Removing PCR primers in targeted sequencing with nested amplicons

Question

[Amitm]

Hi everyone,

I am analyzing a 150x2 MiSeq data where there are multiple amplicons from a gene but nested within each other. I want to remove the PCR primers so that I may not get false calls from within primer regions.

I have tried Cutadapt but I am loosing too many sequences due to the nested amplicons.

A primer seq. for one amplicon sits in the middle of another. See image.

What should be the way out? Any help is much appreciated.

Answer 1

[Pierre Lindenbaum]

I wrote a tool to clip the reads from a BED of amplicons. https://github.com/lindenb/jvarkit/wiki/PcrClipReads (see also : Limiting variant calls to amplicon target regions? ). But it only work with non-overlapping BED fragments, you could create a set of BED file containing non-overlapping intervals and try to clip the reads using several steps.

[Amitm]

Thanks Pierre. I am testing the tool and would update how far I could go.

[Pierre Lindenbaum]

if the amplified regions without the primers don't overlap, then you shouldn't have any problem.

[Amitm]

hi Pierre,

That is the trouble. They overlap even *after* removing primer regions.

[Pierre Lindenbaum]

again, you can create two bed files of non-overlapping regions and extract two bams.

Answer 2

[Tommy Au]

BAMClipper is designed to remove PCR primers in nested amplicon NGS setting (Scientific Reports 7:1567).