Preparation of metagenomic libraries from naturally occurring marine viruses

Sergei A. Solonenko, Matthew Sullivan

Research output: Chapter in Book/Report/Conference proceedingChapter

14 Citations (Scopus)

Abstract

Microbes are now well recognized as major drivers of the biogeochemical cycling that fuels the Earth, and their viruses (phages) are known to be abundant and important in microbial mortality, horizontal gene transfer, and modulating microbial metabolic output. Investigation of environmental phages has been frustrated by an inability to culture the vast majority of naturally occurring diversity coupled with the lack of robust, quantitative, culture-independent methods for studying this uncultured majority. However, for double-stranded DNA phages, a quantitative viral metagenomic sample-to-sequence workflow now exists. Here, we review these advances with special emphasis on the technical details of preparing DNA sequencing libraries for metagenomic sequencing from environmentally relevant low-input DNA samples. Library preparation steps broadly involve manipulating the sample DNA by fragmentation, end repair and adaptor ligation, size fractionation, and amplification. One critical area of future research and development is parallel advances for alternate nucleic acid types such as single-stranded DNA and RNA viruses that are also abundant in nature. Combinations of recent advances in fragmentation (e.g., acoustic shearing and tagmentation), ligation reactions (adaptor-to-template ratio reference table availability), size fractionation (non-gel-sizing), and amplification (linear amplification for deep sequencing and linker amplification protocols) enhance our ability to generate quantitatively representative metagenomic datasets from low-input DNA samples. Such datasets are already providing new insights into the role of viruses in marine systems and will continue to do so as new environments are explored and synergies and paradigms emerge from large-scale comparative analyses.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
Pages143-165
Number of pages23
Volume531
DOIs
StatePublished - 2013

Publication series

NameMethods in Enzymology
Volume531
ISSN (Print)00766879
ISSN (Electronic)15577988

Fingerprint

Metagenomics
Viruses
Bacteriophages
Amplification
Ligation
DNA
Fractionation
High-Throughput Nucleotide Sequencing
Horizontal Gene Transfer
DNA Viruses
Workflow
Single-Stranded DNA
RNA Viruses
DNA Fragmentation
Gene transfer
Gene Library
DNA Sequence Analysis
Acoustics
Nucleic Acids
Libraries

Keywords

  • Bacteriophage
  • Environmental
  • Library amplification
  • Linker amplification
  • Metagenomics
  • Next-generation sequencing
  • Sequencing library
  • Viral ecology
  • Viral metagenomics
  • virology
  • Viromics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Solonenko, S. A., & Sullivan, M. (2013). Preparation of metagenomic libraries from naturally occurring marine viruses. In Methods in Enzymology (Vol. 531, pp. 143-165). (Methods in Enzymology; Vol. 531). https://doi.org/10.1016/B978-0-12-407863-5.00008-3

Preparation of metagenomic libraries from naturally occurring marine viruses. / Solonenko, Sergei A.; Sullivan, Matthew.

Methods in Enzymology. Vol. 531 2013. p. 143-165 (Methods in Enzymology; Vol. 531).

Research output: Chapter in Book/Report/Conference proceedingChapter

Solonenko, SA & Sullivan, M 2013, Preparation of metagenomic libraries from naturally occurring marine viruses. in Methods in Enzymology. vol. 531, Methods in Enzymology, vol. 531, pp. 143-165. https://doi.org/10.1016/B978-0-12-407863-5.00008-3
Solonenko SA, Sullivan M. Preparation of metagenomic libraries from naturally occurring marine viruses. In Methods in Enzymology. Vol. 531. 2013. p. 143-165. (Methods in Enzymology). https://doi.org/10.1016/B978-0-12-407863-5.00008-3
Solonenko, Sergei A. ; Sullivan, Matthew. / Preparation of metagenomic libraries from naturally occurring marine viruses. Methods in Enzymology. Vol. 531 2013. pp. 143-165 (Methods in Enzymology).
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