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.
Original language | English (US) |
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Article number | 85 |
Journal | Genome biology |
Volume | 20 |
Issue number | 1 |
DOIs | |
State | Published - Apr 29 2019 |
Bibliographical note
Publisher Copyright:© 2019 The Author(s).
Keywords
- ATAC-Seq
- DNA library preparation
- Genotyping by sequencing
- Illumina
- Next-generation sequencing
- PCR-free
- RAD-Seq
- RNA-Seq
- Size bias