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.

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…145,146 However, before wholegenome sequencing information can be used in routine clinical practice, several important issues need to be resolved. Whole-genome sequencing is complicated by the need for culture before DNA extraction, 147 incomplete knowledge of all resistance-conferring mutations, 59,148 and the need for a validated pipeline to accurately predict resistance. 149 A relational sequencing tuberculosis data platform (ReSeqTB) is being developed in a partnership between the Foundation for Innovative Diagnostics and Critical Path to TB Drug Regimens to catalogue genotypic and phenotypic data and to provide a validated pipeline for the analysis of whole-genome sequencing.…”

“…Strategies being investigated include selective removal of human DNA and enrichment of samples for M tuberculosis DNA. 147,268 However, selective whole-genome amplification approaches, which have been used across a range of other pathogens might provide a cost-effective alternative. 269 The use of whole-genome sequencing for the rapid drug susceptibility profiling of M tuberculosis for tuberculosis treatment management will be dependent on a routinely curated high-quality drug resistance database.…”

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis

“…Instead of performing WGS according to the standard procedure, i.e., from purified DNA obtained from M. tuberculosis subcultured in Lowenstein-Jensen medium, we purified DNA directly from the primary mycobacterial growth indicator tube (MGIT) liquid culture. We included a pretreatment step to minimize interference from human DNA (14). Briefly, the samples were sonicated, inactivated, centrifuged, and, after removing the supernatant, samples were resuspended in 1 ml of saline wash.…”

Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

“…Therefore, faster and reliable methods for DNA isolation would enable a considerable decrease in the time needed to perform WGS analyses. In this regard, a recent study proposed a WGS workflow starting from early positive liquid cultures of the MGIT system (15), potentially enhancing the speed of WGS procedures as part of routine diagnostics.…”

“…All were processed according to a standard lysis protocol with heat inactivation and sonication as is usually used for PCR (17,18). Lysates were concentrated with Microcon filters (Merck KGaA, Darmstadt, Germany), followed by a purification with ethanol (EtOH) precipitation and bead clean up (AMPure XP bead; Beckman Coulter, Krefeld, Germany) (15). For culture, aliquots of frozen stocks were put on solid Löwenstein-Jensen (LJ) slants and incubated at 35°C.…”