Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance

• Published in: Scientific reports Vol. 13; no. 1; p. 19656
• Main Authors: Bloemen, Bram, Gand, Mathieu, Vanneste, Kevin, Marchal, Kathleen, Roosens, Nancy H. C., De Keersmaecker, Sigrid C. J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11-11-2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/514/2254; 631/326/171; 631/326/1762; 631/326/22; 631/326/2521; 631/326/2522; 631/326/2565/2134; 631/326/2565/2142; 631/326/325; 631/326/41; 631/326/421; 631/61/212; Antimicrobial agents; Antimicrobial resistance; Deoxyribonucleic acid; DNA; DNA sequencing; Drug resistance; Humanities and Social Sciences; Laboratories; Lysis; Metagenomics; multidisciplinary; Onsite; Pathogens; Sample preparation; Science; Science (multidisciplinary); Surveillance
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-46771-z

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings.