Quantifying and correcting bias in transcriptional parameter inference from single-cell data

The snapshot distribution of mRNA counts per cell can be measured using single molecule FISH or single-cell RNA sequencing. These distributions are often fit to the steady-state distribution of the two-state telegraph model to estimate the three transcriptional parameters for a gene of interest: mRNA synthesis rate, the switching on rate (the on state being the active transcriptional state) and the switching off rate. This model assumes no extrinsic noise, i.e. parameters do not vary between cells, and thus estimated parameters are to be understood as approximating the average values in a population. The accuracy of this approximation is currently unclear. Here we develop a theory that explains the size and sign of estimation bias when inferring parameters from single-cell data using the standard telegraph model. We find specific bias signatures depending on the source of extrinsic noise (which parameter is most variable across cells) and the mode of transcriptional activity. If gene expression is not bursty then the population averages of all three parameters are overestimated if extrinsic noise is in the synthesis rate; underestimation occurs if extrinsic noise is in the switching on rate; both underestimation and overestimation can occur if extrinsic noise is in the switching off rate. We find that some estimated parameters tend to infinity as the size of extrinsic noise approaches a critical threshold. In contrast when gene expression is bursty, we find that in all cases, the mean burst size (ratio of the synthesis rate to the switching off rate) is overestimated while the mean burst frequency (the switching on rate) is underestimated. We estimate the size of extrinsic noise from the covariance matrix of sequencing data and use this together with our theory to correct published estimates of transcriptional parameters for mammalian genes.