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

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Science China(Life Sciences) 2013年第2期56卷 134-142页

作者： QING Tao YU Ying DU TingTing SHI LeMing Center for Pharmacogenomics School of PharmacyFudan University

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 ***,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 *** 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 *** entire collection of datasets consisted of 15650143175 short sequence reads,131603796(i.e.,0.84%) of which were mapped to the 92 ERCC *** 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 *** level of fluctuation may prevent the ERCC controls from being used for cross-sample normalization in ***,we observed striking biases of quantification between poly(A) and RiboZero which are *** example,ERCC-00116 showed a 7.3-fold under-enrichment in poly(A) compared to *** care is needed in integrative analysis of multiple datasets and technical artifacts of protocol differences should not be taken as true biological findings.

关键词： RNA-Seq External RNA Control Consortium (ERCC) MAQC/SEQC mRNA enrichment protocol quality control reproducibility quantification bias poly(A) versus RiboZero

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