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.
Bibliographical noteFunding Information:
This work was supported by a grant from the University of Minnesota-Mayo Translational Product Development Fund to D.M.G. and K.B.B. (National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114). The Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley was supported by an NIH S10 OD018174 Instrumentation Grant.
- DNA library preparation
- Genotyping by sequencing
- Next-generation sequencing
- Size bias