Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.
J Clin Microbiol
; 53(4): 1137-43, 2015 Apr.
Article
en En
| MEDLINE
| ID: mdl-25631807
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Tuberculosis
/
ADN Bacteriano
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Técnicas Bacteriológicas
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Genoma Bacteriano
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Mycobacterium tuberculosis
Tipo de estudio:
Guideline
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Clin Microbiol
Año:
2015
Tipo del documento:
Article
País de afiliación:
Reino Unido