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

Author:

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

Abstract

AbstractLibrary 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 artifact, 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 datasets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined 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 minor role for this artifact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any dataset containing PCR duplicates. We discuss how amplification-related artifacts 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.

Publisher

Cold Spring Harbor Laboratory

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