RAPID, FLEXIBLE, AND SCALABLE ALTERNATIVES TO CONVENTIONAL HIGH-THROUGHPUT WHOLE GENOMA SEQUENCING TO ENHANCE SURVEILLANCE OF TUBERCULOSIS TRANSMISSION

Whole genome sequencing (WGS) is essential for monitoring tuberculosis (TB) transmission, enabling precise clustering of cases. Illumina sequencing, while accurate, depends on batch processing and often introduces delays that limit timely interventions. Nanopore sequencing offers a flexible, case-by-case alternative that can accelerate genomic data availability. We prospectively applied nanopore sequencing to 33 primary cultures and 51 clinical sputa during active contact tracing. All cultures achieved optimal WGS performance (≥90% genome coverage at ≥20X), allowing integration of 27 new cases into genomic networks within the 21-day tracing window. Beyond SNP clustering, evolutionary network analysis improved interpretation of transmission dynamics, revealing ongoing chains, reactivations, and diagnostic delays. Among sputa, 31.3% achieved optimal coverage and 15.7% suboptimal results, but these were still usable through a rescue pipeline that exploited circulating marker SNPs, enabling assignment of suboptimal sequences to ongoing clusters. To accelerate responses during high-risk events, we designed a targeted amplicon-based nanopore approach focusing on cluster-defining SNPs. Using 8 marker SNPs, we tracked potential cases linked to an interterritorial cluster associated with a super-spreader, highlighting further cluster expansion without requiring full WGS. By integrating real-time nanopore sequencing, rescue pipelines, and targeted SNP-based amplicon sequencing, we demonstrate rapid, flexible, and scalable alternatives to Illumina-based high-throughput WGS. These strategies strengthen genomic epidemiology and enable more effective, timely TB control interventions.