Transcriptome profiling of Mesoplasma florum strain L1 using RNA sequencing (RNA-Seq) and genome-wide 5'-rapid amplification of cDNA ends (5'-RACE)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mesoplasma florum
PROVIDER: GSE152985 | GEO | 2020/11/02
REPOSITORIES: GEO
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Project description:Long non-coding RNAs (lncRNA) constitute a large fraction of mammalian transcriptomes that still remains unexplored, mainly due to the lack of comprehensive, high-quality lncRNA annotation that limits the possibility to fully explore their functional capacity. We have developed RACE-seq, an experimental workflow based on RACE (Rapid Amplification of cDNA Ends) and long read RNA sequencing, aimed at both rare isoform discovery and better definition of gene boundaries. We applied 3â and 5â RACE-seq on 398 low-expressed GENCODE v7 lncRNA genes in seven human tissues (brain, testis, heart, kidney, liver, lung and spleen). The sequences obtained led to the discovery of 2,641 on-target, previously unknown alternative transcripts. Novel isoforms extended 60% of the 398 targeted lncRNA loci further in either 5' or 3', and often reached genome hallmarks typical of gene boundaries. In parallel, we used nested RACE-seq, and confirmed that nested RACE-seq has overwhelmingly better sensitivity than its standard counterpart.
2016-06-01 | E-MTAB-3912 | biostudies-arrayexpress
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2012-08-31 | E-MTAB-1222 | biostudies-arrayexpress
Project description: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.
2012-08-31 | E-MTAB-1226 | biostudies-arrayexpress
Project description:The green algal Botryococcus braunii (Chlorophyte) is known for accumulating high levels of hydrocarbons that are a useful alternative to fossil fuels. B. braunii is categorized into three groups based on types of their accumulated hydrocarbons: alkadiene/triene in race A, botryococcenes in race B, and lycopadiene in race L. Transcriptomic studies in race A and race B have discovered tremendous information related to the genes encoding proteins involved in hydrocarbon biosynthesis. However, transcriptome of race L has not been reported. In this study, we report a transcriptome of race L B. braunii AC768 through the de novo assembly using Hiseq platform. Our analyses indicate that photosynthesis and protein biosynthesis are the most abundantly transcribed in actively growing race L B. braunii. We show that the transcriptome of race L shares similar amounts (~20%) of mutual best-hits with that of race A or race B. Sequence homologous analyses indicate that enzymes involved in squalene and phytoene biosynthesis are well separated into geranyl-diphosphate synthase, farnesyl-diphosphate synthase, geranylgeranyl-diphosphate synthase, phytoene synthase, and squalene synthase. Both B. braunii specific enzymes botryococcene synthase SSL3 and lycopaoctaene synthase LOS are found to form distinctive subgroups in the group of squalene synthase. One of the ESTs in AC768 transcriptome that falls into the subgroup with LOS and shares >88% identity with that of LOS. Together, our results show that SSL and LOS are unique to race B and race L B. braunii subspecies, respectively. We propose that phytoene synthase in race L shares higher homolog to squalene synthase than phytoene synthase in other algae.
2019-11-21 | GSE96585 | GEO
Project description: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.
2013-05-17 | E-MTAB-1665 | biostudies-arrayexpress
Project description:RACE (Rapid Amplification of cDNA Ends) is a widely used approach for transcript identification. However, the dynamic range in the population of RACE transcript isoforms may be very large, and random clone selection -the typical approach- may be ineffective in sampling the different transcript species present in the population. Here, we describe an effective RACE sampling strategy. The products of the RACE reaction are hybridized onto high-density tiling arrays, and the exons detected are then used to delineate a series of RT-PCR reactions, through which the original RACE mixture is segregated into a number of simpler RT-PCR reactions. These are independently cloned, and randomly selected clones are sequenced. This approach is superior to the direct cloning and sequencing of the RACE products: it specifically targets novel transcripts, and often leads to the overall normalization of their abundances. We indeed show theoretically that this strategy leads to a very efficient sampling of the novel transcript species associated to annotated loci. In a pilot experiment, we used this approach to discover many novel transcripts for a few otherwise well-characterized protein coding genes. Finally we investigate how this strategy can be multiplexed for large-scale transcript discovery by high-density pooling of RACE reactions prior to hybridization. Our results indicate that through the interrogation of a limited number of exons per gene on a limited number of cell types, it is possible to recover a large fraction of the transcript diversity associated to protein coding loci. These loci, however, could be occupying a much larger genomic space than previously expected, implying that efficient multiplexing requires non-trivial pooling optimization.
2008-05-25 | GSE11433 | GEO
Project description:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS. S. oneidensis MR-1 was grown to mid-log phase in Luria-Bertani broth (LB) or defined lactate minimal medium, and total RNA was isolated and used for differential RNA-sequencing (dRNA-seq) by next-generation sequencing, which is used to verify genome-wide transcription start sites. For dRNA-seq, total RNA was partially treated with Terminator Exonuclease (TEX) to digest processed RNA and thereby enrich for primary transcript ends.
2014-06-12 | E-GEOD-58292 | biostudies-arrayexpress
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
2013-05-11 | GSE46636 | GEO
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
2013-05-11 | GSE46635 | GEO
Project description:The purpose of these experiments was to determine the 5' and 3' transcriptional termini of genes on chromosomes 21-22. Towards this end, we first carried out 5’ and 3’ RACE reactions from 1193 exons from 492 genes in these chromosomes using RNA isolated from 11 normal tissues and 5 cell lines. An average of 3 to 4 RACE reactions (5’ and 3’) was carried out from each gene. The products of the RACE reactions were analyzed by hybridization to tiling arrays interrogating the non-repeat portions of these chromosomes at 17 nucleotide resolution. A total of 26,688 RACE reactions were performed and pooled to be placed on to 1020 tiling arrays For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2009-08-13 | GSE17600 | GEO