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.

DOI: 10.1128/JCM.03073-14

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@inproceedings{Votintseva2015MycobacterialDE, title={Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures}, author={Antonina A. Votintseva and Louise J. Pankhurst and Luke W. Anson and Marcus R. Morgan and Deborah M. Gascoyne-Binzi and Timothy M. Walker and T Phuong Quan and David H. Wyllie and Carlos Del Ojo Elias and Mark H . Wilcox and A. Sarah Walker and Tim E. A. Peto and Derrick W. Crook}, booktitle={Journal of clinical microbiology}, year={2015} }