<p>You can use SIFT in two different ways. One way is manually via their website. This is done by downloading the protein sequence (fasta) from a reliable repository (e.g., uniprot or NCBI), and change the amino acid of interest. Then calculate the SIFT score.</p>

<p>The other way, which is probably what you are interested in is with NGS data. Here normally SIFT is already known (precalculated) for all coding SNPs, and you can use that info directly during annotation of your variants. I don't know which variant annotation pipeline you are using, but for example annovar has SIFT data available at their <a href="http://annovar.openbioinformatics.org/en/latest/user-guide/download/" rel="nofollow noreferrer">databases</a> repository.</p>

CC BY-SA 4.0
2018-05-16T04:41:44.400Z
You mean to say that for each variant at specific position (SNP especially), SIFT database has already determined which protein it will affect or what will be its corresponding protein sequence and how much similar it is with reference protein sequence and what will be its similarity score.  Is SIFT score valid for INDELs also.
Lot_to_learn
CC BY-SA 4.0
2018-05-16T07:49:15.053Z
Yes SIFT scores for the human genome are already precalculated, and you can use these scores for annotation of your variants (that is more efficient than to calculate them over and over again with each analysis, doesn't it?). SIFT works only for an amino acid substitution, that means nonsynonomous SNV. So no indels.
benn
CC BY-SA 4.0
2018-05-15T18:26:18.370Z
2018-05-15T19:35:31.977Z
2018-05-15T19:35:31.977Z
benn
<p>Ensembl now have a tool called <a href="https://github.com/Ensembl/ensembl-vep#haplo" rel="nofollow noreferrer">Haplosaurus</a> (still in beta) which will convert your phased VCF into actual protein sequences. </p>

<blockquote>
  <h2>Haplosaurus</h2>
  
  <p><code>haplo</code> is a local tool implementation of the same functionality that
  powers the Ensembl transcript haplotypes view. It takes phased
  genotypes from a VCF and constructs a pair of haplotype sequences for
  each overlapped transcript; these sequences are also translated into
  predicted protein haplotype sequences. Each variant haplotype sequence
  is aligned and compared to the reference, and an HGVS-like name is
  constructed representing its differences to the reference.</p>
  
  <p>This approach offers an advantage over VEP's analysis, which treats
  each input variant independently. By considering the combined change
  contributed by all the variant alleles across a transcript, the
  compound effects the variants may have are correctly accounted for.</p>
  
  <p><code>haplo</code> shares much of the same command line functionality with <code>vep</code>, and
  can use VEP caches, Ensembl databases, GFF and GTF files as sources of
  transcript data; all <code>vep</code> command line flags relating to this
  functionality work the same with <code>haplo</code>.</p>
  
  <h2>Usage</h2>
  
  <p>Input data must be a VCF containing phased genotype data for at
  least one individual; no other formats are currently supported.</p>
  
  <p>When using a VEP cache as the source of transcript annotation, the
  first time you run haplo with a particular cache it will spend some
  time scanning transcript locations in the cache.</p>

<pre><code>./haplo -i input.vcf -o out.txt -cache
</code></pre>
</blockquote>

CC BY-SA 4.0
2018-05-17T12:22:21.060Z
,@terdon : Thanks to both. I tried this. But I got some errors. Error is `Attempt to reload Bio/EnsEMBL/VEP/TranscriptTree.pm aborted.`. Although this file is present. I have checked  and confirmed this. I have also checked the similar issue at github (https://github.com/Ensembl/ensembl-vep/issues/169) which is still unsolved. Can you help me with this. Thanks.
Lot_to_learn
CC BY-SA 4.0
2018-05-17T15:24:00.853Z
You're best sticking with Irina on GitHub with this. She's working directly on developing the tool so will have the best insight and, if it's down to a bug, she can fix it.
Emily_Ensembl
CC BY-SA 4.0
2018-05-18T09:58:19.727Z
I see that Irina has now solved this on GitHub. If anyone else has a similar problem, click through to the linked GitHub issue to see the resolution.
Emily_Ensembl
CC BY-SA 4.0
2018-05-21T11:09:00.703Z
Thanks for software support. One last problem is stopping me to mark this as correct answer. I am not able to interpret output file. From your answer, I was expecting real protein sequence but I am getting many lines like this `ENST00000342066 ENST00000342066:1027T>C ENSP00000342313:343W>R rs6672356 SM_5:2` in output file. Where is the protein sequence. How to read or interpret the output of `./haplo`. Thanks.
Lot_to_learn
CC BY-SA 4.0
2018-05-22T08:50:54.300Z
The json option will give you sequence
Emily_Ensembl
CC BY-SA 4.0
2018-05-23T06:48:38.250Z
Thanks for reply. I tried this too. The output file contains lots of sequences but it is very heavily packed with data or lots of data which is difficult to read. BTW, as you said, protein sequence can be obtained from phased vcf file which I got now. I have raised another issue related to performance of this tool please take a look at https://github.com/Ensembl/ensembl-vep/issues/214 . Thanks. Now I can mark it as right answer. Thanks again.
Lot_to_learn
CC BY-SA 4.0
2018-05-16T08:30:22.740Z
2018-05-17T11:45:12.590Z
2018-05-17T11:45:12.590Z
Emily_Ensembl
<p>Currently, I am working on NGS data and my aim is to get significance prediction of variants present in the vcf file. As we know about <a href="http://sift.jcvi.org/" rel="nofollow noreferrer">SIFT Score</a> for significance score prediction, I am trying to understand how this score works. When I read its <a href="http://sift.jcvi.org/" rel="nofollow noreferrer">reference paper</a>, in the method section, the procedure to determine the sift score starts with patient protein query sequence. </p>

