Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

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  • dc.contributor.author Howald, Cédricca
  • dc.contributor.author Tanzer, Andreaca
  • dc.contributor.author Chrast, Jacquelineca
  • dc.contributor.author Kokocinski, Felixca
  • dc.contributor.author Derrien, Thomasca
  • dc.contributor.author Walters, Nathalieca
  • dc.contributor.author González, José M.ca
  • dc.contributor.author Frankish, Adamca
  • dc.contributor.author Aken, Bronwenca
  • dc.contributor.author Hourlier, Thibautca
  • dc.contributor.author Vogel, Jan-Hinnerkca
  • dc.contributor.author White, Simonca
  • dc.contributor.author Searle, Stephenca
  • dc.contributor.author Harrow, Jenniferca
  • dc.contributor.author Hubbard, Tim J.ca
  • dc.contributor.author Guigó Serra, Rodericca
  • dc.contributor.author Reymond, Alexandreca
  • dc.date.accessioned 2014-07-18T08:38:44Z
  • dc.date.available 2014-07-18T08:38:44Z
  • dc.date.issued 2012ca
  • dc.description.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.
  • dc.description.sponsorship This work was funded by National Human Genome Research Institute (NHGRI)/National Institutes of Health (NIH) grants to GENCODE (U54 HG004555) and Cold Spring Harbor Laboratories (U54 HG004557) subgroups of the ENCODE project. We acknowledge grants from the Swiss National Science Foundation to A.R., from the Spanish Ministry of Science (RD07/0067/0012, BIO2006-03380, and CSD2007-00050) to R.G., and from the Wellcome Trust (WT077198/Z/05/Z) to J.H., A.F., J.M.G., F.K., and T.J.H.
  • dc.format.mimetype application/pdfca
  • dc.identifier.citation Howald C, Tanzer A, Chrast J, Kokocinski F, Derrien T, Walters N et al. Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome. Genome Res. 2012;22(9):1698-710. DOI: 10.1101/gr.134478.111ca
  • dc.identifier.doi http://dx.doi.org/10.1101/gr.134478.111
  • dc.identifier.issn 1088-9051ca
  • dc.identifier.uri http://hdl.handle.net/10230/22634
  • dc.language.iso engca
  • dc.publisher Cold Spring Harbor Laboratory Press (CSHL Press)ca
  • dc.relation.ispartof Genome Research. 2012;22(9):1698-710
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PN/RD2007-0067
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PN/BIO2006-03380
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PN/CSD2007-00050
  • dc.rights © 2012 Cédric Howald et al. This is an Open Access article distributed under the terms of a Creative Commons License (Attribution-NonCommercial 3.0 Unported License)ca
  • dc.rights.accessRights info:eu-repo/semantics/openAccessca
  • dc.rights.uri http://creativecommons.org/licenses/by-nc/3.0/
  • dc.subject.other Expressió gènica -- Mètodes
  • dc.subject.other Genoma humà
  • dc.title Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genomeca
  • dc.type info:eu-repo/semantics/articleca
  • dc.type.version info:eu-repo/semantics/publishedVersionca

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