A rapid shotgun metagenome protocol based on Oxford Nanopore Technology applied to soil biodiversity analysis

Beneficial microorganisms are increasingly used in modern agriculture with significant benefits on yields and sustainability of the agricultural system. However, the massive introduction of non-indigenous bacterial and fungal species in the form of biofertilizers and bioinoculants can result in their uncontrolled spread with undesirable effects on the ecology of agricultural soils and nearby environments. This study aims to develop a protocol for monitoring soil biodiversity after applying bioinoculants with beneficial action. The basis of the experimentation is a third generation DNA sequencer (Oxford Nanopore Technology, ONT) and a shotgun metagenome sequencing of the microbial community (technique that relies on massive sequencing of total DNA extractable from the soil). With ONT sequencing, a single molecule of DNA can be "read" without the need for PCR amplification or chemical labelling of the sample. The sequence of nucleic acids is inferred from changes in the ionic current across a membrane as a single DNA molecule passes through a protein nanopore. Such sequencing technology is of interest for studying soil communities because the it allows sequencing of longer DNA fragments compared to Illumina platforms. The ONT sequencing is particularly suitable for tracing and quantifying single species of fungi or other organisms in complex samples as it allows matching with more comprehensive diagnostic markers, either found in public databases or appropriately selected for diagnostic purposes.