title: Comparison of the Equine Reference Sequence with Its Sanger Source Data and New Illumina Reads.
abstract: The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight's half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects' and Twilight's genome or due to errors in the reference. EquCab2 is regarded as "The Twilight Assembly." The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies between new, high throughput genomic sequence data generated for Twilight and the EquCab2 reference assembly. Most of these represent errors in the assembly, while approximately 10,000 are demonstrated to be contributions from another horse. Other results are presented that include the binary alignment map file of the mapped Sanger reads, a list of variants identified as discrepancies between the source data and resulting reference, and a BED annotation file that lists the regions of the genome whose consensus was likely derived from low coverage alignments.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Jovan
authors.initials: J
authors.lastname: Rebolledo-Mendez
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Hestand
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Coleman
authors.affiliation: Department of Computer Science, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Orlando
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: James N
authors.initials: JN
authors.lastname: MacLeod
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, Kentucky, United States of America; Intrepid Bioinformatics, Louisville, Kentucky, United States of America.
authors.forename: Ted
authors.initials: T
authors.lastname: Kalbfleisch
mesh_terms: D000818:Animals; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D006736:Horses / Q000235:genetics*; D017422:Sequence Analysis, DNA