Abstract
Genomics has greatly transformed our understanding of phage biology; however, traditional methods of DNA isolation for whole genome sequencing have required phages to be grown to high titers in large-scale preparations, potentially selecting for only those phages that can grow efficiently under laboratory conditions. This may also select for mutations or deletions that enable more efficient growth in culture. The ability to sequence a bacteriophage genome from a single isolated plaque reduces these risks while decreasing the time and complexity of bacteriophage genome sequencing. A method of amplification and library preparation is described, utilizing Sequence Independent Single Primer Amplification (SISPA), that can be used for whole genome shotgun sequencing of bacteriophages from a single isolated plaque.
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Acknowledgments
The author wishes to thank Galina Koroleva, Jessica DePew, Janaki Purushe, Bin Zhou, and Manolito Torralba for their contributions to the development of these protocols. This work was supported with funds from the United States National Institutes of Health (R21-DE018063 and U54-AI84844) and in part with funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract number HHSN272200900007C.
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Fouts, D.E. (2018). Amplification for Whole Genome Sequencing of Bacteriophages from Single Isolated Plaques Using SISPA. In: Clokie, M., Kropinski, A., Lavigne, R. (eds) Bacteriophages. Methods in Molecular Biology, vol 1681. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7343-9_12
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DOI: https://doi.org/10.1007/978-1-4939-7343-9_12
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