We are trying to detect variants at very low frequency in human tissues using WES (UMI-UDI libraries), aiming for a mean depth of 3000x, but we are getting very high duplication rates. In our first experiments we started with very low inputs, 20 ng into the library construction (8 PCR cycles), and got 70% duplication rate. Now we have used inputs of 80 ng (with 4 PCR cycles), and 500 ng (no PCR), followed by 160 ng input of each sample into the pool (equimass for 10 samples) for exome capture. For sequencing they just loaded 1/4 of the capture output (around 12 ng) into the Novaseq 6000 S2 (850 Gb output). The surprise came that the duplication rate was still around 45-50%, and minimal difference between 80 and 500 ng input. Do you have a hint on what could cause this apparent lack of complexity? Need more PCR cycles, to pool samples asymmetrically depending on the input, to load all the sample in the sequencer? Thanks!
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