PCR duplicates in deep sequencing experiments and potential biasing

Published on the August 7th (2014) in the journal of Genome Biology:

Accurate allele frequencies are important for measuring subclonal heterogeneity and
clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positivhiseqe rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

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