Assembling the Marine Metagenome, One Cell at a Time

Abstract

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa.

Ecology of marine Bacteroidetes: a comparative genomics approach

“…First, they had more paralogous families and a larger percent of genes in such families (Table 1; Figure 1). The percentage of genes in paralogous families follows an already recognized linear relationship with genome size for a large selection of bacteria ( Figure 1; Pushker et al, 2004;Woyke et al, 2009). Gene duplication and subsequent modifications to carry out novel functions by paralogous proteins is a well-known mechanism of bacterial evolution (review in Andersson and Hughes, 2009).…”

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

“…MDA reactions are sensitive to DNA contamination present in the sample, reagents or contamination introduced through sample handling. The effect of DNA contamination can be controlled by establishing clean cell-sorting procedures 12,20,24,26 , decontaminating commercially available MDA reagents 1,20,23 , expressing a custom ultrapure Phi29 enzyme 27 or by using commercial precleaned MDA kits that are specifically designed for single-cell templates. Volume reduction can also limit the impact of reagent contamination (Box 1).…”

“… crItIcal step Although previous reports suggest debranching of the MDA products with S1 nuclease before sequencing 15 , we do not recommend this step. Our previous results suggest that S1 debranching has no effect on the chimera rate 12 , and we routinely sequence and phylogenetically screen single-cell MDA products without debranching.  pause poInt The amplified DNA is ready for phylogenetic screening and sequencing (Steps 24-30), and it can be stored for several months at −80 °C.…”

“…Mapping of metagenome sequences to single-cell genomes has not only been applied to validating metagenome assembly and binning 7 but also to subsequently improving the single-cell assemblies, as has been shown for SR1 bacteria, atribacteria and ammoniaoxidizing archaea [8][9][10] . Other studies successfully used fragment recruitment by single-cell genomes to investigate biogeographic distribution of uncultivated, marine taxa 11,12 . Although the bulk of the studies using FACS-enabled SCG have focused on bacteria and archaea, this protocol can also be adapted to microbial eukaryotes 13 and mammalian cells 14 .…”

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

“…Moreover, these assemblies are actually consensus genomes derived from closely related species or strains, which makes many interesting analyses of genomes and populations impossible. Single-cell sequencing, enabled by the whole-genome amplification technique of multiple displacement amplification (MDA) (Dean et al 2001(Dean et al , 2002, has allowed researchers to sequence and assemble a significant portion of a single genome (Zhang et al 2006;Woyke et al 2009). However, MDA from a single cell suffers from amplification bias.…”