Genome assembly. chromosomal organization and comparative genomics of multiple bird species: beyond "catalogues of genes"

Lead Research Organisation: Aberystwyth University
Department Name: IBERS

Abstract

Imagine trying to navigate through a town with only the index from your A-Z to guide you and not the street map. In essence, this is what happens when the genome of a new species is sequenced. Old fashioned technology used to "sketch the outline of the streets" first before filling in the gaps with the gene sequence. The new technology (so called "Next Generation Sequencing") is much cheaper and quicker but is quite poor at sorting out where the genes are in relation to one another. Essentially, Next Generation Sequencing assemblies are little more than catalogues of genes with little structure of the overall genome apparent. Fortunately this problem can be rectified using a technique called "FISH" that can take the data from next generation sequencing projects and visualise directly the genes as they appear in their rightful place in the genome. In this project we intend to do this with 25-30 recently completed genome sequences from birds. Birds are among the most diverse animals with around 10,000 living species. Many are models for human disease and development and are critical to agriculture (both meat and eggs). Others are threatened or endangered and, with impending global warming, molecular tools for the study of ecology and conservation of birds are essential. It is also possible to compare the overall structure of one genome with another, somewhat like comparing the maps of towns with similar layouts. We have developed online tools that can directly visualize the similarities and differences between the genomes of several birds at a time. Of course the differences between these bird genomes came about through changes that happened during evolution. One of the main aims of this project is to find out how and why this occurred. We have a number of ideas such as we think there may be different "gene signatures" at the places in the genome where evolutionary rearrangements are more or less likely to occur. We think that the whole process may be related to the mechanisms by which our genes recombine in our germ cells. We also think there may be a role for small pieces of DNA that "float around" these bird genomes. Finally, we have received overwhelming support from numerous laboratories all over the world, all of whom are interested in bird genomics and who would wish to use our online resource to ask biological questions of their own. For this reason we feel that this project not only will help us understand evolution in birds but also establish the UK as a central international hub for work of this kind.

Technical Summary

Every genome sequence needs a good map however modern next generation sequencing (NGS) technologies struggle fully to assemble whole genomes de-novo. This is disappointing as mapping information on a chromosomal basis brings the opportunity to address many fundamental biological questions of genome evolution. Modern genomic research generally focuses on the role of individual genes but the role of chromosomes and homologous synteny blocks (HSBs) is often disregarded. This is despite the ubiquity of HSBs and the phenotypic consequences/evolutionary implications of chromosome rearrangement. In mammals, there are sufficient assembled reference genomes to make assembly by comparative analysis. For birds however only three such reference genomes exist and chromosomal assembly needs to be achieved by other means. Classical approaches involving karyotyping and FISH coupled with comparative genomics browser technology provide a straightforward solution for the many avian genomes that are assembled to the point of 100+ large scaffolds. The aim of the project will thus be to generate a freely available comparative genomic resource that can be used to compare the genome structures of 25-30 bird species. HSBs and Evolutionary Breakpoint Regions (EBRs) will be displayed using the "Evolution Highway" browser. We will anchor each of the scaffolds physically to chromosomes by FISH. We will address at least four fundamental questions pertaining to genome evolution: We will test the hypotheses that HSB lengths are non-randomly distributed in birds, that there are specific gene ontology signatures of HSBs and EBRs, that EBRs coincide with recombination hotspots in birds (but not in mammals) and that transposable element density in increased in EBRs. The extensive letters of support from potential users indicate that this will be a widely used resource addressing a myriad of further biological questions in this most diverse and biologically important group of vertebrates.

Planned Impact

Exploitation and Application.
Technology Transfer activity at both Universities is administered by Enterprise offices. Collaborative agreement already exist with Digital Scientific and CytoCell. These separately capture exploitation and collaborative output issues going forward. Cytocell, Aviagen and Digital Scientific UK see benefit to this project either through the identification of commercially important traits or through the sales of products and have thus offered generous in kind support. A specific strategic advisory board has been established to identify potential impact issues from this and other projects.

