{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T13:12:54Z","timestamp":1784034774878,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,10]]},"group-title":"Bioinformatics","reference-count":46,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T00:00:00Z","timestamp":1783641600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>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\u2013based 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\u2014including sex differences in killifish liver\u2014and 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\u2013based analysis of next-generation sequencing data.<\/jats:p>","DOI":"10.64898\/2026.07.06.736868","type":"posted-content","created":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:12Z","timestamp":1783728012000},"source":"Crossref","is-referenced-by-count":0,"title":["MKMC enables reference-free transcriptomic analysis using k-mer representations"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-5047-5351","authenticated-orcid":false,"given":"Lajoyce","family":"Mboning","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of California, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5986-4979","authenticated-orcid":false,"given":"Maciej","family":"D-lugosz","sequence":"additional","affiliation":[{"name":"Department of Algorithmics and Software, Silesian University of Technology, Gliwice, Poland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6420-1587","authenticated-orcid":false,"given":"Marek","family":"Kokot","sequence":"additional","affiliation":[{"name":"Department of Algorithmics and Software, Silesian University of Technology, Gliwice, Poland."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7320-8652","authenticated-orcid":false,"given":"Jingxun","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Genetics, Stanford University, School of Medicine, California, United States."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9431-6852","authenticated-orcid":false,"given":"Emma K.","family":"Costa","sequence":"additional","affiliation":[{"name":"Department of Neurology and Neurological Sciences, Stanford University, California, United States."},{"name":"Neurosciences Interdepartmental Program, Stanford University School of Medicine, California, United States."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Man-Ru","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Mary M. and Sash A. 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