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

Sophie George,Samantha Thulborn,Gillian Rodger,Derrick W Crook,Esther Robinson,Marcus Morgan,A Sarah Walker,Yifei Xu,Nicholas D Sanderson,Priti Rathod,Kate E Dingle,Sarah J Hoosdally,Timothy E A Peto,Geoffrey A Land

doi:10.1128/jcm.00670-20

Abstract

Mycobacterium tuberculosis 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 M. tuberculosis-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 105Mycobacterium bovis 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 M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis 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 M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

Highlights

Sophie George and Yifei Xu contributed to this work
The application of DNA sequencing to mycobacteria molecular diagnostics yields clinically and epidemiologically valuable information
A model system comprising standardized mock clinical samples was established by pooling infection-negative human sputum samples and spiking with enumerated Mycobacterium bovis BCG Pasteur strain at known concentrations

Summary

Introduction

Sophie George and Yifei Xu contributed to this work. Author order was decided on the basis of amount of time spent performing wet-lab experiments versus time spent developing bioinformatics analysis. Mock clinical samples (infection-negative human sputum spiked with 0 to 105 Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. Mycobacterium tuberculosis is the leading bacterial cause of death from infection, with the World Health Organization (WHO) estimating that 10 million new tuberculosis (TB) cases and 1.2 million deaths occurred worldwide in 2018 [1]. The utility increases with the proportion of genome obtained, from detection to speciation and antimicrobial resistance prediction to phylogenetic and evolutionary insights. This allows whole-genome sequencing (WGS) to outperform other rapid molecular methods (such as GeneXpert MTB/RIF; Cepheid, Solna, Sweden) [6,7,8,9]

Objectives

Methods

Results

Conclusion