Detecting Differential Expression from RNA-seq Data with Expression Measurement Uncertainty

High-throughput RNA sequencing (RNA-seq) has emerged as a revolutionary and powerful technology for expression profiling. Most proposed methods for detecting differentially expressed (DE) genes from RNA-seq are based on statistics that compare normalized read counts between conditions. However, there are few methods considering the expression measurement uncertainty into DE detection. Moreover, most methods are only capable of detecting DE genes, and few methods are available for detecting DE isoforms. In this paper, a Bayesian framework (BDSeq) is proposed to detect DE genes and isoforms with consideration of expression measurement uncertainty. This expression measurement uncertainty provides useful information which can help to improve the performance of DE detection. Three real RAN-seq data sets are used to evaluate the performance of BDSeq and results show that the inclusion of expression measurement uncertainty improves accuracy in detection of DE genes and isoforms. Finally, we develop a GamSeq-BDSeq RNA-seq analysis pipeline to facilitate users, which is freely available at the website http://parnec.nuaa.edu.cn/liux/GSBD/GamSeq-BDSeq.html.Comment: 20 pages, 9 figure

A statistical framework for the analysis of microarray probe-level data

In microarray technology, a number of critical steps are required to convert the raw measurements into the data relied upon by biologists and clinicians. These data manipulations, referred to as preprocessing, influence the quality of the ultimate measurements and studies that rely upon them. Standard operating procedure for microarray researchers is to use preprocessed data as the starting point for the statistical analyses that produce reported results. This has prevented many researchers from carefully considering their choice of preprocessing methodology. Furthermore, the fact that the preprocessing step affects the stochastic properties of the final statistical summaries is often ignored. In this paper we propose a statistical framework that permits the integration of preprocessing into the standard statistical analysis flow of microarray data. This general framework is relevant in many microarray platforms and motivates targeted analysis methods for specific applications. We demonstrate its usefulness by applying the idea in three different applications of the technology.Comment: Published in at http://dx.doi.org/10.1214/07-AOAS116 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

A Model Based Background Adjustment for Oligonucleotide Expression Arrays

High density oligonucleotide expression arrays are widely used in many areas of biomedical research. Affymetrix GeneChip arrays are the most popular. In the Affymetrix system, a fair amount of further pre-processing and data reduction occurs following the image processing step. Statistical procedures developed by academic groups have been successful at improving the default algorithms provided by the Affymetrix system. In this paper we present a solution to one of the pre-processing steps, background adjustment, based on a formal statistical framework. Our solution greatly improves the performance of the technology in various practical applications. Affymetrix GeneChip arrays use short oligonucleotides to probe for genes in an RNA sample. Typically each gene will be represented by 11-20 pairs of oligonucleotide probes. The first component of these pairs is referred to as a perfect match probe and is designed to hybridize only with transcripts from the intended gene (specific hybridization). However, hybridization by other sequences (non-specific hybridization) is unavoidable. Furthermore, hybridization strengths are measured by a scanner that introduces optical noise. Therefore, the observed intensities need to be adjusted to give accurate measurements of specific hybridization. One approach to adjusting is to pair each perfect match probe with a mismatch probe that is designed with the intention of measuring non-specific hybridization. The default adjustment, provided as part of the Affymetrix system, is based on the difference between perfect match and mismatch probe intensities. We have found that this approach can be improved via the use of estimators derived from a statistical model that use probe sequence information. The model is based on simple hybridization theory from molecular biology and experiments specifically designed to help develop it. A final step in the pre-processing of these arrays is to combine the 11-20 probe pair intensities, after background adjustment and normalization, for a given gene to define a measure of expression that represents the amount of the corresponding mRNA species. In this paper we illustrate the practical consequences of not adjusting appropriately for the presence of nonspecific hybridization and provide a solution based on our background adjustment procedure. Software that computes our adjustment is available as part of the Bioconductor project (http://www.bioconductor

Empirical Bayes models for multiple probe type microarrays at the probe level

<p>Abstract</p> <p>Background</p> <p>When analyzing microarray data a primary objective is often to find differentially expressed genes. With empirical Bayes and penalized t-tests the sample variances are adjusted towards a global estimate, producing more stable results compared to ordinary t-tests. However, for Affymetrix type data a clear dependency between variability and intensity-level generally exists, even for logged intensities, most clearly for data at the probe level but also for probe-set summarizes such as the MAS5 expression index. As a consequence, adjustment towards a global estimate results in an intensity-level dependent false positive rate.</p> <p>Results</p> <p>We propose two new methods for finding differentially expressed genes, Probe level Locally moderated Weighted median-t (PLW) and Locally Moderated Weighted-t (LMW). Both methods use an empirical Bayes model taking the dependency between variability and intensity-level into account. A global covariance matrix is also used allowing for differing variances between arrays as well as array-to-array correlations. PLW is specially designed for Affymetrix type arrays (or other multiple-probe arrays). Instead of making inference on probe-set summaries, comparisons are made separately for each perfect-match probe and are then summarized into one score for the probe-set.</p> <p>Conclusion</p> <p>The proposed methods are compared to 14 existing methods using five spike-in data sets. For RMA and GCRMA processed data, PLW has the most accurate ranking of regulated genes in four out of the five data sets, and LMW consistently performs better than all examined moderated t-tests when used on RMA, GCRMA, and MAS5 expression indexes.</p

