Freebayes - not detecting some ALT alleles

Question

[z.klinovska]

Hello, I am a newbie to freebayes and I came across a problem when detecting alternative alleles on SNPs variants. What I am trying to achieve is a .vcf file that would show all ALT alleles found in .bam file on a specific position. I checked a small .bam file by eye and counted how many C,G,T,A should freebayes report on a specific position.

For instance on file.bam on a position chr2:200 there should be base C according to the reference genome. File.bam reports on 2 reads C, 131 reads base G, 2 reads T and no A - I checked this manually, excluding reads with lower mapping quality than 20.

Now, freebayes gave me a .vcf file that reports only 2-C reads and 2-G reads. Where are the reads that reported T? Should I change default options? Is there a parameter that I overlooked? Thank you so much for any help.

My call:

freebayes --min-mapping-quality 20  -f /hg19/hg19.fa  DHCR7-p_R352Q.bam  >varQ.vcf

Note: I tried to use -r chr2... in my call, but since the results are not satisfying I tried to make it as simple as possible.

Answer 1

[finswimmer]

Hello,

AFAIK freebayes assumes by default, that your sample is diploid. So it will never give you more than two alleles for a specific position.

Of course there are some parameter you can use, to get more information. freebayes --help is mentioning a combination of parameters that looks promising:

# naive variant calling: simply annotate observation counts of SNPs and indels
    freebayes -f ref.fa --haplotype-length 0 --min-alternate-count 1 \ 
        --min-alternate-fraction 0 --pooled-continuous --report-monomorphic >var.vcf

fin swimmer