remove primer sequences from BAM file
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3.2 years ago
J.F.Jiang ▴ 860

Hi all,

I am dealing with amplicon data, which was obtained from multiplex PCR.

Original primers can largely increase the mapping accuracy, since some amplicons are highly homologous with little difference within primer region.

And since these primers may introduce FAKE SNPs when they are within inserted regions from other amplicons, we want to remove the primers from BAM file.

https://github.com/tommyau/bamclipper can clip the primer sequence, however, it is time-consuming when amplicon is sequenced more than 1000X.

GATK can softly clip the sequence. However, it can also clip the sequence that is similar with the primers, especially those are not the real primers, but the insert sequence.

I am wondering if there is any tool that can quickly clip the real primers.

Best, Junfeng

bam primer • 1.6k views
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An alternative solution would be to use the -L argument of GATK to specify variant calling to a certain region (bed file) within the amplicons, excluding the primers.

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Take the scenario above as consideration, if position * is always T, but C in the primer region of the second amplicon. The calling result will give T/C SNP, however, the C allele is just the PCR error or synthesis error. If we remove the primer sequence of the amplicons, the final calling will be T/T as expected.

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Oh, so you mean you have overlapping amplicons? Right... then my suggestion doesn't work.

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yes, -L option can handle with non-overlapped regions.

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Hi Dr. Jiang,

did you find any better solutions?