PCR Bias and Depth of Coverage for NHEJ Quantification with NGS

23 May 2017 1 8K Report

Hi,

I would like to use NGS to quantify the levels of NHEJ after Cas9 cutting of a genomic locus. I have gDNA from pools of transfected cells, on which I run a PCR#1 of about 150bp for sequencing (including overhangs for barcode addition (PCR#2)). I have 2 questions regarding this:

PCR cycle bias: in a pilot experiment of only 2 samples, I've noticed that the number of amplification cycles in the genomic PCR#1 affects the levels of NHEJ quantified from the NGS data. The more cycles, the less editing I observe, which suggests a positive selection for the WT allele. This makes sense, and is known from NGS for library validation, but I could not find any guidelines or recommendations for optimising this. Do any exist? I plan on doing a PCR cycle "titration" first, to see if it plots on a curve and if an optimal amount can be deduced. Obviously I will use the same amount of cycles for all experiments consistently, but do you have any suggestions for this?

Depth of coverage: how should depth of coverage be calculated for proper representation of level of editing? Most resources seem to focus on WGS/WES, RNA-seq, etc., as opposed to targeted seq. of a pool of cells.

I'm using the Illumina HiSeq2500 on HiSeq Rapid Run mode, if it's relevant.

Thanks,

Yuval

Rachel E Gate

I think I also might be noticing something similar in my editing, did you find a solution for your PCR?

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