Abstract

Traditional RNA-seq analysis depends heavily on genome alignment and gene annotation, limiting its utility in non-model organisms and introducing biases that can obscure regulatory complexity. We present MKMC (Multi-sample Kmer Counter), a scalable, reference-free toolkit for RNA-seq analysis that leverages k-mer-based statistics to detect biological variation without requiring alignment. MKMC integrates fast k-mer counting, abundance matrix generation, normalization, dimensionality reduction, and differential analysis into a unified workflow. Across diverse datasets, MKMC recapitulates key biological signals, including sex differences in killifish liver, and matches alignment-based pipelines in differential expression analysis and transcriptomic age prediction. Notably, MKMC detects isoform-specific events missed by traditional methods, one of which we validated using in situ hybridization. These results reveal previously hidden isoform-level regulatory events that contribute to sex- and age-associated transcriptional programs. MKMC offers a robust, extensible alternative to alignment-based approaches, enabling transcriptomic discovery across both model and non-model systems. While we focus here on RNA-seq as a primary application, MKMC is broadly applicable to any k-mer-based analysis of next-generation sequencing data.