in sync with your
          reference. Here are the tables:</div>
        <div>analysis</div>
        <div>assembly</div>
        <div>assembly_exception</div>
        <div>coord_system</div>
        <div>seq_region</div>
        <div>seq_region_attrib</div>
        <div>meta</div>
        <div>attrib_type</div>
        <div>seq_region_synonym</div>
        <div>
          <div><br>
          </div>
        </div>
        <div><br>
          <blockquote type="cite">
            <div bgcolor="#FFFFFF" text="#000000">
              <div class="moz-cite-prefix"> <br>
                For the unigene blast, adding the blastall program
                option manually into the parameter string solved that
                problem, but I still wonder if there is a bug in the
                code (it should do this automatically).<br>
              </div>
            </div>
          </blockquote>
          As it is an blastall option and can be set in the analysis
          configuration I don't see it as a bug</div>
        <div><br>
        </div>
        <div>Regards</div>
        <div>Thibaut</div>
        <div> <br>
          <blockquote type="cite">
            <div bgcolor="#FFFFFF" text="#000000">
              <div class="moz-cite-prefix"> <br>
                Anyway, thanks again!<br>
                <br>
                /Marc<br>
              </div>
              <blockquote cite="mid:30269147-DC7E-41DE-8828-14B070152902@sanger.ac.uk" type="cite">
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                Hi Marc,
                <div><br>
                  <div>
                    <div>On 27 Aug 2013, at 13:53, Marc Hoeppner <<a moz-do-not-send="true" href="mailto:mphoeppner@gmail.com">mphoeppner@gmail.com</a>>

                      wrote:</div>
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                        <div class="moz-cite-prefix">Hi Thibaut,<br>
                          <br>
                          thanks for the feedback. Answers to your
                          comments in line:<br>
                          <br>
                          <br>
                        </div>
                        <blockquote cite="mid:8721613D-12DB-4AA8-8B36-141F116838B6@sanger.ac.uk" type="cite">
                          <meta http-equiv="Content-Type" content="text/html; charset=ISO-8859-1">
                          Hi Marc,
                          <div><br>
                            <div>
                              <div>On 26 Aug 2013, at 10:59, Marc
                                Hoeppner <<a moz-do-not-send="true" href="mailto:mphoeppner@gmail.com">mphoeppner@gmail.com</a>>


                                wrote:</div>
                              <br class="Apple-interchange-newline">
                              <blockquote type="cite">
                                <meta http-equiv="content-type" content="text/html;
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                                  <div class="moz-text-flowed" style="font-family: -moz-fixed;
                                    font-size: 12px;" lang="x-western">Hi


                                    EnsEMBL team, <br>
                                    <br>
                                    been playing with the pipeline
                                    again, but am having problems
                                    (again). Please see below for
                                    details - am happy about any
                                    suggestions. <br>
                                    <br>
                                    Cheers, <br>
                                    <br>
                                    Marc <br>
                                    <br>
                                    ######## <br>
                                    1) Pmatch <br>
                                    ######## <br>
                                    <br>
                                    I set up a pmatch analysis as by the
                                    documentation and it runs fine on my
                                    test dataset (small chicken
                                    chromosome) when I try it with
                                    test_RunnableDB. However, when I run
                                    the pipeline, I get this: <br>
                                    <br>
                                    TARGET  0.064u 0.008s 0+0k 0pf 0sw <br>
                                    BUILD   0.116u 0.040s 0+0k 0pf 0sw <br>
                                    SEARCH  22.949u 0.172s 0+0k 0pf 0sw
                                    <br>
                                    WARN: For multiple species use
                                    species attribute in
                                    DBAdaptor->new() <br>
                                    WRITING: Lost the will to live Error
                                    <br>
                                    Job 1198 failed: [ <br>
                                    -------------------- EXCEPTION
                                    -------------------- <br>
                                    MSG: Problems for Pmatch writing
                                    output for
                                    chromosome:vchicken_test:10:1:19911089:1
                                    [Can't call method "version" on an
                                    undefined value at
                                    /opt/bioinformatics/ensembl-70/ensembl/modules/Bio/EnsEMBL/DBSQL/MetaContainer.pm



