DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples

<jats:title>ABSTRACT</jats:title> <jats:p><jats:named-content content-type="genus-species">Mycobacterium tuberculosis</jats:named-content> is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content>-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 10<jats:sup>5</jats:sup> <jats:named-content content-type="genus-species">Mycobacterium bovis</jats:named-content> BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> detection at 10<jats:sup>1</jats:sup> BCG cells/ml, with 31 to 59 <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> complex reads. Maximal genome coverage (&gt;97% at 5× depth) occurred at 10<jats:sup>4</jats:sup> BCG cells/ml; &gt;91% coverage (1× depth) occurred at 10<jats:sup>3</jats:sup> BCG cells/ml. Final validation employed <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content>-positive clinical samples (<jats:italic>n</jats:italic> = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave &gt;96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved &gt;99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> genomes was facilitated by a low-cost thermo-protection buffer.</jats:p>