Communications and engagement with the identified beneficiaries.
The main beneficiaries of this grant will be the avian genomics community in industry, third sector and academia. In 2002 the Kent lab was set up as a resource centre for avian molecular cytogenetics (FARMACRHOM). Through these activities we developed a mature and functioning collaborative network with most of the major players in avian genomics and biology many of whom have attached letters of support. Evolution Highway the "industry standard" for browser comparative genomics in the mammalian chromosome world with over ~50 regular users 16,000 hits from academia, industry and third sector in the last year. Inherently used for collaborative research it is, in itself, a tool to facilitate communication and engagement. Another beneficiary is the poultry breeding industry through which the Kent lab is, and has been, supported and funded through BBSRC grants and PhD CASE studentships. In the past we have provided genome maps for the species of interest (turkey and duck). A local zoo (Wingham Wildlife Park) was engaged as another and benefitted by subsequent broadcast media coverage and by provision of a display on the collaborative work performed (this is currently on display to the general public). We will participate in company-managed events and community activities with specialist audiences and the general public to disseminate findings and will develop as a CPD module for delivery to outside engagers. We will contribute to the Bioscience KTN newsletter, to "BBSRC business," newsletters published by individual companies and by the Departments/Universities and to media coverage as well as online activities. Bioscience KTN provide further help and advice on collaboration and their website has >3,000 registered members working in industry and academia The PI's have a healthy relationship with the University press offices and are regular contributor to public engagement activities including "Café Scientifique," Science Fairs and public lectures. Thus far, this work has led to several press releases in addition to appearances in the national and local broadcast media (BBC's "Big Questions," "The World Tonight", Radio 2 Simon Mayo show" BBC radio Kent etc.). Both Universities have active outreach programmes to which the PIs will contribute.

Capacity and Involvement
The two PIs will be the primary participants in impact activities aided by by PhD students and post-doctoral researchers. Professor Griffin has extensive training and experience in communication activities and commercial exploitation of research. The communications and development office in both Universities provide technical expertise and help in writing publications, web pages and user-friendly interfaces and it is expected that post-docs and PhD students will be provided with significant training in both dissemination and exploitation activities through Departmental, University and Bioscience KTN related activities. In conclusion therefore, this project has a range of expertise and ambitious, but achievable plans for impact at local, regional national and international levels.

See complete impact plan (attached) for further details

Publications

10 25 50

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/K008226/1 16/05/2013 14/08/2013 £280,392
BB/K008226/2 Transfer BB/K008226/1 15/08/2013 14/08/2017 £268,643
 
Title mapping avian genomes 
Description The artistic work was designed that demonstrates our approach to assemble avian genomes combining RACA and universal FISH probes. The work has been used on multiple websites together with our press release. 
Type Of Art Artwork 
Year Produced 2016 
Impact Our press releases have been given a higher attention because of this artwork piece. 
URL http://www.rvc.ac.uk/Media/Default/Press%20Release/genomics-article-illo.jpg
 
Description During the 4 years of this project we focused on producing a comprehensive resource and tools for chromosome mapping and comparative analysis in birds and other reptiles and on questions related to patterns of chromosome evolution in birds.

We aligned 21 avian genomes that were made available to us through the international avian genome project to chicken chromosomes and made results of these alignments available to our collaborators through the Avian Evolution Highway chromosome browser: http://eh-demo.ncsa.uiuc.edu/birds/. We further used this resource to contribute and lead the chromosome evolution section of the avian genomes comparative paper (Zhang et al. Science, 2014), perform sequence-based comparison of chromosome evolution in birds (Farre et al., GBE, 2016), and to reconstruct the ancestral avian chromosome structure (Romanov et al., BMC Genomics, 2014). Together these papers formed a significant contribution to knowledge of avian chromosome evolution: (i) showing that the rates of chromosomal rearrangements in birds are differing in evolutionary time, (ii) stability of avian karyotypes could be explained by a low fraction and distribution of transposable elements and conserved non-coding elements in avian genomes, (iii) evolutionary stable regions of avian chromosomes are enriched for genes responsible for ancestral phenotypes, (iv) genes found in lineage-specific rearrangements of avian chromosomes are often connected to lineage-specific characteristics, such as forebrain development in parrots.

From a technical perspective, we were able to establish a new computational resource: a six node computer cluster at the RVC that was used by our group to achieve these and other deliverables of the grant. We attracted a talented Ph.D. student who built RACA assemblies for 18 avian genomes (RACA of avian genomes was performed as part of the grant BB/J0101170) and published them on Evolution Highway (http://eh-demo.ncsa.uiuc.edu/birds/).

