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Genome Biol. 2011;12(2):R18. doi: 10.1186/gb-2011-12-2-r18. Epub 2011 Feb 21.

Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.

Author information

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

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.

PMID:
21338519
[PubMed - indexed for MEDLINE]
PMCID:
PMC3188800
Free PMC Article

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