Reducing haystacks to needles-ViralClust: a Nextflow pipeline to cluster viral sequences

The rapid accumulation of viral genome sequences presents major challenges for downstream analysis tools, including tools for multiple sequence alignments, phylogeny, and genome/alignment visualization, due to computational constraints and sampling biases caused by outbreak-driven over-representation. Selecting representative genomes through clustering offers a principled alternative to random subsampling, yet choosing appropriate clustering strategies remains non-trivial and context-dependent.

Here, we present ViralClust, a modular Nextflow pipeline for bias-aware representative selection from large viral genome datasets. ViralClust integrates 5 distinct clustering algorithms (CD-HIT-EST, SUMACLUST, VSEARCH, MMSeqs2, and HDBSCAN) within a unified workflow, enabling direct comparison of clustering outcomes and flexible adaptation to diverse biological questions, considering a balanced phylogenetic distribution of the selected sequences. We evaluated ViralClust on 6 RNA and DNA virus datasets ranging from 632 to 156,586 sequences and spanning genome lengths from 890 to 197,185 nucleotides. Across all datasets, clustering reduced dataset size by ~95% or more while preserving genetic diversity across species, genera, and families, and effectively mitigating biases introduced by outbreaks, partial genomes, and sequence orientation artifacts.

By supporting whole-genome clustering and scalable representative selection, ViralClust enables efficient and reproducible downstream analyses that would otherwise be computationally infeasible. Rather than offering a prescriptive, guided analysis engine, our framework functions as a flexible comparative collection of complementary strategies, allowing users to empirically evaluate trade-offs and choose the ideal method tailored to their specific analytical endpoints.