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Optimizing fungal DNA extraction and purification for Oxford Nanopore untargeted shotgun metagenomic sequencing from simulated hemoculture specimens
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Metadata
Document Title
Optimizing fungal DNA extraction and purification for Oxford Nanopore untargeted shotgun metagenomic sequencing from simulated hemoculture specimens
Author
Langsiri N.
Name from Authors Collection
Affiliations
Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Clinical School, Sydney Medical School, Faculty of Medicine and Health, Sydney Infectious Diseases Institute, University of Sydney, Westmead Hospital, Research and Education Network, Westmead, NSW, Australia; Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand; Research Unit of Medical Mycology Diagnosis, Chulalongkorn University, Bangkok, Thailand; Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; Department of Microbiology and Plant Pathology, University of California, Riverside, CA, United States; Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States; Division of Medical Bioinformatics, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand; Center of Excellence in Antimicrobial Resistance and Stewardship, Chulalongkorn University, Bangkok, Thailand
Type
Article
Source Title
mSystems
ISSN
23795077
Year
2025
Volume
10
Issue
6
Open Access
All Open Access; Gold Open Access; Green Open Access
Publisher
American Society for Microbiology
DOI
10.1128/msystems.01166-24
Abstract
Long-read metagenomics provides a promising alternative approach to fungal identification, circumventing methodological biases, associated with DNA amplification, which is a prerequisite for DNA barcoding/metabarcoding based on the primary fungal DNA barcode (Internal Transcribed Spacer (ITS) region). However, DNA extraction for long-read sequencing-based fungal identification poses a significant challenge, as obtaining long and intact fungal DNA is imperative. Comparing different lysis methods showed that chemical lysis with CTAB/SDS generated DNA from pure fungal cultures with high yields (ranging from 11.20 ± 0.17 µg to 22.99 ± 2.22 µg depending on the species) while preserving integrity. Evaluating the efficacy of human DNA depletion protocols demonstrated an 88.73% reduction in human reads and a 99.53% increase in fungal reads compared to the untreated yeast-spiked human blood control. Evaluation of the developed DNA extraction protocol on simulated clinical hemocultures revealed that the obtained DNA sequences exceed 10 kb in length, enabling a highly efficient sequencing run with over 80% active pores. The quality of the DNA, as indicated by the 260/280 and 260/230 ratios obtained from NanoDrop spectrophotometer readings, exceeded 1.8 and 2.0, respectively. This demonstrated the great potential of the herein optimized protocol to extract high-quality fungal DNA from clinical specimens enabling long-read metagenomics sequencing. Copyright © 2025 Langsiri et al.
License
CC BY
Rights
Authors
Publication Source
Scopus