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Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

Antonina A. Votintseva, Phelim Bradley, Louise Pankhurst, Carlos del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, E. Grace Smith, Nicholas Sanderson, Timothy M. Walker, Marcus R. Morgan, David H. Wyllie, A. Sarah Walker, Tim E. A. Peto, Derrick W. Crook, View ORCID ProfileZamin Iqbal
doi: https://doi.org/10.1101/094789
Antonina A. Votintseva
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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  • For correspondence: a.votintseva@gmail.com zam@well.ox.ac.uk
Phelim Bradley
2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
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Louise Pankhurst
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Carlos del Ojo Elias
2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
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Matthew Loose
3School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH
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Kayzad Nilgiriwala
4Foundation for Medical Research, Mumbai
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Anirvan Chatterjee
4Foundation for Medical Research, Mumbai
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E. Grace Smith
5Regional Centre for Mycobacteriology, PHE Public Health Laboratory Birmingham. Heartlands Hospital, Birmingham B9 5SS, UK
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Nicholas Sanderson
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Timothy M. Walker
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Marcus R. Morgan
6Microbiology Laboratory, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, OX3 9DU, United Kingdom
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David H. Wyllie
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
7The Jenner Institute, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom
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A. Sarah Walker
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
8National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Tim E. A. Peto
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
8National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Derrick W. Crook
1Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
8National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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Zamin Iqbal
2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
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  • ORCID record for Zamin Iqbal
  • For correspondence: a.votintseva@gmail.com zam@well.ox.ac.uk
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Abstract

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.

We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of £96/£198 per sample.

We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted December 19, 2016.
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Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples
Antonina A. Votintseva, Phelim Bradley, Louise Pankhurst, Carlos del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, E. Grace Smith, Nicholas Sanderson, Timothy M. Walker, Marcus R. Morgan, David H. Wyllie, A. Sarah Walker, Tim E. A. Peto, Derrick W. Crook, Zamin Iqbal
bioRxiv 094789; doi: https://doi.org/10.1101/094789
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Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples
Antonina A. Votintseva, Phelim Bradley, Louise Pankhurst, Carlos del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, E. Grace Smith, Nicholas Sanderson, Timothy M. Walker, Marcus R. Morgan, David H. Wyllie, A. Sarah Walker, Tim E. A. Peto, Derrick W. Crook, Zamin Iqbal
bioRxiv 094789; doi: https://doi.org/10.1101/094789

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