<html><head><style>body{font-family:Helvetica,Arial;font-size:13px}</style></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;">VEP can do this for you with the ProteinSeqs plugin [1]. I did not recommend it initially as I supposed you might be extracting sequences genome-wide.</div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;"><br></div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;">The plugin generates two files, reference.fa and mutated.fa, containing one mutated sequence *per variant*. The sequences should be accurate WRT to the modifications made via the BAM files. I can’t say from experience how well this plugin will perform on a genome-wide analysis; you could expect that the resultant FASTA files would be very large at least.</div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;"><br></div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;">The plugin does not currently support outputting cDNAs or CDS, but it would be fairly straightforward to add this functionality to the plugin.</div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;"><br></div><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;">Note that if you wish to incorporate the effects of multiple variants together, you can look into using VEP’s sister tool Haplosaurus [2]; this can output CDS and protein sequences in its JSON format output.</div> <div><br></div>Cheers<div><br></div><div>Will</div><div><br></div><div>[1] https://github.com/Ensembl/VEP_plugins/blob/release/90/ProteinSeqs.pm</div><div>[2] https://github.com/Ensembl/ensembl-vep#haplo<br> <div id="bloop_sign_1512475937958374144" class="bloop_sign"></div> <br><p class="airmail_on">On 5 December 2017 at 12:05:41 pm, Luke Goodsell (<a href="mailto:l.goodsell@achillestx.com">l.goodsell@achillestx.com</a>) wrote:</p> <blockquote type="cite" class="clean_bq"><span><div bgcolor="white" lang="EN-GB" link="blue" vlink="purple"><div></div><div>
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<p class="MsoNormal">Thanks, Will,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Unfortunately, I cannot find cDNA from RefSeq – their sequences contain UTRs. Is there an easy way to identify the start and stop codons? The longest ORF is not always the correct one, unfortunately.<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Incidentally, being able to get the sequences used by VEP is very important for us; we’re trying to construct the new protein sequences that result from variants using consequence information annotated by VEP. We’d very much appreciate
the corrected sequences being incorporated into the otherfeatures database as soon as possible.
<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="color:black">Kind regards,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:black">Luke</span><o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><b><span style="font-size:12.0pt;color:black">From: </span></b><span style="font-size:12.0pt;color:black">William McLaren <wm2@ebi.ac.uk><br>
<b>Date: </b>Tuesday, 5 December 2017 at 09:15<br>
<b>To: </b>Luke Goodsell <l.goodsell@achillestx.com>, Ensembl developers list <dev@ensembl.org>, Alessandro Vullo <avullo@ebi.ac.uk><br>
<b>Subject: </b>Re: [ensembl-dev] GRCh37 Protein sequence has asterisks<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">Hi Luke,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">There is no straightforward way to do this via Ensembl at the moment; I’d suggest you download the relevant files from NCBI.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">The BAM files we use are obtained from
<a href="ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens/GRCh37.p13_interim_annotation/">
ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens/GRCh37.p13_interim_annotation/</a>; it seems there’s a protein and rna FASTA file in there which may have what you need.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">Otherwise you may find what you need in the parent directory <a href="ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens">ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens</a>.
I’m not familiar with NCBI’s FASTA layout so you’d have to investigate yourself!<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">Regards<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">Will McLaren<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Helvetica",sans-serif">Ensembl Variation<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="airmailon">On 4 December 2017 at 5:55:38 pm, Luke Goodsell (<a href="mailto:l.goodsell@achillestx.com">l.goodsell@achillestx.com</a>) wrote:<o:p></o:p></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black">Hi Allessandro,<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black">Is there a way to extract the BAM-edited sequences? I'd simply like to get FASTA files of the RefSeq cDNA and proteins as used by VEP.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-family:"Helvetica",sans-serif;color:black">Kind regards,<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black">Luke<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black"><br>
<br>
<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-family:"Helvetica",sans-serif;color:black">From: Alessandro Vullo<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-family:"Helvetica",sans-serif;color:black">Sent: Monday, 4 December, 17:44<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-family:"Helvetica",sans-serif;color:black">Subject: Re: [ensembl-dev] GRCh37 Protein sequence has asterisks<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black">To: Ensembl developers list, Luke Goodsell<br>
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<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><span style="font-family:"Helvetica",sans-serif;color:black">Hi Luke, The problem is likely to depend on RefSeq differing from the reference. Are you using VEP and then retrieving the sequence as annotated by
it? Quoting the relevant people (VEP): "VEP uses BAMs to correct RefSeqs that differ from the reference, and without those the API can give incorrect translations. This will hopefully be fixed in future when the SeqEdit objects that VEP creates from the BAMs
are incorporated directly into the otherfeatures DB." Hope that helps, Alessandro<o:p></o:p></span></p>
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