MirMachine 2: a scalable, evolutionarily informed pipeline for microRNA annotation and comparative genomics across thousands of animal genomes

Genome sequencing is rapidly outpacing the annotation of conserved regulatory elements, limiting the evolutionary and comparative insights that can be extracted from expanding genome collections. MicroRNAs are among the most conserved and phylogenetically informative genes, yet automated annotation has remained difficult to scale while preserving evolutionary interpretability. Here we present MirMachine 2, an evolutionarily informed framework that combines curated reference models, lineage-aware scoring, and adaptive filtering to enable robust genome-wide microRNA annotation at scale. Applying this to thousands of animal genomes reveals that many apparent absences of conserved microRNAs reflect methodological bias rather than biological loss, particularly in underrepresented lineages. By enabling consistent and interpretable comparison of microRNA complements across large datasets, MirMachine 2 establishes scalable microRNA annotation as a practical foundation for genome-scale evolutionary and comparative genomics.