DNA extraction, target amplification and next-generation sequencing

DNA extraction and tNGS were performed as described in the Deeplex-MycTB kit (GenoScreen). Selected gene targets were amplified using ultra-deep sequencing of a single, 24-plexed amplicon mix. Amplicons were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using Qubit dsDNA BR assay (Life Technologies, Paisley, UK). Paired-end libraries (150 base pair [bp]) were prepared using Nextera XT DNA Sample Preparation kits (Illumina Inc, San Diego, CA, USA) and sequenced on an Illumina MiSeq platform. Resistance information was extrapolated from sequence data using a cloud-based analytical platform provided by GenoScreen,⁷ which was informed by published reference databases of genetic variants associated with drug resistance.^10–14 Samples that did not present a clear resistance pattern required further review by a TB sequencing data specialist during post-sequencing data analysis. Uncharacterised mutations with resistant phenotypes were updated according to recent literature or evidence supporting clinical relevance. No patient-related information was linked with the FASTQ (Welcome Trust Sanger Institute, Hinxton, UK) sequence files uploaded or shared in the analysis process.