Experimental and computational applications of microarray technology for malaria eradication in Africa

Various mutation assisted drug resistance evolved in Plasmodium falciparum strains and insecticide resistance to female Anopheles mosquito account for major biomedical catastrophes standing against all efforts to eradicate malaria in Sub-Saharan Africa. Malaria is endemic in more than 100 countries and by far the most costly disease in terms of human health causing major losses among many African nations including Nigeria. The fight against malaria is failing and DNA microarray analysis need to keep up the pace in order to unravel the evolving parasite’s gene expression profile which is a pointer to monitoring the genes involved in malaria’s infective metabolic pathway. Huge data is generated and biologists have the challenge of extracting useful information from volumes of microarray data. Expression levels for tens of thousands of genes can be simultaneously measured in a single hybridization experiment and are collectively called a “gene expression profile”. Gene expression profiles can also be used in studying various state of malaria development in which expression profiles of different disease states at different time points are collected and compared to each other to establish a classifying scheme for purposes such as diagnosis and treatments with adequate drugs. This paper examines microarray technology and its application as supported by appropriate software tools from experimental set-up to the level of data analysis. An assessment of the level of microarray technology in Africa, its availability and techniques required for malaria eradication and effective healthcare in Nigeria and Africa in general were also underscored

Robust Detection and Genotyping of Single Feature Polymorphisms from Gene Expression Data

It is well known that Affymetrix microarrays are widely used to predict genome-wide gene expression and genome-wide genetic polymorphisms from RNA and genomic DNA hybridization experiments, respectively. It has recently been proposed to integrate the two predictions by use of RNA microarray data only. Although the ability to detect single feature polymorphisms (SFPs) from RNA microarray data has many practical implications for genome study in both sequenced and unsequenced species, it raises enormous challenges for statistical modelling and analysis of microarray gene expression data for this objective. Several methods are proposed to predict SFPs from the gene expression profile. However, their performance is highly vulnerable to differential expression of genes. The SFPs thus predicted are eventually a reflection of differentially expressed genes rather than genuine sequence polymorphisms. To address the problem, we developed a novel statistical method to separate the binding affinity between a transcript and its targeting probe and the parameter measuring transcript abundance from perfect-match hybridization values of Affymetrix gene expression data. We implemented a Bayesian approach to detect SFPs and to genotype a segregating population at the detected SFPs. Based on analysis of three Affymetrix microarray datasets, we demonstrated that the present method confers a significantly improved robustness and accuracy in detecting the SFPs that carry genuine sequence polymorphisms when compared to its rivals in the literature. The method developed in this paper will provide experimental genomicists with advanced analytical tools for appropriate and efficient analysis of their microarray experiments and biostatisticians with insightful interpretation of Affymetrix microarray data

An introduction to low-level analysis methods of DNA microarray data

This article gives an overview over the methods used in the low--level analysis of gene expression data generated using DNA microarrays. This type of experiment allows to determine relative levels of nucleic acid abundance in a set of tissues or cell populations for thousands of transcripts or loci simultaneously. Careful statistical design and analysis are essential to improve the efficiency and reliability of microarray experiments throughout the data acquisition and analysis process. This includes the design of probes, the experimental design, the image analysis of microarray scanned images, the normalization of fluorescence intensities, the assessment of the quality of microarray data and incorporation of quality information in subsequent analyses, the combination of information across arrays and across sets of experiments, the discovery and recognition of patterns in expression at the single gene and multiple gene levels, and the assessment of significance of these findings, considering the fact that there is a lot of noise and thus random features in the data. For all of these components, access to a flexible and efficient statistical computing environment is an essential aspect

Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data

A large number of computational methods have been developed for analyzing differential gene expression in RNA-seq data. We describe a comprehensive evaluation of common methods using the SEQC benchmark dataset and ENCODE data. We consider a number of key features, including normalization, accuracy of differential expression detection and differential expression analysis when one condition has no detectable expression. We find significant differences among the methods, but note that array-based methods adapted to RNA-seq data perform comparably to methods designed for RNA-seq. Our results demonstrate that increasing the number of replicate samples significantly improves detection power over increased sequencing depth

Robust mixtures in the presence of measurement errors

We develop a mixture-based approach to robust density modeling and outlier detection for experimental multivariate data that includes measurement error information. Our model is designed to infer atypical measurements that are not due to errors, aiming to retrieve potentially interesting peculiar objects. Since exact inference is not possible in this model, we develop a tree-structured variational EM solution. This compares favorably against a fully factorial approximation scheme, approaching the accuracy of a Markov-Chain-EM, while maintaining computational simplicity. We demonstrate the benefits of including measurement errors in the model, in terms of improved outlier detection rates in varying measurement uncertainty conditions. We then use this approach in detecting peculiar quasars from an astrophysical survey, given photometric measurements with errors.Comment: (Refereed) Proceedings of the 24-th Annual International Conference on Machine Learning 2007 (ICML07), (Ed.) Z. Ghahramani. June 20-24, 2007, Oregon State University, Corvallis, OR, USA, pp. 847-854; Omnipress. ISBN 978-1-59593-793-3; 8 pages, 6 figure