Question: Confirming Identified Seq Mutations By Pcr
3
gravatar for Raju
5.0 years ago by
Raju30
Raju30 wrote:

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

sequence variation • 1.6k views
ADD COMMENTlink modified 5.0 years ago by Alex Paciorkowski3.3k • written 5.0 years ago by Raju30
3
gravatar for Alex Paciorkowski
5.0 years ago by
Rochester, NY USA
Alex Paciorkowski3.3k wrote:

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.

ADD COMMENTlink modified 5.0 years ago • written 5.0 years ago by Alex Paciorkowski3.3k
1
gravatar for Lee Katz
5.0 years ago by
Lee Katz2.9k
Atlanta, GA
Lee Katz2.9k wrote:

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.

ADD COMMENTlink written 5.0 years ago by Lee Katz2.9k
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