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

Abstract

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 sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M 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 experiments employed Mycobacteria DNA, either 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 achieved MTB detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) occurred at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealing initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 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.

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

“…A wide range of sequencing kits and library preparation approaches are available for ONT sequencing, each recommended for specific applications, and requiring various quantities of input material. For example, native DNA or RNA sequences can be detected in workflows without PCR amplification but require 400 to 1000 ng of input DNA ( 11 , 12 ). Amplification-based approaches are recommended by ONT when input DNA is limited in quantity or quality, when control over the read length is required, and for targeted amplicon sequencing.…”

“…We identified 58 articles of which 12 were eligible. Five articles focused on whole-genome sequencing (WGS) ( 8 , 12 , 21 – 23 ), three on targeted sequencing ( Table 1 ) ( 11 , 24 , 25 ), and four on software development for long-read ONT sequencing data with applications for M. tuberculosis ( Table 2 ) ( 16 , 18 , 19 , 26 ).…”

“…The first study of culture-free ONT sequencing of bronchioalveolar lavage and lymph node aspirate specimens was also published in 2020 ( 12 ). George et al ( 12 ) achieved a mean genome coverage for M. tuberculosis clinical specimens ranging from 0.55× to 81×.…”

Does Mycobacterium tuberculosis var. bovis Survival in the Environment Confound Bovine Tuberculosis Control and Eradication? A Literature Review

“…Despite the well-documented, relatively low read accuracy of the MinION, the outputs, in terms of genera and species detected, were remarkably similar, with MinION reporting greater taxonomic resolution [ 152 ]. In addition, proof of concept ultradeep microbiome preparation protocols have been described which facilitate the direct genome sequencing of pathogens from clinical samples [ 153 ], and refinements and adaptations continue to be made [ 154 ]. As above, while the protocols make sense to a molecular microbiologist, they are not trivial and have not been adapted for application directly to the environmental samples that we might include in our proposed studies.…”

Targeted next-generation sequencing: a Swiss army knife for mycobacterial diagnostics?

“…Theoretically, the most flexible solution to mycobacterial diagnostics would be whole genome sequencing (WGS) directly from the clinical sample. Yet, despite recent advances in next-generation sequencing (NGS) and sample preparation, this remains unreliable and prohibitively expensive for routine use in the short term [20]. Even if direct NGS were cost-effective for samples with mycobacteria, it would unlikely be suitable for detection in the foreseeable future, particularly in settings with low rates of positivity [5].…”