Hi,
I'm searching for methods to validate somatic mutations in pancreatic beta cells. The mutations are found by Next Generation Sequencing and I have try Sanger Sequencing with no results.
What is the % of somatic mutation? Sanger sequencing does have lower limit than NGS.
I am not expert of somatic mutations. but being working with Sanger sequencing and NGS (HTS) I learnt few things.
1. Sanger sequencing is always the aggregate of all th copies in the cell or i your case maybe tissues.
2. In HTS you can get individual copies of each gene.
So if you are looking for mutation in the sanger sequence, you can check for the chromatograms to see if there are small peaks hiding in between the large ones at the place of you mutations.
Technically, it would be very difficult to see a site mutation in one cell if you use sanger. Maybe if you do cloning, and then sequencing you may find them in samger sequences.
Sanger sequencing would have a limit of detecting somatic variants of 5-10%, maybe higher and it will require optimization to get good quality sequences. You may try digital PCR to estimate the number of copies of you mutated allele. Design a probe to detect the variant and get statistics of total number of copies of mutated vs normal.
A few different technologies that are more sensitive than Sanger Sequencing for confirming somatic variants are:
Pyrosequencing https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/
Measures both wild-type and mutant alleles. It is great for things like KRAS codons 12 & 13 where there are more than 1 possible mutation. The product information says it is sensitive down to 2%. In my assays, it was closer to 3-4%.
Droplet Digital PCR (dPCR)
http://www.bio-rad.com/en-us/applications-technologies/droplet-digital-pcr-ddpcr-technology?ID=MDV31M4VY
cast-PCR
https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2012/bioprobes-68-november-2012/castpcr-taqman-mutation-detection-assays.html
cast-PCR blocks amplification of the wild-type allele so that you only detects the mutant allele (unless you order a separate assay for the mutant allele.) It is supposedly very sensitive, detecting 0.1% mutant allele.
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