Analysis and design of RNA sequencing experiments for identifying isoform regulation.
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.
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA
Yarden
Y
Katz
Eric T
ET
Wang
Edoardo M
EM
Airoldi
Christopher B
CB
Burge
D017398:Alternative Splicing; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry; D006801:Humans; D007438:Introns / Q000235:genetics; D050778:NFATC Transcription Factors / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017423:Sequence Analysis, RNA / Q000379:methods*
D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D050778:NFATC Transcription Factors; C494010:NFATC4 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D012313:RNA