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

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... ng 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 crosslinking-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. The distinct isoforms expressed from metazoan genes through alternative splicing can be important in development, differentiation and disease 1 . For example, the pyruvate kinase gene produces two distinct tissue-specific spliced isoforms that differ in their enzymatic activity, allosteric regulation and ability to support tumor growth 2 . Conservative estimates predict 2-12 mRNA isoforms for most mammalian genes ( Supplementary Fig. 1) , though some genes, including neurexins, may express more than 1,000 isoforms each 3 . ). AUTHOR CONTRIBUTIONS Y.K., development of MISO model and software, analyses involving MISO, writing of main text and methods; E.T.W., hnRNP H CLIP-seq experiments and associated computational analyses, CUGBP1 knockdown RNA-seq experiments and associated computational analyses; E.M.A., development of model and statistical analysis, writing of methods; C.B.B., development of MISO model, contributions to computational analyses, writing of main text.