Abstract
New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.
Highlights
The development of long read sequencing technologies, such as the Oxford Nanopore Technology MinION and Pacific Biosciences SMRT are presenting new opportunities for the effective recovery of complete, closed genomes[1,2]
We (1) describe new methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes; (2) assess the influence of different correction procedures on genome quality and predicted gene quality; (3) contribute 22 new closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses and (4) compare and contrast the use of long read metagenome assembly to the results obtained from hybrid metagenome assembly workflows
We identified 96 long read assembled contig (LRAC) sequences less than 1Mbp in length that were classified as circular constructs, and these are plausible candidates for being plasmids[58], phages/viruses[59] or small microbial genomes characteristic of the Candidate phyla radiation (CPR) group[60]
Summary
The development of long read sequencing technologies, such as the Oxford Nanopore Technology MinION and Pacific Biosciences SMRT are presenting new opportunities for the effective recovery of complete, closed genomes[1,2]. While these new approaches have been mostly applied to single species isolates[3,4], the ability of this new methodology to recover genomes of member taxa from complex microbial communities (microbiome) data is actively being explored. We (1) describe new methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes; (2) assess the influence of different correction procedures on genome quality and predicted gene quality; (3) contribute 22 new closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses and (4) compare and contrast the use of long read metagenome assembly to the results obtained from hybrid metagenome assembly workflows
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