DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples by the Nanopore platform

Mycobacterium tuberculosis (MTB) 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 Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture).

Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30 min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing.

Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-10⁵ 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 each demonstrated detection at 10¹ BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 10⁴ BCG cells/ml; >91% coverage (1x depth) at 10³ BCG cells/ml.

Final validation employed MTB positive clinical samples (n=20), and revealed initial sample volumes ≥1 ml typically yielded higher mean depth of MTB genome coverage, the overall range being 0.55-81.02. A mean depth of 3x gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15x achieved >99% five-fold genome coverage (in 9/20 clinical samples).

In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.