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

Nat Biotechnol. 2013 Nov;31(11):1015-22. doi: 10.1038/nbt.2702. Epub 2013 Sep 15.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Female
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Nucleotide Sequencing / standards*
  • Humans
  • Male
  • MicroRNAs / analysis
  • MicroRNAs / chemistry*
  • MicroRNAs / genetics
  • RNA, Messenger / analysis
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods
  • Sequence Analysis, RNA / standards*


  • MicroRNAs
  • RNA, Messenger