Rapid construction of reference chromosome-level mammalian genome assemblies and insights into the mechanisms of gross genomic rearrangement

We live in an era in which the genomes of new species are being sequenced all the time. The most modern ways to sequence DNA have many advantages over older approaches (the prominent one being a vastly reduced cost) but a problem that arises each time the genome of a new species is sequenced is that assigning large blocks of sequence to an overall genomic "map" can be problematic and/or very expensive. It's a little like finding your location on Google Maps but not being able to "zoom out" to establish where that position is in relation to the whole country. In essence the aim of this project is to rectify this problem at one fifth of the current cost. Using our experience with birds we have developed a high-throughput approach and the tools for assigning the sequences to their proper positions in chromosomes. This involves our own adaptations to a technique called "FISH" that can take the data from sequenced genomes and visualize directly blocks of DNA sequence as they appear in their rightful place in the genome. In this study we will focus our attention on 25 newly sequenced mammal species. More importantly however we will provide the means through which this can be achieved for any of the 5,000 living mammalian species. Mammals are important to our lives in that many are models for human disease and development and are critical to agriculture (both meat and milk). Others are threatened or endangered and, with impending global warming, molecular tools for the study of their ecology and conservation are essential. Our combined efforts have also developed computer-based browser methods to compare the overall structure of one genome with another, directly visualizing the similarities and differences between the genomes of several animals at a time, something we can share widely amongst the scientific community and general public via the world wide web. The differences between mammalian genomes arose through changes that happened during evolution. One of the main aims of this project is to find out how this occurred and what are implications of these changes. We have a number of ideas such as we think there may be different "signatures" that classify why blocks of genes tend to stay together during evolution. Armed with this information, we fully intend to take it out into the world. The devices that we will develop can be adapted for the screening of individual animals for genomic rearrangements that may cause e.g. breeding problems. Moreover, the resources we will develop provide a source for public information and student learning through a dedicated, outwardly-facing web site. We have received overwhelming support from numerous laboratories all over the world who are interested in using the resources that we will develop to ask biological questions of their own. For this reason, we feel that this project will help us understand evolution in mammals and contribute to establishing the UK as a central international hub of mammalian genomics.

Unless a whole genome sequence is assembled to the level of one "(super)scaffold" per chromosome, the resultant assembly can be studied for gene structure and function but cannot be used effectively to address biological questions pertaining to critical aspects of evolutionary and applied biology. Multiple letters of support for this application attest to this. Contemporary genome sequencing projects however usually fall short of this "chromosome level" assembly unless supported by extensive funding resources (~$100,000/genome). In reality, with the genomes of more animals being sequenced but with limited resources, this problem will only increase unless lower cost solutions can be found. Recently we have, in birds, developed means of taking sub-chromosomal sized scaffold based assemblies (e.g. enhanced by Dovetail or bioinformatically by RACA) and "upgrading" them to chromosome level at a fraction ~20% of the cost. This approach involves a novel method of selecting BAC clones that will hybridise to any mammalian metaphase then multiplex adaptations of FISH approaches. Mammals are the most studied phylogenetic Class, however only ~25/5000 species have sequenced genomes assembled to chromosome level. Indeed, most recent de-novo sequencing projects typically produce assemblies of several super-scaffolds per chromosome. Our approach will upgrade 25 further genomes and provide both proof of principle and the practical means through which many hundreds more can be mapped and compared. Our approach will allow easy comparative visualization of multiple genome assemblies and testing of fundamental hypothesis pertaining to the importance of overall chromosome structure in the formation of lineage-specific and ancestral phenotypes and the conservation of blocks of homologous synteny who's functional and sequence features define phenotypic traits with medical, veterinary or agricultural relevance.

At the core of this application is a commitment to high impact activity, specifically benefitting industry (UK plc), academia, the third sector and the general public (academic beneficiaries are dealt with in another section). The primary industrial supporter (and beneficiary) of this research is Cytocell Ltd who specialize in the development of multiple hybridization FISH probes. Building on a long-standing collaboration initiated by a Knowledge Transfer Partnership for the development of non-human probes, the company is very interested in our approach as it will lead to new product development and maximize the potential of the human BAC collection present in the company. After extensive market research we have collectively identified "chromosome evolution Multiprobe devices" and a range of individual animal translocation screening devices. Cytocell's generous in-kind contribution is outlined in the application and, as clearly stated, represents a genuine partnership incorporating real cash-equivalent contributions designed to maximize our collective skills to bring cross species hybridization probes to market and thus ultimately to the scientific community. Going into partnership with a company in this way means that the highest possible quality product can reach the widest market worldwide.

Digital Scientific UK have identified considerable benefit in collaboration on this project through the development of its new animal karyotyping software suites as a contribution to this project. They have generously agreed to provide these free of charge. Their new "Batch Capture" protocols integrating microscope hardware with their in house algorithms for multiple FISH capture normally are charged to customers at market price but the company have kindly donated unlimited use software to this project. Both these companies also see this project as means of working together with one anther more closely, adding to their R&D portfolio and thereby increasing their share value and the value of UK plc. Finally, Dovetail see value to their company and efforts to generate contiguous chromosome assemblies, seeing our approach as entirely complementary to theirs.

A gap in perception exists in understanding the role of gross chromosomal evolution in academia and industry. While in academia it is accepted that chromosome structures play an important role in gene regulation, industry application is still focused mostly on protein changes and ignores many other features of the genome. Our project will aim to start changing this perception by providing popular resources and outreach activities for non-scientists. These resources and events will hopefully have influence on the general public including the future policy makers (see Pathways to impact for details). Therefore, we expect to have an impact on future policies in animal sciences.

The third sector (museums) will benefit from our project through the inclusion of mammalian chromosome evolution histories into the interactive tools aiming at student education and popular science exhibitions in museums. One of such tools we recently built with ESEB is called 'Evolution Factory' which teaches schoolchildren the principles of chromosome and genome evolution. A more advanced version of the tool is interactive screen that we develop with a group from the University of California at Davis to be displayed in San Francisco Exploratorium. After the tool is developed and tested we will also approach the London Science Museum to investigate their interest in using this and other interactive games we develop for their exhibitions.