Removing PCR primers in targeted sequencing with nested amplicons
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6.5 years ago
Amitm ★ 2.1k

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.

sequencing alignment MiSeq ampicon-seq fastq • 3.0k views
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6.5 years ago

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.

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

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

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hi Pierre,

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

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

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5.2 years ago
Tommy Au ▴ 70

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

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