Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

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


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Artifacts*
  • Base Composition / genetics*
  • Escherichia coli / genetics
  • Genetic Loci
  • Genome, Human*
  • Genomic Library
  • Genomics / methods*
  • Humans
  • Nucleic Acid Denaturation
  • Plasmodium falciparum / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA
  • Temperature