TRUmiCount: correctly counting absolute numbers of molecules using unique molecular identifiers

TRUmiCount: correctly counting absolute numbers of molecules using unique molecular identifiers

Abstract

Motivation Counting molecules using next-generation sequencing (NGS) suffers from PCR amplification bias, which reduces the accuracy of many quantitative NGS-based experimental methods such as RNA-Seq. This is true even if molecules are made distinguishable using unique molecular identifiers (UMIs) before PCR amplification, and distinct UMIs are counted instead of reads: Molecules that are lost entirely during the sequencing process will still cause underestimation of the molecule count, and amplification artifacts like PCR chimeras create phantom UMIs and thus cause over-estimation. Results We introduce the TRUmiCount algorithm to correct for both types of errors. The TRUmiCount algorithm is based on a mechanistic model of PCR amplification and sequencing, whose two parameters have an immediate physical interpretation as PCR efficiency and sequencing depth and can be estimated from experimental data without requiring calibration experiments or spike-ins. We show that our model captures the main stochastic properties of amplification and sequencing, and that it allows us to filter out phantom UMIs and to estimate the number of molecules lost during the sequencing process. Finally, we demonstrate that the phantom-filtered and loss-corrected molecule counts computed by TRUmiCount measure the true number of molecules with considerably higher accuracy than the raw number of distinct UMIs, even if most UMIs are sequenced only once as is typical for single-cell RNA-Seq. Availability and implementation TRUmiCount is available at http://www.cibiv.at/software/trumicount and through Bioconda (http://bioconda.github.io).

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Authors
  • Pflug, Florian G
  • von Haeseler, Arndt
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Shortfacts
Category
Journal Paper
Divisions
Bioinformatics and Computational Biology
Journal or Publication Title
Bioinformatics
ISSN
1367-4803
Publisher
Oxford University Press
Place of Publication
Oxford OX1 2JD
Page Range
pp. 3137-3144
Number
18
Volume
34
Date
16 April 2018
Official URL
http://dx.doi.org/10.1093/bioinformatics/bty283
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