Optimizing fungal DNA extraction and purification for Oxford Nanopore untargeted shotgun metagenomic sequencing from simulated hemoculture specimens
A novel streamlined DNA extraction protocol was developed to efficiently isolate high molecular weight fungal DNA from hemoculture samples, which is crucial for long-read sequencing applications. By eliminating the need for labor-intensive and shear-force-inducing steps, such as liquid nitrogen grinding or bead beating, the protocol is more user-friendly and better suited for clinical laboratory settings. The automation of cleanup and extraction steps further shortens the overall turnaround time to under 6 hours. Although not specifically designed for ultra-long DNA extraction, this protocol effectively supports fungal identification through Oxford Nanopore Technology (ONT) sequencing. It yields high molecular weight DNA, resulting in longer sequence fragments that improve the number of fungal reads over human reads. Future improvements, including adaptive sampling technology, could further simplify the process by reducing the need for human DNA depletion, paving the way for more automated, bioinformatics-driven workflows.
Authors
- Piroon Jenjaroenpun (ORCID: https://orcid.org/0000-0002-1555-401X)
- Navaporn Worasilchai (ORCID: https://orcid.org/0000-0001-5055-6830)
- Nuttapon Pombubpa (ORCID: https://orcid.org/0000-0003-3385-5331)
- Thidathip Wongsurawat (ORCID: https://orcid.org/0000-0002-3659-2074)
- Wieland Meyer (ORCID: https://orcid.org/0000-0001-9933-8340)
- Ariya Chindamporn (ORCID: https://orcid.org/0000-0002-9761-1670)
- László Irinyi (ORCID: https://orcid.org/0000-0002-4453-5539)
- Nattapong Langsiri (ORCID: https://orcid.org/0000-0002-1889-7122)
- Janet Jennifer Luangsa-ard (ORCID: https://orcid.org/0000-0001-6801-2145)
Institutions
- The University of Sydney (AU)
- National Science and Technology Development Agency (TH)
- Siriraj Hospital (TH)
- Chulalongkorn University (TH)
- Mahidol University (TH)
- Westmead Hospital (AU)
- Westerdijk Fungal Biodiversity Institute (NL)
- Infectious Diseases Institute (UG)
- Plant (United States) (US)
- University of Arkansas for Medical Sciences (US)
- National Center for Genetic Engineering and Biotechnology (TH)
Publication Details
- Journal
- mSystems
- Published
- 2025-04-08
- DOI
- https://doi.org/10.1128/msystems.01166-24
- Citations
- 6
- Primary Topic
- Plant Pathogens and Fungal Diseases
- Type
- article
- Field-Weighted Citation Impact
- 6.91
Funders
- Chulalongkorn University
- National Research Council of Thailand
- Faculty of Medicine, Chulalongkorn University