DNA thermo-protection facilitates whole-genome sequencing of mycobacteria direct from clinical samples

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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. tuberculosispositive 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 105 Mycobacterium 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.

Original languageEnglish
Article numbere0067020
JournalJournal of Clinical Microbiology
Volume58
Issue number10
DOIs
Publication statusPublished - Oct 2020
Externally publishedYes

Bibliographical note

Funding Information:
The study was funded by the NIHR Oxford Biomedical Research Centre. Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre.

Funding Information:
The study was funded by the NIHR Oxford Biomedical Research Centre. Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, the Department of Health, or Public Health England.

Publisher Copyright:
Copyright © 2020 George et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Keywords

  • Clinical diagnostics
  • Direct-from-sample sequencing
  • DNA sequencing
  • Mycobacteria
  • Mycobacterium tuberculosis
  • Nanopore DNA sequencing

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