Small RNAs (sRNAs) are important regulatory elements in eukaryotic organisms and comprise the functional elements of RNA interference. Numerous classes of sRNAs have been annotated, however they vary greatly in their ease of annotation and compatibility with most annotators. Significant challenges exist for the annotation process, including variation in sRNA library quality, alignment depth, and poorly defined loci, collectively making this process difficult. Additionally, few annotators are fully agnostic to sRNA classes and may struggle identifying loci in less explored organisms. To address these problems, we present the annotation tool YASMA-tradeoff (YTO), which is specifically suited to finding reliable thresholds for locus annotation which balance sensitivity with specificity. We compared YTO with other annotators, we show that it and other pipelines based on coverage-normalization methods have great advantages, balancing many metrics to produce a more reproducible annotation. We also demonstrate that YTO produces more contiguous and representative loci, through the aggressive merging of similar expressed regions. Finally, we also show that the tool produces much more descriptive locus dimensions, a major advantage in species where sRNAs may be distinct or unique. Overall, we demonstrate substantial improvements in annotation accuracy, reproducibility, and description, particularly in non-model organisms and less-explored clades.
Keywords: Eukaryotes; MiRNAs; Non-model organisms; PiRNAs; SiRNAs; TinyRNAs; Total small RNAs.
© 2025 The Authors.