calling phased variants

Question

[mark.rose]

Hi All

I’m trying to call phased variants in 400-450bp amplicons derived from pooled samples that may contain multiple haplotypes.

To do thismy plan is to sequence amplicons in a 2X300 bp MiSeq run, merged reads 1 & 2, and then aligned to a reference.

For haplotype calling, my initial plan was to use freebayes, which I have used quite successfully in the past to call (unphased) variants. According to its documentation and some posts I’ve read online, it is capable of calling haplotypes (ostensibly through the use of the “-E” flag) and, furthermore, does not assume (if properly set) that samples are from diploid sources (which they are not). Unfortunately, all my attempts to use freebayes to call haplotypes on simulated data (where haplotypes are known) have failed, though the component variants are called. I’ve written to the freebayes author and also posted my problem online ( freebayes problem example ), but have received no response.

So, if no one can offer a solution to my freebayes problem, can anyone suggest a tool well suited to my need? I was considering Platypus, however it assumes samples come from diploid organisms and so may not have the sensitivity I need. Also, I cannot use GATK.

Thanks for your help

Mark

[Fabio Marroni]

Does each pool have its own barcode?

[arezansoff]

Hi there, I'm having the exact same problem as you, not sure if you ever solved your problem. Would love to know what you found! I have no idea how to get actual haplotype information out of the freebayes vcf output file. Their documentation talks nothing about this that I can find.

[Santosh Anand]

My suggestion would be to not use a s/w whose documentation is not clear and if the authors are not reachable, that's a clear no no. You will save a lot of your future frustrations. That said, I used CRISP in one of our pooled project http://www.nature.com/articles/srep33735. It works like charm and the author is very much available for any doubt clearing. Am not sure if it supports phased calling though

[GenoMax]

Please use ADD REPLY/ADD COMMENT when responding to existing posts to keep threads logically organized.

[Santosh Anand]

Why not GATK? Haplotype caller of GATK supports ploidy