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
 .

/pdf/moderated-estimation-of-fold-change-and-dispersion-for-rna-3ywnbkcan0.pdf

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

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).

http://dmrocke.ucdavis.edu/Class/BST226.2014.Winter/edgeR%20bioinformatics%202010.pdf

edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.

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.

/pdf/limma-powers-differential-expression-analyses-for-rna-4yl7ae3arh.pdf

limma powers differential expression analyses for RNA-sequencing and microarray studies

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.

/pdf/moderated-estimation-of-fold-change-and-dispersion-for-rna-59glcgpwlk.pdf

Increasing quantitative data generated from transcriptomics and proteomics require integrative strategies for analysis Here, we present an R package, clusterProfiler that automates the process of biological-term classification and the enrichment analysis of gene clusters The analysis module and visualization module were combined into a reusable workflow Currently, clusterProfiler supports three species, including humans, mice, and yeast Methods provided in this package can be easily extended to other species and ontologies The clusterProfiler package is released under Artistic-20 License within Bioconductor project The source code and vignette are freely available at http://bioconductororg/packages/release/bioc/html/clusterProfilerhtml

/pdf/clusterprofiler-an-r-package-for-comparing-biological-themes-xbzzr3fbnj.pdf

clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters

High-density oligonucleotide expression arrays are a widely used microarray platform. Affymetrix GeneChip arrays dominate this market. An important distinction between the GeneChip and other technologies is that on GeneChips, multiple short probes are used to measure gene expression levels. This makes preprocessing particularly important when using this platform. This chapter begins by describing how to import probe-level data into the system and how these data can be examined using the facilities of the AffyBatch class. Then we will describe background adjustment, normalization, and summarization methods. Functionality for GeneChip probe-level data is provided by the affy, affyPLM, affycomp, gcrma, and affypdnn packages. All these tools are useful for preprocessing probe-level data stored in an AffyBatch object into expression-level data stored in an exprSet object. Because there are many competing methods for this preprocessing step, it is useful to have a way to assess the differences. In Bioconductor, this can be carried out using the affycomp package, which we discuss briefly.

Preprocessing High-density Oligonucleotide Arrays

Department of Psychiatry, University of California, Irvine, CA, USA; Mental Health Research Institute, University of Michigan, Ann Arbor, MI, USA; Biostatistics, University of California, Berkeley, CA, USA; Stanford Human Genome Center, Stanford University, Palo Alto, CA, USA; Center for Neuroscience, University of California, Davis, CA, USA and University of Hawaii, John A Burns School of Medicine, Honolulu, HI, USA

Mitochondrial related gene expression changes are sensitive to agonal-pH state

Robust Multi-array Analysis (RMA) is the most widely used preprocessing algorithm for Affymetrix and Nimblegen gene-expression microarrays. RMA performs background correction, normalization, and summarization in a modular way. The last two steps require multiple arrays to be analyzed simultaneously. The ability to borrow information across samples provides RMA various advantages. For example, the summarization step fits a parametric model that accounts for probe-effects, assumed to be fixed across arrays, and improves outlier detection. Residuals, obtained from the fitted model, permit the creation of useful quality metrics. However, the dependence on multiple arrays has two drawbacks: (1) RMA cannot be used in clinical settings where samples must be processed individually or in small batches and (2) data sets preprocessed separately are not comparable. We propose a preprocessing algorithm, frozen RMA (fRMA), which allows one to analyze microarrays individually or in small batches and then combine the data for analysis. This is accomplished by utilizing information from the large publicly available microarray databases. In particular, estimates of probe-specific effects and variances are precomputed and frozen. Then, with new data sets, these are used in concert with information from the new array(s) to normalize and summarize the data. We find that fRMA is comparable to RMA when the data are analyzed as a single batch and outperforms RMA when analyzing multiple batches. The methods described here are implemented in the R package frma and are currently available for download from the software section of http://rafalab.jhsph.edu

/pdf/frozen-robust-multi-array-analysis-frma-2rihuothnf.pdf

FROZEN ROBUST MULTI-ARRAY ANALYSIS (fRMA)

Quality Assessment for Short Oligonucleotide Microarray Data. Rejoinder.

Quality of microarray gene expression data has emerged as a new research topic. As in other areas, microarray quality is assessed by comparing suitable numerical summaries across microarrays, so that outliers and trends can be visualized, and poor quality arrays or variable quality sets of arrays can be identified. Since each single array comprises tens or hundreds of thousands of measurements, the challenge is to find numerical summaries which can be used to make accurate quality calls. To this end, several new quality measures are introduced based on probe level and probeset level information, all obtained as a by-product of the low-level analysis algorithms RMA/fitPLM for Affymetrix GeneChips. Quality landscapes spatially localize chip or hybridization problems. Numerical chip quality measures are derived from the distributions of Normalized Unscaled Standard Errors and of Relative Log Expressions. Quality of chip batches is assessed by Residual Scale Factors. These quality assessment measures are demonstrated on a variety of datasets (spike-in experiments, small lab experiments, multi-site studies). They are compared with Affymetrix's individual chip quality report.

Benjamin M. Bolstad

Papers

Preprocessing High-density Oligonucleotide Arrays

Mitochondrial related gene expression changes are sensitive to agonal-pH state

FROZEN ROBUST MULTI-ARRAY ANALYSIS (fRMA)

Quality Assessment for Short Oligonucleotide Microarray Data. Rejoinder.

Quality assessment for short oligonucleotide microarray data