title: Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events.
abstract: Alternative splicing shapes mammalian transcriptomes, with many RNA molecules undergoing multiple distant alternative splicing events. Comprehensive transcriptome analysis, including analysis of exon co-association in the same molecule, requires deep, long-read sequencing. Here we introduce an RNA sequencing method, synthetic long-read RNA sequencing (SLR-RNA-seq), in which small pools (≤1,000 molecules/pool, ≤1 molecule/gene for most genes) of full-length cDNAs are amplified, fragmented and short-read-sequenced. We demonstrate that these RNA sequences reconstructed from the short reads from each of the pools are mostly close to full length and contain few insertion and deletion errors. We report many previously undescribed isoforms (human brain: ∼13,800 affected genes, 14.5% of molecules; mouse brain ∼8,600 genes, 18% of molecules) and up to 165 human distant molecularly associated exon pairs (dMAPs) and distant molecularly and mutually exclusive pairs (dMEPs). Of 16 associated pairs detected in the mouse brain, 9 are conserved in human. Our results indicate conserved mechanisms that can produce distant but phased features on transcript and proteome isoforms.
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Hagen
authors.initials: H
authors.lastname: Tilgner
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Jahanbani
authors.affiliation: Illumina Inc., San Francisco, California, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Blauwkamp
authors.affiliation: Illumina Inc., San Francisco, California, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Moshrefi
authors.affiliation: Illumina Inc., San Francisco, California, USA.
authors.forename: Erich
authors.initials: E
authors.lastname: Jaeger
authors.affiliation: Illumina Inc., San Francisco, California, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Itamar
authors.initials: I
authors.lastname: Harel
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Carlos D
authors.initials: CD
authors.lastname: Bustamante
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Morten
authors.initials: M
authors.lastname: Rasmussen
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Snyder
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001923:Brain Chemistry; D019295:Computational Biology; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D059368:RNA Isoforms / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D059368:RNA Isoforms