The ARCHAIC project delivered several results that extend beyond current virome research standards:
- We systematically evaluated multiple methods for viral detection in ancient metagenomes and demonstrated how different taxonomic classifiers perform under challenging conditions typical of ancient DNA (aDNA), such as short read lengths and damage-induced substitutions. The benchmarking analysis revealed that while genus-level classification is relatively robust across classifiers, species-level annotation remains problematic, especially with comprehensive databases that include bacterial genomes. This insight underscores the need for specialized, virus-focused databases when analyzing ancient samples.
- By applying our optimized pipeline to real ancient datasets from four archaeological sites (Boomerang Shelter, Zape Cave, Arid West Cave, and Hallstatt), we detected over 260 viral species. After rigorous validation through genome breadth coverage and DNA damage profiling, 38 high-confidence viral species were confirmed, all of which were bacteriophages. These findings included both widespread phage taxa and site-specific ones illustrating both ubiquity and local adaptation of ancient phages.
- We also compared assembly-based viral detection using MEGAHIT and metaSPAdes. Although MEGAHIT produced fewer but more complete contigs, both tools contributed to improved viral genome reconstruction when combined with relaxed parameter detection using geNomad. Additionally, our work demonstrated that contig-based analysis could recover viruses missed at the read-level due to sequence degradation, emphasizing the complementary nature of these methods.
Importantly, our approach identified novel viral contigs with no current database match, indicating the presence of ancient viral lineages previously undescribed in modern samples. These findings suggest that ancient samples may harbor unique phage diversity lost in modern times due to changes in lifestyle, environment, or microbiome composition.
Overall, these contributions provide not only a detailed portrait of ancient virome structure and evolution, but also deliver a robust methodological framework for future studies in ancient viral ecology.
