TUCL-Seq for miRNA single molecule sequencing

Background: MicroRNAs (miRNAs) are pivotal regulatory molecules in gene expression, and their precise quantification is essential for elucidating their roles in diverse biological processes. This research paper introduces a pioneering workflow for the single molecule sequencing (SMS) of miRNA, integrating advanced miRNA sample preparation with SMS sequencing. Central to our approach is TUCL-Seq (terminal deoxyribonucleotidyl transferase-assisted uridine connector-mediated 1st cDNA ligation sequencing), a method based on the TACS (TdT-assisted adenylate connector-mediated ssDNA ligation) approach, which is an automated, on-chip miRNA library preparation method designed to streamline the SMS sequencing process.Results: This innovative approach incorporates the addition of sequencing adaptor to RNA samples in solution, and converts it to cDNA on a sequencing flow cell for sequencing. Coupled with SMS instrument and base-call software, our workflow significantly reduces sample-to-result time, and decreases the need for manual interventions compared to traditional SMS and next-generation sequencing methods. We demonstrate the efficacy of TUCL-Seq by accurately quantifying synthetic miRNA samples with a 24-hour workflow, showcasing its potential for high-throughput miRNA profiling.Conclusions: Our findings highlight the promise of this integrated workflow for advancing miRNA research and applications in diagnostics and therapeutics.