Samtools mpileup on multiple samples

[cristae8]

Hi,
I have multiple samples from which I would like to call SNP. I've been trying either to call them separately or all at once

samtools mpileup -uf hg19.fa sample1.bam | bcftools view -bvcg - > out.bcf
or
samtools mpileup -uf hg19.fa sample1.bam sample2.bam | bcftools view -bvcg - > out.bcf

In the first case, I can get coverage (DP) at each position where a SNP is encountered, but only for the sample that has the SNP. That is, I would like to get the coverage at that position in all samples, but I could not.

In the second case, for SNP I know are present in both samples, I found only 1 value for DP, which I assume is the combined coverage of both samples (see below).
Is there any way I can get DP and DP4 for all samples even if the SNP is only in one?

chr1 15211 . T G 51 . DP=16;AF1=1;CI95=0.8333,1;DP4=3,0,10,3;MQ=30;FQ=-37.5;PV4=1,1.3e-05,1,1 GT:PL:GQ 1/1:46,9,0:21 1/1:23,9,0:21

Second question, I've been having a hard time trying to annotate the SNP calls in the vcf file (produced by bcftools view) with its rsID. I tried vcftools

>bgzip UCSC_SNP_v132
>zcat input.vcf.gz | vcftools_0.1.5/bin/fill-rsIDs -r UCSC_SNP_v132.gz | tabix-0.2.5/bgzip -c > out

but kept getting error below which suggests to me that I'm not using the right format for the input dbSNP. I used the BED format downloaded from UCSC. What am I doing wrong here?

tabix ./UCSC_SNP_v132.gz chr1 2>&1 |: No such file or directory at vcftools_0.1.5/bin/fill-rsIDs line 20
main::error('tabix ./UCSC_SNP_v132.gz chr1 2>&1 |: No such file or directory') called at vcftools_0.1.5/bin/fill-rsIDs line 93
main::fill_rsids('HASH(0x169602a0)', './UCSC_SNP_v132.gz') called at vcftools_0.1.5/bin/fill-rsIDs line 11

Thanks!!

[DerSeb]

Regarding your first question: The -D option keeps per sample read depth with mpileup.

[cristae8]

Thanks, DerSeb!

Regarding my 2nd question, I realized that error was because tabix (called inside fill-rsIDs) was not on my path.

[aslihan]

how to annotate VCF files created by bcftools view?

I have 1 big vcf files which compares 12 samples by using samtools mpileup. And I would like annotate them? my reference genome is Hg19. How can annotate VCf files ?

Could you write commands and set up and where to download vcftools?

Thanks in advance.

Are you using only vcftools? What about ANNOVAR?

[swbarnes2]

No, here's no way to run multiple sample .bams together and get the DP4 of each sample. I Which is a pity, since that would be useful. What you get instead are other stats, which can give you an idea of how good the SNP is in each sample.

[DineshCyanam]

For annotating the VCF file you can use the Ensembl Variant Effect Predictor. It is very helpful and can also produce SIFT, PolyPhen and Condel consensus.
http://ensembl.org/tools.html

I would recommend using the Perl script with a pre-built cache. http://ensembl.org/info/docs/variati...ep_script.html

Here is the command that I use to annotate on a Mac:

perl variant_effect_predictor.pl --species homo_sapiens -i SNP.vcf --format vcf -o SNP_effects.txt --sift b --polyphen b --condel b --gene --hgnc --canonical --regulatory --coding_only --no_intergenic --cache --compress gzcat --check_existing

Variant Effect Predictor can also be used to check the SNPs against the 1000 Genomes and HapMap projects but I was not able to get it running. It was always throwing an error.

--Dinesh

[aslihan]

Thank you. Sorry for my late. I will try them.