title: Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.
abstract: BACKGROUND
  Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).
  
  
  METHODS
  Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.
  
  
  RESULTS
  Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.
  
  
  CONCLUSIONS
  Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.
authors.affiliation: Department of Microbiology, The Ohio State University , Columbus , OH , United States.
authors.forename: Simon
authors.initials: S
authors.lastname: Roux
authors.affiliation: Department of Microbiology, The Ohio State University , Columbus , OH , United States.
authors.forename: Natalie E
authors.initials: NE
authors.lastname: Solonenko
authors.affiliation: Department of Microbiology, Ha Long University , Uong Bi , Quang Ninh , Vietnam.
authors.forename: Vinh T
authors.initials: VT
authors.lastname: Dang
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona , Tucson , AZ , United States.
authors.forename: Bonnie T
authors.initials: BT
authors.lastname: Poulos
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona , Tucson , AZ , United States.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Schwenck
authors.affiliation: College of Marine Science, University of South Florida , St. Petersburg , FL , United States.
authors.forename: Dawn B
authors.initials: DB
authors.lastname: Goldsmith
authors.affiliation: Department of the Geophysical Sciences, University of Chicago , Chicago , IL , United States.
authors.forename: Maureen L
authors.initials: ML
authors.lastname: Coleman
authors.affiliation: College of Marine Science, University of South Florida , St. Petersburg , FL , United States.
authors.forename: Mya
authors.initials: M
authors.lastname: Breitbart
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH, United States; Department of Civil, Environmental and Geodetic Engineering, The Ohio State University, Columbus, OH, United States.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Sullivan
keywords: Environmental virology; Viral metagenomics; ssDNA viruses