P30

Molecular assays for detection of (multi) drug-resistant TB are now established in many diagnostic laboratories worldwide. These assays screen for specific drug resistance-associated mutations and are effective. Nonetheless, tests like the line probe assay (LPA) can occasionally return unclear results, in case rare or untargeted mutations are detected. It is desirable to sequence such amplicons to determine the mutations. Nanopore sequencing has long been known as a long-read sequencer with several advantages like robustness, lower cost, and turnaround time, but with lower accuracy. With accuracy now reaching over 99% this sequencing platform becomes competitive also for short-read sequencing. We investigated the utility of nanopore sequencing for sequencing of amplicons that are available from LPAs. Ten PCR products were obtained from culture and two from clinical material. After purification, library preparation and barcoding, the PCR products were subjected to Nanopore sequencing. Sequencing data was analysed in CLC genomics workbench and the WHO catalogue of drug resistance-associated mutations in MTBC was used to identify mutations. In three out of 12 samples, mutations were found in one or more resistance genes. The concordance with the (blinded) LPA results was 100%. The total turnaround time was 19h with the library preparation taking only 2h. We demonstrated the use of the Nanopore technology to sequence LPA PCR products. The method proposed here could be useful for TB reference laboratories that require quick and reliable sequencing of their available PCR products to disclose unknown drug-resistance associated mutations in MTBC.