Journal Article

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

Billstein, Bradley, McDevitt, Shana L., Becker, Aaron, Froehling, Elyse, Beckman, Kenneth B., Garbe, John, Johnson, Darrell M., Auch, Benjamin, Anderson, Shea, Gohl, Daryl M., Magli, Alessandro

2019

Overview

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.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Accuracy / Animal Genetics and Genomics / Animals / Bias / Bioinformatics / Biomedical and Life Sciences / Deoxyribonucleic acid / DNA / DNA library preparation / DNA Restriction Enzymes / DNA sequencing / droplets / economics / Enzymes / Evolutionary Biology / Expected values / Gene expression / Genetic Techniques / Genomes / Genomics / Human Genetics / Humans / Illumina / Life Sciences / Method / Methods / Microbial Genetics and Genomics / Molecular biology / Next-generation sequencing / Nucleotide sequence / PCR-free / Plant Genetics and Genomics / Plasmids / Polymerase chain reaction / RNA-Seq / Sequence Tagged Sites / Size bias / Transgenes / Transposons