RNA-seq_ an assessment of technical reproducibility and comparison with gene expression arrays英文资料.pdfVIP

Methods RNA-seq: An assessment of technical reproducibility and comparison with gene expression arrays John C. Marioni,1,6 Christopher E. Mason,2,3,6 Shrikant M. Mane,4 Matthew Stephens,1,5,7 and Yoav Gilad1,7 1Department of Human Genetics, University of Chicago, Chicago, Illinois 60637, USA; 2Program on Neurogenetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA; 3Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA; 4Keck Biotechnology Laboratory, New Haven, Connecticut 06511, USA; 5Department of Statistics, University of Chicago, Chicago, Illinois 60637, USA Ultra-high-throughput sequencing is emerging as an attractive alternative to microarrays for genotyping, analysis of methylation patterns, and identification of transcription factor binding sites. Here, we describe an application of the Illumina sequencing (formerly Solexa sequencing) platform to study mRNA expression levels. Our goals were to estimate technical variance associated with Illumina sequencing in this context and to compare its ability to identify differentially expressed genes with existing array technologies. To do so, we estimated gene expression differences between liver and kidney RNA samples using multiple sequencing replicates, and compared the sequencing data to results obtained from Affymetrix arrays using the same RNA samples. We find that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane). The information in a single lane of Illumina sequencing data appears comparable to that in a single array in enabling identification of differentially expressed genes, while allowing for additional analyses such as detection of low-expressed genes, alternative spli