Hi all,
I am wondering how to map paired-end amplicon reads to the reference properly and if I really understand amplicon sequencing itself...
Correct me if I'm wrong but paired-end reads must originate from the very same amplicon, right? That means the insert size (of untrimmed reads) must be equal to the amplicon size. And it means that the first base of fwd reads must match the amplicon start position, while the last base of rev reads must match the amplicon stop position.
The question is now how to use this information? Is there a way to tell the mapper (e.g. bwa) which amplicons where used in order to improve mapping?
The problem is, I see a lot of reads starting at positions which do not correlate with the amplicon start/stop positions which tells me that they are indeed mapped to the wrong position! In the end I have a lot of false positive variants...
Thanks for any suggestions!
Sebastian
I am wondering how to map paired-end amplicon reads to the reference properly and if I really understand amplicon sequencing itself...
Correct me if I'm wrong but paired-end reads must originate from the very same amplicon, right? That means the insert size (of untrimmed reads) must be equal to the amplicon size. And it means that the first base of fwd reads must match the amplicon start position, while the last base of rev reads must match the amplicon stop position.
The question is now how to use this information? Is there a way to tell the mapper (e.g. bwa) which amplicons where used in order to improve mapping?
The problem is, I see a lot of reads starting at positions which do not correlate with the amplicon start/stop positions which tells me that they are indeed mapped to the wrong position! In the end I have a lot of false positive variants...
Thanks for any suggestions!
Sebastian
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