High-throughput RNA isoform sequencing using programmable cDNA concatenation

Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses. ### Competing Interest Statement A.M.A., K.V.G., J.S., M.B., P.B., and N.H. have filed a patent on the MAS-seq method. A.A.P. is a Venture Partner and Employee of GV. He has received funding from Verily, Microsoft, Illumina, Bayer, Pfizer, Biogen, Abbvie, Intel, and IBM. M.S.F. receives funding from Bristol-Myers Squibb. G.M.B. has served on SAB and on the steering committee for Nektar Therapeutics. She has SRAs with Olink proteomics and Palleon Pharmaceuticals. She served on SAB and as a speaker for Novartis N.H. holds equity in BioNTech and is a founder and equity holder of Danger Bio. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic or genome editing technologies: 10X Genomics, General Automation Lab Technologies, Celsius Therapeutics, Next Gen Diagnostics LLC, Cache DNA, and Concerto Biosciences. P.C.B. receives funding from industry for unrelated work.