Zihan Cui1, Yuhang Liu1, Jinfeng Zhang1, Xing Qiu2. 1. Department of Statistics, Florida State University, Tallahassee, FL, 32304. 2. Department of Biostatistics and Computational Biology, University of Rochester, Rochester, NY, 14624.
Abstract
MOTIVATION: We developed super-delta2, a differential gene expression analysis pipeline designed for multi-group comparisons for RNA-seq data. It includes a customized one-way ANOVA F-test and a post-hoc test for pairwise group comparisons; both are designed to work with a multivariate normalization procedure to reduce technical noise. It also includes a trimming procedure with bias-correction to obtain robust and approximately unbiased summary statistics used in these tests. We demonstrated the asymptotic applicability of super-delta2 to log-transformed read counts in RNA-seq data by large sample theory based on Negative Binomial Poisson (NBP) distribution. RESULTS: We compared super-delta2 with three commonly used RNA-seq data analysis methods: limma/voom, edgeR, and DESeq2 using both simulated and real datasets. In all three simulation settings, super-delta2 not only achieved the best overall statistical power, but also was the only method that controlled type I error at the nominal level. When applied to a breast cancer dataset to identify differential expression pattern associated with multiple pathologic stages, super-delta2 selected more enriched pathways than other methods, which are directly linked to the underlying biological condition (breast cancer). CONCLUSIONS: By incorporating trimming and bias-correction in the normalization step, super-delta2 was able to achieve tight control of type I error. Because the hypothesis tests are based on asymptotic normal approximation of the NBP distribution, super-delta2 does not require computationally expensive iterative optimization procedures used by methods such as edgeR and DESeq2, which occasionally have convergence issues. AVAILABILITY: Our method is implemented in a R-package, "superdelta2", freely available at: https://github.com/fhlsjs/superdelta2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
MOTIVATION: We developed super-delta2, a differential gene expression analysis pipeline designed for multi-group comparisons for RNA-seq data. It includes a customized one-way ANOVA F-test and a post-hoc test for pairwise group comparisons; both are designed to work with a multivariate normalization procedure to reduce technical noise. It also includes a trimming procedure with bias-correction to obtain robust and approximately unbiased summary statistics used in these tests. We demonstrated the asymptotic applicability of super-delta2 to log-transformed read counts in RNA-seq data by large sample theory based on Negative Binomial Poisson (NBP) distribution. RESULTS: We compared super-delta2 with three commonly used RNA-seq data analysis methods: limma/voom, edgeR, and DESeq2 using both simulated and real datasets. In all three simulation settings, super-delta2 not only achieved the best overall statistical power, but also was the only method that controlled type I error at the nominal level. When applied to a breast cancer dataset to identify differential expression pattern associated with multiple pathologic stages, super-delta2 selected more enriched pathways than other methods, which are directly linked to the underlying biological condition (breast cancer). CONCLUSIONS: By incorporating trimming and bias-correction in the normalization step, super-delta2 was able to achieve tight control of type I error. Because the hypothesis tests are based on asymptotic normal approximation of the NBP distribution, super-delta2 does not require computationally expensive iterative optimization procedures used by methods such as edgeR and DESeq2, which occasionally have convergence issues. AVAILABILITY: Our method is implemented in a R-package, "superdelta2", freely available at: https://github.com/fhlsjs/superdelta2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.