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

• Published in: Journal of clinical microbiology 2020-09, Vol.58 (10)
• Main Authors: George, Sophie, Xu, Yifei, Rodger, Gillian, Morgan, Marcus, Sanderson, Nicholas D, Hoosdally, Sarah J, Thulborn, Samantha, Robinson, Esther, Rathod, Priti, Walker, A Sarah, Peto, Timothy E A, Crook, Derrick W, Dingle, Kate E
• Format: Article
• Language: English
• Subjects: Mycobacteriology and Aerobic Actinomycetes
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.00670-20

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 -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 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 detection at 10 BCG cells/ml, with 31 to 59 complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 10 BCG cells/ml; >91% coverage (1× depth) occurred at 10 BCG cells/ml. Final validation employed -positive clinical samples (  = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of genomes was facilitated by a low-cost thermo-protection buffer.