                                    line 218. <br>
                                    ] <br>
                                    STACK
                                    Bio::EnsEMBL::Pipeline::Job::run_module
/opt/bioinformatics/ensembl-70/ensembl-pipeline/modules/Bio/EnsEMBL/Pipeline/Job.pm:720<br>
                                    STACK (eval)
/opt/bioinformatics/ensembl-70/ensembl-pipeline/modules/Bio/EnsEMBL/Pipeline/runner.pl:219<br>
                                    STACK main::run_jobs_with_lsfcopy
/opt/bioinformatics/ensembl-70/ensembl-pipeline/modules/Bio/EnsEMBL/Pipeline/runner.pl:218<br>
                                    STACK toplevel
/opt/bioinformatics/ensembl-70/ensembl-pipeline/modules/Bio/EnsEMBL/Pipeline/runner.pl:128<br>
                                    Date (localtime)    = Fri Aug 23
                                    14:53:27 2013 <br>
                                    Ensembl API version = 70 <br>
                                    <br>
                                  </div>
                                </div>
                              </blockquote>
                              We would need to see how your coord_system
                              and meta tables are populated.</div>
                            <div>The API complains that it can't find
                              the version of your assembly. Your
                              coord_system table should look like this
                              one:</div>
                            <div>
                              <div>+-----------------------+----------------+------------------+------------+-------+--------------------------------+</div>
                              <div>| coord_system_id | species_id | name
                                              | version   | rank |
                                attrib                         |</div>
                              <div>+-----------------------+----------------+------------------+------------+-------+--------------------------------+</div>
                              <div>|                             1 |    
                                              1 | contig              |
                                NULL      |       3 |
                                default_version,sequence_level |</div>
                              <div>|                             2 |    
                                              1 | scaffold           |
                                oryCun2 |       2 | default_version    
                                           |</div>
                              <div>|                             3 |    
                                              1 | chromosome | oryCun2 |
                                      1 | default_version              
                                 |</div>
                              <blockquote type="cite">
                                <div bgcolor="#FFFFFF" text="#000000">
                                  <div class="moz-text-flowed" style="font-family: -moz-fixed;
                                    font-size: 12px;" lang="x-western">
                                    <br>
                                  </div>
                                </div>
                              </blockquote>
                            </div>
                          </div>
                        </blockquote>
                        This is my coord_system table:<br>
                        <br>
+-----------------+------------+------------+---------------+------+--------------------------------+<br>
                        | coord_system_id | species_id | name       |
                        version       | rank |
                        attrib                         |<br>
+-----------------+------------+------------+---------------+------+--------------------------------+<br>
                        |               1 |          1 | chromosome |
                        vchicken_test |    1 |
                        default_version                |<br>
                        |               2 |          1 | contig     |
                        vchicken_test |    3 |
                        default_version,sequence_level |<br>
+-----------------+------------+------------+---------------+------+--------------------------------+<br>
                        <br>
                        <br>
                        I don't have a supercontig layer, since I am
                        faking contigs from assembled sequences for
                        testing purposes. I think I had that discussed
                        over this mailing list as well and was told that
                        the API code should be able to deal with a
                        contig-chromosome setup. Anything suspicious
                        here?<br>
                      </div>
                    </blockquote>
                    I'm not sure that this is the problem but you should
                    change the rank of your contig, set it to 2. The API
                    might be looking for the coordinate system with the
                    rank 2 and fails to find it at the moment.</div>
                  <div><br>
                    <blockquote type="cite">
                      <div bgcolor="#FFFFFF" text="#000000">
                        <blockquote cite="mid:8721613D-12DB-4AA8-8B36-141F116838B6@sanger.ac.uk" type="cite">
                          <div>
                            <div>
                              <blockquote type="cite">
                                <div bgcolor="#FFFFFF" text="#000000">
                                  <div class="moz-text-flowed" style="font-family: -moz-fixed;
                                    font-size: 12px;" lang="x-western">
                                    ########## <br>
                                    2) Unigene <br>
                                    ########## <br>
                                    <br>
                                    This one really bothers me <span class="moz-smiley-s3" title=";)"></span>
                                    I think everything is set up
                                    correctly (downloaded the unigene
                                    file, header seems to comply with
                                    the reference formatting in Blast.pm
                                    etc), bit I cannot for the life of
                                    me get it to work. Specifically, I
                                    am trying to use ncbi blast and the
                                    command just looks off - seems like
                                    it tries to do a mix of Wublast and
                                    Ncbi blast (works fine with Uniprot
                                    though - so perhaps something with
                                    the BlastGenscanDna module?). <br>
                                    <br>
                                    Running job 1791 <br>
                                    Module is BlastGenscanDNA <br>
                                    Input id is
                                    contig:vchicken_test:10_68:1:50000:1
                                    <br>
                                    Analysis is unigene <br>
                                    Files are
                                    /data2/projects/annotation/EnsEMBL/chicken/output//unigene/0/contig:vchicken_test:10_114:1:50000:1.unigene.55.retry2.out
/data2/projects/annotation/EnsEMBL/chicken/output//unigene/0/contig:vchicken_test:10_114:1:50000:1.unige$



