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Defining the human gut host–phage network through single-cell viral tagging

Abstract

Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses. One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses. To this end, we have adapted the viral tagging approach, which bypasses the need for culture-based methods to identify host–phage pairings. Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells, which are then individually sorted as host–phage pairs, followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus(es). We demonstrate single-cell viral tagging using the faecal microbiome, including cross-tagging of viruses and bacteria between human subjects. A total of 363 unique host–phage pairings were predicted, most of which were subject-specific and involved previously uncharacterized viruses despite the majority of their bacterial hosts having known taxonomy. One-fifth of these pairs were confirmed by multiple individual tagged cells. Viruses targeting more than one bacterial species were conspicuously absent in the host–phage network, suggesting that phages are not major vectors of inter-species horizontal gene transfer in the human gut. A high level of cross-reactivity between phages and bacteria from different subjects was noted despite subject-specific viral profiles, which has implications for faecal microbiota transplant therapy.

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Fig. 1: Method development.
Fig. 2: Workflow.
Fig. 3: Examples of viral clusters detected in multiple SAGs.
Fig. 4: Faecal host–phage network obtained from 11 human subjects.
Fig. 5: Host–phage ratios in native faecal metagenomes.
Fig. 6: Crossover single-cell VT between four subjects.

Data availability

Sequence data (raw reads, MAGs, viral contigs from viromes and single-cell VT) have been deposited in the NCBI Sequence Read Archive under the BioProject number PRJNA492716.

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Acknowledgements

We thank the 11 volunteers for providing samples for this study, M. Nefedov for assistance with operation of the FACSAria III, S. Low for creating the Illumina libraries, R. Webb for assistance with transmission electron microscopy and A. Hassan for advice on phage detection. We also thank A. Pribyl Rinke and N. Angel for providing the longitudinal faecal metagenome series, and R. Edwards and J. Barr for useful feedback on the manuscript. The project was supported by an Australian Research Council Laureate Fellowship (FL150100038) awarded to P.H., Australian Research Council (ARC) Future Fellowship (FT170100213) awarded to C.R., fundings from German Research Foundation (DFG Emmy Noether program; DE2360/1-1) and European Research Council Starting (ERC StG 803077) awarded to L.D., and ‘Microbiome Challenge’ strategic funding (DVCR16003A) from the University of Queensland.

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M.D. and P.H. conceived and designed the experiments and analysis. L.D. and C.R. contributed to the method development. M.D. performed the laboratory experiments. M.D., S.J.L and J.N.D. performed bioinformatic analyses. M.D. and P.H. wrote the paper. All co-authors edited the completed draft of the manuscript.

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Correspondence to Mária Džunková or Philip Hugenholtz.

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Džunková, M., Low, S.J., Daly, J.N. et al. Defining the human gut host–phage network through single-cell viral tagging. Nat Microbiol 4, 2192–2203 (2019). https://doi.org/10.1038/s41564-019-0526-2

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