On the causes, consequences, and avoidance of PCR duplicates: Towards a theory of library complexity

Nicolas C. Rochette, Angel G. Rivera-Colón, Jessica Walsh, Thomas J. Sanger, Shane C. Campbell-Staton, Julian M. Catchen

Research output: Contribution to journalArticlepeer-review

Abstract

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.

Original languageEnglish (US)
Pages (from-to)1299-1318
Number of pages20
JournalMolecular ecology resources
Volume23
Issue number6
DOIs
StatePublished - Aug 2023

Keywords

  • PCR duplicates
  • RAD-seq
  • allele dropout
  • bioinformatics
  • sequencing library

ASJC Scopus subject areas

  • Genetics
  • Ecology, Evolution, Behavior and Systematics
  • Biotechnology

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