Precision and Accuracy in Quantitative Measurement of Gene Expression from Single-cell/nucleus RNA Sequencing Data

Abstract

Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) have become essential tools for profiling gene expression across different cell types in biomedical research. While factors like RNA integrity, cell count, and sequencing depth are known to influence data quality, quantitative benchmarks and actionable guidelines are lacking. This gap contributes to variability in study designs and inconsistencies in downstream analyses. In this study, we systematically evaluated quantitative precision and accuracy in expression measures across 23 sc/snRNA-seq datasets comprising 3,682,576 cells from 339 samples. Precision was assessed using technical replicates based on pseudo-bulks created from subsampling. Accuracy was evaluated using sample-matched scRNA-seq and pooled-cell RNA sequencing data of mononuclear phagocytes from four species. Our results show that precision and accuracy are generally low at the single-cell level, with reproducibility being strongly influenced by cell count and RNA quality. We established data-driven thresholds for optimizing study design, recommending at least 500 cells per cell type per individual to achieve reliable quantification. Furthermore, we showed that signal-To-noise ratio is a key metric for identifying reproducible differentially expressed genes. To support future research, we developed Variability In single-Cell gene Expression (VICE), a tool that evaluates sc/snRNA-seq data quality and estimates the true positive rate of differential expression results based on sample size, observed noise levels, and expected effect size. These findings provide practical, evidence-based guidelines to enhance the reliability and reproducibility of sc/snRNA-seq studies.