Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

Daryl M. Gohl, Aaron Becker, Darrell M Johnson, Shea Anderson, Bradley Billstein, Shana L. McDevitt, Kenny B Beckman

Research output: Contribution to journalArticlepeer-review

Abstract

Quantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification can introduce significant quantitative error into these measurements. Here we describe REcount, a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We further demonstrate that the REcount approach is amenable to multiplexing through the use of orthogonal restriction enzymes. Finally, we use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Aug 9 2018

Keywords

  • clustering
  • DNA library preparation
  • Illumina
  • Next-generation sequencing
  • PCR-free
  • size bias

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