Investigating higher order interactions in single cell data with scHOT

Single-cell RNA-sequencing has transformed our ability to examine cell fate choice. For example, in the context of development and differentiation, computational ordering of cells along ‘pseudotime’ enables the expression profiles of individual genes, including key transcription factors, to be examined at fine scale temporal resolution. However, while cell fate decisions are typically marked by profound changes in expression, many such changes are observed in genes downstream of the initial cell fate decision. By contrast, the genes directly involved in the cell fate decision process are likely to interact in subtle ways, potentially resulting in observed changes in patterns of correlation and variation rather than mean expression prior to cell fate commitment. Herein, we describe a novel approach, scHOT – single cell Higher Order Testing - which provides a flexible and statistically robust framework for identifying changes in higher order interactions among genes. scHOT is general and modular in nature, can be run in multiple data contexts such as along a continuous trajectory, between discrete groups, and over spatial orientations; as well as accommodate any higher order measurement such as variability or correlation. We demonstrate the utility of scHOT by studying embryonic development of the liver, where we find coordinated changes in higher order interactions of programs related to differentiation and liver function. We also demonstrate its ability to find subtle changes in gene-gene correlation patterns across space using spatially-resolved expression data from the mouse olfactory bulb. scHOT meaningfully adds to first order effect testing, such as differential expression, and provides a framework for interrogating higher order interactions from single cell data.