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Phage-inclusive profiling of human gut microbiomes with Phanta

Nature Biotechnology volume 42, pages 651–662 (2024)Cite this article

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Abstract

Due to technical limitations, most gut microbiome studies have focused on prokaryotes, overlooking viruses. Phanta, a virome-inclusive gut microbiome profiling tool, overcomes the limitations of assembly-based viral profiling methods by using customized k-mer-based classification tools and incorporating recently published catalogs of gut viral genomes. Phanta’s optimizations consider the small genome size of viruses, sequence homology with prokaryotes and interactions with other gut microbes. Extensive testing of Phanta on simulated data demonstrates that it quickly and accurately quantifies prokaryotes and viruses. When applied to 245 fecal metagenomes from healthy adults, Phanta identifies ~200 viral species per sample, ~5× more than standard assembly-based methods. We observe a ~2:1 ratio between DNA viruses and bacteria, with higher interindividual variability of the gut virome compared to the gut bacteriome. In another cohort, we observe that Phanta performs equally well on bulk versus virus-enriched metagenomes, making it possible to study prokaryotes and viruses in a single experiment, with a single analysis.

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Fig. 1: Overview of Phanta’s comprehensive, virus-inclusive metagenomic annotation workflow.
Fig. 2: Evaluation of Phanta’s performance using simulated metagenomes.
Fig. 3: Evaluation of Phanta’s performance using shotgun gut metagenomes from 245 healthy human adults.
Fig. 4: Core properties of the healthy adult virome.
Fig. 5: Application of Phanta to n = 143 pairs of virus-enriched and bulk metagenomes from infant gut samples.

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Data availability

Accession numbers of all publicly available metagenomes used for analysis are provided in Supplementary Table 11. Source data for individual figures are provided with this manuscript. Phanta’s databases are available from the links specified at https://github.com/bhattlab/phanta (ref. 45). There are no restrictions on data availability. Source data are provided with this paper.

Code availability

Workflows were used for the preprocessing and assembly steps described in Methods and the workflows are available at https://github.com/bhattlab/bhattlab_workflows. Phanta and its postprocessing scripts are publicly available at https://github.com/bhattlab/phanta (ref. 45) with a detailed tutorial describing installation and usage.

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Acknowledgements

We thank D. Maghini and B. Doyle for thoughtful comments on the manuscript; S. Nayfach, P. Hiseni, S. Salzberg and J. Lu for helpful conversations; B. Doyle and J. Wirbel for testing Phanta; and B. Siranosian, C. Nicolau, K. Bettinger, A. Behr and the Stanford Research Computing Center for computational support. Computing costs were supported, in part, by an NIH S10 Shared Instrumentation under grant 1S10OD02014101. Figure 1 was created using BioRender.com. This study was supported in part by NIH R01AI148623 and R01AI143757, a Stand Up 2 Cancer Grant, the Chan Zuckerberg Initiative, a Sloan Foundation Fellowship and the Allen Distinguished Investigator Award (to A.S.B.). Y.P. is supported by the School of Medicine Dean’s Postdoctoral Fellowship. M.C. was supported by an NIH-funded predoctoral fellowship (5T32HG000044-25) and is supported by the National Defense Science and Engineering Graduate Fellowship (starting September 2022).

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  1. These authors contributed equally: Yishay Pinto, Meenakshi Chakraborty.

Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, CA, USA

    Yishay Pinto, Meenakshi Chakraborty, Navami Jain & Ami S. Bhatt

  2. Department of Medicine, Divisions of Hematology and Blood & Marrow Transplantation, Stanford University, Stanford, CA, USA

    Yishay Pinto, Navami Jain & Ami S. Bhatt

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Contributions

Y.P., M.C. and A.S.B. conceived the study and wrote the manuscript. Y.P. and M.C. developed Phanta. Y.P., M.C., N.J. and A.S.B. analyzed the data. All authors read and approved the final manuscript.

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Correspondence to Ami S. Bhatt.

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Pinto, Y., Chakraborty, M., Jain, N. et al. Phage-inclusive profiling of human gut microbiomes with Phanta. Nat Biotechnol 42, 651–662 (2024). https://doi.org/10.1038/s41587-023-01799-4

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