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Nat Methods. 2010 Dec;7(12):1009-15. doi: 10.1038/nmeth.1528. Epub 2010 Nov 7.

Analysis and design of RNA sequencing experiments for identifying isoform regulation.

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  • 1Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA

Abstract

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV cross-linking-immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression.

PMID:
21057496
[PubMed - indexed for MEDLINE]
PMCID:
PMC3037023
Free PMC Article

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