Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling

Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational “deconvolution” of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of ‘ON’ versus ‘OFF’ cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies.
Author Summary Different cells can make different amounts of biomolecules such as RNA transcripts of genes. New technologies are emerging to measure the transcript level of many genes in single cells. However, accurate quantification of the biological variation from cell to cell can be challenging due to the low transcript level of many genes and the presence of substantial measurement noise. Here we present a flexible, novel computational approach to quantify biological cell-to-cell variation that can use different types of data, namely measurements directly obtained from single cells, and/or those from random pools of k-cells (e.g., k = 10). Assessment of these different inputs using simulated and real data revealed that each data type can offer advantages under different scenarios, but combining both single- and k-cell measurements tend to offer the best of both. Application of our approach to single- and k-cell data obtained from resting and inflammatory macrophages, an important type of immune cells implicated in diverse diseases, revealed interesting changes in cell-to-cell variation in transcript levels upon inflammatory stimulation, thus suggesting that inflammation can shape not only the average expression level of a gene but also the gene’s degree of expression variation among single cells.