A sensitive approach to quantitative analysis of transcriptional regulation in diploid organisms is analysis of allelic imbalance (AI) in RNA sequencing (RNA-seq) data. A near-universal practice in such studies is to prepare and sequence only one library per RNA sample. We present theoretical and experimental evidence that data from a single RNA-seq library is insufficient for reliable quantification of the contribution of technical noise to the observed AI signal; consequently, reliance on one-replicate experimental design can lead to unaccounted-for variation in error rates in allele-specific analysis. We develop a computational approach, Qllelic, that accurately accounts for technical noise by making use of replicate RNA-seq libraries. Testing on new and existing datasets shows that application of Qllelic greatly decreases false positive rate in allele-specific analysis while conserving appropriate signal, and thus greatly improves reproducibility of AI estimates. We explore sources of technical overdispersion in observed AI signal and conclude by discussing design of RNA-seq studies addressing two biologically important questions: quantification of transcriptome-wide AI in one sample, and differential analysis of allele-specific expression between samples.

In mammalian cells, maternal and paternal alleles usually have similar transcriptional activity. Epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. Discovery of autosomal monoallelic expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. Despite MAE prevalence, its mechanistic basis remains unknown. Using an RNA sequencing-based screen for reactivation of silenced alleles, we identified DNA methylation as key mechanism of MAE mitotic maintenance. In contrast with the all-or-nothing allelic choice in XCI, allele-specific expression in MAE loci is tunable, with exact allelic imbalance dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to DNA demethylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance and provide basis for understanding the biological role of MAE.

Thousands of mammalian genes show epigenetically controlled unequal transcription of the parental alleles. Genes subject to autosomal monoallelic expression (MAE) display mitotically stable allelic choice, leading to persistent transcriptional differences between clonal cell lineages. Mechanism of MAE mitotic maintenance is unknown. Using a new screening-by-sequencing strategy, we uncovered a key role for DNA methylation in MAE maintenance. Subset of MAE loci were insensitive to DNA demethylation, suggesting mechanistic heterogeneity of MAE. Genome-wide analyses indicate that MAE is part of a more general mode of gene regulation and reveal a previously unappreciated interplay of genetic and epigenetic control of allele-specific transcription. While cis-acting regulation defines a common underlying state for all cells, DNA methylation plays the role of an allele-specific rheostat and determines multiple regulatory states distinguishing between developmentally equivalent clonal cell lineages. Our findings imply that allele-specific analyses of clonal cell populations can unmask longterm transcriptional responses to drug-driven perturbations.