In year 3 our efforts were dedicated to building a tool that would allow us to use RACA avian assemblies to produce complete and verified chromosome-level genome assemblies for birds. We have used our multiple species alignments of 21 avian genomes to select bacterial artificial chromosomes (BACs) from chicken and zebra finch genomes that have a high probability to hybridise specifically to metaphase chromosomes in majority of bird species. A set of 250 such clones have been selected and used to map RACA assemblies of pigeon and peregrine falcon to chromosomes. We estimated that more than 90% of pigeon and falcon genomes were mapped to chromosomes using this new hybrid assembly approach. The chromosome-level assembly of peregrine falcon (first assembled to chromosomes highly rearranged avian genome) allowed us to find a new possible cause for evolutionary stability of avian genomes - interchromosomal rearrangements in birds are strictly limited to the genome intervals containing long "deserts" of highly conserved elements and this might be related to a unique regulatory landscape of avian genomes. We published a paper in Genome Research describing our new mapping approach and this discovery (Damas et al., Genome Research 2016).

During the year 4 we focused on comparing genome assemblies of avian genomes and applying our novel assembly approach to new species. We assembled 3 additional avian genomes to complete chromosomes: Ostrich, Saker falcon and Budgie. Comparing these genomes with genomes of other birds we confirmed our previous discovery that interchromosomal changes in avian genomes are limited to a small number of sites with low density of conserved elements (O'Connor et al., Genome Biology, 2018). We utilised our and other avian genome assemblies together with conserved BAC clones to answer the question about the probable karyotype structure of the ancestral dinosaur genome (O'Connor et al., Nature Communication, 2018) and finally we reconstructed chromosome structures of 14 avian ancestors in the lineage leading to zebra finch. A striking difference in the rates of rearrangements was detected for macro- and micro-chromosomes between the older and newer ancestors with micro-chromosomes being very stable in avian evolution until the appearance of the Passeri lineage. We also identified several micro-chromosomes that were maintained intact in the avian evolution since the Avian ancestor and linked evolutionary stability of avian chromosomes with density and distribution of evolutionary conserved non-coding elements (Damas et al., Genome Biology, 2018). In appreciation of our work we were invited to contribute a chapter on avian chromosome evolution to a book about avian genomics (Damas et al. in press).

As it follows from the text above we had very successful 4 years of the project: (i) we developed the largest publicly available resource of chromosome structures in birds, (ii) have contributed to a major comparative paper on avian genome evolution published in Science, had another one in BMC Genomics; (iii) Designed and applied a method to foster chromosome mapping and assembling genomes to chromosome level in birds, (iv) published four important papers on patterns of avian genome evolution (Farre et al., 2016; Damas et al., 2016, O'Connor et al., 2018; Damas et al., 2018), (v) secured further funding from BBSRC to apply our assembly approach to 25 mammalian genomes in order to study chromosome evolution in mammals. All members of our lab had a chance to present the results of this project at multiple international conferences.
Exploitation Route Our Avian Evolution Highway chromosome browser became a widely-used database to look for structures of chromosomes and synteny in different birds and reptiles. This database, together with the NCBI and our UCSC Genome Browser hub is the place where we place the new chromosome-level and RACA assemblies we produce for avian genomes. This information facilitates search of chromosome intervals and scaffolds that contain orthologous genes in different species and therefore could be used to find signatures of selection in different avian lineages as well as to transfer the genome annotation from the well-annotated avian genomes like chicken and zebra finch to other, "poorly" annotated genomes (17-18 genomes). By increasing the number of species and improving quality of assemblies we expect to increase the impact of this resource.

A new method to assemble avian genomes to chromosomes is critically important nowadays when sequencing and scaffolding of genomes can be done easily but an error-free chromosome level assembly is very expensive to produce. Our method already caused a lot of interest and will be utilized by the Genome10K consortium. We built contacts with Dovetail Genomics to develop this approach further to make their HiC based chromosome assemblies more accurate.

