Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project

Abstract

Summary The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes and across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

Improved reference genome for the domestic horse increases assembly contiguity and composition

“…The initial phase of this project has produced RNA-sequencing (RNA-seq) and whole-genome shotgun (WGS) sequence data from two Thoroughbred mares that are not the subject of the reference assembly. Data from both horses have been mapped to both EquCab2 and EquCab3 25 . The first phase of the equine FAANG effort was comprised of RNA-seq data from eight tissues.…”

Functionally Annotating Regulatory Elements in the Equine Genome Using Histone Mark ChIP-Seq

“…Along with the regulatory regions identified in this study, the equine FAANG consortium is characterizing the full RNA profile of more than 50 tissues and cell types from the FAANG equine biobank supported by a research community initiative [26] and the DNA methylation profiles for the TOI. Moreover, the equine FAANG consortium is currently mapping a major insulator protein known as CTCF, with the goal of generating tissue-specific chromatin state predictions for the eight TOI.…”

“…Jensen-Shannon distance is a common statistic used to compare two distributions that can be applied to ChIP datasets using deepTools version 2.4.3 [28], and a threshold was determined by agreement among FAANG collaborators. Tissues of interest (TOI) used in the final experiments that generated combined peak-calls were collected from two thoroughbred mares (referred to in this manuscript as ECA_UCD_AH1 for SAMEA104728862 and ECA_UCD_AH2 for SAMEA104728877) as part of the FAANG equine biobank [26], and all protocols for this work were approved by the University of California, Davis Institutional Animal Care and Use Committee (Protocol #19037). Laboratory procedures that varied based on tissue are summarized in Table 1.…”

“…As part of the international FAANG collaboration, we performed ChIP sequencing (ChIP-Seq) on four major histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight equine tissues (adipose, brain (parietal cortex), heart, lamina, liver, lung, (skeletal) muscle, and ovary). These tissues were collected as part of the FAANG equine biobank, which includes samples from 80 tissues, six fluids, and two cell lines that were collected from two healthy adult thoroughbred mares [26]. The eight tissues evaluated in this study were prioritized for thorough investigation based on (1) continuity with other FAANG species to enable across species comparisons and/or (2) the primary needs of the equine community in terms of health, performance, and reproduction.…”

Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

“…Liver and parietal lobe of the cerebrum were collected from two healthy Thoroughbred mares (adult horse 1: AH1 and adult horse 2: AH2) [20]. RNA was isolated and prepared for sequencing with poly-A + selection and rRNA-depletion.…”