Deep Sequencing: Intra-Terrestrial Metagenomics Illustrates The Potential Of Off-Grid Nanopore DNA Sequencing

Genetic and genomic analysis of nucleic acids from environmental samples has helped transform our perception of the subsurface as a major reservoir of microbial novelty. Many of the microbial taxa living in the subsurface are under-represented in culture-dependent investigations. In this regard, metagenomic analyses of subsurface environments exemplify both the utility of metagenomics and its power to explore microbial life in some of the most extreme and inaccessible environments on Earth. Hitherto, the transfer of microbial samples to home laboratories for DNA sequencing and bioinformatics is the standard operating procedure for exploring microbial diversity. This approach incurs logistical challenges and delays the characterization of microbial biodiversity. For selected applications, increased portability and agility in metagenomic analysis is therefore desirable. Here, we describe the implementation of sample extraction, metagenomic library preparation, nanopore DNA sequencing and taxonomic classification using a portable, battery-powered, suite of off-the-shelf tools (the MetageNomad) to sequence ochreous sediment microbiota while within the South Wales Coalfield. While our analyses were frustrated by short read lengths and a limited yield of DNA, within the assignable reads, Proteobacterial taxa dominated, followed by members of Actinobacteria, Firmicutes and Bacteroidetes, all of which have previously been identified in coals. Further to this, the fungal genus Candida was detected, as well as a methanogenic archaeal taxon. To the best of our knowledge, this application of the MetageNomad represents an initial effort to conduct metagenomics within the subsurface, and stimulates further developments to take metagenomics off the beaten track.