Inferring TF activation order in time series scRNA-Seq studies

Methods for the analysis of time series single cell expression data (scRNA-Seq) either do not utilize information about transcription factors (TFs) and their targets or only study these as a post-processing step. Using such information can both, improve the accuracy of the reconstructed model and cell assignments, while at the same time provide information on how and when the process is regulated. We developed the Continuous-State Hidden Markov Models TF (CSHMM-TF) method which integrates probabilistic modeling of scRNA-Seq data with the ability to assign TFs to specific activation points in the model. TFs are assumed to influence the emission probabilities for cells assigned to later time points allowing us to identify not just the TFs controlling each path but also their order of activation. We tested CSHMM-TF on several mouse and human datasets. As we show, the method was able to identify known and novel TFs for all processes, assigned time of activation agrees with both expression information and prior knowledge and combinatorial predictions are supported by known interactions. We also show that CSHMM-TF improves upon prior methods that do not utilize TF-gene interaction.Author summary: An important attribute of time series single cell RNA-Seq (scRNA-Seq) data, is the ability to infer continuous trajectories of genes based on orderings of the cells. While several methods have been developed for ordering cells and inferring such trajectories, to date it was not possible to use these to infer the temporal activity of several key TFs. These TFs are are only post-transcriptionally regulated and so their expression does not provide complete information on their activity. To address this we developed the Continuous-State Hidden Markov Models TF (CSHMM-TF) methods that assigns continuous activation time to TFs based on both, their expression and the expression of their targets. Applying our method to several time series scRNA-Seq datasets we show that it correctly identifies the key regulators for the processes being studied. We analyze the temporal assignments for these TFs and show that they provide new insights about combinatorial regulation and the ordering of TF activation. We used several complementary sources to validate some of these predictions and discuss a number of other novel suggestions based on the method. As we show, the method is able to scale to large and noisy datasets and so is appropriate for several studies utilizing time series scRNA-Seq data.