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Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

ABSTRACT: Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.

SUBMITTER: Gohl DM

PROVIDER: S-EPMC6489363 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

Gohl Daryl M DM Magli Alessandro A Garbe John J Becker Aaron A Johnson Darrell M DM Anderson Shea S Auch Benjamin B Billstein Bradley B Froehling Elyse E McDevitt Shana L SL Beckman Kenneth B KB

Genome biology 20190429 1

Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate ...[more]

PMID: 31036053

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