Sources of PCR-induced distortions in high-throughput sequencing data sets

Nucleic Acids Res. 2015 Dec 2;43(21):e143. doi: 10.1093/nar/gkv717. Epub 2015 Jul 17.

Abstract

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • DNA / chemistry
  • DNA-Directed DNA Polymerase / metabolism
  • High-Throughput Nucleotide Sequencing*
  • Polymerase Chain Reaction*
  • Sequence Analysis, DNA*
  • Stochastic Processes
  • Templates, Genetic

Substances

  • DNA
  • DNA-Directed DNA Polymerase