A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
Authors
Raven, Kathy E.
Girgis, Sophia T.
Akram, Asha
Blane, Beth
Leek, Danielle
Brown, Nicholas
Peacock, Sharon J.
Publication Date
2021-01-08Journal Title
Scientific Reports
Publisher
Nature Publishing Group UK
Volume
11
Issue
1
Language
en
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Raven, K. E., Girgis, S. T., Akram, A., Blane, B., Leek, D., Brown, N., & Peacock, S. J. (2021). A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing. Scientific Reports, 11 (1) https://doi.org/10.1038/s41598-020-80031-8
Abstract
Abstract: Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.
Keywords
Article, /631/208/325/2482, /692/699/255/1318, /692/700/478, /631/326, /631/337, article
Sponsorship
Health Innovation Challenge Fund (WT098600, HICF-T5-342)
Identifiers
s41598-020-80031-8, 80031
External DOI: https://doi.org/10.1038/s41598-020-80031-8
This record's URL: https://www.repository.cam.ac.uk/handle/1810/315931
Rights
Attribution 4.0 International (CC BY 4.0)
Licence URL: https://creativecommons.org/licenses/by/4.0/
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