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Journal ArticleDOI

voom: precision weights unlock linear model analysis tools for RNA-seq read counts

TL;DR: New normal linear modeling strategies are presented for analyzing read counts from RNA-seq experiments, and the voom method estimates the mean-variance relationship of the log-counts, generates a precision weight for each observation and enters these into the limma empirical Bayes analysis pipeline.
Abstract: New normal linear modeling strategies are presented for analyzing read counts from RNA-seq experiments. The voom method estimates the mean-variance relationship of the log-counts, generates a precision weight for each observation and enters these into the limma empirical Bayes analysis pipeline. This opens access for RNA-seq analysts to a large body of methodology developed for microarrays. Simulation studies show that voom performs as well or better than count-based RNA-seq methods even when the data are generated according to the assumptions of the earlier methods. Two case studies illustrate the use of linear modeling and gene set testing methods.

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Citations
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Journal ArticleDOI
TL;DR: This work presents DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates, which enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression.
Abstract: In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html .

47,038 citations


Cites methods from "voom: precision weights unlock line..."

  • ...Other methods compared were the voom normalization method followed by linear modeling using the limma package [35] and the SAMseq permutation method of the samr package [23]....

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Journal ArticleDOI
TL;DR: The philosophy and design of the limma package is reviewed, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
Abstract: limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.

22,147 citations


Cites background or methods or result from "voom: precision weights unlock line..."

  • ...One way is through estimating a mean-variance trend, which can either be incorporated into the empirical Bayes procedure as mentioned above or used to generate observation weights (10)....

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  • ...Both observationlevel (2,10,43) and sample-specific weights (11) can be used in an analysis....

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  • ...The resulting pipeline gives comparable performance to the best of the negative binomial-based software packages but with greater speed and reliability for large data sets (10,21)....

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  • ...First, the global variance estimate can now incorporate a mean-variance trend (10,17,18)....

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Posted ContentDOI
17 Nov 2014-bioRxiv
TL;DR: This work presents DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates, which enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression.
Abstract: In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-Seq data, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data. DESeq2 uses shrinkage estimation for dispersions and fold changes to improve stability and interpretability of the estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression and facilitates downstream tasks such as gene ranking and visualization. DESeq2 is available as an R/Bioconductor package.

17,014 citations


Cites methods from "voom: precision weights unlock line..."

  • ...Other methods compared were the voom normalization method followed by linear modeling using the limma package [35] and the SAMseq permutation method of the samr package [23]....

    [...]

Journal ArticleDOI
TL;DR: FeatureCounts as discussed by the authors is a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments, which implements highly efficient chromosome hashing and feature blocking techniques.
Abstract: MOTIVATION: Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. In many applications, the key information required for downstream analysis is the number of reads mapping to each genomic feature, for example to each exon or each gene. The process of counting reads is called read summarization. Read summarization is required for a great variety of genomic analyses but has so far received relatively little attention in the literature. RESULTS: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications. AVAILABILITY AND IMPLEMENTATION: featureCounts is available under GNU General Public License as part of the Subread (http://subread.sourceforge.net) or Rsubread (http://www.bioconductor.org) software packages.

14,103 citations

Journal ArticleDOI
TL;DR: Treatment with atezolizumab resulted in a significantly improved RECIST v1.1 response rate, compared with a historical control overall response rate of 10%, and Exploratory analyses showed The Cancer Genome Atlas (TCGA) subtypes and mutation load to be independently predictive for response to atezolediazepine.

2,934 citations

References
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Journal ArticleDOI
TL;DR: The Gene Set Enrichment Analysis (GSEA) method as discussed by the authors focuses on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.
Abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

34,830 citations

Journal ArticleDOI
TL;DR: EdgeR as mentioned in this paper is a Bioconductor software package for examining differential expression of replicated count data, which uses an overdispersed Poisson model to account for both biological and technical variability and empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference.
Abstract: Summary: It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data. Availability: The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org).

29,413 citations


"voom: precision weights unlock line..." refers background or methods in this paper

  • ...Specifically we used the goodTuringProportions function of the edgeR package [12], which implements the Good-Turing algorithm [55], to predict the true proportion of total RNA attributable to each gene....

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  • ...One common approach to summarize RNA-seq data is to count the number of sequence reads mapping to each gene or genomic feature of interest [11, 12, 13, 14]....

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Book
01 Jan 1983
TL;DR: In this paper, a generalization of the analysis of variance is given for these models using log- likelihoods, illustrated by examples relating to four distributions; the Normal, Binomial (probit analysis, etc.), Poisson (contingency tables), and gamma (variance components).
Abstract: The technique of iterative weighted linear regression can be used to obtain maximum likelihood estimates of the parameters with observations distributed according to some exponential family and systematic effects that can be made linear by a suitable transformation. A generalization of the analysis of variance is given for these models using log- likelihoods. These generalized linear models are illustrated by examples relating to four distributions; the Normal, Binomial (probit analysis, etc.), Poisson (contingency tables) and gamma (variance components).

23,215 citations

Journal ArticleDOI
TL;DR: FeatureCounts as discussed by the authors is a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments, which implements highly efficient chromosome hashing and feature blocking techniques.
Abstract: MOTIVATION: Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. In many applications, the key information required for downstream analysis is the number of reads mapping to each genomic feature, for example to each exon or each gene. The process of counting reads is called read summarization. Read summarization is required for a great variety of genomic analyses but has so far received relatively little attention in the literature. RESULTS: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications. AVAILABILITY AND IMPLEMENTATION: featureCounts is available under GNU General Public License as part of the Subread (http://subread.sourceforge.net) or Rsubread (http://www.bioconductor.org) software packages.

14,103 citations

Journal ArticleDOI
TL;DR: A method based on the negative binomial distribution, with variance and mean linked by local regression, is proposed and an implementation, DESeq, as an R/Bioconductor package is presented.
Abstract: High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.

13,356 citations


"voom: precision weights unlock line..." refers background or methods or result in this paper

  • ...The performance of voom and limma-trend is compared to that of edgeR, DESeq, baySeq, TSPM, PoissonSeq, and DSS. Simulation studies show that the limma-based pipelines more than hold their own against the count-based RNA-seq meth- ods in terms of power and error rate control even when the data are generated according to the assumptions of the earlier methods....

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  • ...The voom mean-variance trend is shown in Figure 1b. Software The results presented in this article are carried out using R version 3.0.0 and software packages limma 3.16.2, edgeR 3.2.3, baySeq 1.14.1, DESeq 1.12.0, DSS 1.4.0, PoissonSeq 1.1.2 and tweeDEseqCountData 1.0.8....

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  • ...For example, it would be at least as scientifically reasonable to assume that the true expression levels for each gene follow a log-normal distribution between replicates instead of a gamma distribution, and such an assumption would tend to improve the performance of voom relative to edgeR, DESeq, baySeq and DSS....

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  • ...When the library sizes are unequal, DSS and DESeq became extremely conservative....

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  • ...TMM-scale normalization [54] was used for all the analysis methods, except for DESeq and PoissonSeq, which have their own built-in normalization methods....

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