Cell-type deconvolution for bulk RNA-seq data using single-cell reference: a comparative analysis and recommendation guideline.

The accurate estimation of cell type proportions in tissues is crucial for various downstream analyses. With the increasing availability of single-cell sequencing data, numerous deconvolution methods that use single-cell RNA sequencing data as a reference have been developed. However, a unified understanding of how these deconvolution approaches perform in practical applications is still lacking. To address this, we systematically assessed the accuracy and robustness of nine deconvolution methods that use single-cell RNA sequencing data as a reference, evaluating them on real bulk data with cell proportions verified through flow cytometry, as well as simulated bulk data generated from five single-cell RNA sequencing datasets. Our study highlights the importance of several factors-including reference dataset construction strategies, dataset size, cell type subdivision, and cell type inconsistency-on the accuracy and robustness of deconvolution results. We also propose a set of recommended guidelines for software users in diverse scenarios.