Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories

Abstract

RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and guidelines for assessing technical biases in RNA-seq data.

Interethnic DNA Methylation Difference and Its Implications in Pharmacoepigenetics

“…: E-GEUV-1); these data were shared by the Geuvadis RNA sequencing project based on the samples of the 1000 Genomes project [30,31]. These gene expression profiles were generated by RNA sequencing experiments.…”

“…Finally, 53,934 genes were included in each expression profile. The details of data processing and quality control are described in a previous study [31].…”

Integrated Whole Genome and Transcriptome Analysis Identified a Therapeutic Minor Histocompatibility Antigen in a Splice Variant of ITGB2

“…EBV-B cell lines for which RNA-sequence data (.bam files) are available in the GEUVADIS project (18,19) were selected for SNP genotype (þ/þ, þ/À, and À/À) from the 1000 GP (20). For each SNP (rs760462 and rs9945924), two representative individuals per genotype were selected, and bigwig files containing RNAsequence coverage and mapping and split coordinates of individual sequence reads in the region of interest for these EBV-B cell lines were uploaded to the UCSC genome browser (23).…”

“…RNA-sequence data were analyzed as online available in the GEUVADIS project (18,19) for 462 EBV-B cell lines for which corresponding whole genome sequences are available in the 1000 GP. On the basis of SNP genotypes, EBV-B cell lines were selected from individuals who were homozygous positive (A/A; þ/þ), heterozygous (A/G; þ/À), or homozygous negative (G/G; À/À) for associating SNP rs760462.…”

“…In this study, we explored whether RNA-sequence data as available in the GEUVADIS project (18,19) can be used to identify MiHAs encoded by alternative transcripts. With the rapid advances in sequence technology, the GEUVADIS consortium set out to combine whole genome and transcriptome data and sequenced all mRNA expressed in 462 EBV-B cell lines from the 1000 Genomes Project (1000 GP; ref.…”