Georgescu, C. H.; Al-Eryani, G.; Brookhart, A.; Chandrasekar, J.; Yaung, S. J.; Rogers-Peckham, M.; Freer, M.; Kartje, M. E.; Yu, H.; Khorgade, A.; Yang, C.; McGee, L.; Berg, K.; Cech, C.; Barrett, S.; Arryman, A.; Bartlett, D. A.; Slamin, A.; Low, S.; Dubinsky, D.; Cipicchio, M.; Hacohen, N.; Lehmann, T.; Lennon, N. J.; Popic, V.; Zhao, C.; Prindle, M.; Mannion, J.; Nabavi, M.; Haas, B. J.; Kokoris, M.; AlKhafaji, A. M.

Single-cell RNA sequencing has transformed our understanding of cellular systems, yet the reliance on short-read sequencing restricts analysis to gene-level quantification and obscures the immense biological diversity generated by alternative splicing. While long-read sequencing technologies can capture full-length RNA and resolve transcript isoforms, current platforms remain constrained by throughput and high per-base costs, rendering them impractical for modern million-cell applications. To address this critical limitation, we developed and optimized sequencing-by-expansion (SBX) chemistry for high-throughput single-cell RNA isoform profiling. Integrated within the AXELIOS 1 sequencing platform, SBX employs a unique biochemical conversion process that transforms complementary DNA into expanded surrogate high signal-to-noise polymers called Xpandomers which are sequenced via translocation through a dense nanopore array yielding over 9.5 billion reads in a two-hour run. To leverage this unique data type for long-read single-cell RNA isoform sequencing, we developed the Consensus UMI Deduplication using Longest Length (CUDLL) algorithm, which computationally consolidates variable-length raw SBX reads into single, high-fidelity consensus reads, elevating sequence accuracy to 99.83% and maximizing per transcript read length. We demonstrate that this consensus approach successfully captures the vast isoform diversity of single-cell libraries and enables the accurate measure of differential isoform expression across distinct cell types in peripheral blood mononuclear cells. Furthermore, SBX coupled with CUDLL efficiently resolves T-cell and B-cell receptor clonotypes directly from whole-transcriptome libraries without the need for VDJ-specific target enrichment. Ultimately, this work establishes SBX and the AXELIOS 1 as a transformative platform for high-scale single-cell isoform sequencing.