Getting skewed ratios of heterozygosity between Ion Torrent and Sanger sequencing.
Hi,
I have sequenced a sample with a custom AmpliSeq panel. On the ion torrent results I see an already described pathogenic mutation in heterozygosity with 578 reads (285/293). When I do the sanger sequencing to confirm this result I have an heterozygosity, but like 20%/80%.
Can you explain the difference? Do you have any solution? How can I be sure that both alleles are being amplified in the same amount? I have already tried several different primers.
Thank you all.
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What kind of mutation are you looking at (SNV, small InDel, a large InDel, etc.)?
Thank you.
It's a point mutation.
Are you using the same primers for both Ampliseq and the PCR for Sanger sequencing?
No. On AmpliSeq we use the primers that were designed by Life Technologies. But for the Sanger Sequencing, I already tested 3 different primers pairs wit the same kind of final result.
Try using the same primers (or primers as similar as possible). If those don't produce similar results, I'd be concerned with the Ampliseq protocol (assuming you did everything correctly on the Sanger sequencing end).
How did you measure your zygosity on your Sanger read? You probably have preferential amplification of one allele over the other. Do you see the mutation in your chromatographs? Have you done bidirectional sequencing? If it's visible on the chromatographs on bidirectional Sanger sequencing, I wouldn't worry about it too much.