Cargando…

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

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 qua...

Descripción completa

Detalles Bibliográficos
Autores principales: Gohl, Daryl M., Magli, Alessandro, Garbe, John, Becker, Aaron, Johnson, Darrell M., Anderson, Shea, Auch, Benjamin, Billstein, Bradley, Froehling, Elyse, McDevitt, Shana L., Beckman, Kenneth B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489363/
https://www.ncbi.nlm.nih.gov/pubmed/31036053
http://dx.doi.org/10.1186/s13059-019-1691-6
Descripción
Sumario: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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13059-019-1691-6) contains supplementary material, which is available to authorized users.