Isoform prefiltering improves performance of count-based methods for analysis of differential transcript usage

Soneson, Charlotte; Matthes, Katarina L.; Nowicka, Małgorzata; Law, Charity W.; Robinson, Mark D.

doi:10.1186/s13059-015-0862-3

Cited by 130 publications

(206 citation statements)

References 35 publications

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“…Previous studies have shown that the utilization of a reduced, expressed transcriptome as reference for short-read mapping instead of the total reference dramatically impacts transcriptome quantification (Mezlini et al 2013;Soneson et al 2016) and improves replicability of expression level estimates (Au et al 2013). We sought to investigate how the new transcripts impact quantification by short reads.…”

Section: Novel Transcripts Have a Major Impact On Accurate Transcriptmentioning

confidence: 99%

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

et al. 2018

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High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.

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Section: Novel Transcripts Have a Major Impact On Accurate Transcriptmentioning

confidence: 99%

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

et al. 2018

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“…There are various methods designed explicitly to detect DS based on samples from different experimental conditions 19, 22, 23 . Independently, a set of methods was developed for detecting genetic variation associated with changes in splicing (sQTLs).…”

Section: Approaches To Ds and Sqtl Analysesmentioning

confidence: 99%

“…Furthermore, detection of more complex transcript variations remains a challenge for exon junction or PSI methods (see Figure S5 in the paper by Ongen et al 27 ). Soneson et al 23 considered counting which accommodates various types of local splicing events, such as exon paths traced out by paired reads, junction counts or events that correspond to combinations of isoforms; in general, the default exon-based counting resulted in strongest performance for DS gene detection.…”

Section: Approaches To Ds and Sqtl Analysesmentioning

confidence: 99%

DRIMSeq: a Dirichlet-multinomial framework for multivariate count outcomes in genomics

2016

Self Cite

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There are many instances in genomics data analyses where measurements are made on a multivariate response. For example, alternative splicing can lead to multiple expressed isoforms from the same primary transcript. There are situations where differences (e.g. between normal and disease state) in the relative ratio of expressed isoforms may have significant phenotypic consequences or lead to prognostic capabilities. Similarly, knowledge of single nucleotide polymorphisms (SNPs) that affect splicing, so-called splicing quantitative trait loci (sQTL) will help to characterize the effects of genetic variation on gene expression. RNA sequencing (RNA-seq) has provided an attractive toolbox to carefully unravel alternative splicing outcomes and recently, fast and accurate methods for transcript quantification have become available. We propose a statistical framework based on the Dirichlet-multinomial distribution that can discover changes in isoform usage between conditions and SNPs that affect relative expression of transcripts using these quantifications. The Dirichlet-multinomial model naturally accounts for the differential gene expression without losing information about overall gene abundance and by joint modeling of isoform expression, it has the capability to account for their correlated nature. The main challenge in this approach is to get robust estimates of model parameters with limited numbers of replicates. We approach this by sharing information and show that our method improves on existing approaches in terms of standard statistical performance metrics. The framework is applicable to other multivariate scenarios, such as Poly-A-seq or where beta-binomial models have been applied (e.g., differential DNA methylation). Our method is available as a Bioconductor R package called DRIMSeq.

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“…We used recently published sets of simulated data (Soneson et al, 2016) to compare the performances of the three approaches. They consist of six sets of human RNAseq data (two triplicates) where differential splicing has been introduced for a thousand of genes.…”

Section: Comparison Of Exon-centric and Junction-centric Approaches Omentioning

confidence: 99%

“…Soneson and colleagues (Soneson et al, 2016) generated simulated fastq files corresponding to 2 x 3 samples where a thousand genes are differentially spliced between two conditions (array express repository E-MTAB-3766). These 1000 protein-coding genes (ENSG) correspond to the positive sets, and we used them to assess the discriminative power of the different approaches with several rates of annotation degradation.…”

Section: Analysis On Simulated Datamentioning

confidence: 99%

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis

Noiret

Méreau

Angrand

et al. 2017

Developmental Biology

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Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exoncentric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed.We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

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Isoform prefiltering improves performance of count-based methods for analysis of differential transcript usage

Cited by 130 publications

References 35 publications

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

DRIMSeq: a Dirichlet-multinomial framework for multivariate count outcomes in genomics

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis

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