I am trying to detect rare variants in pooled mutated samples. I have barcoded my pools and I have 3 genes I am analyzing in each of 28 pools. Since I am doing this as a test with diluted samples bearing a known mutation I can confirm if the technique is working. I can clearly see the expected mutations (around 2% change), but also see a bunch of sequencing errors, mainly at the end of the reads, which at the end show frequencies close to the real mutation. I been struggling for the last couple of days with SAMtools, CRISP and even the ion torrent variant call plugin without success (this last one only calls alleles with a minimum of 5% frequency and rare mutations are lower than that). I know there are other Biologist-friendly tools out there, but they are usually expensive, so I am also on the lookout for something free and efficient.
I would mainly like to find a way to tell those sequencing errors from the real variants...any advise?
I would mainly like to find a way to tell those sequencing errors from the real variants...any advise?
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