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Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.

ABSTRACT: We herein describe an integrated system for the high-throughput analysis of splicing events and the identification of transcript variants. The system resolves individual splicing events and elucidates transcript variants via a pipeline that combines aspects such as bioinformatic analysis, high-throughput transcript variant amplification, and high-resolution capillary electrophoresis. For the 14?369 human genes known to have transcript variants, minimal primer sets were designed to amplify all transcript variants and examine all splicing events; these have been archived in the ASprimerDB database, which is newly described herein. A high-throughput thermocycler, dubbed GenTank, was developed to simultaneously perform thousands of PCR amplifications. Following the resolution of the various amplicons by capillary gel electrophoresis, two new computer programs, AmpliconViewer and VariantAssembler, may be used to analyze the splicing events, assemble the consecutive exons embodied by the PCR amplicons, and distinguish expressed versus putative transcript variants. This novel system not only facilitates the validation of putative transcript variants and the detection of novel transcript variants, it also semi-quantitatively measures the transcript variant expression levels of each gene. To demonstrate the system's capability, we used it to resolve transcript variants yielded by single and multiple splicing events, and to decipher the exon connectivity of long transcripts.

SUBMITTER: Hsiao TH

PROVIDER: S-EPMC2978383 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.

Hsiao Tzu-Hung TH Lin Chien-Hong CH Lee Te-Tsui TT Cheng Ji-Yen JY Wei Pei-Kuen PK Chuang Eric Y EY Peck Konan K

Nucleic acids research 20100826 20

We herein describe an integrated system for the high-throughput analysis of splicing events and the identification of transcript variants. The system resolves individual splicing events and elucidates transcript variants via a pipeline that combines aspects such as bioinformatic analysis, high-throughput transcript variant amplification, and high-resolution capillary electrophoresis. For the 14 369 human genes known to have transcript variants, minimal primer sets were designed to amplify all tr ...[more]

PMID: 20798177

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Project description:BackgroundRNA-seq is a powerful and cost-effective technology for molecular diagnostics of cancer and other diseases, and it can reach its full potential when coupled with validated clinical-grade informatics tools. Despite recent advances in long-read sequencing, transcriptome assembly of short reads remains a useful and cost-effective methodology for unveiling transcript-level rearrangements and novel isoforms. One of the major concerns for adopting the proven de novo assembly approach for RNA-seq data in clinical settings has been the analysis turnaround time. To address this concern, we have developed a targeted approach to expedite assembly and analysis of RNA-seq data.ResultsHere we present our Targeted Assembly Pipeline (TAP), which consists of four stages: 1) alignment-free gene-level classification of RNA-seq reads using BioBloomTools, 2) de novo assembly of individual targets using Trans-ABySS, 3) alignment of assembled contigs to the reference genome and transcriptome with GMAP and BWA and 4) structural and splicing variant detection using PAVFinder. We show that PAVFinder is a robust gene fusion detection tool when compared to established methods such as Tophat-Fusion and deFuse on simulated data of 448 events. Using the Leucegene acute myeloid leukemia (AML) RNA-seq data and a set of 580 COSMIC target genes, TAP identified a wide range of hallmark molecular anomalies including gene fusions, tandem duplications, insertions and deletions in agreement with published literature results. Moreover, also in this dataset, TAP captured AML-specific splicing variants such as skipped exons and novel splice sites reported in studies elsewhere. Running time of TAP on 100-150 million read pairs and a 580-gene set is one to 2 hours on a 48-core machine.ConclusionsWe demonstrated that TAP is a fast and robust RNA-seq variant detection pipeline that is potentially amenable to clinical applications. TAP is available at http://www.bcgsc.ca/platform/bioinfo/software/pavfinder.

Dataset Information

Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.

Publications

Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.

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