I am analyzing samples for low-grade mosaic mutations using Cap-Seq (Nimblegen capture on HiSeq 2500). I have found point mutaitons and (small, 1-6 bp) indels and need to validate the findings using another method/platform. Sanger will not work because a mutation at 10% or lower will be very difficult (at best) to spot.
I am just trying to collect some ideas here, so:
Which method(s) would you use?
Well, in my experience Sanger can work for low grade mosaicism so I would certainly give it a try. You know where to look so could potentially use ASO to amplify your mutant allele specifically. If you want to be certain (as far as one can ever be in biology) of catching the mosaic, and you're looking in biopsy material, I guess laser capture of the appropriate cell population would be the way to go. Easiest of course if you know where to look, more labor intensive if you want every cellular compartment obviously. If looking in blood, get more samples so more chances to repeat.
Thank you Maurice. We have already tried Sanger many times and for different types of mutation, but it is not suitable for mosaics below 10% (in many cases even below 20%, depending on sequence context). So for Sanger our result is: tested, but not good/sensitive enough.
I forgot to mention that we are looking for the mutation in blood samples only, so laser capture is not a possibility, I am afraid - but it is a good idea that I will keep in mind for future projects.
What exactly do you mean with "get more blood samples"? We have enough of our one blood sample to prep more DNA than we need for our assay(s). We sequence at over 5000x coverage, and the samples that I have repeated for method validation did not give any different results in the repeat runs. (Or are you saying that blood samples taken at different time points would give different results?)
Well, I guess for Sanger YMMV - as said we get better sensitivity but that is on tissue, not on blood. SNaPShot is a nice one; selectively amplifying your mutant DNA would certainly help. Another approach, but more cumbersome, might be to use polony sequencing. Amplification of DNA has the disadvantage that you will only amplify the mutations that you know are there but not sure how much of an issue that would be in this case.
How about this: http://www.ashg.org/2013meeting/abstracts/fulltext/f130123414.htm
thanks for all the feedback and please excuse the delayed response - I was on leave over the holidays .-)
@Maurice: Digital PCR does sound like the best option - I will ask around and see if I can get accesss to such a platform somewhere. I've been wanting to check out that method anyways, and this seems like a good oportunity!
Thanks Maurice :-)
@ Aries: I will take a look into SNaPShot, as we have the equipment do that here. However, we do have quite a few mosaics between 1% and 5%, and I am afraid the method may not be sensitive enough. We will see... thank you Aries!
@ Ekkapong: The DHPLC sounds nice, but I actually like the two-round AS-PCR even more as we have already performed (single-round) AS-PCR in our lab, so the adaptation should be easy. Thank you for the valuable link, Ekkapong!
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