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<p>Dear Kenneth,</p>
<p>Unfortunately we changed significantly the VEP code (and it's
structure) between the version 87 and 88 and we added the BAM-edit
option in version 90 so it won't be easy to find a way to make it
works with VEP version 80.</p>
<p>The RefSeq data has been updated few times since the freeze of
the Ensembl geneset on GRCh37 (version 75) and we regularly update
the Variation data on this assembly.</p>
<p>The "new" VEP (from version 88) is more robust: <a
moz-do-not-send="true"
href="http://www.ensembl.info/2016/12/13/unwrap-the-new-vep-for-christmas/">http://www.ensembl.info/2016/12/13/unwrap-the-new-vep-for-christmas/</a>
and the Ensembl/VEP version 92 has more variant annotations, so I
would recommend to upgrade to the release 92.</p>
<p><br>
Best regards,<br>
</p>
<pre class="moz-signature" cols="72">Laurent</pre>
<div class="moz-cite-prefix">On 14/05/2018 09:00, Kenneth Wong
wrote:<br>
</div>
<blockquote type="cite"
cite="mid:CAHSPU_tQ-QuayBnKX5vLEwn6TDqfNv=-cq9vu+EPuf6ei6GCfw@mail.gmail.com">
<div dir="ltr">Dear Laurent,
<div><br>
</div>
<div>I try to run below command with VEP version 88 (and cache
v88). It can fix the mentioned issue successfully. <br>
</div>
<div><br>
</div>
<div>
<pre class="gmail-code gmail-sh_sh" style="font-family:"Courier New",Courier,monospace;font-size:16px;margin:0px 0px 16px;border:1px solid rgb(204,204,204);background:rgb(240,240,240);overflow:auto;line-height:16px;color:rgb(102,102,102);font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial;padding:8px">./vep -cache -refseq -i variants.vcf --bam interim_GRCh37.p13_knownrefseq_alignments_2017-01-13.bam</pre>
</div>
<div class="gmail_extra"><br>
</div>
<div class="gmail_extra">As t<span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">he
VEP version used in our project is version 80</span>, I try
running the above command in version 80 but it failed due to
unsupported option "--bam". <br
class="gmail-Apple-interchange-newline">
<span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">To
avoid upgrading VEP (code & cache) from version 80 to 92
which will involve tremendous amount of testing work, would
you please advice any workaround approach to use </span>BAM-edited
transcript models in version 80 ?</div>
<div class="gmail_extra"><br>
</div>
<div class="gmail_extra">If there is no simple workaround
approach, do you think it is feasible for us to port the
related scripts from version 92 to version 80?</div>
<div class="gmail_extra"><br>
</div>
<div class="gmail_extra">Many thanks for your kindly help an
advice!</div>
<div class="gmail_extra"><br>
</div>
<div class="gmail_extra">Best Rdgs,</div>
<div class="gmail_extra">Kenneth </div>
<div class="gmail_extra"><br>
<div class="gmail_quote">On Sat, May 12, 2018 at 8:26 AM,
Kenneth Wong <span dir="ltr"><<a
href="mailto:kenneth@l3-bioinfo.com" target="_blank"
moz-do-not-send="true">kenneth@l3-bioinfo.com</a>></span>
wrote:<br>
<blockquote class="gmail_quote" style="margin:0px 0px 0px
0.8ex;border-left:1px solid
rgb(204,204,204);padding-left:1ex">
<div dir="auto">
<div>Dear Laurent,</div>
<div dir="auto"><br>
</div>
<div dir="auto">Thanks a lot for your prompt and
detailed answer! It makes sense to fix such a mismatch
through VEP cache. </div>
<div dir="auto"><br>
</div>
<div dir="auto">Thanks again for yr great help!</div>
<div dir="auto"><br>
</div>
<div dir="auto">Best Rgds,</div>
<div dir="auto">Kenneth</div>
<div>
<div class="gmail-h5">
<div dir="auto"><br>
<br>
<div class="gmail_quote" dir="auto">
<div dir="ltr">在 2018年5月11日週五 23:29，Laurent Gil
<<a href="mailto:lgil@ebi.ac.uk"
target="_blank" moz-do-not-send="true">lgil@ebi.ac.uk</a>>
寫道：<br>
</div>
<blockquote class="gmail_quote"
style="margin:0px 0px 0px
0.8ex;border-left:1px solid
rgb(204,204,204);padding-left:1ex">
<div bgcolor="#FFFFFF">
<p>Dear Kenneth,<br>
<br>
You are right: the VEP cache result is
correct.<br>
<br>
VEP returns annotations based on the
Genomic Reference Assembly sequence.</p>
<p>As you might know, sometimes the RefSeq
sequences don't match the Genomic
Reference Assembly sequence, which is the
case for NM_000348.3:<br>
</p>
<pre class="gmail-m_-4874758897740869537m_6100284029201197083text_sequence" style="font-family:"Courier New",Courier,monospace;font-size:12.8px;margin:0px 0px 16px;color:rgb(102,102,102);font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial">NM_000348.3: 121 <span class="gmail-m_-4874758897740869537m_6100284029201197083adorn gmail-m_-4874758897740869537m_6100284029201197083adorn-2 gmail-m_-4874758897740869537m_6100284029201197083_seq" style="font-size:1em">TGGTCGCCCTTGGGGCACTGGCCTTGTACG<wbr>TCGCGAAGCCCTCCGGCTACGGGAAGCACA</span> NM_000348.3: 180<span class="gmail-m_-4874758897740869537m_6100284029201197083ad-post-2" style="font-size:1em"></span>
121 <span class="gmail-m_-4874758897740869537m_6100284029201197083adorn gmail-m_-4874758897740869537m_6100284029201197083adorn-8" style="font-size:1em">||||||||||||||||||||||||||||||<wbr>|||||||||| |||||||||||||||||||</span> 180<span class="gmail-m_-4874758897740869537m_6100284029201197083ad-post-8" style="font-size:1em"></span>
