title: Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome.
abstract: Within the ENCODE Consortium, GENCODE aimed to accurately annotate all protein-coding genes, pseudogenes, and noncoding transcribed loci in the human genome through manual curation and computational methods. Annotated transcript structures were assessed, and less well-supported loci were systematically, experimentally validated. Predicted exon-exon junctions were evaluated by RT-PCR amplification followed by highly multiplexed sequencing readout, a method we called RT-PCR-seq. Seventy-nine percent of all assessed junctions are confirmed by this evaluation procedure, demonstrating the high quality of the GENCODE gene set. RT-PCR-seq was also efficient to screen gene models predicted using the Human Body Map (HBM) RNA-seq data. We validated 73% of these predictions, thus confirming 1168 novel genes, mostly noncoding, which will further complement the GENCODE annotation. Our novel experimental validation pipeline is extremely sensitive, far more than unbiased transcriptome profiling through RNA sequencing, which is becoming the norm. For example, exon-exon junctions unique to GENCODE annotated transcripts are five times more likely to be corroborated with our targeted approach than with extensive large human transcriptome profiling. Data sets such as the HBM and ENCODE RNA-seq data fail sampling of low-expressed transcripts. Our RT-PCR-seq targeted approach also has the advantage of identifying novel exons of known genes, as we discovered unannotated exons in ~11% of assessed introns. We thus estimate that at least 18% of known loci have yet-unannotated exons. Our work demonstrates that the cataloging of all of the genic elements encoded in the human genome will necessitate a coordinated effort between unbiased and targeted approaches, like RNA-seq and RT-PCR-seq.
authors.affiliation: Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Cédric
authors.initials: C
authors.lastname: Howald
authors.forename: Andrea
authors.initials: A
authors.lastname: Tanzer
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Chrast
authors.forename: Felix
authors.initials: F
authors.lastname: Kokocinski
authors.forename: Thomas
authors.initials: T
authors.lastname: Derrien
authors.forename: Nathalie
authors.initials: N
authors.lastname: Walters
authors.forename: Jose M
authors.initials: JM
authors.lastname: Gonzalez
authors.forename: Adam
authors.initials: A
authors.lastname: Frankish
authors.forename: Bronwen L
authors.initials: BL
authors.lastname: Aken
authors.forename: Thibaut
authors.initials: T
authors.lastname: Hourlier
authors.forename: Jan-Hinnerk
authors.initials: JH
authors.lastname: Vogel
authors.forename: Simon
authors.initials: S
authors.lastname: White
authors.forename: Stephen
authors.initials: S
authors.lastname: Searle
authors.forename: Jennifer
authors.initials: J
authors.lastname: Harrow
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Hubbard
authors.forename: Roderic
authors.initials: R
authors.lastname: Guigó
authors.forename: Alexandre
authors.initials: A
authors.lastname: Reymond
mesh_terms: D019295:Computational Biology / Q000379:methods; D005091:Exons; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D058977:Molecular Sequence Annotation; D016366:Open Reading Frames; D059368:RNA Isoforms; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059467:Transcriptome
chemical_list: D059368:RNA Isoforms; D012333:RNA, Messenger