RNA-seq mixology: Designing realistic control experiments to compare protocols and analysis methods

Abstract

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 samplemultiple 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 (sta..