Differential expression in RNA-seq: A matter of depth
Top Cited Papers
Open Access
- 8 September 2011
- journal article
- research article
- Published by Cold Spring Harbor Laboratory in Genome Research
- Vol. 21 (12), 2213-2223
- https://doi.org/10.1101/gr.124321.111
Abstract
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.This publication has 36 references indexed in Scilit:
- Comparative and demographic analysis of orang-utan genomesNature, 2011
- The developmental transcriptome of Drosophila melanogasterNature, 2010
- A map of human genome variation from population-scale sequencingNature, 2010
- The genome of the domesticated apple (Malus × domestica Borkh.)Nature Genetics, 2010
- Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiationNature Biotechnology, 2010
- ChIP–seq: advantages and challenges of a maturing technologyNature Reviews Genetics, 2009
- Biogenesis of small RNAs in animalsNature Reviews Molecular Cell Biology, 2009
- Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in YeastMolecular Cell, 2008
- Mapping and quantifying mammalian transcriptomes by RNA-SeqNature Methods, 2008
- The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurementsNature Biotechnology, 2006