Mar 26, 2026
Creation and processing of different sample types for Whole Genome Sequencing of Vibrio cholerae
This protocol is a draft, published without a DOI.
- Kirabo Tess Ayazika1,
- Audrey Safir1,
- Nathaniel Matteson2,
- Jerome Ateudjieu3,
- Etienne Guenou3,
- Ketina Hirma Tchio-Nighie3,
- Emmanuel Tize3,
- Ismael Feudjio3,
- Armand Dopgang3,
- Shirlee Wohl2,
- Amanda Debes1
- 1Department of International Health, Johns Hopkins Bloomberg School of Public Health;
- 2Brigham and Women's Hospital, Harvard Medical School;
- 3Meilleur Acces aux Soins de Sante, Yaounde, Cameroon
Protocol Citation: Kirabo Tess Ayazika, Audrey Safir, Nathaniel Matteson, Jerome Ateudjieu, Etienne Guenou, Ketina Hirma Tchio-Nighie, Emmanuel Tize, Ismael Feudjio, Armand Dopgang, Shirlee Wohl, Amanda Debes 2026. Creation and processing of different sample types for Whole Genome Sequencing of Vibrio cholerae. protocols.io https://protocols.io/view/creation-and-processing-of-different-sample-types-jws7cpehp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 25, 2026
Last Modified: March 26, 2026
Protocol Integer ID: 313919
Keywords: cholera, whole genome sequencing, RDT, rectal swab, filter paper, culture, DNA extraction, alternate specimens, stool, surveillance, whole genome sequencing of vibrio cholerae, usable sequences in cholera surveillance, cholera surveillance, cholera outbreak, vibrio cholerae, gold standard sample method for whole genome sequencing, cholera epidemic, whole genome sequencing, laboratory procedures for the creation, laboratory procedure, laboratory setting, laboratory, laboratory resource, preparation of alternate sample type, laboratory resources for culture, controlled laboratory setting, gold standard sample method, effective ways of specimen, outbreak setting, laboratory at johns hopkins university
Funders Acknowledgements:
Gates Foundation
Grant ID: INV-047157
Abstract
This protocol describes laboratory procedures for the creation and preparation of alternate sample types for collection during a cholera outbreak for whole genome sequencing. The purpose of this protocol is to evaluate the implementation of low cost and easy-to-use methods of sample collection and preservation to generate usable sequences in cholera surveillance.
Whole stool and samples enriched by alkaline peptone water were collected in an outbreak setting according to standard operating procedures and transported to our laboratory at Johns Hopkins University. Additional samples were created from whole stool in a controlled laboratory setting. DNA is extracted from the matched samples in parallel according to the procedures described in this protocol.
This protocol was developed to address the high costs associated with the gold standard sample method for whole genome sequencing, which is isolation of V. cholerae through culture. Laboratory resources for culture and cold chain storage are limited in the countries that experience cholera epidemics. Evaluation of culture-independent methods of sample collection and transportation could provide more cost-effective ways of specimen handling.
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Protocol references
1. QIAamp Fast DNA Stool Mini Kit [Internet]. [cited 2026 Feb 18]. Available from: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaamp-fast-dna-stool-mini-kit
2. Manual for Detecting Vibrio cholerae O1 from Fecal Samples Using an Enriched Dipstick Assay – a Low-Cost, Simplified Method of Confirming Cholera.
3. Koskela KA, Matero P, Blatny JM, Fykse EM, Olsen JS, Nuotio LO, et al. A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae. Diagn Microbiol Infect Dis. 2009 Nov 1;65(3):339–44. doi:10.1016/j.diagmicrobio.2009.07.009
4. Qubit 4 Fluorometer - US [Internet]. [cited 2026 Feb 18]. Available from: https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/models/qubit-4.html
5. Illumina DNA Prep [Internet]. [cited 2026 Feb 18]. Available from: https://support-docs.illumina.com/LP/IlluminaDNAPrep/Content/LP/FrontPages/IlluminaDNAPrep.htm
6. Mboowa, G., Matteson, N.L., Tanui, C.K., Kasonde, M., Kamwiziku, G.K., Akanbi, O.A., Chitio, J.J.E., Kagoli, M., Essomba, R.G., Ayitewala, A. and Ssewanyana, I., 2026. Multicountry genomic analysis underscores regional cholera spread in Africa. Nature Communications.