The domestic cat now has one of the best genome assemblies for mammals, termed telomere-to-telomere (T2T). Whole genome and exome sequencing is becoming commonplace to identify causal variants for various phenotypes and diseases in any cat, a process that is part of Precision Medicine. But as like whole genome and exome sequencing studies in humans, only 40-50% of the attempts to find causal variants are successful in cats. One cause for failure is poor annotation of gene structure in the cat reference assembly. Most mammals have approximately 21,000 coding genes but over 100,000 transcripts, each gene averaging three transcripts. The transcript expression is dependent on the tissue type, time of development, and disease state. Thus, if a transcript with an alternative exonic structure or a long transcript has not been previously or fully sequenced, this transcript’s structure is likely missed in the genome assembly annotation. Hence, improved knowledge of alternative transcripts will improve genome assembly and thereby improve the success rate for causal variant discovery. Other species have the same variant discovery problems and thus a consortium called FAANG – Functional Annotation of ANimal Genomes – was developed. This consortium is based on the human Genotype Tissue Expression project (GTEx) in that specific tissues are collected for RNA sequencing studies to identify alternative transcript expression. This proposal will follow FAANG and GTEx consortium recommendations to improve the cat genome annotation and to allow cross-species comparisons. Short-read RNA sequencing has been conducted previously as part of Cornell Feline Health Center funded projects and also by the collaborators. However, short-read RNA sequencing is inadequate for the proper annotation of medium to larger genes, which tend to have more alternative transcripts. The goal of this project is to improve the annotation of the genome assembly by improving alternative transcript identification and completeness using long-read RNA sequencing. The hypothesis is that better genome annotation will facilitate DNA variant discovery for both simple Mendelian and complex diseases and traits, and will support cross-species genomic studies. The specific aims are as follows: Aim 1: to perform whole transcriptome sequencing on RNA isolated from 62 feline tissues to create a resource for the improvement of the cat genome annotation; Aim 2: to demonstrate the usability of the improved feline annotation from aim 1 for ongoing feline disease studies, for example, diabetes mellitus.