TY - CHAP
T1 - Preparation of metagenomic libraries from naturally occurring marine viruses
AU - Solonenko, Sergei A.
AU - Sullivan, Matthew B.
N1 - Funding Information:
We thank Christine Schirmer for assistance with figures and tables and technical discussions as well as Jennifer Brum and Natalie Solonenko for comments on the chapter. Funding was provided by the Gordon and Betty Moore Foundation to M. B. S. and an NSF IGERT Comparative Genomics Training Grant to S. A. S.
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
KW - Bacteriophage
KW - Environmental
KW - Library amplification
KW - Linker amplification
KW - Metagenomics
KW - Next-generation sequencing
KW - Sequencing library
KW - Viral ecology
KW - Viral metagenomics
KW - Viromics
KW - virology
UR - http://www.scopus.com/inward/record.url?scp=84884546451&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884546451&partnerID=8YFLogxK
U2 - 10.1016/B978-0-12-407863-5.00008-3
DO - 10.1016/B978-0-12-407863-5.00008-3
M3 - Chapter
C2 - 24060120
AN - SCOPUS:84884546451
SN - 9780124078635
T3 - Methods in Enzymology
SP - 143
EP - 165
BT - Microbial Metagenomics, Metatranscriptomics, and Metaproteomics
PB - Academic Press Inc.
ER -