Relative effect size-based profiles as an alternative to differentiation analysis in multi-species single-cell transcriptional studies.

Combining data from experiments on multispecies studies provides invaluable contributions to the understanding of basic disease mechanisms and pathophysiology of pathogens crossing species boundaries. The task of multispecies gene expression analysis, however, is often challenging given annotation inconsistencies and in cases of small sample sizes due to bias caused by batch effects. In this work we aim to demonstrate that an alternative approach to standard differential expression analysis in single cell RNA-sequencing (scRNA-seq) based on effect size profiles is suitable for the fusion of data from small samples and multiple organisms. The analysis pipeline is based on effect size metric profiles of samples in specific cell clusters. The effect size substitutes standard differentiation analyses based on p-values and profiles identified based on these effect size metrics serve as a tool to link cell type clusters between the studied organisms. The algorithms were tested on published scRNA-seq data sets derived from several species and subsequently validated on own data from human and bovine peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis. Correlation of the effect size profiles between clusters allowed for the linkage of human and bovine cell types. Moreover, effect size ratios were used to identify differentially regulated genes in control and stimulated samples. The genes identified through effect size profiling were confirmed experimentally using qPCR. We demonstrate that in situations where batch effects dominate cell type variation in single cell small sample size multispecies studies, effect size profiling is a valid alternative to traditional statistical inference techniques.