Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries
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
Genome Biology 2011, 12:R18 doi:10.1186/gb-2011-12-2-r18Published: 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 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.