title: Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.
abstract: We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom votintse@stats.ox.ac.uk.
authors: Antonina A
authors: AA
authors: Votintseva
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Louise J
authors: LJ
authors: Pankhurst
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Luke W
authors: LW
authors: Anson
authors: Microbiology Laboratory, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
authors: Marcus R
authors: MR
authors: Morgan
authors: Department of Microbiology, Leeds General Infirmary, Leeds, United Kingdom.
authors: Deborah
authors: D
authors: Gascoyne-Binzi
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Timothy M
authors: TM
authors: Walker
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors: T Phuong
authors: TP
authors: Quan
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom The Jenner Institute, University of Oxford, Oxford, United Kingdom.
authors: David H
authors: DH
authors: Wyllie
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Carlos
authors: C
authors: Del Ojo Elias
authors: Department of Microbiology, Leeds General Infirmary, Leeds, United Kingdom.
authors: Mark
authors: M
authors: Wilcox
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom National Institute for Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
authors: A Sarah
authors: AS
authors: Walker
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom National Institute for Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Tim E A
authors: TE
authors: Peto
authors: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom National Institute for Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
authors: Derrick W
authors: DW
authors: Crook
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D016680:Genome, Bacterial / Q000235:genetics*; D006801:Humans; D058889:Molecular Typing / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial
references: Lancet Infect Dis. 2013 Feb;13(2):137-46
references: 23158499
references: J Clin Microbiol. 2013 Apr;51(4):1272-7
references: 23345298
references: Mol Diagn Ther. 2009;13(3):137-51
references: 19650669
references: J Clin Microbiol. 2014 Apr;52(4):1052-9
references: 24430455
references: PLoS One. 2013;8(1):e54587
references: 23349933
references: PLoS Med. 2013;10(2):e1001387
references: 23424287
references: Tuberculosis (Edinb). 2014 Jul;94(4):434-40
references: 24888866
references: J Clin Microbiol. 2014 Jan;52(1):139-46
references: 24172157
references: J Clin Microbiol. 2014 May;52(5):1501-10
references: 24574290
references: Trends Genet. 2014 Sep;30(9):401-7
references: 25096945
references: Clin Microbiol Infect. 2013 Sep;19(9):796-802
references: 23432709
references: PLoS One. 2014;9(6):e99129
references: 24922084
references: BMC Infect Dis. 2013;13:352
references: 23895665
references: PeerJ. 2014 Sep 23;2:e585
references: 25279265
references: PLoS One. 2014;9(2):e88626
references: 24586360
references: Lancet Respir Med. 2014 Apr;2(4):285-92
references: 24717625
references: Clin Chem. 2005 Jan;51(1):113-8
references: 15539466
references: BMC Infect Dis. 2005;5:62
references: 16050959
references: PLoS Med. 2012;9(11):e1001347
references: 23185139
references: Clin Microbiol Rev. 2003 Apr;16(2):319-54
references: 12692101
references: J Clin Microbiol. 2014 Dec;52(12):4305-8
references: 25297335