Program for Amplicon (MHC) Analysis?

Question

[joelslade]

Hi there,

I have been trying out different programs to analyze amplicons sequenced by an Illumina MiSeq. Originally I gave AmpliSAS a try, and the program seems to eliminate too many MHC alleles from each individual, and only allows uploads of 500mb files.

I thought mothur may be a good program to cluster my reads, do quality control, and assign my alleles as OTUs, which wouldn't be a problem, but I ran into trouble with that too as their SOP is designed for microbiome data, and I just can't get it to work for my data.

So, does anyone know of any program that would work with amplicons by merging paired-end files, trim the oligos (barcodes were trimmed by my genomics center already), do a quality control check, and then assign the amplicons to each individual?

Any help is greatly appreciated!

[jean.elbers]

I would recommend mothur, but you seem to have discarded the idea.

Maybe this will help. Here is a README for using an older version of mothur that I used to do part of a MHC analysis for a bird species. I say part because we had a bad 454 run and didn't really have hundreds of reads to support each allele. Let me know if it is useful.

https://raw.githubusercontent.com/jelber2/sosp_mhc/master/README.md

[joelslade]

I see that your files were SOSP MHC -- that is what I did for my PhD. Neat.

[jean.elbers]

Interesting. Are those data (your SOSP MHC) published? It might make for an interesting comparison when we eventually try to publish ours.

Answer 1

[Vitis]

For amplicon-based SNP assays, I've tried simple "grep" (string search) and tallying the read numbers supporting the different alleles. You would get less accurate counts due to sequencing errors, but it's fairly straightforward for calling homozygosity and heterozygosity with these less accurate counts (read counts do not mean much as a result of amplicon anyway). A more complicated approach would be mapping the reads and querying the CIGAR strings for known alleles with SNPs/insertions/deletions at specific locations if you know the possible alleles. But I don't have any experience with MHC alleles so don't know whether these would work for your amplicons.

Answer 2

[joelslade]

Unfortunately, these aren't SNPs but entire sequences. I know there are some HLA-specific programs, but many of them are only based for human MHC.