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<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="text_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; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); text-decoration-style: initial; text-decoration-color: initial;">NM_000348.3: 121 <span class="adorn adorn-2 _seq" style="font-size: 1em;">TGGTCGCCCTTGGGGCACTGGCCTTGTACGTCGCGAAGCCCTCCGGCTACGGGAAGCACA</span> NM_000348.3: 180<span class="ad-post-2" style="font-size: 1em;"></span>
121 <span class="adorn adorn-8 " style="font-size: 1em;">|||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||</span> 180<span class="ad-post-8" style="font-size: 1em;"></span>
2:31805920 <span class="adorn adorn-14 " style="font-size: 1em;"><span style="font-size: 1em;">TGGTCGCCCTTGGGGCACTGGCCTTGTACGTCGCGAAGCC-</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="ad-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="moz-signature" cols="72">Laurent</pre>
<div class="moz-cite-prefix">On 11/05/2018 10:45, Kenneth Wong
wrote:<br>
</div>
<blockquote type="cite"
cite="mid:CAHSPU_uBS3ET9fMei-4f635GRMd84AjzmGBx9=wXkgHKiGLhYA@mail.gmail.com">
<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.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_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-Apple-interchange-newline">
</div>
</div>
<br>
<fieldset class="mimeAttachmentHeader"></fieldset>
<br>
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</pre>
</blockquote>
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