Heterogeneous pseudobulk simulation enables realistic benchmarking of cell-type deconvolution methods

Computational cell type deconvolution enables the estimation of cell type abundance from bulk tissues and is important for understanding tissue microenviroment, especially in tumor tissues. With rapid development of deconvolution methods, many benchmarking studies have been published aiming for a comprehensive evaluation for these methods. Benchmarking studies rely on cell-type resolved single-cell RNA-seq data to create simulated pseudobulk datasets by adding individual cells-types in controlled proportions. In our work, we show that the standard application of this approach, which uses randomly selected single cells, regardless of the intrinsic difference between them, generates synthetic bulk expression values that lack appropriate biological variance. We demonstrate why and how the current bulk simulation pipeline with random cells is unrealistic and propose a heterogeneous simulation strategy as a solution. The heterogeneously simulated bulk samples match up with the variance observed in real bulk datasets and therefore provide concrete benefits for benchmarking in several ways. We demonstrate that conceptual classes of deconvolution methods differ dramatically in their robustness to heterogeneity with reference-free methods performing particularly poorly. For regression-based methods, the heterogeneous simulation provides an explicit framework to disentangle the contributions of reference construction and regression methods to performance. Finally, we perform an extensive benchmark of diverse methods across eight different datasets and find BayesPrism and a hybrid MuSiC/CIBERSORTx approach to be the top performers. Our heterogeneous bulk simulation method and the entire benchmarking framework is implemented in a user friendly package https://github.com/humengying0907/deconvBenchmarking and https://doi.org/10.5281/zenodo.8206516 , enabling further developments in deconvolution methods.