Dec 28, 2015

The impact of amplification on differential expression analyses by RNA-seq

BioRxiv : the Preprint Server for Biology
Swati ParekhInes Hellmann

Abstract

Background Currently quantitative RNA-Seq methods are pushed to work with increasingly small starting amounts of RNA that require PCR amplification to generate libraries. However, it is unclear how much noise or bias amplification introduces and how this effects precision and accuracy of RNA quantification. To assess the effects of amplification, reads that originated from the same RNA molecule (PCR-duplicates) need to be identified. Computationally, read duplicates are defined via their mapping position, which does not distinguish PCR- from natural duplicates that are bound to occur for highly transcribed RNAs. Hence, it is unclear how to treat duplicate reads and how important it is to reduce PCR amplification experimentally. Here, we generate and analyse RNA-Seq datasets that were prepared with three different protocols (Smart-Seq, TruSeq and UMI-seq). We find that a large fraction of computationally identified read duplicates can be explained by sampling and fragmentation bias. Consequently, the computational removal of duplicates does not improve accuracy, power or false discovery rates, but can actually worsen them. Even when duplicates are experimentally identified by unique molecular identifiers (UMIs), power and false ...Continue Reading

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Mentioned in this Paper

Positioning Attribute
Abnormal Fragmented Structure
Genes
Sequence Determinations, RNA
Hemoglobin Kokura
Gene Amplification Technique
Base Sequence
Analysis
cDNA Library
Gene Amplification Abnormality

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