AuPairWise: A Method to Estimate RNA-Seq Replicability through Co-expression

In addition to detecting novel transcripts and higher dynamic range, a principal claim for RNA-sequencing has been greater replicability, typically measured in sample-sample correlations of gene expression levels. Through a re-analysis of ENCODE data, we show that replicability of transcript abundances will provide misleading estimates of the replicability of conditional variation in transcript abundances (i.e., most expression experiments). Heuristics which implicitly address this problem have emerged in quality control measures to obtain ‘good’ differential expression results. However, these methods involve strict filters such as discarding low expressing genes or using technical replicates to remove discordant transcripts, and are costly or simply ad hoc. As an alternative, we model gene-level replicability of differential activity using co-expressing genes. We find that sets of housekeeping interactions provide a sensitive means of estimating the replicability of expression changes, where the co-expressing pair can be regarded as pseudo-replicates of one another. We model the effects of noise that perturbs a gene’s expression within its usual distribution of values and show that perturbing expression by only 5% within that range is readily detectable (AUROC~0.73). We have made our method available as a set of easily implemented R scripts.Author Summary: RNA-sequencing has become a popular means to detect the expression levels of genes. However, quality control is still challenging, requiring both extreme measures and rules which are set in stone from extensive previous analysis. Instead of relying on these rules, we show that co-expression can be used to measure biological replicability with extremely high precision. Co-expression is a well-studied phenomenon in which two genes that are known to form a functional unit are also expressed at similar levels, and change in similar ways across conditions. Using this concept, we can detect how well an experiment replicates by measuring how well it has retained the co-expression pattern across defined gene-pairs. We do this by measuring how easy it is to detect a sample to which some noise has been added. We show this method is a useful tool for quality control.