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Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.

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.

SUBMITTER: Aird D

PROVIDER: S-EPMC3188800 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.

Aird Daniel D Ross Michael G MG Chen Wei-Sheng WS Danielsson Maxwell M Fennell Timothy T Russ Carsten C Jaffe David B DB Nusbaum Chad C Gnirke Andreas A

Genome biology 20110221 2

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 i ...[more]

PMID: 21338519

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