RT Journal Article SR Electronic T1 RNA-seq mixology: designing realistic control experiments to compare protocols and analysis methods JF bioRxiv FD Cold Spring Harbor Laboratory SP 063008 DO 10.1101/063008 A1 Aliaksei Z. Holik A1 Charity W. Law A1 Ruijie Liu A1 Zeya Wang A1 Wenyi Wang A1 Jaeil Ahn A1 Marie-Liesse Asselin-Labat A1 Gordon K. Smyth A1 Matthew E. Ritchie YR 2016 UL http://biorxiv.org/content/early/2016/07/19/063008.abstract AB Carefully designed control experiments provide a gold standard for benchmarking different genomics research tools. A shortcoming of many gene expression control studies is that replication involves profiling the same reference RNA sample multiple times. This leads to low, pure technical noise that is atypical of regular studies. To achieve a more realistic noise structure, we generated a RNA-sequencing mixture experiment using two cell lines of the same cancer type. Variability was added by extracting RNA from independent cell cultures and degrading particular samples. The systematic gene expression changes induced by this design allowed benchmarking of different library preparation kits (standard poly-A versus total RNA with Ribozero depletion) and analysis pipelines. Data generated using the total RNA kit had more signal for introns and various RNA classes (ncRNA, snRNA, snoRNA) and less variability after degradation. For differential expression analysis, voom with quality weights marginally outperformed other popular methods, while for differential splicing, DEXSeq was simultaneously the most sensitive and the most inconsistent method. For sample deconvolution analysis, DeMix outperformed IsoPure convincingly. Our RNA-sequencing dataset provides a valuable resource for benchmarking different protocols and data pre-processing workflows. The extra noise mimics routine lab experiments more closely, ensuring any conclusions are widely applicable.