title: Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.
abstract: The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Brown
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom j.bryant@ucl.ac.uk.
authors.forename: Josephine M
authors.identifier: http://orcid.org/0000-0002-0149-5768
authors.initials: JM
authors.lastname: Bryant
authors.affiliation: Qiagen-AAR, Aarhus, Denmark.
authors.forename: Katja
authors.initials: K
authors.lastname: Einer-Jensen
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Jolyon
authors.initials: J
authors.lastname: Holdstock
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Darren T
authors.initials: DT
authors.lastname: Houniet
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Jacqueline Z M
authors.initials: JZ
authors.lastname: Chan
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Depledge
authors.affiliation: National Mycobacterium Reference Laboratory (NMRL), ICMS, London, United Kingdom.
authors.forename: Vladyslav
authors.initials: V
authors.lastname: Nikolayevskyy
authors.affiliation: National Mycobacterium Reference Laboratory (NMRL), ICMS, London, United Kingdom.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Broda
authors.affiliation: Dept. Microbiology, Frimley Health NHS Foundation Trust, Wexham Park Hospital, Berkshire, United Kingdom.
authors.forename: Madeline J
authors.initials: MJ
authors.lastname: Stone
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Mette T
authors.initials: MT
authors.lastname: Christiansen
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Rachel
authors.initials: R
authors.lastname: Williams
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Michael B
authors.initials: MB
authors.lastname: McAndrew
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Helena
authors.initials: H
authors.lastname: Tutill
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Julianne
authors.initials: J
authors.lastname: Brown
authors.affiliation: Barts Health NHS Trust, West Smithfield, London, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Melzer
authors.affiliation: Barts Health NHS Trust, West Smithfield, London, United Kingdom.
authors.forename: Caryn
authors.initials: C
authors.lastname: Rosmarin
authors.affiliation: Centre for Clinical Microbiology, UCL, Royal Free Campus, London, United Kingdom.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: McHugh
authors.affiliation: Centre for Clinical Microbiology, UCL, Royal Free Campus, London, United Kingdom Specialist Microbiology Network, Public Health Laboratory Manchester, Manchester Royal Infirmary, Manchester, United Kingdom.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Shorten
authors.affiliation: National Mycobacterium Reference Laboratory (NMRL), ICMS, London, United Kingdom.
authors.forename: Francis
authors.initials: F
authors.lastname: Drobniewski
authors.affiliation: Oxford Gene Technology, Oxford, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: Speight
authors.affiliation: UCL, Division of Infection and Immunity, London, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Breuer
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008097:Lithuania; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology*; D006113:United Kingdom