Journal
JOURNAL OF CLINICAL MICROBIOLOGY
Volume 53, Issue 4, Pages 1137-1143Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JCM.03073-14
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Funding
- Health Innovation Challenge Fund grants [G0800778, 087646/Z/08/Z, HICF-T5-358]
- National Institute for Health Research (NIHR) Oxford Biomedical Research Centre
- Wellcome Trust
- Medical Research Council, Biotechnology and Biological Sciences Research Council on behalf of the Department of Health
- MRC [MR/J011398/1, G0800778] Funding Source: UKRI
- Medical Research Council [G0800778, MR/J011398/1] Funding Source: researchfish
- National Institute for Health Research [NF-SI-0512-10047, NF-SI-0513-10110, NF-SI-0508-10279] Funding Source: researchfish
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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/mu l 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.
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