A benchmarking of workflows for detecting differential splicing and differential expression at isoform level in human RNA-seq studies

MERINO, GABRIELA A.; CONESA, ANA; FERNÁNDEZ, ELMER A.

BRIEFINGS IN BIOINFORMATICS

OXFORD UNIV PRESS

Año: 2017

1467-5463

Over the last few years, RNA-seq has been used to study alterations in alternative splicing related to several diseases.Bioinformatics workflows used to perform these studies can be divided into two groups, those finding changes in the abso-lute isoform expression and those studying differential splicing. Many computational methods for transcriptomics analysishave been developed, evaluated and compared; however, there are not enough reports of systematic and objective assess-ment of processing pipelines as a whole. Moreover, comparative studies have been performed considering separately thechanges in absolute or relative isoform expression levels. Consequently, no consensus exists about the best practices andappropriate workflows to analyse alternative and differential splicing. To assist the adequate pipeline choice, we presenthere a benchmarking of nine commonly used workflows to detect differential isoform expression and splicing. We eval-uated the workflows performance over different experimental scenarios where changes in absolute and relative isoformexpression occurred simultaneously. In addition, the effect of the number of isoforms per gene, and the magnitude ofthe expression change over pipeline performances were also evaluated. Our results suggest that workflow performance isinfluenced by the number of replicates per condition and the conditions heterogeneity. In general, workflows based onDESeq2, DEXSeq, Limma and NOISeq performed well over a wide range of transcriptomics experiments. In particular,we suggest the use of workflows based on Limma when high precision is required, and DESeq2 and DEXseq pipelines toprioritize sensitivity. When several replicates per condition are available, NOISeq and Limma pipelines are indicated.