P033

1:The University of Oxford; 2:Oxford University Hospitals NHS Foundation Trust; 3:National Institute of Communicable Disease, Johannesburg, South Africa

Next-generation sequencing using Illumina has emerged as the standard for Mycobacterial genomic analysis. Mechanical DNA extraction methods have often yielded inadequate concentrations for Oxford Nanopore Technologies (ONT) sequencing kits. We report substantial improvements to the workflow using simplified, automation-aided extraction and a modified Rapid PCR Barcoding protocol, resulting in an ONT-compatible process that enables faster, more cost-effective, high-throughput sequencing. 

178 samples collected by a routine NHS laboratory were cultured in mycobacterial growth indicator tubes, then heat-inactivated in a convection oven before removal from containment level 3. Mycobacterial cells were mechanically lysed, and DNA purified through an automated platform. We report on the initial 82 samples sequenced by both Illumina and ONT. One sample failed to amplify through the modified ONT protocol.  

All samples had human reads removed prior to processing by an online cloud analysis platform. 80/81 samples were identified as mycobacterial species by both sequencing platforms. One sample (from tissue) failed due to mostly human reads. We identified 17 M. tuberculosis and 63 non-tuberculosis mycobacteria; 67/80 were concordant for species and lineage across both sequencing platforms. Eight subspecies differences were observed between platforms, five of which were M. intracellulare. Two samples also showed mixed species discordance. Results for all 178 samples will be reported. Clear differences in nucleotide substitutions and indels were observed for the 17 M. tuberculosis isolates, which will be further reported in a separate abstract of 400 isolates. These results highlight the need for further robust comparison of ONT and Illumina sequencing.