Unknown

Dataset Information

0

Modeling Enzyme Processivity Reveals that RNA-Seq Libraries Are Biased in Characteristic and Correctable Ways.


ABSTRACT: Experimental procedures for preparing RNA-seq and single-cell (sc) RNA-seq libraries are based on assumptions regarding their underlying enzymatic reactions. Here, we show that the fairness of these assumptions varies within libraries: coverage by sequencing reads along and between transcripts exhibits characteristic, protocol-dependent biases. To understand the mechanistic basis of this bias, we present an integrated modeling framework that infers the relationship between enzyme reactions during library preparation and the characteristic coverage patterns observed for different protocols. Analysis of new and existing (sc)RNA-seq data from six different library preparation protocols reveals that polymerase processivity is the mechanistic origin of coverage biases. We apply our framework to demonstrate that lowering incubation temperature increases processivity, yield, and (sc)RNA-seq sensitivity in all protocols. We also provide correction factors based on our model for increasing accuracy of transcript quantification in existing samples prepared at standard temperatures. In total, our findings improve our ability to accurately reflect in vivo transcript abundances in (sc)RNA-seq libraries.

SUBMITTER: Archer N 

PROVIDER: S-EPMC5167349 | BioStudies | 2016-01-01

REPOSITORIES: biostudies

Similar Datasets

2019-01-01 | S-EPMC6538698 | BioStudies
2014-01-01 | S-EPMC4197826 | BioStudies
2019-01-01 | S-EPMC6366399 | BioStudies
2016-11-09 | GSE84785 | GEO
2016-01-01 | S-EPMC4793214 | BioStudies
1000-01-01 | S-EPMC3428589 | BioStudies
2020-01-01 | S-EPMC7722316 | BioStudies
2014-01-01 | S-EPMC4070569 | BioStudies
2011-01-01 | S-EPMC3166838 | BioStudies
1000-01-01 | S-EPMC5976456 | BioStudies