Precise determination of the genome of a species is important to understanding disease and for the development of diagnostic and screening tests that will improve disease treatment. The genome has been sequenced in humans, dogs, horses, cattle, cats, and several other species. Unfortunately, the genomic sequence has only been determined in a single cat. Cinnamon was an Abyssinian cat from the University of Missouri whose genome was sequenced in 2007. Because the genome was mapped in a single animal, and since less robust methodologies were used, we lack the information needed to develop tests that may help in the identification of mutations and treatment of disease.

A research community-based project has developed that will sequence the genome of 99 or more cats in order to:

 Improve coverage of the cat genome

 Improve future assemblies of the cat genome

 Identify normal and abnormal genetic variation

 Identify causative mutations for specific health concerns

 Allow veterinary hospitals to provide individual genome sequencing for cats for state-of-the-art health care (similar to the developing standard of care for humans)

The 99 Lives project is being coordinated by the Lyons Feline Genetics Laboratory at the University of Missouri - Columbia to help ensure that:

 Different cats from different breeds and locations are sequenced to maximize variation

 Similar technologies are used to facilitate efficient data management, transfer and variant calling

 Data quality is high and consistent

 Data are publically available so that all members of the research community can benefit in a non-competitive and collaborative manner

 Researchers less familiar with genomics research and large-scale bioinformatics will have a resource for data interpretation

 Follow standards and precedence set by human genetics efforts

All data from the project will become publically available; however, participants have access to data prior to publications.

99 Lives Researcher Participation Expectations

Participants in the project are expected to provide high-quality sequence and deep coverage of a unique cat genome as part of the collaboration.

 Currently, Illumina HiSeq 100 bp paired end sequencing reads are supported by the project. Sequencing should be performed from two PCR-free libraries per cat of 350 bp and 550 bp for at least 15x coverage; 20x coverage for standard libraries. Contributors must provide the sequencing details for each genome and can perform sequencing at their own preferred facilities.

 The data need to be transferred to Maverix Biomics at a cost of $1,000 per cat.

 Maverix Biomics (www.maverixbio.com) is based in San Mateo, CA. They will align the reads to the cat genome Felis_catus-6.2 sequence (www.ncbi.nlm.nih.gov/genome/78?genome_assembly_id=22791) and will use FreeBayes and/or Platypus to call SNPs. The cat reads and SNPs are overlaid onto a UCSC type browser for easy viewing of the data. Data tables can be accessed that provide the identified SNPs in specific genes, regions, chromosomes, or the entire genome. Maverix will provide a cumulative analysis of the variant calls for all cats after large groups or specific milestones (20, 40, 60, 80 and 99) of new cat genomes have been added to the database. Variant calling to new cat genome assemblies will be conducted when the annotated assemblies are available.

 Once a genome dataset is transferred to Maverix and funds accepted, the contributor will be given access to the website for all the data in the 99 Lives cat genome data.

 The Lyons' Laboratory may be able to subsidize some data contributions.

Contributors are expected to provide the basic signalment of the cats when possible, such as gender, breed, place of origin, and coat color.

 One of the most significant concerns of a shared resource is the potential for overlapping projects and performing data analyses on contributed genomes that would interfere or "scoop" the research of another contributor. Therefore, the specific phenotype(s) of interest can be withheld from the cat identification information. However, we fully expect a highly cooperative and collaborative effort for participation in the resource.

 Researchers can privately contact a subset of contributors to ask for phenotypes of interest and for collaborative efforts.

 All cats are welcome, including exotic and wild felids.

All contributors to the 99 Lives data resource are expected to be co-authors on the first publication that is produced by each contributor who uses the data resource.

 All funding sources need to be provided for acknowledgements and included in each publication and presentation using the 99 Lives resource data.

 All the cat genome data can be downloaded by other investigators who are interested in other data analyses of the cat genome provided they make a contribution to the resource.

 NIH-funded cat sequences will be deposited into NCBI Short Read Archive (SRA) as well as any other sequences volunteered by the researchers.

99 Lives Cat Genome Sequencing Initiative Contributors

The 99 Lives project has developed as collaboration between the University of Missouri - Columbia, College of Veterinary Medicine and the University of California - Davis, but is extended to all researchers interested in feline health care. The Feline Genetics Laboratory of Leslie Lyons, PhD, at the University of Missouri is coordinating the project. For details, please e-mail: lyonsla@missouri.edu or felinegenome@missouri.edu or call 01 573 884 2287.