<p>My question is how to get patient protein sequence from patient data (either vcf file or bam file) (which is not explained in the paper). Rest I can follow by other algorithms like PSI-BLAST &amp; MOTIF.</p>

<p>Any suggestions....</p>

<p>Thanks</p>

<vcf><ngs><variation>
Protein sequence from patient data
# Protein sequence from patient data

(Asked by: username_0 on 2018-05-15)

Currently, I am working on NGS data and my aim is to get significance prediction of variants present in the vcf file. As we know about [SIFT Score](http://sift.jcvi.org/) for significance score prediction, I am trying to understand how this score works. When I read its [reference paper](http://sift.jcvi.org/), in the method section, the procedure to determine the sift score starts with patient protein query sequence.

My question is how to get patient protein sequence from patient data (either vcf file or bam file) (which is not explained in the paper). Rest I can follow by other algorithms like PSI-BLAST & MOTIF.

Any suggestions....

Thanks

**Tags:** vcf, ngs, variation

---

## 2 answers

### Answer by username_2 on 2018-05-16. Score: 5 | Accepted answer

Ensembl now have a tool called Haplosaurus (still in beta) which will convert your phased VCF into actual protein sequences. 

Haplosaurus
haplo is a local tool implementation of the same functionality that
  powers the Ensembl transcript haplotypes view. It takes phased
  genotypes from a VCF and constructs a pair of haplotype sequences for
  each overlapped transcript; these sequences are also translated into
  predicted protein haplotype sequences. Each variant haplotype sequence
  is aligned and compared to the reference, and an HGVS-like name is
  constructed representing its differences to the reference.
This approach offers an advantage over VEP's analysis, which treats
  each input variant independently. By considering the combined change
  contributed by all the variant alleles across a transcript, the
  compound effects the variants may have are correctly accounted for.
haplo shares much of the same command line functionality with vep, and
  can use VEP caches, Ensembl databases, GFF and GTF files as sources of
  transcript data; all vep command line flags relating to this
  functionality work the same with haplo.
Usage
Input data must be a VCF containing phased genotype data for at
  least one individual; no other formats are currently supported.
When using a VEP cache as the source of transcript annotation, the
  first time you run haplo with a particular cache it will spend some
  time scanning transcript locations in the cache.
./haplo -i input.vcf -o out.txt -cache

> **Comments:**
> **username_0**: ,@terdon : Thanks to both. I tried this. But I got some errors. Error is `Attempt to reload Bio/EnsEMBL/VEP/TranscriptTree.pm aborted.`. Although this file is present. I have checked  and confirmed this. I have also checked the similar issue at github (https://github.com/Ensembl/ensembl-vep/issues/169) which is still unsolved. Can you help me with this. Thanks.  
> **username_2**: You're best sticking with Irina on GitHub with this. She's working directly on developing the tool so will have the best insight and, if it's down to a bug, she can fix it.  
> **username_2**: I see that Irina has now solved this on GitHub. If anyone else has a similar problem, click through to the linked GitHub issue to see the resolution.  
> **username_0**: Thanks for software support. One last problem is stopping me to mark this as correct answer. I am not able to interpret output file. From your answer, I was expecting real protein sequence but I am getting many lines like this `ENST00000342066 ENST00000342066:1027T>C ENSP00000342313:343W>R rs6672356 SM_5:2` in output file. Where is the protein sequence. How to read or interpret the output of `./haplo`. Thanks.  
> **username_2**: The json option will give you sequence  
> **username_0**: Thanks for reply. I tried this too. The output file contains lots of sequences but it is very heavily packed with data or lots of data which is difficult to read. BTW, as you said, protein sequence can be obtained from phased vcf file which I got now. I have raised another issue related to performance of this tool please take a look at https://github.com/Ensembl/ensembl-vep/issues/214 . Thanks. Now I can mark it as right answer. Thanks again.  

---

### Answer by username_1 on 2018-05-15. Score: 3

You can use SIFT in two different ways. One way is manually via their website. This is done by downloading the protein sequence (fasta) from a reliable repository (e.g., uniprot or NCBI), and change the amino acid of interest. Then calculate the SIFT score.

The other way, which is probably what you are interested in is with NGS data. Here normally SIFT is already known (precalculated) for all coding SNPs, and you can use that info directly during annotation of your variants. I don't know which variant annotation pipeline you are using, but for example annovar has SIFT data available at their [databases](http://annovar.openbioinformatics.org/en/latest/user-guide/download/) repository.

> **Comments:**
> **username_0**: You mean to say that for each variant at specific position (SNP especially), SIFT database has already determined which protein it will affect or what will be its corresponding protein sequence and how much similar it is with reference protein sequence and what will be its similarity score.  Is SIFT score valid for INDELs also.  
> **username_1**: Yes SIFT scores for the human genome are already precalculated, and you can use these scores for annotation of your variants (that is more efficient than to calculate them over and over again with each analysis, doesn't it?). SIFT works only for an amino acid substitution, that means nonsynonomous SNV. So no indels.  

---

