Acquiring Reference Genomes from Uncultured Microbes by Micromanipulation and Low-complexity Metagenomics

A pre-requisite for many of the –omics approaches applied in environmental microbiology today are high quality reference genomes. Until recently such genomes have been difficult to obtain from unculturable, complex microbial communities. However, lately the ‘single cell genomics’ approach based on isolation and amplification of genomic DNA from a single or few clonal cells has proven efficient for this purpose although very tedious. The aim of this study was to apply the methodology of single cell genomics to filamentous organisms and microcolonies of specific species from microbial communities in order to obtain low complexity metagenomes from which high quality draft genomes could be assembled.
Microorganisms were visualized with FISH in samples from Danish wastewater treatment plants and single filaments and microcolonies were isolated using an epifluorescense microscope equipped with a Skerman micromanipulator mounted with µm sized glass hooks. The isolated cells were lysed and genomic DNA was amplified using multiple displacement amplification. The amplified DNA was validated by 16/18S PCR and Sanger sequencing before being sequenced on an Illumina platform. Metagenome assembly was performed in CLC Workbench. Number of species in assemblies was assessed by screening for 16/23S and 18/28S genes and 100 essential single copy bacterial genes. Contigs were binned by using principle component analysis of tetranucleotide frequencies, blast best hit classification and explorative mapping. Draft genomes were assembled from contig bins and validated by screening for essential single copy genes. Draft genomes were annotated using RAST and IMG/ER.
Forty five single filaments/microcolonies have been isolated of which 13 samples passed validation and proceeded to sequencing. In the initial analysis several samples showed promising assembly statistics, low metagenome complexity and good binning capabilities. Species obtained belong to a number phylogenetic groups which are highly relevant in wastewater treatment: Chloroflexi, Thiothrix and Microthrix.