Single cell RNA-seq by mostly-natural sequencing by synthesis

Massively parallel single cell RNA-seq (scRNA-seq) for diverse applications, from cell atlases to functional screens, is increasingly limited by sequencing costs, and large-scale low-cost sequencing can open many additional applications, including patient diagnostics and drug screens. Here, we adapted and systematically benchmarked a newly developed, mostly-natural sequencing by synthesis method for scRNA-seq. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5' and 3' scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq. Our data show comparable results to existing technology, including compatibility with state-of-the-art scRNA-seq libraries independent of the sequencing technology used -- thus providing an enhanced cost-effective path for large scale scRNA-seq. ### Competing Interest Statement A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and until July 31, 2020 was an S.A.B. member of Thermo Fisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov. From August 1, 2020, A.R. is an employee of Genentech, a member of the Roche Group, and has equity in Roche. From November 16, 2020, K.G.S. is an employee of Genentech. From March 22, 2021, P.I.T. is an employee of Genentech. From October 19, 2020, O.R.R. is an employee of Genentech. O.R.R. is a co-inventor on patent applications filed by the Broad Institute for inventions related to single cell genomics. She has given numerous lectures on the subject of single cell genomics to a wide variety of audiences and in some cases, has received remuneration to cover time and costs. G.L.Y., F.O, N.I., O.B., G.A., and D.L. are employees and shareholders of Ultima Genomics. The other authors declare that they have no competing interests.