title: 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.
authors.affiliation: Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Aird
authors.forename: Michael G
authors.initials: MG
authors.lastname: Ross
authors.forename: Wei-Sheng
authors.initials: WS
authors.lastname: Chen
authors.forename: Maxwell
authors.initials: M
authors.lastname: Danielsson
authors.forename: Timothy
authors.initials: T
authors.lastname: Fennell
authors.forename: Carsten
authors.initials: C
authors.lastname: Russ
authors.forename: David B
authors.initials: DB
authors.lastname: Jaffe
authors.forename: Chad
authors.initials: C
authors.lastname: Nusbaum
authors.forename: Andreas
authors.initials: A
authors.lastname: Gnirke
mesh_terms: D016477:Artifacts; D001482:Base Composition / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D056426:Genetic Loci; D015894:Genome, Human; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D010963:Plasmodium falciparum / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013696:Temperature