E-MTAB-3766 - Simulation-based assessment of differential transcript usage using RNA-seq data: a matter of counting
Submitted on 10 June 2015, released on 23 July 2015, last updated on 30 July 2015
'Background: Large-scale sequencing of cDNA (RNA-seq) has been a boon to the quantitative analysis of transcriptomes. A notable application of significant biomedical relevance is the detection of changes in transcript usage between experimental conditions. For example, discovery of pathological alternative splicing may allow the development of new treatments or better management of patients. From an analysis perspective, there are several ways to represent RNA-seq data to unravel differential transcript usage, such as annotation-based exon-level counting, differential analysis of the `percent spliced in'' measure or quantitative analysis of assembled transcripts. The goal of this research is to compare and contrast current state-of-the-art methods, as well as to suggest improvements to commonly used workflows. Results: We assess the performance of representative workflows using synthetic data, and explore the effect of using non-standard counting bin definitions as input to a state-of-the-art inference engine (DEXSeq). Although the canonical counting provided the best results overall, several non-canonical approaches were as good or better in specific aspects, and most counting approaches outperformed the evaluated event- and assembly-based methods. We show that an incomplete annotation catalog can have a detrimental effect on the ability to detect differential transcript usage in transcriptomes with few isoforms per gene, and that isoform-level pre-filtering can considerably improve the false discovery rate (FDR) control. Conclusion: Count-based methods generally perform well in detection of differential transcript usage. Controlling the FDR at the imposed threshold is difficult, mainly in complex organisms, but can be improved by pre-filtering of the annotation catalog.'
RNA-seq of coding RNA, cell type comparison design
Isoform prefiltering improves performance of count-based methods for analysis of differential transcript usage. Soneson C, Matthes KL, Nowicka M, Law CW, Robinson MD. :12 (2016), PMID:26813113