Quantitative comparison of single-cell RNA sequencing versus single-molecule RNA imaging for quantifying transcriptional noise

SUMMARY Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability. However, how best to quantify genome-wide noise, remains unclear. Here we utilize a small-molecule perturbation (IdU) to amplify noise and assess noise quantification from numerous scRNA-seq algorithms on human and mouse datasets, and then compare to noise quantification from single-molecule RNA FISH (smFISH) for a panel of representative genes. We find that various scRNA-seq analyses report amplified noise, without altered mean-expression levels, for ∼90% of genes and that smFISH analysis verifies noise amplification for the vast majority of genes tested. Collectively, the analyses suggest that most scRNA-seq algorithms are appropriate for quantifying noise including a simple normalization approach, although all of these systematically underestimate noise compared to smFISH. From a practical standpoint, this analysis argues that IdU is a globally penetrant noise-enhancer molecule—amplifying noise without altering mean-expression levels—which could enable investigations of the physiological impacts of transcriptional noise. MOTIVATION Cell-to-cell variability in isogenic populations is predominantly attributed to the stochastic fluctuations (i.e., noise) in transcription. However, the quantification of this noise, particularly on a genome-wide scale, remains an open question. To address this general question, here we utilize a small-molecule perturbation reported to amplify transcriptional noise. Previous single-cell RNA-sequencing (scRNA-seq) analysis indicated that the pyrimidine nucleobase 5′-iodo-2′-deoxyuridine (IdU) amplifies noise but technical drawbacks of scRNA-seq may have obscured the penetrance and degree of noise amplification. Consequently, here we assess numerous scRNA-seq algorithms, on two different scRNA-seq datasets for a human and mouse cell type, for their ability to quantify noise amplification and then compare the results to noise quantification from single-molecule RNA FISH (smFISH) imaging for a panel of representative genes. The specific questions addressed are whether IdU amplifies noise in a globally or partially penetrant manner, and what scRNA-seq algorithm is most appropriate for quantifying transcriptional noise. Graphical Abstract