title: A comparative study of techniques for differential expression analysis on RNA-Seq data.
abstract: Recent advances in next-generation sequencing technology allow high-throughput cDNA sequencing (RNA-Seq) to be widely applied in transcriptomic studies, in particular for detecting differentially expressed genes between groups. Many software packages have been developed for the identification of differentially expressed genes (DEGs) between treatment groups based on RNA-Seq data. However, there is a lack of consensus on how to approach an optimal study design and choice of suitable software for the analysis. In this comparative study we evaluate the performance of three of the most frequently used software tools: Cufflinks-Cuffdiff2, DESeq and edgeR. A number of important parameters of RNA-Seq technology were taken into consideration, including the number of replicates, sequencing depth, and balanced vs. unbalanced sequencing depth within and between groups. We benchmarked results relative to sets of DEGs identified through either quantitative RT-PCR or microarray. We observed that edgeR performs slightly better than DESeq and Cuffdiff2 in terms of the ability to uncover true positives. Overall, DESeq or taking the intersection of DEGs from two or more tools is recommended if the number of false positives is a major concern in the study. In other circumstances, edgeR is slightly preferable for differential expression analysis at the expense of potentially introducing more false positives.
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Zong Hong
authors.initials: ZH
authors.lastname: Zhang
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Dhanisha J
authors.initials: DJ
authors.lastname: Jhaveri
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Vikki M
authors.initials: VM
authors.lastname: Marshall
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia; CSIRO Preventative Health Flagship and CSIRO Computational Informatics, Sydney, New South Wales, Australia.
authors.forename: Denis C
authors.initials: DC
authors.lastname: Bauer
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia; The University of Queensland, Diamantina Institute, Brisbane, Queensland, Australia.
authors.forename: Janette
authors.initials: J
authors.lastname: Edson
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Narayanan
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Robinson
authors.affiliation: Swedish University of Agricultural Sciences, Department of Clinical Sciences, Uppsala, Sweden.
authors.forename: Andreas E
authors.initials: AE
authors.lastname: Lundberg
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Perry F
authors.initials: PF
authors.lastname: Bartlett
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Naomi R
authors.initials: NR
authors.lastname: Wray
authors.affiliation: The University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.
authors.forename: Qiong-Yi
authors.initials: QY
authors.lastname: Zhao
mesh_terms: D000818:Animals; D019985:Benchmarking; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods; D012984:Software
chemical_list: D018076:DNA, Complementary; D012313:RNA