Dataset Information

TrueSight: a new algorithm for splice junction detection using RNA-seq.

ABSTRACT: RNA-seq has proven to be a powerful technique for transcriptome profiling based on next-generation sequencing (NGS) technologies. However, due to the short length of NGS reads, it is challenging to accurately map RNA-seq reads to splice junctions (SJs), which is a critically important step in the analysis of alternative splicing (AS) and isoform construction. In this article, we describe a new method, called TrueSight, which for the first time combines RNA-seq read mapping quality and coding potential of genomic sequences into a unified model. The model is further utilized in a machine-learning approach to precisely identify SJs. Both simulations and real data evaluations showed that TrueSight achieved higher sensitivity and specificity than other methods. We applied TrueSight to new high coverage honey bee RNA-seq data to discover novel splice forms. We found that 60.3% of honey bee multi-exon genes are alternatively spliced. By utilizing gene models improved by TrueSight, we characterized AS types in honey bee transcriptome. We believe that TrueSight will be highly useful to comprehensively study the biology of alternative splicing.

SUBMITTER: Li Y

PROVIDER: S-EPMC3575843 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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TrueSight: a new algorithm for splice junction detection using RNA-seq.

Li Yang Y Li-Byarlay Hongmei H Burns Paul P Borodovsky Mark M Robinson Gene E GE Ma Jian J

Nucleic acids research 20121218 4

RNA-seq has proven to be a powerful technique for transcriptome profiling based on next-generation sequencing (NGS) technologies. However, due to the short length of NGS reads, it is challenging to accurately map RNA-seq reads to splice junctions (SJs), which is a critically important step in the analysis of alternative splicing (AS) and isoform construction. In this article, we describe a new method, called TrueSight, which for the first time combines RNA-seq read mapping quality and coding pot ...[more]

PMID: 23254332

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Project description:Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Eighty million paired-end Illumina reads and ?500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing.

Dataset Information

TrueSight: a new algorithm for splice junction detection using RNA-seq.

Publications

TrueSight: a new algorithm for splice junction detection using RNA-seq.

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