Manual resolution of virome dark matter uncovers hundreds of viral families in the infant gut

The gut microbiome (GM) is shaped through infancy and plays a major role in determining susceptibility to chronic diseases later in life. Bacteriophages (phage) are known to modulate bacterial populations in numerous ecosystems, including the gut. However, virome data is difficult to analyse because it mostly consists of unknown viruses, i.e. viral dark matter. Here, we manually resolved the viral dark matter in the largest human virome study published to date. Fecal viromes from a cohort of 647 infants at 1 year of age were deeply sequenced and analysed through successive rounds of clustering and curation. This uncovered more than ten thousand viral species distributed over 248 viral families falling within 17 viral order-level clusters. Most of the defined viral families and orders were novel and belonged to the Caudoviricetes viral class. Bacterial hosts were predicted for 79% of the viral species using CRISPR spacers in metagenomes from the same fecal samples. While Bacteroides-infecting Crassphages were present, novel viral families were more predominant, including phages infecting Clostridiales and Bifidobacterium. Phage lifestyles were determined for more than three thousand caudoviral species. Lifestyles were homogeneous at the family level for 149 caudiviral families. 32 families were found to be virulent, while 117 families were temperate. Virulent phage families were more abundant but temperate phage families were more diverse and widespread. Together, the viral families found in this study represent a major expansion of current bacteriophage taxonomy, and the sequences have been put online for use and validation by the community. Introduction The establishment of the gut microbiome (GM) during the first years of life plays a pivotal role in the maturation of the infant immune system1,2. Early-life GM dysbiosis has been linked to a series of chronic diseases occurring later in life, indicative of an immune system thrown off balance3–6. Most existing research has been on the bacterial component of the GM but in recent years it has become evident that other microbes and especially viruses are prominent GM members. They populate the gut during the first months of life following a patterned trajectory resembling the establishment of gut bacteria7–10. Bacteriophages (phages) are viruses that infect bacteria in a host specific manner. Virulent phages multiply by killing their host bacteria. Temperate phages can integrate into the bacterial chromosome as prophages, thereby postponing an attack on the host until conditions are favourable. Some phages also cause chronic infections leading to continuous shedding of viral particles11. Bacteria will defend themselves using defence systems such as Clustered Regularly Interspaced Palindromic Repeats (CRISPR), an adaptive immune mechanism where records of past infections saved on the chromosome help combat any future attacks12. Lately, it has become clear that phages are not only abundant in the gut, but also possess the ability to alter GM composition and function8,13. Moreover, the reported interactions between phage proteins and the host immune response14–16 suggest a tripartite interaction