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Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome
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 systematica...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431487/ https://www.ncbi.nlm.nih.gov/pubmed/22955982 http://dx.doi.org/10.1101/gr.134478.111 |
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author | Howald, Cédric Tanzer, Andrea Chrast, Jacqueline Kokocinski, Felix Derrien, Thomas Walters, Nathalie Gonzalez, Jose M. Frankish, Adam Aken, Bronwen L. Hourlier, Thibaut Vogel, Jan-Hinnerk White, Simon Searle, Stephen Harrow, Jennifer Hubbard, Tim J. Guigó, Roderic Reymond, Alexandre |
author_facet | Howald, Cédric Tanzer, Andrea Chrast, Jacqueline Kokocinski, Felix Derrien, Thomas Walters, Nathalie Gonzalez, Jose M. Frankish, Adam Aken, Bronwen L. Hourlier, Thibaut Vogel, Jan-Hinnerk White, Simon Searle, Stephen Harrow, Jennifer Hubbard, Tim J. Guigó, Roderic Reymond, Alexandre |
author_sort | Howald, Cédric |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3431487 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-34314872012-09-08 Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome Howald, Cédric Tanzer, Andrea Chrast, Jacqueline Kokocinski, Felix Derrien, Thomas Walters, Nathalie Gonzalez, Jose M. Frankish, Adam Aken, Bronwen L. Hourlier, Thibaut Vogel, Jan-Hinnerk White, Simon Searle, Stephen Harrow, Jennifer Hubbard, Tim J. Guigó, Roderic Reymond, Alexandre Genome Res Method 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. Cold Spring Harbor Laboratory Press 2012-09 /pmc/articles/PMC3431487/ /pubmed/22955982 http://dx.doi.org/10.1101/gr.134478.111 Text en © 2012, Published by Cold Spring Harbor Laboratory Press This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/. |
spellingShingle | Method Howald, Cédric Tanzer, Andrea Chrast, Jacqueline Kokocinski, Felix Derrien, Thomas Walters, Nathalie Gonzalez, Jose M. Frankish, Adam Aken, Bronwen L. Hourlier, Thibaut Vogel, Jan-Hinnerk White, Simon Searle, Stephen Harrow, Jennifer Hubbard, Tim J. Guigó, Roderic Reymond, Alexandre Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title | Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title_full | Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title_fullStr | Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title_full_unstemmed | Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title_short | Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome |
title_sort | combining rt-pcr-seq and rna-seq to catalog all genic elements encoded in the human genome |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431487/ https://www.ncbi.nlm.nih.gov/pubmed/22955982 http://dx.doi.org/10.1101/gr.134478.111 |
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