DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples
<p><em>Mycobacterium tuberculosis</em> (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 p...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
American Society for Microbiology
2020
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| _version_ | 1797051230608949248 |
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| author | George, S Xu, Y Rodger, G Morgan, M Sanderson, N Hoosdally, SJ Thulborn, S Robinson, E Rathod, P Walker, AS Peto, TEA Crook, DW Dingle, K |
| author_facet | George, S Xu, Y Rodger, G Morgan, M Sanderson, N Hoosdally, SJ Thulborn, S Robinson, E Rathod, P Walker, AS Peto, TEA Crook, DW Dingle, K |
| author_sort | George, S |
| collection | OXFORD |
| description | <p><em>Mycobacterium tuberculosis</em> (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 0-10<sup>5</sup> 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 10<sup>1</sup> BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) occurred at 10<sup>4</sup> BCG cells/ml; >91% coverage (1x depth) at 10<sup>3</sup> 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.</p>
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| first_indexed | 2024-03-06T18:16:59Z |
| format | Journal article |
| id | oxford-uuid:04f2ff2f-f90b-48a9-90cc-aef982763810 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:16:59Z |
| publishDate | 2020 |
| publisher | American Society for Microbiology |
| record_format | dspace |
| spelling | oxford-uuid:04f2ff2f-f90b-48a9-90cc-aef9827638102022-03-26T08:54:33ZDNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samplesJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:04f2ff2f-f90b-48a9-90cc-aef982763810EnglishSymplectic ElementsAmerican Society for Microbiology2020George, SXu, YRodger, GMorgan, MSanderson, NHoosdally, SJThulborn, SRobinson, ERathod, PWalker, ASPeto, TEACrook, DWDingle, K<p><em>Mycobacterium tuberculosis</em> (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 0-10<sup>5</sup> 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 10<sup>1</sup> BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) occurred at 10<sup>4</sup> BCG cells/ml; >91% coverage (1x depth) at 10<sup>3</sup> 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.</p> |
| spellingShingle | George, S Xu, Y Rodger, G Morgan, M Sanderson, N Hoosdally, SJ Thulborn, S Robinson, E Rathod, P Walker, AS Peto, TEA Crook, DW Dingle, K DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title | DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title_full | DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title_fullStr | DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title_full_unstemmed | DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title_short | DNA thermo-protection facilitates whole genome sequencing of Mycobacteria direct from clinical samples |
| title_sort | dna thermo protection facilitates whole genome sequencing of mycobacteria direct from clinical samples |
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