2:31805920 <span class="gmail-m_-4874758897740869537m_6100284029201197083adorn gmail-m_-4874758897740869537m_6100284029201197083adorn-14" style="font-size:1em"><span style="font-size:1em">TGGTCGCCCTTGGGGCACTGGCCTTGTACG<wbr>TCGCGAAGCC-</span><span title="ENSE00001559957
ENSE00001070145" style="font-size:1em;color:rgb(221,0,0)">T</span><span title="ENSE00001559957" style="font-size:1em;color:rgb(221,0,0)">CC</span><span title="ENSE00001559957
ENSE00000932452" style="font-size:1em;color:rgb(221,0,0)">GGCTACGGGAAGCACA</span></span> 2:31805861<span class="gmail-m_-4874758897740869537m_6100284029201197083ad-post-14" style="font-size:1em"></span>
</pre>
<br>
<p>We started to develop a way to alter the
Genomic Reference Assembly sequence for
RefSeq sequence (when the sequences don't
match) in order to provide a more accurate
prediction/annotation of variants on
RefSeq data.<br>
<br>
At the moment this is only available
through the VEP cache, so we strongly
recommend to use VEP cache when you use
VEP with the RefSeq dataset.</p>
<p>I hope this makes sense.</p>
<p>Best regards,<br>
</p>
<pre class="gmail-m_-4874758897740869537m_6100284029201197083moz-signature" cols="72">Laurent</pre>
<div
class="gmail-m_-4874758897740869537m_6100284029201197083moz-cite-prefix">On
11/05/2018 10:45, Kenneth Wong wrote:<br>
</div>
<blockquote type="cite">
<div dir="ltr">
<div>The scenario is described as below
:</div>
<div>1. input.vcf :</div>
<div> ##fileformat=VCFv4.0</div>
<div> #CHROM POS ID REF
ALT QUAL FILTER INFO</div>
<div> 2 31754395 .
C T . . .</div>
<div><br>
</div>
<div>2. Execute below commands with
versions 92</div>
<div><br>
</div>
<div> /* Run VEP by connecting to remote
Ensembl DB */</div>
<div>
<div> ./vep -i input.vcf -o
vep-online.out\</div>
<div>
<div> --fork 4 --buffer_size 100\</div>
<div> --refseq --assembly "GRCh37"\</div>
<div> --database --port 3337 --db
92\</div>
<div> --hgvs --numbers --symbol
--flag_pick --no_stats\</div>
<div> --species homo_sapiens\</div>
<div> --fields $VEP_FIELDS</div>
</div>
</div>
<div><br>
</div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline"> /*
Run VEP via local cache */</span><br>
</div>
<div><span
style="text-align:start;text-indent:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">
<div> ./vep -i input.vcf -o
vep-cache.out\</div>
<div> --fork 4 --buffer_size 1000\</div>
<div> --offline --cache --dir_cache
$VEP_CACHE_DIR\</div>
<div> --refseq --fasta <span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">~/</span><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">Homo_sapiens.GRCh37.75.dna.<wbr>primary_assembly.fa</span>\</div>
<div> --hgvs --numbers --symbol
--flag_pick --no_stats\</div>
<div> --species homo_sapiens\</div>
<div> --fields $VEP_FIELDS\</div>
<div> --use_given_ref</div>
</span></div>
<div><br>
</div>
<div>, where </div>
<div>$VEP_FIELDS=Uploaded_<wbr>variation,
Location, Allele, Gene, SYMBOL,
SYMBOL_SOURCE, HGNC_ID, Feature,
Consequence, Protein_position,
Amino_acids, Codons, HGVSc, HGVSp</div>
<div><br>
</div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">$VEP_CACHE_DIR=~/.vep/cache/</span></div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline"><br>
</span></div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">3.
Below discrepancies are found
between the output file
"vep-cache.out" and "vep-online.out"
(as attached)</span></div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline"><br>
</span></div>
<div><span
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;background-color:rgb(255,255,255);text-decoration-style:initial;text-decoration-color:initial;float:none;display:inline">vep-cache.out
:</span></div>
<div>- Amino_acids = R/Q</div>
<div>- Codons = cGa/cAa</div>
<div>- HGVSp = NP_000339.2:p.Arg227Gln</div>
<div><br>
</div>
<div>vep-online.out :</div>
<div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">-
Amino_acids = E/K</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">-
Codons = Gag/Aag</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">-
HGVSp = NP_000339.2:p.Glu227Lys</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial"><br>
</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">Assume
the result in vep-cache.out is
correct, would you please advice
what's wrong in the parameters when
running VEP via remote database?</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial"><br>
</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">Many
thanks for your help!</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial"><br>
</div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial">Kenneth
Wong </div>
<div
style="color:rgb(34,34,34);font-family:arial,sans-serif;font-size:small;font-style:normal;font-variant-ligatures:normal;font-variant-caps:normal;font-weight:400;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration-style:initial;text-decoration-color:initial"><br>
</div>
<br
class="gmail-m_-4874758897740869537m_6100284029201197083gmail-Apple-interchange-newline">
</div>
</div>
<br>
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<br>
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</pre>
</blockquote>
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