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Analysis and design of RNA sequencing experiments for identifying isoform regulation Katz Y; Wang ET; Airoldi EM; Burge CBNat Methods 2010[Dec]; 7 (12): 1009-15Through 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.|Alternative Splicing[MESH]|Base Sequence[MESH]|DNA-Binding Proteins/chemistry/genetics/metabolism[MESH]|Exons/genetics[MESH]|Heterogeneous-Nuclear Ribonucleoproteins/chemistry[MESH]|Humans[MESH]|Introns/genetics[MESH]|NFATC Transcription Factors/genetics[MESH]|Protein Isoforms/chemistry/genetics[MESH]|RNA, Messenger/chemistry/genetics[MESH]|RNA/*chemistry/genetics[MESH]|Reverse Transcriptase Polymerase Chain Reaction/methods[MESH]|Sequence Analysis, RNA/*methods[MESH] |
