Dissecting and improving gene regulatory network inference using single-cell transcriptome data.

Single-cell transcriptome data has been widely used to reconstruct gene regulatory networks (GRNs) controlling critical biological processes such as development and differentiation. Although a growing list of algorithms has been developed to infer GRNs using such data, achieving an inference accuracy consistently higher than random guessing has remained challenging. To address this, it is essential to delineate how the accuracy of regulatory inference is limited. Here, we systematically characterized factors limiting the accuracy of inferred GRNs and demonstrated that using pre-mRNA information can help improve regulatory inference compared to the typically used information (i.e., mature mRNA). Using kinetic modeling and simulated single-cell data sets, we showed that target genes' mature mRNA levels often fail to accurately report upstream regulatory activities because of gene-level and network-level factors, which can be improved by using pre-mRNA levels. We tested this finding on public single-cell RNA-seq data sets using intronic reads as proxies of pre-mRNA levels and can indeed achieve a higher inference accuracy compared to using exonic reads (corresponding to mature mRNAs). Using experimental data sets, we further validated findings from the simulated data sets and identified factors such as transcription factor activity dynamics influencing the accuracy of pre-mRNA-based inference. This work delineates the fundamental limitations of gene regulatory inference and helps improve GRN inference using single-cell RNA-seq data.