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Old 07-12-2016, 05:35 AM   #1
melop
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Location: college station

Join Date: Jan 2012
Posts: 22
Default Always get the same insert size after PCR despite initial size selection on gDNA

Dear All, I am trying to make our own WGS library with dual barcodes. It involves a very common workflow including fragmentation (we use a Shearase from Zymo instead of covaris), End-Repair + A-tailing, Y-shape adapter ligation and PCR amplification.

After shearing, I cleaned up the gDNA fragments with 1:1 SPRI beads. A tapestation run shows that this shifts the peak distribution from 170bp to 271 bp. I then continued with the other steps. After PCR however, the fragment size turns out to be around 303 bp, minus the adapters it's around 160bp, despite the fact that I have done a size selection at the beginning.

Based on your experience, is this mainly due to PCR bias towards small fragments (we use KAPA Hifi mix)? I also included a negative control from the start of the workflow to make sure that these small fragments are not contaminants or some other random amplicons.
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