Also, Avian Evolution Highway is an excellent educational resource that could be used to teach students about the patterns and principles of chromosome evolution in birds and other reptiles in a visually appealing interactive way.
Sectors Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education

URL http://eh-demo.ncsa.uiuc.edu/birds/
 
Description Evolution Highway freely available from http://evolutionhighway.nsca.uiuc.edu/birds is used as an educational aid by Dr. Larkin and other groups to introduce students to ideas of comparative genomics during lectures and practicals, as well as to develop tutorial student projects related to comparative and "informatics-paleogenomics". The database build during the four years of this project provides a new excellent educational material for students and the general public to study bird and mammalian genomes and ancestral amniote chromosomes. We expect that the special issue of Science dedicated to bird genomics (and associated media coverage of the issue) will have large societal impact making the general public aware of the variety and unique biology of birds. The new assembly method we designed for making better avian genomes resulted in five avian genomes being assembled (5 published). The list includes the Ostrich and Pigeon genomes that are from economically important species. Their genome assemblies could be used by breeding companies to design new or improve old breeds. Our new assembly method allowed us to build connections to apply this approach to additional genomes including the Bustard genome together with the UAE University. We developed contacts with the Dovetail Genomics company to use our approach as part of their HiC genome assembly pipeline to end up with better assembly results from their pipeline.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education
Impact Types Societal

 
Description Responsive mode
Amount £387,536 (GBP)
Funding ID BB/P020062/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Responsive mode
Amount 6,000,000 руб. (RUB)
Organisation Russian Foundation for Basic Research 
Sector Academic/University
Country Russian Federation
Start 10/2017 
End 09/2020
 
Description Royal Veterinary College Ph.D. studentship award
Amount £50,000 (GBP)
Organisation Royal Veterinary College (RVC) 
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 04/2016
 
Title Avian ancestors 
Description Reconstruction of 14 avian ancestor chromosome structures were obtained. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact The paper describing this resource in under review in Genome Biology 
URL http://eh-demo.ncsa.uiuc.edu/birds/#/SynBlocks
 
Title Chromosome-level assemblies of the saker falcon, budgie, and ostrich 
Description Chromosome-level assemblies were generated for the saker falcon, budgie, and ostrich genomes using a combination of Reference-Assisted Chromosome Assembly and the conserved avian BAC clones 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact A paper was published in Genome Biology describing patterns of chromosome evolution in saker falcon, budgie, and ostrich genomes. 
URL https://datadryad.org/resource/doi:10.5061/dryad.q70q40m
 
Title Peregrine and pigeon chromosome level assemblies 
Description Chromosomal level genome assemblies of peregrine falcon and rock pigeon have been generated combining our RACA method with the universal FISH probes designed by our group. These assemblies have been submitted to NCBI under accession numbers MLQY00000000 and MLQZ00000000, our avian chromosome level-assembly UCSC Genome Browser hub: http://sftp.rvc.ac.uk/rvcpaper/birdsHUB/hub.txt and our Evolution Highway comparative chromosome browser: http://eh-demo.ncsa.uiuc.edu/birds/ 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact Our new method of assembling genomes to chromosome levels has a potential to change the way how genome assemblies are done (reducing the cost drastically). We are making additional assemblies using this method and expect these to have a large impact of the genetic studies of the species we assemble to chromosome levels. 
URL http://eh-demo.ncsa.uiuc.edu/birds/
 
Title Universal set of 250 BAC clones to map avian genomes 
Description A set of >200 chicken and zebra finch bacterial artificial chromosome (BAC) clones equally spaced across the avian genomes were chosen to produce specific high quality hybridization signal across diverse avian genomes. The set was built based on multiple-sequence alignments of multiple (21) avian genomes and considering the level of inter-species sequence conservation within the BAC clones. The resulting set of clones works with >90% success rate on any avian species chromosomes. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact Five avian genomes (pigeon, peregrine falcon, chicken, zebra finch and turkey) were already mapped with this set of clones. For two genomes (pigeon and peregrine falcon) chromosome-level genome maps and assemblies were built. A paper describing the tool and genome assemblies was published in Genome Research (Damas et al., 2016). Three more avian assemblies are currently being generated (saker falcon, budgerigar, ostrich). These clones have been used to map a total of 25+ avian and other reptile genomes. 
URL http://sftp.rvc.ac.uk/rvcpaper/birdsHUB/hub.txt
 
