Question: Getting skewed ratios of heterozygosity between Ion Torrent and Sanger sequencing.
0
gravatar for svalpereira
5.0 years ago by
Switzerland
svalpereira0 wrote:

Hi,

I have sequenced a sample with a custom AmpliSeq pannel. On the ion torrent results I see an already described pathogenic mutation in heterozigosity with 578 reads (285/293). When I do the sanger sequencing to confirm this result I have an heterozigosity, but like 20%/80%.

 

Can you explain the difference? Do you have any solution? How can I be sure that both alleles are being ampplified in the same amount? I have already tried several different primers.

Thank you all.

 

sequencing next-gen • 1.6k views
ADD COMMENTlink modified 5.0 years ago by Istvan Albert ♦♦ 81k • written 5.0 years ago by svalpereira0

What kind of mutation are you looking at (SNV, small InDel, a large InDel, etc.)?

ADD REPLYlink written 5.0 years ago by Devon Ryan91k

Thank you.

It's a point mutation.

ADD REPLYlink written 5.0 years ago by svalpereira0

Are you using the same primers for both Ampliseq and the PCR for Sanger sequencing?
 

ADD REPLYlink written 5.0 years ago by Devon Ryan91k

No. On AmpliSeq we use the primeres that were designed by LifeTechnogies. But for the Sanger Sequencing, I already tested 3 different primers pairs wit the same kind of final result.

ADD REPLYlink written 5.0 years ago by svalpereira0

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).

ADD REPLYlink written 5.0 years ago by Devon Ryan91k

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.

ADD REPLYlink written 5.0 years ago by Alex Paciorkowski3.3k
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