Deng, L.; Silins, R.; Castro-Mejía, J.L.; Kot, W.; Jessen, L.; Thorsen, J.; Shah, S.; Stokholm, J.; Bisgaard, H.; Moineau, S.; Nielsen, D.S. A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples. Viruses2019, 11, 667.
Deng, L.; Silins, R.; Castro-Mejía, J.L.; Kot, W.; Jessen, L.; Thorsen, J.; Shah, S.; Stokholm, J.; Bisgaard, H.; Moineau, S.; Nielsen, D.S. A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples. Viruses 2019, 11, 667.
Deng, L.; Silins, R.; Castro-Mejía, J.L.; Kot, W.; Jessen, L.; Thorsen, J.; Shah, S.; Stokholm, J.; Bisgaard, H.; Moineau, S.; Nielsen, D.S. A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples. Viruses2019, 11, 667.
Deng, L.; Silins, R.; Castro-Mejía, J.L.; Kot, W.; Jessen, L.; Thorsen, J.; Shah, S.; Stokholm, J.; Bisgaard, H.; Moineau, S.; Nielsen, D.S. A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples. Viruses 2019, 11, 667.
Abstract
The human gut microbiome (GM) plays an important role in human health and diseases. However, while substantial progress has been made in understanding the role of bacterial inhabitants of the gut, much less is known regarding the viral component of the GM. Bacteriophages (phages) are viruses attacking specific host bacteria and likely play important roles in shaping the GM. Although metagenomic approaches have led to the discoveries of many new viruses, they largely remain uncultured as their hosts have not been identified, which hampers our understanding of their biological roles. Existing protocols for isolation of viromes generally require relatively high input volumes and are generally more focused on extracting nucleic acids of good quality and purity for down-stream analysis and less on purification of still infective viruses. Here we report the development of an efficient protocol requiring low sample input yielding purified viromes containing still infective phages which also are of sufficient purity for genome sequencing. We validated the method through spiking of known phages followed by plaque assays, qPCR and metagenomic sequencing. The protocol should facilitate the culturing of novel viruses from the gut as well as large scale studies on gut viromes.
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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