Comprehensive unbiased metagenomics biodiversity analysis of environmental DNA using Oxford Nanopore sequencing for eukaryotes

Environmental DNA (eDNA) technology is essential for monitoring living organisms in ecological research. The study of symbiotic organisms from different classes or kingdoms, including those previously unknown, is possible with simultaneous and equally efficient eDNA analysis of these species. For metagenomics biodiversity, various targeted primer sets are used, including those universal for certain families, classes, or kingdoms. The most universal for all existing cellular organisms is the presence of ribosomal RNA encoding gene sequences. For eukaryotic sequences, these are 16S and 23s rDNA, for eukaryotic sequences of the nuclear (18S and 28S) and mitochondrial (12S and 16S) ribosomal RNA. Here we present the application of an eDNA approach to the simultaneous, quantitative, and unbiased identification of most eukaryotic species (excluding archaea, cyanobacteria, and mycoplasmas). To achieve this, we developed a universal PCR assay that targets the nuclear most conservative ribosomal gene regions for all cellular organisms (excluding mitochondria and chloroplasts genomes), including plants, algae, fungi, protists, insects, and animals. The amplification product contains polymorphic regions of both ribosomal genes and intergenic spacer. The size of PCR products varies from class, kingdom, or domain, ranging from 2 kB for fungi to 7 kB for birds. This assay is also adapted for using the Oxford Nanopore Rapid Barcoding library kit that allows metagenomics biodiversity analysis. Our approach provides a quick, sensitive, and equally efficient way to study the composition of eDNA of mixer species in the environment. This protocol reduces the time and cost of metagenomics biodiversity analysis by sequencing using the Oxford Nanopore. We can efficiently analyze the biodiversity of mixed species present in environmental samples.