RT Journal
A1 Howald, Cédric
A1 Tanzer, Andrea
A1 Chrast, Jacqueline
A1 Kokocinski, Felix
A1 Derrien, Thomas
A1 Walters, Nathalie
A1 Gonzalez, Jose M.
A1 Frankish, Adam
A1 Aken, Bronwen L.
A1 Hourlier, Thibaut
A1 Vogel, Jan-Hinnerk
A1 White, Simon
A1 Searle, Stephen
A1 Harrow, Jennifer
A1 Hubbard, Tim J.
A1 Guigó, Roderic
A1 Reymond, Alexandre
T1 Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome
JF Genome Research 
JO Genome Research 
YR 2012 
FD September 01 
VO 22 
IS 9 
SP 1698 
OP 1710 
DO 10.1101/gr.134478.111 
UL http://genome.cshlp.org/content/22/9/1698.abstract 
AB 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.