What is the best way to perform ultra-deep targeted sequencing of few samples?

[jocognito]

Any help would be appreciated?

Our group have performed recent whole genome/exome sequencing in tumor samples and now I would like to take a few of these variants (say maximum 50 amplicons) to:

1) Validate them in the original samples
2) Perform ultra-deep sequencing in an extension cohort of samples but using different amplicons for different samples (hope that makes sense!)

How would one do this...as majority of the targeted sequencing approaches focuses on a large panel of genes/exons in a large number of samples. We have experience of using Fluidigm but I don't need to multiplex that number of amplicons.

So for example, say I want to screen just 2 exons of KRAS in 10 samples but want a coverage of say 10,000x so I can study the clonal complexity and evolution. How would one do this?

Thank you!

[MrGuy]

Optimize your PCRs in singleplex to give the same peak height and same mastermix/thermalcycling program. Then pool the non-overlapping amplicons in one tube, then the others in another tube. Step out with a barcode/sequencing adapters as appropriate. The PCR products should all be the same length as this is your library size selection process. If you see uneven coverage, you can limit some of the PCRs as necessary. It's pretty straightforward and usually works with minimal optimization. If you want, you could probably throw some error free tags in there to remove PCR, amplification and sequencing biases.