Integrated omics analyses reveal insights into microbial dark matter associated with deep-sea water in Central Indian Ocean.

Deep-sea environments represent one of the most complex ecosystems on Earth and harbour extensive microbial dark matter (MDM) that remains poorly characterized due to limitations of culture-dependent approaches. Here, we present a pilot-scale multi-omics investigation to profile MDM from a deep-sea water sample collected at 1600 m, employing an integrated workflow that combine Illumina and Nanopore hybrid metagenomics sequencing, fluorescence-activated cell sorting-based single-cell genomics and untargeted metabolomics using LC(Orbitrap) -MS and GC-MS. Hybrid sequencing yielded metagenome-assembled genomes (MAGs) from well-reported clades Pseudomondota, Actinomycetota, and Bacteriodota. Taxonomic profiling revealed rare groups viz. Candidatus Omnitrophota, Saccharibacteria, and Abscondibacteria. A total of 150 single amplified genomes (SAGs) were generated which were found to be affiliated to MDM candidate phyla including Patescibacteria, Armatimondota, J088, and Thermoproteota. Annotation of SAGs suggested the critical role of MDM in complex carbohydrate metabolism, methyl-linked methanogenesis and methanotrophy. SAGs from potentially novel MDM exhibited a high density of Biosynthetic Gene Clusters (BGCs). Further exometabolomic analysis divulged the presence of complex fatty acid biosynthesis, polyketide synthesis and terpenoid pathway networks. This study has provided a significant pointer on applying integrated metagenome and single-cell genome-based exploration of complex microbiomes which could potentially be a precursor to high-throughput culturomics and downstream bio-prospection studies.