Biodiversity is declining on a planetary scale at an alarming rate due
to anthropogenic factors. Classical biodiversity monitoring approaches
are time-consuming, resource-intensive, and not scalable to address the
current biodiversity crisis. The environmental DNA-based next-generation
biomonitoring framework provides an efficient, scalable, and holistic
solution for evaluating changes in various ecological entities. However,
its scope is currently limited to monitoring targeted groups of
organisms using metabarcoding, which suffers from various PCR-induced
biases. To utilise the full potential of next-generation biomonitoring,
we intended to develop PCR-free genomic technologies that can deliver
unbiased biodiversity data across the tree of life in a single assay.
Here, we describe a novel metagenomic workflow comprising of a
customised extracellular DNA enrichment protocol from large-volume
filtered water samples, a completely PCR-free library preparation step,
an ultra-deep next-generation sequencing, and a pseudo-taxonomic
assignment strategy using the dual lowest common ancestor algorithm. We
demonstrate the utility of our approach in a pilot-scale
spatially-replicated experimental setup in Chilika, a large
hyper-diverse brackish lagoon ecosystem in India. Using incidence-based
statistics, we show that biodiversity across the tree of life, from
microorganisms to the relatively low-abundant macroorganisms such as
Arthropods and Fishes, can be effectively detected with about one
billion paired-end reads using our reproducible workflow. With
decreasing costs of sequencing and the increasing availability of
genomic resources from the earth biogenome project, our approach can be
tested in different ecosystems and adapted for large-scale rapid
assessment of biodiversity across the tree of life
A Novel Metagenomic Workflow for Biomonitoring across the Tree of Life using PCR-free Ultra-deep Sequencing of Extracellular eDNA