Confirming Identified Seq Mutations By Pcr

Question

[Raju]

I have identified a set of mutations specific for tumor samples from WGS. I would like to confirm such mutations in large number of tumor samples. I was wondering if there are any special points for which I may have to be careful or just deisgn regular primers spanning across the variation. I am dealing with single bases pair to several (5-10 bp) changes. Is there any other alternative approach (other than PCR) to validate identified seq variations.

Thanks

Answer 1

[Alex Paciorkowski]

Sanger sequencing is the most accepted method of confirming any next-gen sequencing results, especially if you plan to build any further experiments off of those results, write a grant including those variants as preliminary data, or try to publish them. For any of these purposes in silico analyses just don't do the job. And to answer your specific question, standard Sanger methods using (with a little luck ) appropriate primers designed in Primer3 or similar should do the trick.

Answer 2

[Lee Katz]

PCR/Sanger sequencing is a great way. However one totally in-silico method is to look at the short-read pileup in a browser such as IGV or Tablet. Visually inspecting in this way is one method for confirming these bp changes. I wouldn't say that this is the gold standard way but it's a pretty good method.