title: The variability and reproducibility of whole genome sequencing technology for detecting resistance to anti-tuberculous drugs.
abstract: The emergence of resistance to anti-tuberculosis drugs is a serious and growing threat to public health. Next-generation sequencing is rapidly gaining traction as a diagnostic tool for investigating drug resistance in Mycobacterium tuberculosis to aid treatment decisions. However, there are few little data regarding the precision of such sequencing for assigning resistance profiles.
  
  
  
  We investigated two sequencing platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (TBProfiler, Mykrobe predictor) using a well characterised reference strain (H37Rv) and clinical isolates from patients with tuberculosis resistant to up to 13 drugs. Results were compared to phenotypic drug susceptibility testing. To assess analytical robustness individual DNA samples were subjected to repeated sequencing.
  
  
  
  The MiSeq and Ion PGM systems accurately predicted drug-resistance profiles and there was high reproducibility between biological and technical sample replicates. Estimated variant error rates were low (MiSeq 1 per 77 kbp, Ion PGM 1 per 41 kbp) and genomic coverage high (MiSeq 51-fold, Ion PGM 53-fold). MiSeq provided superior coverage in GC-rich regions, which translated into incremental detection of putative genotypic drug-specific resistance, including for resistance to para-aminosalicylic acid and pyrazinamide. The TBProfiler bioinformatics pipeline was concordant with reported phenotypic susceptibility for all drugs tested except pyrazinamide and para-aminosalicylic acid, with an overall concordance of 95.3%. When using the Mykrobe predictor concordance with phenotypic testing was 73.6%.
  
  
  
  We have demonstrated high comparative reproducibility of two sequencing platforms, and high predictive ability of the TBProfiler mutation library and analytical pipeline, when profiling resistance to first- and second-line anti-tuberculosis drugs. However, platform-specific variability in coverage of some genome regions may have implications for predicting resistance to specific drugs. These findings may have implications for future clinical practice and thus deserve further scrutiny, set within larger studies and using updated mutation libraries.
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT, London, UK.
authors.forename: Jody
authors.initials: J
authors.lastname: Phelan
authors.affiliation: Molecular Biology, LGC Ltd, Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Denise M
authors.initials: DM
authors.lastname: O'Sullivan
authors.affiliation: Unidade de Microbiologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, UNL, Lisbon, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Machado
authors.affiliation: Unidade de Microbiologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, UNL, Lisbon, Portugal.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramos
authors.affiliation: Molecular Biology, LGC Ltd, Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
authors.forename: Justin
authors.initials: J
authors.lastname: O'Grady
authors.affiliation: Division of Pulmonary Medicine and UCT Lung Institute, Lung Infection and Immunity Unit, University of Cape Town, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa.
authors.forename: Keertan
authors.initials: K
authors.lastname: Dheda
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT, London, UK.
authors.forename: Susana
authors.initials: S
authors.lastname: Campino
authors.affiliation: Division of Pulmonary Medicine and UCT Lung Institute, Lung Infection and Immunity Unit, University of Cape Town, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa.
authors.forename: Ruth
authors.initials: R
authors.lastname: McNerney
authors.affiliation: Unidade de Microbiologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, UNL, Lisbon, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
authors.affiliation: Molecular Biology, LGC Ltd, Queens Road, Teddington, Middlesex, TW11 0LY, UK.|School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford, GU2 7XH, UK.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT, London, UK. taane.clark@lshtm.ac.uk.|Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, WC1E 7HT, London, UK. taane.clark@lshtm.ac.uk.
authors.forename: Taane G
authors.initials: TG
authors.lastname: Clark
mesh_terms: D000995:Antitubercular Agents; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000995:Antitubercular Agents
keywords: Diagnostics; Drug resistance; Drug-susceptibility testing; Next-generation sequencing; Tuberculosis; XDR-TB