Fiorini, M. R.; Amiri, S.; Dilliott, A. A.; Yde Ohki, C. M.; Smigielski, L.; Walitza, S.; Fon, E. A.; Grunblatt, E.; Thomas, R. A.; Farhan, S. M. K.

Multiplexing samples from distinct individuals prior to sequencing is a promising step toward achieving population-scale single-cell RNA sequencing by reducing the restrictive costs of the technology. Individual genetic demultiplexing tools resolve the donor-of-origin identity of pooled cells using natural genetic variation but present diminished accuracy on highly multiplexed experiments, impeding the analytic potential of the dataset. In response, we introduce Ensemblex: an accuracy-weighted, ensemble genetic demultiplexing framework that integrates four distinct algorithms to identify the most probable subject labels. Using computationally and experimentally pooled samples, we demonstrate Ensemblex\'s superior accuracy and illustrate the implications of robust demultiplexing on biological analyses.