Abstract
Most of Earth’s bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning are usually highly fragmented genomes with heterogeneous sequence contamination. In this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs—six novel bacterial orders, 20 families, 66 genera, and 154 species—from Lake Shunet, a secluded meromictic lake in Siberia. With our workflow, the average N50 of reconstructed MAGs greatly increased 10–40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia.
Highlights
Most of Earth’s bacteria have yet to be cultivated
To recover novel metagenome-assembled genomes (MAGs) with high contiguity without compromising accuracy, 3.0, 5.0, and 5.5 m depth Lake Shunet samples were sequenced by Nanopore machines individually, and the resulting long reads (LRs) were analyzed together with short reads (SRs) using a workflow we developed for this study (Fig. 1a and Supplementary Table 1)
From the 5.5 m dataset, we identified two MAGs belonging to candidate novel families under Methanomassiliicoccales and Iainarchaeales (MAG ID: M55A1 and M55A2, respectively) based on the relative evolutionary divergence (RED) and phylogenetic placement determined by Genome Taxonomy Database (GTDB)-Tk (Supplementary Data 2), and one MAG belonging to a potential novel species under Nanoarchaeota based on a 95% Average Nucleotide Identities (ANIs) cutoff for species boundary (Fig. 3 and Supplementary Data 2)
Summary
Most of Earth’s bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. Our previous 16 rRNA amplicon survey showed that the lake contains diverse microbial communities with a higher Shannon diversity index and Chao[1] richness estimator than Lake Shira and Lake Oigon, two another saline meromictic lakes near the center of Asia[13] It showed that the lake comprises at least hundreds of unknown bacteria and archaea[14], highlighting the importance of mining microbial MAGs from this rarely explored lake. In this study, we developed a new workflow combining Illumina and Nanopore sequencing reads by integrating several cutting-edge bioinformatics tools to recover and reconstruct MAGs with high contiguity and accuracy. We demonstrate that our newly built workflow can be used to reconstruct hundreds of complete highquality MAGs from environmental samples in a high-complexity microbial community
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