Next-generation sequencing (NGS) technologies are revolutionizing genome research, and in particular, their application to transcriptomics (RNA-seq) is increasingly being used for gene expression profiling as a replacement for microarrays. However, the properties of RNA-seq data have not been yet fully established, and additional research is needed for understanding how these data respond to differential expression analysis. In this work, we set out to gain insights into the characteristics of RNA-seq data analysis by studying an important parameter of this technology: the sequencing depth. We have analyzed how sequencing depth affects the detection of transcripts and their identification as differentially expressed, looking at aspects such as transcript biotype, length, expression level, and fold-change. We have evaluated different algorithms available for the analysis of RNA-seq and proposed a novel approach-NOISeq-that differs from existing methods in that it is data-adaptive and nonparametric. Our results reveal that most existing methodologies suffer from a strong dependency on sequencing depth for their differential expression calls and that this results in a considerable number of false positives that increases as the number of reads grows. In contrast, our proposed method models the noise distribution from the actual data, can therefore better adapt to the size of the data set, and is more effective in controlling the rate of false discoveries. This work discusses the true potential of RNA-seq for studying regulation at low expression ranges, the noise within RNA-seq data, and the issue of replication.

As the use of RNA-seq has popularized, there is an increasing consciousness of the importance of experimental design, bias removal, accurate quantification and control of false positives for proper data analysis. We introduce the NOISeq R-package for quality control and analysis of count data. We show how the available diagnostic tools can be used to monitor quality issues, make pre-processing decisions and improve analysis. We demonstrate that the non-parametric NOISeqBIO efficiently controls false discoveries in experiments with biological replication and outperforms state-of-the-art methods. NOISeq is a comprehensive resource that meets current needs for robust data-aware analysis of RNA-seq differential expression.

In this work, we propose a statistical procedure to identify genes that show different gene expression profiles across analytical groups in time-course experiments. The method is a two-regression step approach where the experimental groups are identified by dummy variables. The procedure first adjusts a global regression model with all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study differences between groups and to find statistically significant different profiles. The methodology is illustrated on both a real and a simulated microarray dataset.

This paper addresses the problem of using future multivariate observations with missing data to estimate latent variable scores from an existing principal component analysis (PCA) model. This is a critical issue in multivariate statistical process control (MSPC) schemes where the process is continuously interrogated based on an underlying PCA model. We present several methods for estimating the scores of new individuals with missing data: a so-called trimmed score method (TRI), a single-component projection method (SCP), a method of projection to the model plane (PMP), a method based on the iterative imputation of missing data, a method based on the minimization of the squared prediction error (SPE), a conditional mean replacement method (CMR) and various least squared-based methods: one based on a regression on known data (KDR) and the other based on a regression on trimmed scores (TSR). The basis for each method and the expressions for the score estimators, their covariance matrices and the estimation errors are developed. Some of the methods discussed have already been proposed in the literature (SCP, PMP and CMR), some are original (TRI and TSR) and others are shown to be equivalent to methods already developed by other authors: iterative imputation and SPE methods are equivalent to PMP; KDR is equivalent to CMR. These methods can be seen as different ways to impute values for the missing variables. The efficiency of the methods is studied through simulations based on an industrial data set. The KDR method is shown to be statistically superior to the other methods, except the TSR method in which the matrix to be inverted is of a much smaller size.

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Supplementary data are available at Bioinformatics online.

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