Combining denoising of RNA-seq data and flux balance analysis for cluster analysis of single cells

Background: Sophisticated methods to properly pre-process and analyze the increasing collection of single-cell RNA sequencing (scRNA-seq) data are increasingly being developed. On the contrary, the best practices to integrate these data into metabolic networks, aiming at describing metabolic phenotypes within a heterogeneous cell population, have been poorly investigated. In this regard, a critical factor is the presence of false zero values in reactions essential for a fundamental metabolic function, such as biomass or energy production. Here, we investigate the role of denoising strategies in mitigating this problem. Methods: We applied state-of-the-art denoising strategies - namely MAGIC, ENHANCE, and SAVER - on three public scRNA-seq datasets. We then associated a metabolic flux distribution with every single cell by embedding its noise-free transcriptomics profile in the constraints of the optimization of a core metabolic model. Finally, we used the obtained single-cell optimal metabolic fluxes as features for cluster analysis. We compared the results obtained with different techniques, and with or without the use of denoising. We also investigated the possibility of applying denoising directly on the Reaction Activity Scores, which are metabolic features extracted from the read counts, rather than on the read counts. Results: We show that denoising of transcriptomics data improves the clustering of single cells. We also illustrate that denoising restores important metabolic properties, such as the correlation between cell cycle phase and biomass accumulation, and between the RAS scores of reactions belonging to the same metabolic pathway. We show that MAGIC performs better than ENHANCE and SAVER, and that, denoising applied directly on the RAS matrix could be an effective alternative in removing false zero values from essential metabolic reactions. Conclusions: Our results indicate that including denoising as a pre-processing operation represents a milestone to integrate scRNA-seq data into Flux Balance Analysis simulations and to perform single-cell cluster analysis with a focus on metabolic phenotypes.