Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

In Genomeweb February 24, 2011

Broad Team IDs, Improves PCR Amplification Bias in Illumina Sequencing Libraries

A team of Broad Institute researchers has developed a panel of qPCR assays designed to identify the primary causes of PCR amplification bias in Illumina sequencing libraries, and built an optimized protocol that can help reduce such bias, according to a recent paper.
The scientists said they expect the new protocol to amplify sequencing libraries more evenly than the standard Illumina protocol, and to minimize bias introduced by factors such as choice of thermocycler and PCR amplification enzyme, and temperature ramp rate.
And although their optimized protocol does not minimize bias in all scenarios, it is expected to improve sequencing of GC-rich regions of the human genome, which contain important information for cancer and medical genetics studies, the researchers said.
 

Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

Daniel Aird, Michael G Ross, Wei-Sheng Chen, Maxwell Danielsson, Timothy Fennell, Carsten Russ, David B Jaffe, Chad Nusbaum and Andreas Gnirke

Genome Biology 2011, 12:R18 doi:10.1186/gb-2011-12-2-r18
Published: 21 February 2011

 

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 qPCR. 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.