Deconvolution of complete microbial genomes from shotgun metagenomes

It is estimated that only ˜0.1% of environmental microbes have ever been cultured in the laboratory [1], meaning that the biosynthetic potential of the remaining ˜99.9% is largely unexplored except through culture-independent sequencing (metagenomics). Although it is believed that small molecules mediate microbial interspecies interactions in nature [2], the high-resolution details of these ecosystems largely remains obscure. To alleviate these problems, we devised a bioinformatics workflow to efficiently deconvolute complete microbial genomes from single shotgun metagenomes. Existing techniques such as differential coverage analysis [3] require multiple DNA extractions or multiple related metagenomes in order to assemble divergent bacteria without reference sequences. By contrast, our technique is able to assemble divergent genomes from single metagenomic datasets, allowing the study of dynamic changes in metagenomes involving rare bacteria and the genetic diversity of individual species common to multiple metagenomes. We used this technique to examine two marine sponges with high microbial diversity (both Hippospongia lachne de Laubenfels (Spongiidae), collected from Florida, U.S.A), and one sponge with low microbial diversity (unidentified species, collected from Florida, U.S.A). This yielded up to ˜100 high quality genomes per metagenome (see Table 1). Six genomes were unclassified at the phylum level, and in both FL20 – 3 and FL20 – 9 significant numbers were unclassified at the class, order and family levels (average 12.0%, 25.5% and 35.4%, respectively). In both FL20 – 3 and FL20 – 9, the most abundant phylum was Proteobacteria, and these samples also contained a handful of genomes classified as Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Candidatus Tectomicrobia. AntiSMASH [4] analysis revealed that secondary metabolite pathways were distributed fairly evenly amongst genomes in H. lachne, with no particular phylogenetic group possessing high concentrations of biosynthetic pathways.

Tab. 1: Characteristics of genomes assembled from three marine sponge metagenomes

					Median values
Sample	Species	No. genomes assembled	Completeness*	Duplicated markers†	Size	n50	No. contigs
FL20 – 3	H. lachne	77	82%	1	3.1 Mbp	46.6 kbp	84
FL20 – 9	H. lachne	103	81%	2	2.9 Mbp	31.3 kbp	94
FL48 – 1	Unknown	5	52%	4	3.4 Mbp	14.1 kbp	199
*Completeness calculated from the number of universal single-copy marker genes present [5]. †The number of single-copy marker genes found more than once (out of 139).

Acknowledgements: This research was performed in part using the compute resources and assistance of the UW-Madison Center For High Throughput Computing (CHTC) in the Department of Computer Sciences. The CHTC is supported by UW-Madison, the Advanced Computing Initiative, the Wisconsin Alumni Research Foundation, the Wisconsin Institutes for Discovery, and the National Science Foundation, and is an active member of the Open Science Grid, which is supported by the National Science Foundation and the U.S. Department of Energy's Office of Science. This work was supported by the National Institutes of Health, NIAID Grant R21AI121704, and the School of Pharmacy, the Graduate School, and the Institute for Clinical & Translational Research at the University of Wisconsin-Madison.

Keywords: shotgun metagenomics, binning, bioinformatics method.

References:

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