Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

Authors: Daniel Aird, Michael Ross, Wei‐Sheng Chen, Maxwell Danielsson, Timothy R. Fennell, Carsten Russ, David B. Jaffe, Chad Nusbaum, Andreas Gnirke - Genome biology 2011 cited by 1,238

Despite the ever-increasing output of Illumina sequencing data, loci with extreme base compositions are often under-represented or absent. To evaluate sources of base-composition bias, we traced genomic sequences ranging from 6% to 90% GC through the process by quantitative PCR. We identified PCR during library preparation as a principal source of bias and optimized the conditions. Our improved protocol significantly reduces amplification bias and minimizes the previously severe effects of PCR instrument and temperature ramp rate.