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Preparation of Metagenomic Libraries from Naturally Occurring Marine Viruses

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 h... Full description

Journal Title: Methods in Enzymology 2013, Vol.531, p.143-165
Main Author: Solonenko, Sergei A
Other Authors: Sullivan, Matthew B
Format: Electronic Article Electronic Article
Language: English
Subjects:
Publisher: United States: Elsevier Science & Technology
ID: ISSN: 0076-6879
Link: https://www.ncbi.nlm.nih.gov/pubmed/24060120
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recordid: cdi_gale_vrl_7005800019
title: Preparation of Metagenomic Libraries from Naturally Occurring Marine Viruses
format: Article
creator:
  • Solonenko, Sergei A
  • Sullivan, Matthew B
subjects:
  • Aquatic Organisms - virology
  • Bacteriophage
  • Bacteriophages - genetics
  • DNA, Single-Stranded
  • Environmental virology
  • Evolutionary biology
  • Gene Library
  • Genome, Viral
  • High-Throughput Nucleotide Sequencing
  • Library amplification
  • Linker amplification
  • Metagenome
  • Metagenomics
  • Molecular biology
  • Next-generation sequencing
  • Sequencing library
  • Viral ecology
  • Viral metagenomics
  • Viromics
  • Viruses - genetics
ispartof: Methods in Enzymology, 2013, Vol.531, p.143-165
description: 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.
language: eng
source:
identifier: ISSN: 0076-6879
fulltext: no_fulltext
issn:
  • 0076-6879
  • 1557-7988
url: Link


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descriptionMicrobes 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.
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subjectAquatic Organisms - virology ; Bacteriophage ; Bacteriophages - genetics ; DNA, Single-Stranded ; Environmental virology ; Evolutionary biology ; Gene Library ; Genome, Viral ; High-Throughput Nucleotide Sequencing ; Library amplification ; Linker amplification ; Metagenome ; Metagenomics ; Molecular biology ; Next-generation sequencing ; Sequencing library ; Viral ecology ; Viral metagenomics ; Viromics ; Viruses - genetics
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abstractMicrobes 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.
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