A low-cost Mycobacterium tuberculosis complete genome using nanopore sequencing | LC26

Abstract
Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) costs ~US$63/sample and produces incomplete assemblies, as Illumina short reads struggle with repetitive regions, limiting the clinical and epidemiological utility of WGS in high-burden settings. This study optimised an end-to-end nanopore long-read pipeline for complete, high-quality Mtb genomes, improving DNA preparation, sequencing output, and affordability in Lima, Peru. Using H37Rv reference strain, we evaluated inactivation temperatures, DNA isolation (commercial kit vs. phenol-chloroform), and fragmentation for yield, quality, and sequencing metrics. The optimal pipeline sequenced 377 clinical Mtb isolates, comparing MinION/PromethION Flow Cells, Native/Rapid Barcoding Kits, and loading strategies. Down-sampling determined minimum required reads for assemblies, resistance detection, and lineage; costs modelled multiplexing. High temperatures (80–95°C) inefficiently inactivated Mtb and degraded DNA, reducing read length. Commercial extraction kit with DNA fragmentation and high loading concentration (85 fmol) tripled sequencing depth (217x vs. 68x, p=0.001) and slowed pore decay (2%/hour). PromethION yielded 5.6x more data than MinION (93.6 Gb vs. 16.6 Gb); rapid barcoding reduced costs/labour vs. native barcoding. Ten thousand reads per sample sufficed for high-quality assemblies and reliable resistance calling. Ultimately, multiplexing 96 samples achieved complete genomes for US$26, less than half the cost of prior methods, while exceeding WHO criteria for genetic drug resistance diagnosis. Nanopore variant detection showed 98.1% sensitivity (95% CI 95.9–99.1%), 94.8% PPV, and per-drug sensitivity and specificity ranged from 97.2–100% and 94.7–100%, respectively (Illumina reference). This pipeline delivers high-confidence Mtb complete genomes at unprecedented low cost, suitable for deployment in reference labs in high-burden settings.

Biography
Alice Osmaston is a PhD candidate at University College London researching the genomics of Mycobacterium tuberculosis, with a focus on transmission dynamics and drug resistance. Based in Lima, Peru, for the past three years, she collaborates with Universidad Peruana Cayetano Heredia, combining field studies, laboratory work, method development, and population-level analyses to investigate genomic factors of treatment outcome, disease transmission, and antibiotic resistance.