Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

<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>

Original publication




Journal article


Journal of Clinical Microbiology


American Society for Microbiology

Publication Date