The impact of amplification on di↵erential expression analyses by RNA-seq

Correspondence: [email protected] Anthropology and Human Genomics, Department of Biology II, Ludwig Maximilians University Munich, Grosshaderner Str. 2, D-82152 Martinsried, Germany Full list of author information is available at the end of the article 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 e↵ects precision and accuracy of RNA quantification. To assess the e↵ects 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 PCRfrom 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. Results Here, we generate and analyse RNA-Seq datasets that were prepared with three di↵erent 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 discovery rate are only mildly improved. However, we do find that power does improve with fewer PCR amplification cycles across datasets and that early barcoding of samples and hence PCR amplification in one reaction can restore this loss of power. Conclusions Computational removal of read duplicates is not recommended for di↵erential expression analysis. However, the pooling of samples as made possible by the early barcoding of the UMI-protocol leads to an appreciable increase in the power to detect di↵erentially expressed genes.