Accelerating genomic epidemiology of tuberculosis through immediate nanopore sequencing
Background: Genomic analysis has enhanced our understanding of Mycobacterium tuberculosis (MTB) transmission. Short-read sequencing in reference labs ensures full population coverage but delays local data access. We assessed a case-by-case genomic approach using primary cultures from each incident case.  Methods: We prospectively evaluated 53 TB cases in Almeria, Spain, over two periods: an initial stage of 4 months (March–July 2023), when we collected solid cultures (LJ, 23 cases) and to improve turnaround time for genomic results and evaluate the impact of interfering human/other bacterial content, for 9 months (April–December 2024) we adopted liquid cultures (MGIT, 30 cases). DNA was purified from LJ when sufficient growth was observed and from MGIT at positivity. Nanopore sequencing was performed on 1–4 cases per run, with flow-cells reused up to six times.  Results: In 74% of LJ, adequate growth was reached within 21 days. All LJ cultures achieved optimal genome coverage (>90% at >20X), 61% with runs under 2.5 hours. Nearly all MGIT cultures analysed at positivity (all but six within 21 days), achieved optimal genome coverage, despite interfering DNA. Four cases had slightly lower but acceptable coverage (73–88%, >20X). Same-day classification as clustered or orphan was possible. Nanopore sequencing correlated with high-throughput short-read data, enabling quicker correction of epidemiological errors and identification of transmission links.  Conclusions: Our strategy offers a flexible, and rapid workflow for decentralized alternative, accelerating cluster information contact tracing.