Performance of computational algorithms to deconvolve heterogeneous bulk tumor tissue depends on experimental factors

Background Single-cell gene expression profiling provides unique opportunities to understand tumor heterogeneity and the tumor microenvironment. Because of cost and feasibility, profiling bulk tumors remains the primary population-scale analytical strategy. Many algorithms can deconvolve these tumors using single-cell profiles to infer their composition. While experimental choices do not change the true underlying composition of the tumor, they can affect the measurements produced by the assay. Results We generated a dataset of high-grade serous ovarian tumors with paired expression profiles from using multiple strategies to examine the extent to which experimental factors impact the results of downstream tumor deconvolution methods. We find that pooling samples for single-cell sequencing and subsequent demultiplexing has a minimal effect. We identify dissociation-induced differences that affect cell composition, leading to changes that may compromise the assumptions underlying some deconvolution algorithms. We also observe differences across mRNA enrichment methods that introduce additional discrepancies between the two data types. We also find that experimental factors change cell composition estimates and that the impact differs by method. Conclusions Previous benchmarks of deconvolution methods have largely ignored experimental factors. We find that methods vary in their robustness to experimental factors. We provide recommendations for methods developers seeking to produce the next generation of deconvolution approaches and for scientists designing experiments using deconvolution to study tumor heterogeneity.