Striving towards improved full-length single-cell RNA-sequencing

Abstract Full-length single-cell RNA-sequencing (scRNA-seq) methods provide superior transcript coverage and isoform resolution compared to their 3’-end counterparts, but are typically limited to short-read platforms. Here, we report efforts to improve the FLASH-seq protocol and adapt it for long-read sequencing on the Oxford Nanopore Technologies (ONT) platform (FLASH-seq-ONT). We developed two plate-barcoding strategies enabling higher multiplexing: a custom PCR-ligation approach (PCR-LIG) and ONT native barcoding (NB-ONT). To support data processing, we built FSNanoporeR, a comprehensive bioinformatics pipeline for barcode demultiplexing, chimeric read detection and splitting, UMI extraction, and transcript quantification. Both barcoding strategies produced high-quality transcriptomic data from HEK293T cells, with notable differences in read length distributions. We further demonstrated that monomeric and trimeric UMIs can be reliably detected in >82% of reads, enabling accurate molecular counting at isoform resolution. However, both multiplexing approaches exhibited also critical limitations, including high chimeric read rates and index-swapping artifacts. Our results highlight both the promise and current technical hurdles of full-length single-cell long-read sequencing, and provide a practical framework for researchers considering ONT-based scRNA-seq workflows.