Unlocking the abundance of information within environmental microbiomes

Microbiomes are broad communities of microbial species, including bacteria, archaea, fungi, and viruses, that live together in a specific habitat. Environmental microbiomes — present across ecosystems, including in the air, soil, and water — play essential ecological roles, such as nitrogen fixation, mineral recycling, and the production of vitamins and secondary metabolites¹. Understanding these hugely diverse and complex communities on a molecular level offers insight into how ecosystems function and respond to environmental change. This, in turn, means that environmental microbiomes can be used as indicators to track the impacts of climate change, as well as in pathogen surveillance.

Until recently, efforts to study microbiomes were limited by biased or incomplete methods, such as culture-based analysis or targeted 16S ribosomal RNA (rRNA) gene sequencing, which capture only a fraction of microbial diversity and provide limited genomic context. Even when metagenomic approaches using short-read sequencing became available, their restricted read lengths struggled to deconvolute or resolve complex and repetitive regions of microbial genomes, leading to heavy reliance on computational prediction of metagenome-assembled-genomes (MAGs) and often resulting in gaps.

Oxford Nanopore metagenomic sequencing overcomes these limitations by generating reads of any length, without the need for amplification or primers, for comprehensive characterisation of whole genomes within microbial communities. Using this technology to study all the genetic material in mixed microbial samples, researchers are now gaining a deeper understanding of environmental microbiomes than was previously possible.

Discovering bioactive molecules in the ‘dark matter’ of soil microbiomes

Soil microbiomes contain an immense diversity of microorganisms, with some capable of producing bioactive molecules that could form the basis of new antibiotics. In the fight against multidrug-resistant pathogens, finding those with new modes of action could be key. With this aim, Burian et al. investigated soil samples to search for these important microbes².

The team used a high-throughput PromethION Flow Cell to sequence high molecular-weight DNA extracted from a forest soil sample. The data enabled them to generate megabase-scale assemblies, uncovering hundreds of circular, complete and near-complete microbial genomes from a single sample. From these assemblies, the researchers identified non-ribosomal peptide biosynthetic gene clusters (BGCs), and chemically synthesised their encoded products. In contrast, the authors explained that the fragmented MAGs previously generated using short-read sequencing precluded complete BGC analysis.

Within the identified BGCs, the group discovered products with rare modes of antibiotic action against multidrug-resistant pathogens. Two were significantly potent: one demonstrated broad-spectrum antibiotic activity and lacked cytotoxicity, whilst the other demonstrated potent activity against Staphylococcus aureus. The authors emphasised how their method ‘advances metagenomic access to the vast genetic diversity of the uncultured bacterial majority and provides a means to convert it to bioactive molecules’.

The potential of air microbiome monitoring

As well as below the ground, wide microbial diversity is also present in the air. In a study by Reska et al., the authors noted that though air microbiomes play significant roles in human health and ecosystem resilience, they remain understudied³. Citing the benefits of Oxford Nanopore sequencing for metagenomic assembly, combined with the portability of the MinION device and fast workflows, the team deployed this technology to investigate these neglected microbiomes³.

To test their workflow, the researchers first sampled air from greenhouse and natural outdoor environments, extracted DNA, and performed multiplexed nanopore sequencing. They then assembled and annotated the microbial genomes present. This revealed detailed information about the microbiomes, including ecosystem functions and the presence of antimicrobial resistance and virulence genes.

Having validated their approach, the group applied the method to a pilot study in Barcelona, Spain. From air samples taken across 19 days from five sites, their metagenomic Oxford Nanopore sequencing data revealed ‘surprisingly stable location-specific signatures of microbial composition and diversity’ (Figure 1). A core urban microbiome was detected across all samples, whilst the only natural environment sampled featured several unique microbial taxa with known associations commonly found in the soil, forests, and green spaces.

Figure 1. Metagenomic sequencing of air samples from five areas across Barcelona, Spain, revealed stable relative abundances of the top 20 bacterial genera in each location (top), with principal component analysis showing site-specific clustering (bottom). Figure adapted from Reska et al.³ and available under Creative Commons license (creativecommons.org/licenses/by/4.0/)

Analysing the air to safeguard public health

A deeper dive into the data revealed the power of environmental metagenomic sequencing for pathogen surveillance. The researchers used the data to annotate antimicrobial resistance genes, which showed the vancomycin resistance gene VanR-O to be most common, whilst the highest resistance gene density was present at their urban beach sampling location.

Generating highly contiguous MAGs from the data also allowed the researchers to confidently identify pathogenic microorganisms. In the urban air samples, this included the difficult-to-treat emerging pathogen Stenotrophomonas maltophilia, and Salmonella enterica, which can transmit to humans via zoonosis or contaminated food. Interestingly, the authors hypothesised that the nanopore read length distributions could be used to indicate viability, as more fragmented material could have originated from non-viable microorganisms.

With new antimicrobial candidates identified from a single soil sample, and ecosystem and pathogen surveillance insights generated from three hours of air sampling, these studies showcase the power of Oxford Nanopore metagenomics to reveal what was previously hidden within the microbiomes around us. They transform the unseen microbial world into a rich source of ecological and biomedical information.

Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.