mRNA enrichment protocols determine the quantification characteristics of external RNA spike-in controls in RNA-Seq studies

摘要

RNA-Seq promises to be used in clinical settings as a gene-expression profiling tool; however, questions about its variability and biases remain and need to be addressed. Thus, RNA controls with known concentrations and sequence identities originally developed by the External RNA Control Consortium ( ERCC ) for microarray and qPCR platforms have recently been proposed for RNA-Seq platforms, but only with a limited number of samples. In this study, we report our analysis of RNA-Seq data from 92 ERCC controls spiked in a diverse collection of 447 RNA samples from eight ongoing studies involving five species (human, rat, mouse, chicken, and Schistosoma japonicum ) and two mRNA enrichment protocols, i.e., poly(A) and RiboZero. The entire collection of datasets consisted of 15650143175 short sequence reads, 131603796 (i.e., 0.84%) of which were mapped to the 92 ERCC references. The overall ERCC mapping ratio of 0.84% is close to the expected value of 1.0% when assuming a 2.0% mRNA fraction in total RNA, but showed a difference of 2.8-fold across studies and 4.3-fold among samples from the same study with one tissue type. This level of fluctuation may prevent the ERCC controls from being used for cross-sample normalization in RNA-Seq. Furthermore, we observed striking biases of quantification between poly(A) and RiboZero which are transcript-specific. For example, ERCC -00116 showed a 7.3-fold under-enrichment in poly(A) compared to RiboZero. Extra care is needed in integrative analysis of multiple datasets and technical artifacts of protocol differences should not be taken as true biological findings.