                                    <br>
                                    <br>
                                    -------------------- WARNING
                                    ---------------------- <br>
                                    MSG: Error running Blast cmd
                                    </usr/bin/blastall -d
                                    /data2/projects/annotation/EnsEMBL/chicken/refseqs/unigene.fa
                                    -i /tmp/seq.22305.24863.fa -cpus=1
                                    2>&1 >
                                    /tmp/unigene.fa.22305.5651.blast.out>.
                                    Returned error 256 BLAST EXIT: '1',
                                    SIGNA$ <br>
                                    FILE: Analysis/Runnable/Blast.pm
                                    LINE: 380 <br>
                                    CALLED BY:
                                    EnsEMBL/Analysis/Runnable.pm  LINE:
                                    729 <br>
                                    Date (localtime)    = Fri Aug 23
                                    14:54:47 2013 <br>
                                    Ensembl API version = 70 <br>
                                  </div>
                                </div>
                              </blockquote>
                              Have you tried to run the command by
                              itself to see if it works? The error
                              message you have seems to be from the ncbi
                              blast program.</div>
                            <div>As the module dies the temporary file
                              containing your chicken sequence should
                              still exists. If not, you will need to
                              comment a line in the run method of
ensembl-analysis/modules/Bio/EnsEMBL/Analysis/Runnable.pm:</div>
                            <div><br>
                            </div>
                            <div>  #$self->delete_files;</div>
                            <div><br>
                            </div>
                            <div>You probably need to change your
                              parameters in the analysis table of your
                              reference database. We use WU blast at the
                              moment.</div>
                            <div><br>
                            </div>
                            <div>Also, the parameters for blast should
                              be "-cpus 1 -hitdist 40" instead of "<span style="font-family: -moz-fixed;
                                font-size: 12px; ">-cpus => 1,
                                -hitdist => 40"</span></div>
                            <div><br>
                            </div>
                            <div>Regards</div>
                            <div>Thibaut</div>
                            <div><br>
                            </div>
                          </div>
                        </blockquote>
                        I think the problem is that the blastall string
                        is mal-formatted. It should be<br>
                        <br>
                        blastall -i input.fasta -d database -p blastn <br>
                        <br>
                        So it failed to determine which blast program to
                        use. Interestingly, it works fine for
                        protein-protein blast, but fails in this
                        protein-dna configuration. Hence my question
                        whether this may be a problem in the
                        BlastGenscanDna module. I can try wublast also,
                        but I think I had serious trouble getting that
                        to work. Are you guys calling your executables
                        wublastp, wublastn etc? Because the only thing I
                        could find was blastp, blastn etc. I assume this
                        would still work if I specify these binary names
                        in the configs..? Gave up at some point because
                        it keep whining about something, so went the
                        ncbi route..<br>
                      </div>
                    </blockquote>
                    Maybe blast look at the database to know which type
                    of search it will do by default.<br>
                    For the moment you need to change the parameters and
                    add '-p blastn' and it should work.</div>
                  <div><br>
                  </div>
                  <div>If your blastn is similar to blastall -p blastn
                    then you can change your program to be blastn and
                    you don't need to add '-p blastn' to your parameters<br>
                    <blockquote type="cite">
                      <div bgcolor="#FFFFFF" text="#000000"> <br>
                        Oh and thanks for pointing out the parameter
                        issue, I actually took those from the
                        documentation, sooo... ;) But will update my
                        scripts. <br>
                      </div>
                    </blockquote>
                    Unfortunately the documentation available is a bit
                    old and updating it take a lot of time.</div>
                  <div><br>
                  </div>
                  <div>Regards</div>
                  <div>Thibaut</div>
                  <div><br>
                    <blockquote type="cite">
                      <div bgcolor="#FFFFFF" text="#000000"> <br>
                        All the best,<br>
                        <br>
                        Marc<br>
                        <br>
                        <br>
                        <blockquote cite="mid:8721613D-12DB-4AA8-8B36-141F116838B6@sanger.ac.uk" type="cite">
                          <div>
                            <div>
                              <blockquote type="cite">
                                <div bgcolor="#FFFFFF" text="#000000">
                                  <div class="moz-text-flowed" style="font-family: -moz-fixed;
                                    font-size: 12px;" lang="x-western">
                                    <br>
                                    And here the config for the unigene
                                    search: <br>
                                    <br>
                                    [unigene] <br>
                                    db=unigene <br>
                                    db_file=/data2/projects/annotation/EnsEMBL/chicken/refseqs/unigene.fa



                                    <br>
                                    program=blastall <br>
                                    program_file=blastall <br>
                                    parameters=-cpus => 1, -hitdist
                                    => 40 <br>
                                    module=BlastGenscanDNA <br>
                                    input_id_type=CONTIG <br>
                                    <br>
                                    (Blast.pm is configured to use
                                    'ncbi' as default type, so unigene
                                    should inherit that, no?)<br>
                                    <br>
                                  </div>
                                </div>
                              </blockquote>
                              <blockquote type="cite">
                                <div bgcolor="#FFFFFF" text="#000000">
                                  <div class="moz-text-flowed" style="font-family: -moz-fixed;
                                    font-size: 12px;" lang="x-western">
                                    <br>
                                    <div class="moz-txt-sig"><span class="moz-txt-tag">-- <br>
                                      </span>Marc P. Hoeppner, PhD <br>
                                      Department of Medical Biochemistry
                                      and Microbiology <br>
                                      Uppsala University, Sweden <br>
                                      <a moz-do-not-send="true" class="moz-txt-link-abbreviated" href="mailto:marc.hoeppner@imbim.uu.se">marc.hoeppner@imbim.uu.se</a> <br>
                                      <br>
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              </blockquote>
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            </div>
          </blockquote>
        </div>
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