Title Avian Genomes Interactive Evolution Highway Chromosome Browser 
Description We built an interactive Evolution Highway website that contains results of alignments of chromosome structure of 21 avian and 5 non-avian species. This site provides user is easy means for visualising and comparing chromosome structures of different bird species, detections of the regions of conserved synteny, and evolutionary breakpoint regions. The browser was upgraded to contain reconstructed chromosomes of 14 avian ancestors. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact This database was used to produce data and visualise the results of chromosome analysis for 4 papers: Zhang et al. (Science, 2014); Romanov et al (BMC Genomics, 2014); Farré et al. (GBE, 2016); Damas et al. (Genome Research 2017); Damas et al. (Genome Biology 2018); O'Connor et al. (Genome Biology 2018). 
URL http://eh-demo.ncsa.uiuc.edu/birds/
 
Description Houbara genome assembly 
Organisation United Arab Emirates University
Country United Arab Emirates 
Sector Academic/University 
PI Contribution We started a collaboration with the UAEU on assembling the houbara genome in order to help conservation and restore population of this bird species So far my research team instructed the group in the United Arab Emirates to perform HiC assembly of the genome in addition to the Ion Nanapore assembly they already had. After the assembly was constructed we preformed comparison of HiC scaffolds to zebra finch and chicken chromosomes in order find miss-asemblies and structural differences between genomes.
Collaborator Contribution They performed initial sequencing of the houbara genome as well as prayed for the HiC assembly.
Impact Comparative map of the houbara genome has been delivered to the UAEU group.
Start Year 2018
 
Description Muntjac rearrangements 
Organisation 3i Consortium
Country United Kingdom 
Sector Multiple 
PI Contribution The group of Dr. Helder Maiato is specialising in the study of mitosis using a combination of cytogenetic and molecular biology techniques. They are using Indian Muntjac as a model to study mechanisms of mitosis. Dr. Larkin's group own genome assemblies of two muntjac species. They were used to design anti-sense RNA sequences to knock-out muntjac genes related to mitosis. Dr. Larkin's group did identification of muntjac sequences for 200+ mitotic genes and passed that information to Dr. Maiato's group.
Collaborator Contribution Dr. Maiato's group helps Dr. Larkin to identify genes that have contributed to extremely high level of chromosomal rearrangements found in the Indian Muntjac (2n=6). They are performing knock-out of the genes in muntjac cell lines that were identified as top candidates for this process in the Dr. Larkin's lab from RACA assisted chromosome assemblies of muntjac genomes.
Impact This is a multidisciplinary collaboration bringing together cell molecular geneticists and bioinformaticians. So far 200+ mitotic genes were knocked out and tested on Indian muntjac cell lines to reveal their effect on mitosis. The paper was published in Current Biology (PMID: 29706521) In addition, five top candidate genes are being currently investigated for the effects on contribution to spontaneous chromosome fusions in muntjac cell lines.
Start Year 2016
 
Title Chromosome level assembly method for ainmal genomes 
Description A novel technique has been developed and published allowing to assemble fragmented animal genomes to chromosome level using a combination of RACA and universal FISH probes. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact Several avian genomes have been upgraded to chromosome level using this approach and a larger number of genomes is currently in our pipeline to be upgraded. 
URL http://genome.cshlp.org/content/early/2016/11/30/gr.213660.116.abstract
 
Description Night at the RVC 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact About 300 people have attended our boost at the Night at the RVC event. We presented our software tool that teaches the general public the principles of the chromosome evolution.
Year(s) Of Engagement Activity 2015
 
Description School for young scientists 2016 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact More than 100 participants from the former soviet union counties have attended the School for young scientists held in Zvenigorod, Russia in 2016. Dr Larkin gave an invited lecture on the current status of animal genome studies resulting in a lot of questions from the audience and the follow up discussions. The organising committee has requested a review paper to be written and published based on the lecture given.
Year(s) Of Engagement Activity 2016
 
Description School of young scientists 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact More than 100 participants from the former soviet union counties have attended the School for young scientists held in Zvenigorod, Russia in 2018. Dr Larkin gave an invited lecture on the current status of animal genome studies resulting in a lot of questions from the audience and the follow up discussions. The organising committee has requested a review paper to be written and published based on the lecture given.
Year(s) Of Engagement Activity 2018
 
Description School visit (Stevenage) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact about 150 pupils from regional schools attended the RVC event at which we presented our software tool aiming at teaching kids the principles of chromosome evolution
Year(s) Of Engagement Activity 2015