Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

GENCODE Batch VIII c: determining lncRNA transcript 5' and 3'-ends by RACE-PCR / 454 sequencing

ABSTRACT: As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. In the current phase of ENCODE we have found strong evidence that many lncRNAs transcript termini are still unknown. This experiment aims to set up an experimental validation strategy to accurately determine the 5' and 3' ends of transcripts, which is based on semi-nested RACE extensions of annotated 5' and 3' ends followed by high throughput sequencing. A total of 400 highly expressed lncRNA transcript models from Gencode 7 which did not have any CAGE/PET support were selected as the test set whereas 25 transcripts with transcript start site (TSS) supported by CAGE tags and transcript termination site (TTS) supported by PET ditags formed the positive control set. Transcript ends were amplified by RACE-PCR from RNA samples from five different tissues (heart, kidney, liver, lung and spleen) and sequenced using the Roche 454 platform. The sequencing was performed at the Andalusian Human Genome Sequencing Centre (CASEGH), Seville, Spain.

INSTRUMENT(S): 454 GS FLX+

ORGANISM(S): Homo sapiens

SUBMITTER: Jose Gonzalez

PROVIDER: E-MTAB-1665 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

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

Howald Cédric C Tanzer Andrea A Chrast Jacqueline J Kokocinski Felix F Derrien Thomas T Walters Nathalie N Gonzalez Jose M JM Frankish Adam A Aken Bronwen L BL Hourlier Thibaut T Vogel Jan-Hinnerk JH White Simon S Searle Stephen S Harrow Jennifer J Hubbard Tim J TJ Guigó Roderic R Reymond Alexandre A

Genome research 20120901 9

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 perce ...[more]

PMID: 22955982

Publication: 1/2

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Project description:Transcriptional profiling by high throughput sequencing of RACE-PCR-amplified human brain and testis samples to determine lncRNA transcript 5' and 3'-ends (GENCODE Batch VIII b) As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. In the current phase of ENCODE we have found strong evidence that many lncRNAs transcript termini are still unknown. This experiment aims to set up an experimental validation strategy to accurately determine the 5' and 3' ends of transcripts, which is based on semi-nested RACE extensions of annotated 5' and 3' ends followed by high throughput sequencing. A total of 400 highly expressed lncRNA transcript models from Gencode 7 which did not have any CAGE/PET support were selected as the test set whereas 25 transcripts with transcript start site (TSS) supported by CAGE tags and transcript termination site (TTS) supported by PET ditags formed the positive control set. Transcript ends were amplified by RACE-PCR from brain and testis RNA samples and sequenced using the Roche 454 platform. The sequencing was performed at the Centre for Genomic Regulation (CRG), Barcelona, Spain. ArrayExpress Release Date: 2012-08-31 Person Roles: submitter Person Last Name: Gonzalez Person First Name: Jose Person Mid Initials: M Person Email: jmg@sanger.ac.uk Person Phone: -498006 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Hubbard Person First Name: Tim Person Mid Initials: Person Email: th@sanger.ac.uk Person Phone: -498055 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Reymond Person First Name: Alexandre Person Mid Initials: Person Email: Alexandre.Reymond@unil.ch Person Phone: Person Address: Lausanne, Switzerland Person Affiliation: University of Lausanne Person Roles: investigator Person Last Name: Guigo Person First Name: Roderic Person Mid Initials: Person Email: roderic.guigo@crg.cat Person Phone: Person Address: Barcelona, Spain Person Affiliation: Centre for Genomic Regulation (CRG) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Project description:Transcriptional profiling by high throughput sequencing of RACE-PCR-amplified human brain and testis samples to determine lncRNA transcript 5' and 3'-ends (GENCODE Batch VIII a) As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. In the current phase of ENCODE we have found strong evidence that many lncRNAs transcript termini are still unknown. This experiment aims to set up an experimental validation strategy to accurately determine the 5' and 3' ends of transcripts, which is based on semi-nested RACE extensions of annotated 5' and 3' ends followed by high throughput sequencing. A total of 400 highly expressed lncRNA transcript models from Gencode 7 which did not have any CAGE/PET support were selected as the test set whereas 25 transcripts with transcript start site (TSS) supported by CAGE tags and transcript termination site (TTS) supported by PET ditags formed the positive control set. Transcript ends were amplified by RACE-PCR from brain and testis RNA samples and sequenced using the Roche 454 platform. The sequencing was performed at the Andalusian Human Genome Sequencing Centre (CASEGH), Seville, Spain. ArrayExpress Release Date: 2012-08-31 Person Roles: submitter Person Last Name: Gonzalez Person First Name: Jose Person Mid Initials: M Person Email: jmg@sanger.ac.uk Person Phone: -498006 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Hubbard Person First Name: Tim Person Mid Initials: Person Email: th@sanger.ac.uk Person Phone: -498055 Person Address: Wellcome Trust Genome Campus, Hinxton, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Reymond Person First Name: Alexandre Person Mid Initials: Person Email: Alexandre.Reymond@unil.ch Person Phone: Person Address: Lausanne, Switzerland Person Affiliation: University of Lausanne Person Roles: investigator Person Last Name: Guigo Person First Name: Roderic Person Mid Initials: Person Email: roderic.guigo@crg.cat Person Phone: Person Address: Barcelona, Spain Person Affiliation: Centre for Genomic Regulation (CRG) Person Roles: investigator Person Last Name: Santoyo Person First Name: Javier Person Mid Initials: Person Email: jsantoyo@bioinfomgp.org Person Phone: Person Address: Seville, Spain Person Affiliation: Andalusian Human Genome Sequencing Centre (CASEGH) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Project description:Rationale: Asthma and atopy shares common features including Th2-inflammation. However, impairment of airway function seems to be absent in atopy. Increased understanding of the complex cellular and molecular pathways defining the similarities and differences between asthma and atopy may be achieved by transcriptomic analysis (RNA-Seq). Hypothesis and Aims: As the airway smooth muscle (ASM) layer plays an important role in airway function, we hypothesized that the transcriptomic profile of the ASM layer in endobronchial biopsies is different between atopic asthma patients and atopic healthy controls. First, we examined the differences in transcriptomic profiles of the ASM layer in endobronchial biopsies between atopic mild, steroid-free asthma patients, and atopic and non-atopic healthy controls. Second, we investigated the association between the transcriptomic profiles of the ASM layer and airway function. Methods: This cross-sectional study included 12 steroid-free atopic asthma patients, 6 atopic, and 6 non-atopic healthy controls. RNA of ASM from 4 endobronchial biopsies per subject was isolated and sequenced (GS FLX+, 454/Roche). Ingenuity Pathway Analysis was used to identify gene networks. Comparison of the numbers of reads per gene in asthma and controls was based on the negative binomial distribution. At the current sample size the estimated false discovery rate was approximately 1%. Results: Yield of isolated RNA was 30-821ng. We identified 174 differentially expressed genes between asthma and atopic controls, 108 between asthma and non-atopic controls, and 135 between atopic and non-atopic controls. A set of 8 genes was identified, which seems to define asthma patients from non-asthmatic controls regardless of atopy. Four of these genes were significantly associated with airway hyperresponsiveness. Conclusion: A difference in transcriptomic profile of the airway smooth muscle layer in asthma patients compared to atopic and non-atopic healthy controls may lead to a different regulation of inflammatory pathways and of airway smooth muscle function and development resulting in impaired airway function. This cross-sectional transcriptomics study consisted of 2 visits. At visit 1, asthma patients (n=12), and healthy atopic (n=6) and non-atopic (n=6) controls were screened for eligibility to participate according to the in- and exclusion criteria. Spirometry and a methacholine bronchoprovocation test were performed. At visit 2, FEV1 reversibility was measured and 4 endobronchial biopsies per subject were collected during a bronchoscopy. Airway smooth muscle was collected from the biopsies by laser capture microdissection and total RNA isolated. cDNA was prepared using the Ovation RNA-Seq System (NuGEN). RNA-Seq was performed using the GS FLX+ instrument (454/Roche). Sequence reads were mapped against the human genome (hg19; UCSC). Comparison of the numbers of reads per gene between asthma and healthy controls was based on the negative binomial distribution and carried out with the R package DESeq including correction for multiple testing.