The Lyons' Feline Genetics Laboratory will provide periodic updates regarding the project to the contributors and the research community.

The contributors listed below have agreed to a Memorandum of Understanding (MOU) and have submitted or are anticipating the contribution of cat genome sequences to the 99 Lives Cat Genome Sequencing Initiative. Several other investigators are interested in contributing and are raising funds for sequencing.

The Winn Feline Foundation supported the first whole genome sequences of nine cats - the 9 Lives Project. Zoetis also made a significant donation to support the project. Via support from National Geographic, 12 random bred cats have been whole genome sequenced to help identify the normal cat genetic variation throughout its worldwide range.

To date, 51 domestic cats and 4 exotic cats, including a trio of Black-footed cats with a retinal degeneration and a Pallas cat with polycystic kidney disease, have been submitted to the project. The publically available sequences for two lions, a tiger, an Amur leopard, and a Snow leopard are being retrieved. The Felid taxonomic advisory group (TAG) is being approached to support the effort.

Via funding from the Feline Health Center at Cornell (www.vet.cornell.edu/fhc), funding is available to perform in-depth phenotype for health, such as MRIs, CTs, echocardiograms, ultrasound, radiographs, blood work, urinalysis, and other diagnostic techniques.

Table 1 presents the investigators who have coordinated the submission of samples, which is supporting the genetic studies for many students, postdoctoral fellows and researchers.

To date, the Lyons' Laboratory has been successful with the identification of the variant causing Devon Rex "spasticity," a model for congenital myasthenic syndrome, retinal degenerations in the Bengal, Persian and Black-footed cats, polycystic kidney disease in the Pallas cat, and cutaneous asthenia in Burmese, a type of Ehlers-Danlos syndrome. Other diseases are under investigation, such as inherited mediastinal lymphoma in Oriental shorthair cats, dwarfism, coat colors and fur types, and feline orofacial pain.

A crowdfunding site has been developed to help raise support for the project from the lay public. Please visit: felinegenetics.Missouri.edu/99lives.

Public funding has been raised to sequence Lil' Bub and four private donor cats. Breed groups are working with the Winn Feline Foundation to support breeds with specific health concerns - such as Oriental and Siamese cats with amyloidosis. Other investigators are initiating detailed projects on hypertrophic cardiomyopathy and studies on small wild felids. The Lyons laboratory hopes to support studies on amyloidosis in Black-footed cats, transition cell carcinoma in Fishing cats, and Vitamin A deficiency in lions.

Table 1. 99 Lives cat genome sequencing participants

How Cat Veterinarians and Breeders Help?

All genetic projects need the cooperation and continued assistance of veterinarians and cat owners and breeders. Veterinarians are key to the ascertainment of cats that have health concerns with a significant genetic component. Proper and in-depth diagnoses are critical to the project. We welcome any new disease or trait presentations to the study. We hope concerned individuals will help us to raise funds to sequence cats with interesting health conditions. Breeders and cat owners can launch their own crowdfunding efforts to raise funds, as have the Siamese and Oriental breeders, to help sequence a cat of every breed!

Healthy cats are just as important to the project as cats with afflictions as the healthy cats help to identify the normal, non-malicious DNA variant in the genome.

Please help us spread our interests and the website for the 99 Lives Cat Genome Sequencing Initiative!

Acknowledgements

Feline Genetics Laboratory at MU

Barbara Gandolfi, Erica Creighton, Nick Gustafson, Madison Bullock, Victoria Spreyer, Sara Shippey, Destiny Monroe, Jena Grahn and Ashley Bullock.

Devon Rex Project

Peter Dickinson, Beverly Sturges, D. Colette Williams, Robert A. Grah - UC Davis; C. Diane Shelton - UC San Diego; Maria Longeri - University of Milan; Peter Leegwater - Utrecht University.

The 99 Lives Participants and Maverix Biomics, Inc.

Max Rothschild, Matthew Ellinwood, Dorian Garrick, Niels Pedersen, Tosso Leeb, Hannes Lohi, William Swanson, Greg Barsh/Chris Kaelin, William Murphy, Rory Todhunter/Adam Boyko/Marta Castelhano/Colin Parrish.