3.2 Low-abundant non-microbial Eukaryotes can be effectively detected by PCR-free deep sequencing of extracellular eDNA
We examined the taxonomic composition of the samples under all the domains of life. The taxonomic resolution of the classified reads progressively decreased from 80.61% at the phylum level to 21.34% at the species level (Supplementary Fig. 6). We then inspected the taxonomic assignments of reads classified at least up to the family level. After filtering out the low-abundant families, 2.14 billion reads were retained from all the samples classified under 1001 families across the tree of life (Fig. 3). Interestingly, despite the high abundance of Bacterial reads, the highest proportion of classified families belonged to Eukaryota (73%) followed by Bacteria (23%), Archaea (1%), and DNA Viruses (3%). Upon further investigation, about 62% of the viral diversity was found to be composed of DNA Phages from the kingdom Heunggongvirae. Whereas, Archaea majorly consisted of families under the Phylum Euryarchaeota (63%) and Crenarchaeota (19%). Under the domain Bacteria, Proteobacteria was the richest phylum with 43% of the families, followed by Actinobacteria (13%), and Firmicutes (9%). Surprisingly, Eukaryotic families were majorly distributed under Metazoa (54%), followed by Fungi (20%), Viridiplantae (13%), and Protists (13%). As the non-microbial organisms are relatively less abundant in an ecosystem than the microbes, the probability of their detection in an environmental sample is generally low. The significant presence of animal and plant reads in the data indicates that the low-abundant non-microbial organisms can be effectively detected from PCR-free deep sequencing of extracellular eDNA.