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Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

Daniel Aird1, Michael G Ross1, Wei-Sheng Chen2, Maxwell Danielsson2, Timothy Fennell3, Carsten Russ1, David B Jaffe1, Chad Nusbaum1 and Andreas Gnirke1*

Author Affiliations

1 Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA

2 Learning Community C, Cambridge Rindge and Latin School, 459 Broadway, Cambridge, MA 02138, USA

3 Genome Sequencing Platform, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA

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

Published: 21 February 2011

Abstract

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.