TAP: a targeted clinical genomics pipeline for detecting transcript variants using RNA-seq data

Abstract

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/pavfinderElectronic supplementary materialThe online version of this article (10.1186/s12920-018-0402-6) contains supplementary material, which is available to authorized users.

Base excision repair deficiency signatures implicate germline and somatic MUTYH aberrations in pancreatic ductal adenocarcinoma and breast cancer oncogenesis

“…1A). The targeted assembly pipeline (TAP) (Chiu et al 2018) on the transcriptome showed the synonymous germline variant (c.996G>A, p.Ser332Ser) to create a novel canonical AG acceptor splice site removing 42 bp and 14 amino acids (Fig. 1B; Supplemental Table S2E).…”

“…The targeted assembly pipeline (TAP) (Chiu et al 2018) on the transcriptome showed two mechanisms of abnormal splicing associated with this germline variant. The first mechanism leads to a 9-bp deletion due to the canonical splice site acceptor disruption and selection of a downstream canonical AG acceptor splice site at Chr 1:45797750–45797751 (new exon start at Chr 1:45797749).…”

A clinical transcriptome approach to patient stratification and therapy selection in acute myeloid leukemia

“…To detect inter- and intra-gene SVs, we performed de novo assembly on all the RNA-Seq libraries, using Trans-ABySS (version 1.5.2) 70 and PAVfinder (versions 0.2.0 and 0.3.0) 71 . PAVfinder relied on GMAP (version 2014-12-28) 58 and BWA MEM (version 0.7.12-r1039) 72 for contig-to-genome, contig-to-transcript, and read-to-contig alignments.…”

Mismatch-tolerant, alignment-free sequence classification using multiple spaced seeds and multiindex Bloom filters

“…This suggests that a spaced seed approach may be superior in terms of overall specificity when using incomplete or missing reference sequences. The practical speed and favorable sensitivity of miBFs have prompted the use of BBT in the targeted transcriptome assembly pipeline called TAP (43) and may be a good fit for other pipelines performing read binning.…”