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

Introduction: Metagenomics, the genomic analysis of all species present within a mixed population, is an important tool used for the exploration of microbiomes in clinical and environmental microbiology. Whilst the development of next-generation sequencing, and more recently third generation long-read approaches such as nanopore sequencing, have greatly advanced the study of metagenomics, recovery of unbiased material from microbial populations remains challenging. One promising advancement in genomic sequencing from Oxford Nanopore Technologies (ONT) is adaptive sampling, which enables real-time enrichment or depletion of target sequences. As sequencing technologies continue to develop, and advances such as adaptive sampling become common techniques within the microbiological toolkit, it is essential to evaluate the benefits of such advancements to metagenomic studies, and the impact of methodological choices on research outcomes. Aim and methods: Given the rapid development of sequencing tools and chemistry, this study aimed to demonstrate the impacts of choice of DNA extraction kit and sequencing chemistry on downstream metagenomic analyses. We first explored the quality and accuracy of 16S rRNA amplicon sequencing for DNA extracted from the ZymoBIOMICS Microbial Community Standard, using a range of commercially available DNA extraction kits to understand the effects of different kit biases on assessment of microbiome composition. We next compared the quality and accuracy of metagenomic analyses for two nanopore-based ligation chemistry kits with differing levels of base-calling error; the older and more error-prone (~ 97% accuracy) LSK109 chemistry, and newer more accurate (~ 99% accuracy) LSK112 Q20 + chemistry. Finally, we assessed the impact of the nanopore sequencing chemistry version on the output of the novel adaptive sampling approach for real-time enrichment of the genome for the yeast Saccharomyces cerevisiae from the microbial community. Results: Firstly, DNA extraction kit methodology impacted the composition of the yield, with mechanical bead-beating methodologies providing the least biased picture due to efficient lysis of Gram-positive microbes present in the community standard, with differences in bead-beating methodologies also producing variation in composition. Secondly, whilst use of the Q20 + nanopore sequencing kit chemistry improved the base-calling data quality, the resulting metagenomic assemblies were not significantly improved based on common metrics and assembly statistics. Most importantly, we demonstrated the effective application of adaptive sampling for enriching a low-abundance genome within a metagenomic sample. This resulted in a 5-7-fold increase in target enrichment compared to non-adaptive sequencing, despite a reduction in overall sequencing throughput due to strand-rejection processes. Interestingly, no significant differences in adaptive sampling enrichment efficiency were observed between the older and newer ONT sequencing chemistries, suggesting that adaptive sampling performs consistently across different library preparation kits. Conclusion: 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.