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

Details

Serval ID
serval:BIB_E1581D2AC4AD
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome.
Journal
Genome Research
Author(s)
Howald C., Tanzer A., Chrast J., Kokocinski F., Derrien T., Walters N., Gonzalez J.M., Frankish A., Aken B.L., Hourlier T., Vogel J.H., White S., Searle S., Harrow J., Hubbard T.J., Guigó R., Reymond A.
ISSN
1549-5469 (Electronic)
ISSN-L
1088-9051
Publication state
Published
Issued date
2012
Volume
22
Number
9
Pages
1698-1710
Language
english
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.
Pubmed
Web of science
Open Access
Yes
Create date
19/12/2012 16:55
Last modification date
20/08/2019 17:05
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