Dataset Information

DiPhiSeq: robust comparison of expression levels on RNA-Seq data with large sample sizes.

ABSTRACT: MOTIVATION:In the analysis of RNA-Seq data, detecting differentially expressed (DE) genes has been a hot research area in recent years and many methods have been proposed. DE genes show different average expression levels in different sample groups, and thus can be important biological markers. While generally very successful, these methods need to be further tailored and improved for cancerous data, which often features quite diverse expression in the samples from the cancer group, and this diversity is much larger than that in the control group. RESULTS:We propose a statistical method that can detect not only genes that show different average expressions, but also genes that show different diversities of expressions in different groups. These 'differentially dispersed' genes can be important clinical markers. Our method uses a redescending penalty on the quasi-likelihood function, and thus has superior robustness against outliers and other noise. Simulations and real data analysis demonstrate that DiPhiSeq outperforms existing methods in the presence of outliers, and identifies unique sets of genes. AVAILABILITY AND IMPLEMENTATION:DiPhiSeq is publicly available as an R package on CRAN: https://cran.r-project.org/package=DiPhiSeq. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

SUBMITTER: Li J

PROVIDER: S-EPMC6596898 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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DiPhiSeq: robust comparison of expression levels on RNA-Seq data with large sample sizes.

Li Jun J Lamere Alicia T AT

Bioinformatics (Oxford, England) 20190701 13

<h4>Motivation</h4>In the analysis of RNA-Seq data, detecting differentially expressed (DE) genes has been a hot research area in recent years and many methods have been proposed. DE genes show different average expression levels in different sample groups, and thus can be important biological markers. While generally very successful, these methods need to be further tailored and improved for cancerous data, which often features quite diverse expression in the samples from the cancer group, and ...[more]

PMID: 30452547

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Project description:BACKGROUND:High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that deviate from it. Robust statistics have been widely used in multivariate data analysis for outlier detection in chemometrics and engineering. Here we apply robust statistics on RNA-seq data analysis. RESULTS:We report the use of two robust principal component analysis (rPCA) methods, PcaHubert and PcaGrid, to detect outlier samples in multiple simulated and real biological RNA-seq data sets with positive control outlier samples. PcaGrid achieved 100% sensitivity and 100% specificity in all the tests using positive control outliers with varying degrees of divergence. We applied rPCA methods and classical principal component analysis (cPCA) on an RNA-Seq data set profiling gene expression of the external granule layer in the cerebellum of control and conditional SnoN knockout mice. Both rPCA methods detected the same two outlier samples but cPCA failed to detect any. We performed differentially expressed gene detection before and after outlier removal as well as with and without batch effect modeling. We validated gene expression changes using quantitative reverse transcription PCR and used the result as reference to compare the performance of eight different data analysis strategies. Removing outliers without batch effect modeling performed the best in term of detecting biologically relevant differentially expressed genes. CONCLUSIONS:rPCA implemented in the PcaGrid function is an accurate and objective method to detect outlier samples. It is well suited for high-dimensional data with small sample sizes like RNA-seq data. Outlier removal can significantly improve the performance of differential gene detection and downstream functional analysis.

Project description:Normalization is an essential step with considerable impact on high-throughput RNA sequencing (RNA-seq) data analysis. Although there are numerous methods for read count normalization, it remains a challenge to choose an optimal method due to multiple factors contributing to read count variability that affects the overall sensitivity and specificity. In order to properly determine the most appropriate normalization methods, it is critical to compare the performance and shortcomings of a representative set of normalization routines based on different dataset characteristics. Therefore, we set out to evaluate the performance of the commonly used methods (DESeq, TMM-edgeR, FPKM-CuffDiff, TC, Med UQ and FQ) and two new methods we propose: Med-pgQ2 and UQ-pgQ2 (per-gene normalization after per-sample median or upper-quartile global scaling). Our per-gene normalization approach allows for comparisons between conditions based on similar count levels. Using the benchmark Microarray Quality Control Project (MAQC) and simulated datasets, we performed differential gene expression analysis to evaluate these methods. When evaluating MAQC2 with two replicates, we observed that Med-pgQ2 and UQ-pgQ2 achieved a slightly higher area under the Receiver Operating Characteristic Curve (AUC), a specificity rate > 85%, the detection power > 92% and an actual false discovery rate (FDR) under 0.06 given the nominal FDR (?0.05). Although the top commonly used methods (DESeq and TMM-edgeR) yield a higher power (>93%) for MAQC2 data, they trade off with a reduced specificity (<70%) and a slightly higher actual FDR than our proposed methods. In addition, the results from an analysis based on the qualitative characteristics of sample distribution for MAQC2 and human breast cancer datasets show that only our gene-wise normalization methods corrected data skewed towards lower read counts. However, when we evaluated MAQC3 with less variation in five replicates, all methods performed similarly. Thus, our proposed Med-pgQ2 and UQ-pgQ2 methods perform slightly better for differential gene analysis of RNA-seq data skewed towards lowly expressed read counts with high variation by improving specificity while maintaining a good detection power with a control of the nominal FDR level.

Dataset Information

DiPhiSeq: robust comparison of expression levels on RNA-Seq data with large sample sizes.

Publications

DiPhiSeq: robust comparison of expression levels on RNA-Seq data with large sample sizes.

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