Differential analysis of RNA-Seq incorporating quantification uncertainty

Pimentel, Harold; Bray, Nicolas; Puente, Suzette; Melsted, Páll; Pachter, Lior

doi:10.1101/058164

Cited by 61 publications

(30 citation statements)

References 36 publications

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“…We used Kallisto (43) to perform read pseudoalignment and performed differential analysis using Sleuth (44). We fit a GLM for an isoform t in sample i: y t,i = β t,0 + βt, genotype · X t,i + β t, batch · Y t,i + t,i…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements

Angeles-Albores

Robinson

Williams

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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RNA-sequencing (RNA-seq) is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Also, quantifying and interpreting epistatic effects using expression profiles remains a challenge. We developed a single coefficient to quantify transcriptome-wide epistasis that reflects the underlying interactions and which can be interpreted intuitively. To demonstrate our approach, we sequenced four single and two double mutants of Caenorhabditis elegans. From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes with HIF-1-dependent expression that have opposite changes in expression in mutants of two genes that cooperate to negatively regulate HIF-1 abundance; however, the double mutant of these genes exhibits suppression epistasis. This class violates the classical model of HIF-1 regulation but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.hypoxia | transcriptomics | epistasis | genetics | gene expression

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Section: Methodsmentioning

confidence: 99%

“…After fitting the GLM, we tested isoforms for differential expression using the built-in Wald test in Sleuth (44), which outputs a q value that has been corrected for multiple hypothesis testing.…”

Section: Methodsmentioning

confidence: 99%

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements

Angeles-Albores

Robinson

Williams

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

“…However, this is apparently beyond the scope of this paper and the experimental results are in principle geared towards convincing the reader that DRIMSeq improves on existing approaches to discover changes in isoform usage, as suggested in the abstract. In my view, the experimental results do not address this question and I would suggest the authors to compare DRIMSeq with methods that also work with transcript-quantification values and assess differential isoform usage such as, for instance, Cuffdiff 3 or sleuth 4 .…”

mentioning

confidence: 99%

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

2016

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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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“…As a result, the expression change in minor isoform ENST00000375512 is masked at the gene level. Therefore, ignoring transcript isoforms and counting reads at the gene level does not always give correct answers [17,18].…”

Section: Gene Quantificationmentioning

confidence: 99%

“…By appropriately accounting for uncertainty in quantification, more accurate downstream differential analyses were obtained at both the gene and isoform levels. Sleuth [18] is a downstream analyses tool specifically developed for differential analyses at the transcript level. Figure 2: Minor isoform changes are masked at the gene level.…”

Section: Isoform Quantification Of Known Transcriptsmentioning

confidence: 99%

Bioinformatics Tools for RNA-seq Gene and Isoform Quantification

Zhang¹,

Zhang²,

Vincent³

et al. 2016

Next Generat Sequenc & Applic

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In recent years, RNA-seq has emerged as a powerful technology in estimation of gene or transcript expression. 'Union-exon' and transcript based approaches are widely used in gene quantification. The 'Union-exon' based approach is simple, but it does not distinguish between isoforms when multiple alternatively spliced transcripts are expressed from the same gene. Because a gene is expressed in one or more transcript isoforms, the transcript based approach is more biologically meaningful than the 'union exon'-based approach. However, estimating the expression of individual isoform is intrinsically challenging because different isoforms of a gene usually have a high proportion of genomic overlap. Recently, a number of tools have been developed for RNA-seq isoform quantification. We review those methods and their main features to serve as guidance for users to choose suitable tools for their specific projects.

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Differential analysis of RNA-Seq incorporating quantification uncertainty

Cited by 61 publications

References 36 publications

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements

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

Bioinformatics Tools for RNA-seq Gene and Isoform Quantification

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