Many papers working with CRISPR method present their indel mutations result by sequencing TA clones. If we can identify one mutation by direct sequencing of DNA sample, is it necessary to identify multiple mutations?
Hello Masita,
the problem with the direct sequencing analysis is that it is not able to rule-out the presence of subclones carrying different variants. For instance Sanger sequencing is not able to reliably detect variants below 25-30%. Therefore, even if you're confident that your CRISPR cell line is clonal (i.e. you did FACS single-cell sorting or similar) , it is always better to double check it with TA clones or, if a NGS facility is available, by doing deep-sequencing.
Dear Masita,
I totally agree with Rocco. So far, there is no better opportunity than that as long as you can´t be sure to have a single cell clone.
Thank you Bjorn.
But, in case of low variants as mentioned by Rocco, even with TA cloning, wouldn't the number of clones carrying mutant sequenced will also be low? Thus, even TA cloning will not be efficient since we have to sequence many clones from it. Also, how can we make sure that PCR amplification for TA cloning insert will also amplify mutant variants, not only wild type?
Dear Mandasari,
of course this is a lot of work to do. With each TA cloning you have 50:50 chance to get an allele. Normally,we sequence at least 5 colonies after TA cloning. In case, you have a very low efficiency of mutation, you would have to increase the number of colonies to be sequenced.Though TA cloning is not very efficient, you don't have other opportunities or you would have to use single cell sequencing.
Dear Masita,
do not rule out the possibility of doing deep-seq by NGS. In case you're not familiar with these techniques, deep-seq (or ultradeep-seq) means that you do a PCR, possibly with a high-fidelity enzyme, and send it to a NGS company/NGS facility (if available) where they will generate a library and NGS-sequence it. Given that NGS sequencing is clonal by definition you will end up with many individual clones each representing one of your alleles. Doing it for a single sample would be cheap, fast and would allow you to easily screen even thousands of clones in a single shot.
Dear Bjorn and Rocco,
Thank you very much for sharing your knowledge. I will take your suggestions into consideration and check whether my institution owned NGS facility. Again, thank you very much. It has been very helpful.
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