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. Competing Interest Statement The authors have declared no competing interest. Footnotes Disclaimer: Please note that this manuscript is NOT going to be sent for peer review. Given some of the limitations observed, we decided to discontinue the approach presented here and focus on novel ideas. Moreover, these observations were generated between 2022 and 2023. As technology evolves fast in the long-read sequencing field, some of our observations and pitfalls may not apply to the latest long-read technology or Isoquant pipeline. We nonetheless wanted to report our preliminary results, observations, and failed attempts, as we believe they can still benefit the research community.