Question: Removing PCR primers in targeted sequencing with nested amplicons
1
gravatar for Amitm
3.8 years ago by
Amitm1.6k
UK
Amitm1.6k wrote:

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.

ADD COMMENTlink modified 2.4 years ago by Tommy Au70 • written 3.8 years ago by Amitm1.6k
1
gravatar for Pierre Lindenbaum
3.8 years ago by
France/Nantes/Institut du Thorax - INSERM UMR1087
Pierre Lindenbaum123k wrote:

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.

 

ADD COMMENTlink written 3.8 years ago by Pierre Lindenbaum123k

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

ADD REPLYlink written 3.8 years ago by Amitm1.6k

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

ADD REPLYlink written 3.8 years ago by Pierre Lindenbaum123k

hi Pierre,

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

ADD REPLYlink written 3.8 years ago by Amitm1.6k

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

ADD REPLYlink written 3.8 years ago by Pierre Lindenbaum123k
0
gravatar for Tommy Au
2.4 years ago by
Tommy Au70
Hong Kong
Tommy Au70 wrote:

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

ADD COMMENTlink written 2.4 years ago by Tommy Au70
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