Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation

Mycobacterium tuberculosis whole-genome sequencing (WGS) using Illumina technology has been widely adopted for genotypic drug susceptibility testing (DST) and outbreak investigation. Oxford Nanopore Technologies is reported to have higher error rates but has not been thoroughly evaluated for these applications. We analyse 151 isolates from Madagascar, South Africa and England with phenotypic DST and matched Illumina and Nanopore data. Using PacBio assemblies, we select Nanopore filters for BCFtools (software) detection of single nucleotide polymorphisms (SNPs). We compare transmission clusters identified by Nanopore and the United Kingdom Health Security Agency Illumina pipeline (COMPASS). We compare Illumina and Nanopore WGS-based DST predictions using Mykrobe (software). Nanopore/BCFtools identifies SNPs with median precision/recall of 99.5/90.2% compared with 99.6/91.9% for Illumina/COMPASS. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identifies 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduces this distribution with addition of 5 singleton isolates to distinct clusters and merging of two cluster pairs. Illumina-based clusters are also replicated using a 5 SNP threshold. Clustering accuracy is maintained using mixed Illumina/Nanopore datasets. Genotyping resistance variants is highly concordant, with 0(4) discordant SNPs (indels) across 151 isolates genotyped at >3000 (60,000) SNPs (indels). We conclude that Illumina and Nanopore sequence data provide comparable cluster-identification and DST results.