[ensembl-dev] GRCh37 Protein sequence has asterisks

Luke Goodsell l.goodsell at achillestx.com
Tue Dec 5 13:13:22 GMT 2017


Thanks, Will, those look like very useful tools.

I still think it’d be good to make sure the otherfeatures DB is accurate, but those tools address our immediate needs.

Kind regards,
Luke

From: William McLaren <wm2 at ebi.ac.uk>
Date: Tuesday, 5 December 2017 at 12:23
To: Alessandro Vullo <avullo at ebi.ac.uk>, Ensembl developers list <dev at ensembl.org>, Luke Goodsell <l.goodsell at achillestx.com>
Subject: Re: [ensembl-dev] GRCh37 Protein sequence has asterisks

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.

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.

The plugin does not currently support outputting cDNAs or CDS, but it would be fairly straightforward to add this functionality to the plugin.

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.

Cheers

Will

[1] https://github.com/Ensembl/VEP_plugins/blob/release/90/ProteinSeqs.pm
[2] https://github.com/Ensembl/ensembl-vep#haplo


On 5 December 2017 at 12:05:41 pm, Luke Goodsell (l.goodsell at achillestx.com<mailto:l.goodsell at achillestx.com>) wrote:
Thanks, Will,

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.

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.

Kind regards,
Luke

From: William McLaren <wm2 at ebi.ac.uk>
Date: Tuesday, 5 December 2017 at 09:15
To: Luke Goodsell <l.goodsell at achillestx.com>, Ensembl developers list <dev at ensembl.org>, Alessandro Vullo <avullo at ebi.ac.uk>
Subject: Re: [ensembl-dev] GRCh37 Protein sequence has asterisks

Hi Luke,

There is no straightforward way to do this via Ensembl at the moment; I’d suggest you download the relevant files from NCBI.

The BAM files we use are obtained from ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens/GRCh37.p13_interim_annotation/; it seems there’s a protein and rna FASTA file in there which may have what you need.

Otherwise you may find what you need in the parent directory ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/H_sapiens. I’m not familiar with NCBI’s FASTA layout so you’d have to investigate yourself!

Regards

Will McLaren
Ensembl Variation



On 4 December 2017 at 5:55:38 pm, Luke Goodsell (l.goodsell at achillestx.com<mailto:l.goodsell at achillestx.com>) wrote:
Hi Allessandro,
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.
Kind regards,
Luke



From: Alessandro Vullo
Sent: Monday, 4 December, 17:44
Subject: Re: [ensembl-dev] GRCh37 Protein sequence has asterisks
To: Ensembl developers list, Luke Goodsell


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
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