Long-read RNA-seq resolves isoform-level and context-specific regulatory architecture of complex traits in cattle

Zheng, W.; Zhang, Q.; He, J.; Zhu, X.; Gong, M.; Li, H.; Liu, L.; Li, J.; Ma, Z.; Han, B.; Zhang, Y.; Lu, J.; Lin, B.; An, B.; Guo, Z.; Liu, Y.; Du, A.; Zhu, D.; Liu, X.; Lu, X.; Fang, L.; Sun, D.

doi:10.64898/2026.06.04.730050

bioRxiv · 10.64898/2026.06.04.730050

Long-read RNA-seq resolves isoform-level and context-specific regulatory architecture of complex traits in cattle

Zheng, W.·Zhang, Q.·He, J.·Zhu, X.·Gong, M.·Li, H.·Liu, L.·Li, J.·Ma, Z.·Han, B.·Zhang, Y.·Lu, J.·Lin, B.·An, B.·Guo, Z.·Liu, Y.·Du, A.·Zhu, D.·Liu, X.·Lu, X.·Fang, L.·Sun, D.

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Abstract

Traditional short-read RNA-seq lacks the resolution to fully capture full-length isoforms and complex alternative splicing, leaving a "missing regulation" gap in existing molecular atlases. Here, we present a population-scale, multi-omics dissection of bovine complex traits using matched deep whole-genome sequencing (30x), long-read (ONT) and short-read RNA-seq, as well as metabolome data from 432 dairy cows across four distinct lactation stages. We expanded the bovine transcript atlas with 13,177 novel isoforms and fine-mapped 1,088,900 regulatory effects across 11 diverse molecular phenotypes. Notably, long-read mapping uncovered 606 genes with isoform expression quantitative trait loci (12.8%, eQTL) and 1,116 genes with detailed splicing QTL (sQTL) (31.8%) that were missed by conventional short-read mapping. We further showed that lactation-specific regulation was profoundly mediated by the metabolic microenvironment (46.8%) and cellular composition (4.3%). Across genome-wide associations (GWAS) of 90 bovine complex traits, isoform-level and context-specific regulatory effects captured GWAS loci missed by short-read eQTL and sQTL analyses, increasing colocalized loci from 35.2% to 60.3%. These regulatory variants also exhibited evolutionary constraints with human immune and metabolic loci. Our study establishes an invaluable resource (https://cattleblr.farmgtex.org) demonstrating that long-read transcriptomics is essential for resolving the complexity of regulatory mechanisms underlying mammalian physiology.

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Zheng, W., Zhang, Q., He, J., Zhu, X., Gong, M., Li, H., Liu, L., Li, J., Ma, Z., Han, B., Zhang, Y., Lu, J., Lin, B., An, B., Guo, Z., Liu, Y., Du, A., Zhu, D., Liu, X., Lu, X., Fang, L., Sun, D.. 2026-06-05. Long-read RNA-seq resolves isoform-level and context-specific regulatory architecture of complex traits in cattle. https://doi.org/10.64898/2026.06.04.730050

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