Impact of microbiological molecular methodologies on adaptive sampling using nanopore sequencing in metagenomic studies

Our findings underscore the importance of selecting a DNA extraction methodology that minimises bias to ensure an accurate representation of microbial diversity in metagenomic studies. Additionally, despite the improved base-calling accuracy provided by newer Q20 + sequencing chemistry, we demonstrate that even older ONT sequencing chemistries can achieve reliable metagenomic sequencing results, enabling researchers to confidently use these approaches depending on their specific experimental needs. Critically, we highlight the significant potential of ONT's adaptive sampling technology for targeted enrichment of specific genomes within metagenomic samples. This approach offers broad applicability for enriching target organisms or genetic elements (e.g., pathogens or plasmids) or depleting unwanted DNA (e.g., host DNA) in diverse sample types from environmental and clinical studies. However, researchers should carefully weigh the benefits of adaptive sampling against the potential trade-offs in sequencing throughput, particularly for low-abundance targets, where strand rejection can lead to pore blocking. These results provide valuable guidance for optimising adaptive sampling in metagenomic workflows to achieve specific research objectives.