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Old 03-15-2013, 11:23 AM   #1
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Location: N.California

Join Date: Mar 2012
Posts: 7
Default Lib prep - Splitting PCR to reduce PCR bias

I want to reduce my high levels of duplicate reads (>50%) in my illumina HiSeq 100bp Paired end sequences.
The normal solutions aren't possible as I am using the max DNA available per sample (80-200ng), and only with 10-12 PCR cycles, my library is strong enough to sequence.

I wondered if I could reduce PCR bias by splitting the PCR into two tubes, and then pool after PCR? This idea is based on the assumption that the bias generated by PCR is random, and therefore different PCRs will over/under represent different parts of the genome.

A similar approach is used in some protocols for RADseq - but their library prep does differ.
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