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Refining mouse annotations via targeted RNA sequencing

ABSTRACT: In this work we aim to improve the understanding of the mouse transcriptome complexity, investigate the expressed fraction of the genome and ameliorate the available mouse annotations. We utilized CatureSeq, a recently described strategy meant to enhance the sequencing coverage of low abundant genes. In our experimental design we generated oligonucleotide probes to select annotated and putative long-noncoding RNA and splice junctions. This allowed us to improve dramatically the sequencing throughput of the targeted regions. As a consequence our approach permitted the simultaneous identification of thousands of exons and the expansion of the already known ones

ORGANISM(S): Mus musculus

PROVIDER: GSE72311 | GEO | 2016/04/04

SECONDARY ACCESSION(S): PRJNA293710

REPOSITORIES: GEO

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Refining mouse annotations via targeted RNA sequencing

Project description:In this work we aim to improve the understanding of the mouse transcriptome complexity, investigate the expressed fraction of the genome and ameliorate the available mouse annotations. We utilized CatureSeq, a recently described strategy meant to enhance the sequencing coverage of low abundant genes. In our experimental design we generated oligonucleotide probes to select annotated and putative long-noncoding RNA and splice junctions. This allowed us to improve dramatically the sequencing throughput of the targeted regions. As a consequence our approach permitted the simultaneous identification of thousands of exons and the expansion of the already known ones The mouse gene assembly is anlaysed by targeted RNA sequencing of lncRNA and splice junctions. 8 mouse tissues and 16 samples are considered in the analysis. Each sample was added with external RNA controls. The controls are polyadenylated transcripts of known concentration designed to be added to an RNA analysis experiment after sample isolation.

2016-04-04 | E-GEOD-72311 | biostudies-arrayexpress

Validation of alternatively spliced protein isoforms in human mesenchymal stem cells

Project description:Alternative splicing of mRNA diversifies the function of human proteins, with tissue- and cell-specific protein isoforms being the most difficult to validate. While transcriptomic experiments enable the detection of many alternatively spliced transcripts, it is not known if these transcripts have protein-coding potential. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and exon-exon junctions of spliced transcripts by integrating transcriptomics and proteomics data. Using the PG Nexus, we analyzed undifferentiated human mesenchymal stem cells and compared the number of protein isoforms validated using different protein sequence database, including public online databases and RNA-seq derived databases. With significant overlaps with other databases, we identified 8,011 exons and 3,824 splice junctions with the Ensembl database. Both exonic and junction peptides were important for protein isoform validation. The Ensembl database consistently outperformed the other data sources, but predicted open reading frames from RNA-seq derived transcripts were comparable, with only 6 less splice junctions validated. Using proteotypic and isoform-specific peptides, we validated 462 protein isoforms. This number increases to 1,083 if multiple proteotypic peptides per protein are included. Multiplexing proteotypic peptides in SRM assays or similar experiments will increase the confidence and coverage of protein isoform validation experiments.

2015-05-20 | PXD001449 | Pride

Non-canonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in cancer patients

Project description:ost characterized tumor antigens are ‘genomic’, i.e. encoded by canonical, non-canonical or somatically mutated genomic sequences. We investigate here the presentation and immunogenicity of tumor antigens derived from non-canonical mRNA splicing events between coding exons and transposable elements (TEs). Comparing non-small cell lung cancer (NSCLC), an immunogenic tumor type, and diverse non-tumor tissues, we identify several thousand splicing junctions between exons and diverse TE classes. A subset of these junctions is both tumor-specific and shared across patients. HLA-I peptidomic identifies peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides are immunogenic in vitro and CD8+ T cells specific for junction-encoded epitopes are present in tumors and tumor-draining lymph nodes from NSCLC patients. We conclude that non-canonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in NSCLC cancer patients.

2023-02-27 | PXD034820 | Pride

The Drosophila melanogaster Transcriptome by Paired-End RNA-Sequencing

Project description:RNA-seq was used to generate an extensive map of the Drosophila melanogaster transcriptome by broad sampling of 10 developmental stages. In total, 142.2 million uniquely mapped 64-100bp paired-end reads were generated on the Illumina GA II yielding 356x sequencing coverage. More than 95% of FlyBase genes and 90% of splicing junctions were observed. Modifications to 30% of FlyBase gene models were made by extension of untranslated regions, inclusion of novel exons, and identification of novel splicing events. A total of 319 novel transcripts were identified, representing a 2% increase over the current annotation. Alternate splicing was observed in 31% of D. melanogaster genes, a 38% increase over previous estimations, but significantly less than that observed in higher organisms. Much of this splicing is subtle such as tandem alternate splice sites.

2010-09-24 | GSE24324 | GEO

The Drosophila melanogaster Transcriptome by Paired-End RNA-Sequencing

2010-09-23 | E-GEOD-24324 | biostudies-arrayexpress

Epigenetically-controlled tumor antigens derived from splice junctions between exons and transposable elements

Project description:Epigenetically-controlled tumor antigens derived from splice junctions between exons and transposable elements

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Half decimal place rule

Project description:Many MS2 spectra in bottom-up proteomics experiments remain unassigned. To improve proteome coverage, we applied the half decimal place rule (HDPR) to remove non-peptidic molecules. The HDPR considers the ratio of the first digit after the decimal point to the full molecular mass and results in a relatively small permitted mass window for most peptides. Although the HDPR has been described previously, it has never been integrated into an acquisition strategy using high resolution mass spectrometers. The HDPR was applied to three technical replicates of an in-solution tryptic digest of HeLa cells which were analysed by LC-MS using a Q Exactive mass spectrometer.

2022-02-28 | PXD003308 | Pride

Half decimal place rule - Application of the half decimal place rule to increase the peptide identification rate

2016-12-23 | PXD004900 | Pride

A long-read RNA-seq approach to identify novel transcripts of very large genes

Project description:RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon-junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap - 5kb), large (Nebulin - 22 kb) and very-large (Titin - 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type specific differential expression of these novel transcripts. The improved transcript identification and quantification demonstrated by our approach removes previous impediments to studies aimed at quantitative differential expression of ultra-long transcripts.

2020-05-17 | GSE138362 | GEO

GENCODE PCR-Seq Batch XVII

Project description:As part of the ENCODE consortium, the GENCODE project is producing a reference gene set through manual and automated gene prediction. Selected transcript models are verified experimentally by RT-PCR amplification of at least one of their unique splice junctions, followed by high-throughput sequencing (RT-PCR-seq). The experimental targets are manually annotated transcripts with novel or putative status, non-pseudogene biotype, and unique splice junctions which have not been validated previously and are not supported by RNAseq data from the ENCODE and GTEx projects. For Batch XVII, 1159 splice junctions which failed this experimental verification in previous experiments were tested again. On this occasion, newly designed PCR primers were used and compared with the original ones. In addition, the original tissue panel was widened with the inclusion of eight new tissues.

2017-03-31 | E-MTAB-5527 | biostudies-arrayexpress

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