Andrews-Pfannkoch, C., Fadrosh, D. W., Thorpe, J., Williamson, S. J.
Hydroxyapatite Mediated Separation of DsDNA, SsDNA and RNA Genotypes from Natural Viral Assemblages
Appl Environ Microbiol. 2010 Jun 11; 76(15): 5039-45.
Metagenomics can be used to determine the diversity of complex, often unculturable, viral communities containing various nucleic acid compositions. Here we report the use of hydroxyapatite chromatography to efficiently fractionate dsDNA, ssDNA, dsRNA, and ssRNA genomes from known bacteriophages. Linker-amplified shotgun libraries were constructed to generate sequencing reads from each hydroxyapatite fraction. Greater than 90% of the reads displayed significant similarity to the expected genomes at the nucleotide level. These methods were applied to marine viruses collected from the Chesapeake Bay and the Dry Tortugas National Park. Isolated nucleic acids were fractionated using hydroxyapatite chromatography followed by linker-amplified shotgun library construction and sequencing. Taxonomic analysis demonstrated that the majority of environmental sequences, regardless of their source nucleic acid, were most similar to dsDNA viruses, reflecting the bias of viral metagenomic sequence databases.