title: A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.
abstract: Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary.
  
  
  
  We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels.
  
  
  
  This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.
authors.affiliation: British Columbia Public Health Laboratory, British Columbia Centre for Disease Control, Vancouver, BC, V5Z 4R4, Canada.|Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Miguel I
authors.initials: MI
authors.lastname: Uyaguari-Diaz
authors.affiliation: British Columbia Public Health Laboratory, British Columbia Centre for Disease Control, Vancouver, BC, V5Z 4R4, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Chan
authors.affiliation: South Kensington Campus, Imperial College London, Sir Ernst Chain Building, London, SW7 2AZ, UK.
authors.forename: Bonnie L
authors.initials: BL
authors.lastname: Chaban
authors.affiliation: British Columbia Public Health Laboratory, British Columbia Centre for Disease Control, Vancouver, BC, V5Z 4R4, Canada.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Croxen
authors.affiliation: Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.|Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.|Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Jan F
authors.initials: JF
authors.lastname: Finke
authors.affiliation: Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Janet E
authors.initials: JE
authors.lastname: Hill
authors.affiliation: Department of Molecular Biology and Biochemistry, South Science Building, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Peabody
authors.affiliation: Department of Molecular Biology and Biochemistry, South Science Building, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Thea
authors.initials: T
authors.lastname: Van Rossum
authors.affiliation: Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.|Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.|Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.|Integrated Microbial Biodiversity Program, Canadian Institute for Advanced Research, Toronto, ON, M5G 1Z8, Canada.
authors.forename: Curtis A
authors.initials: CA
authors.lastname: Suttle
authors.affiliation: Department of Molecular Biology and Biochemistry, South Science Building, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Fiona S L
authors.initials: FS
authors.lastname: Brinkman
authors.affiliation: British Columbia Public Health Laboratory, British Columbia Centre for Disease Control, Vancouver, BC, V5Z 4R4, Canada.|Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Judith
authors.initials: J
authors.lastname: Isaac-Renton
authors.affiliation: British Columbia Public Health Laboratory, British Columbia Centre for Disease Control, Vancouver, BC, V5Z 4R4, Canada.|Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Prystajecky
authors.affiliation: Department of Pathology, Sidra Medical and Research Center, PO Box 26999, Doha, Qatar. ptang@sidra.org.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tang
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D001955:British Columbia; D021901:DNA, Intergenic / Q000235:genetics; D056890:Eukaryota / Q000145:classification* / Q000235:genetics; D005618:Fresh Water / Q000382:microbiology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D054892:Metagenome / Q000235:genetics; D064307:Microbiota / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D014780:Viruses / Q000145:classification* / Q000235:genetics; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis*
chemical_list: D021901:DNA, Intergenic; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S
keywords: Amplicon sequencing; Metagenomes; Metagenomics; Microbial fractions; Microbiome; Watersheds