Project description:Rationale: The cellular and molecular pathways in asthma are highly complex. Increased understanding can be obtained by unbiased transcriptomic analysis (RNA-Seq). Hypothesis and Aims: We hypothesized that the transcriptomic profile of whole human endobronchial biopsies differs between patients with asthma and controls. First, we investigated the feasibility to obtain RNA from whole endobronchial biopsies suitable for RNA-Seq. Second, we examined the difference in transcriptomic profiles between asthma and controls. Methods: This cross-sectional study compared 4 steroid-free atopic asthma patients and 5 healthy non-atopic controls. RNA of ASM from 4 endobronchial biopsies per subject was isolated and sequenced (GS FLX+, 454/Roche). Ingenuity Pathway Analysis was used to identify gene networks. Comparison of the numbers of reads per gene in asthma and controls was based on the Poisson distribution. At the current sample size the estimated false discovery rate was 4%. Results: Yield of isolated RNA was 900-9,300ng. We identified 10,167 and 11,006 unique genes for asthma and controls, respectively. Forty-six genes were differentially expressed between asthma and controls, including pendrin, periostin, and BCL2. Ten gene networks involved in cellular morphology, movement, and development had an IPA network score ≥2. Conclusion:RNA isolated from whole human endobronchial biopsies is suitable for RNA-Seq, showing different transcriptomic profiles between asthma and controls. Novel and confirmative genes were found to be linked to asthma. These results indicate that biological processes in the airways of asthma patients are differently regulated compared to healthy controls, which may be relevant for the pathogenesis and treatment of the disease. This cross-sectional transcriptomics study consisted of 2 visits. At visit 1, atopic asthma patients (n=4), and healthy non-atopic controls (n=5) were screened for eligibility to participate according to the in- and exclusion criteria. Spirometry and a methacholine bronchoprovocation test were performed. At visit 2, FEV1 reversibility was measured and 4 endobronchial biopsies per subject were collected during a bronchoscopy. Total RNA from whole endobronchial biopsies was collected. cDNA was prepared using the Ovation RNA-Seq System (NuGEN). RNA-Seq was performed using the GS FLX+ instrument (454/Roche). Sequence reads were mapped against the human genome (hg19; UCSC). Comparison of the numbers of reads per gene between asthma and healthy controls was based on the Poisson distribution.

Dataset Information

GENCODE Batch VIII c: determining lncRNA transcript 5' and 3'-ends by RACE-PCR / 454 sequencing

Publications

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

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