Complex microbial communities typically contain a large number of low abundance species, which collectively, comprise a considerable proportion of the community. This 'rare biosphere' has been speculated to contain keystone species and act as a repository of genomic diversity to facilitate community adaptation. Many environmental microbes are currently resistant to cultivation, and can only be accessed via culture-independent approaches. To enhance our understanding of the role of the rare biosphere, we aimed to improve their metagenomic representation using DNA normalization methods, and assess normalization success via shotgun DNA sequencing. A synthetic metagenome was constructed from the genomic DNA of five bacterial species, pooled in a defined ratio spanning three orders of magnitude. The synthetic metagenome was fractionated and thermally renatured, allowing the most abundant sequences to hybridize. Double-stranded DNA was removed either by hydroxyapatite chromatography, or by a duplex-specific nuclease (DSN). The chromatographic method failed to enrich for the genomes present in low starting abundance, whereas the DSN method resulted in all genomes reaching near equimolar abundance. The representation of the rarest member was increased by approximately 450-fold. De novo assembly of the normalized metagenome enabled up to 18.0% of genes from the rarest organism to be assembled, in contrast to the un-normalized sample, where genes were not able to be assembled at the same sequencing depth. This study has demonstrated that the application of normalization methods to metagenomic samples is a powerful tool to enrich for sequences from rare taxa, which will shed further light on their ecological niches.
Keywords: DNA normalization; duplex-specific nuclease; high-throughput sequencing; hydroxyapatite chromatography; metagenomics; microbial community; rare biosphere.
© 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.