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Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 to 6 MYA, but that are absent in the Hominidae. In fact, Hominidae show between four-and seven-fold lower rates of nucleotide change and feature turnover in both neutral and functional sequences suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. For example, recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli. This process resulted in thousands of novel, species-specific CTCF binding sites. Our results demonstrate that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

The human reference genome assembly plays a central role in nearly all aspects of today’s basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009 and reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that while the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model., The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 249894 (SelSweep)

In mammals, Immunoglobulin light chain (IgL) are localized to two chromosomal regions (designated kappa and lambda). Here we report a genome-wide survey of IgL genes in the zebrafish revealing (V(L)-J(L)-C(L)) clusters spanning 5 separate chromosomes. To elucidate IgL loci present in the zebrafish genome assembly (Zv6), conventional sequence similarity searches and a novel scanning approach based on recombination signal sequence (RSS) motifs were applied. RT-PCR with zebrafish cDNA was used to confirm annotations, evaluate VJ-rearrangement possibilities and show that each chromosomal locus is expressed. In contrast to other vertebrates in which IgL exon usage has been studied, inversional rearrangement between (V(L)-J(L)-C(L)) clusters were found. Inter-cluster rearrangements may convey a selective advantage for editing self-reactive receptors and poise zebrafish by virtue of their extensive numbers of V(L), J(L) and C(L) to have greater potential for immunoglobulin gene shuffling than traditionally studied mice and human models.

Animals and plants have evolved a range of mechanisms for recognizing noxious substances and organisms. A particular challenge, most successfully met by the adaptive immune system in vertebrates, is the specific recognition of potential pathogens, which themselves evolve to escape recognition. A variety of genomic and evolutionary mechanisms shape large families of proteins dedicated to detecting pathogens and create the diversity of binding sites needed for epitope recognition. One family involved in innate immunity are the NACHT-domain-and Leucine-Rich-Repeat-containing (NLR) proteins. Mammals have a small number of NLR proteins, which are involved in first-line immune defense and recognize several conserved molecular patterns. However, there is no evidence that they cover a wider spectrum of differential pathogenic epitopes. In other species, mostly those without adaptive immune systems, NLRs have expanded into very large families. A family of nearly 400 NLR proteins is encoded in the zebrafish genome. They are subdivided into four groups defined by their NACHT and effector domains, with a characteristic overall structure that arose in fishes from a fusion of the NLR domains with a domain used for immune recognition, the B30.2 domain. The majority of the genes are located on one chromosome arm, interspersed with other large multi-gene families, including a new family encoding proteins with multiple tandem arrays of Zinc fingers. This chromosome arm may be a hot spot for evolutionary change in the zebrafish genome. NLR genes not on this chromosome tend to be located near chromosomal ends. Extensive duplication, loss of genes and domains, exon shuffling and gene conversion acting differentially on the NACHT and B30.2 domains have shaped the family. Its four groups, which are conserved across the fishes, are homogenised within each group by gene conversion, while the B30.2 domain is subject to gene conversion across the groups. Evidence of positive selection on diversifying mutations in the B30.2 domain, probably driven by pathogen interactions, indicates that this domain rather than the LRRs acts as a recognition domain. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system.

Optical mapping is a technology that gathers long-range information on genome sequences similar to ordered restriction digest maps. Because it is not subject to cloning, amplification, hybridisation or sequencing bias, it is ideally suited to the improvement of fragmented genome assemblies that can no longer be improved by classical methods. In addition, its low cost and rapid turnaround make it equally useful during the scaffolding process of de novo assembly from high throughput sequencing reads. We describe how optical mapping has been used in practice to produce high quality vertebrate genome assemblies. In particular, we detail the efforts undertaken by the Genome Reference Consortium (GRC), which maintains the reference genomes for human, mouse, zebrafish and chicken, and uses different optical mapping platforms for genome curation.

We describe the generation and analysis of an integrated sequence map of a 2.4-Mb region of pig chromosome 7, comprising the classical class I region, the extended and classical class II regions, and the class III region of the major histocompatibility complex (MHC), also known as swine leukocyte antigen (SLA) complex. We have identified and manually annotated 151 loci, of which 121 are known genes (predicted to be functional), 18 are pseudogenes, 8 are novel CDS loci, 3 are novel transcripts, and 1 is a putative gene. Nearly all of these loci have homologues in other mammalian genomes but orthologues could be identified with confidence for only 123 genes. The 28 genes (including all the SLA class I genes) for which unambiguous orthology to genes within the human reference MHC could not be established are of particular interest with respect to porcine-specific MHC function and evolution. We have compared the porcine MHC to other mammalian MHC regions and identified the differences between them. In comparison to the human MHC, the main differences include the absence of HLA-A and other class I-like loci, the absence of HLA-DP-like loci, and the separation of the extended and classical class II regions from the rest of the MHC by insertion of the centromere. We show that the centromere insertion has occurred within a cluster of BTNL genes located at the boundary of the class II and III regions, which might have resulted in the loss of an orthologue to human C6orf10 from this region.

We have generated an improved assembly and gene annotation of the pig X chromosome, and a first draft assembly of the pig Y chromosome, by sequencing BAC and fosmid clones, and incorporating information from optical mapping and fibre-FISH. The X chromosome carries 1,014 annotated genes, 689 of which are protein-coding. Gene order closely matches that found in Primates (including humans) and Carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X chromosome were absent from the pig (e.g. the cancer/testis antigen family) or inactive (e.g. AWAT1), and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y chromosome assembly focussed on two clusters of male-specific low-copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. The long arm of the chromosome is almost entirely repetitive, containing previously characterised sequences. Many of the ancestral X-related genes previously reported in at least one mammalian Y chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes - both single copy and amplified - on the pig Y, to compare the pig X and Y chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y chromosome evolution.

Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

I have read the journal's policy and have the following conflicts: Paul Flicek is married to the deputy editor of PLoS Medicine, Melissa Norton. Evan Eichler is on the board of Pacific Biosciences. Support for this work came from the Intramural Research Program of the NIH, The National Library of Medicine, the European Molecular Biology Laboratory, the Wellcome Trust (grant number 077198), and the Howard Hughes Medical Institute (EEE). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Ensembl (http://www.ensembl.org) integrates genomic information for a comprehensive set of chordate genomes with a particular focus on resources for human, mouse, rat, zebrafish and other high-value sequenced genomes. We provide complete gene annotations for all supported species in addition to specific resources that target genome variation, function and evolution. Ensembl data is accessible in a variety of formats including via our genome browser, API and BioMart. This year marks the tenth anniversary of Ensembl and in that time the project has grown with advances in genome technology. As of release 56 (September 2009), Ensembl supports 51 species including marmoset, pig, zebra finch, lizard, gorilla and wallaby, which were added in the past year. Major additions and improvements to Ensembl since our previous report include the incorporation of the human GRCh37 assembly, enhanced visualisation and data-mining options for the Ensembl regulatory features and continued development of our software infrastructure.

The zebrafish genome, which consists of 25 linkage groups and is ~1.4Gb in size, is being sequenced, finished and analysed in its entirety at the Wellcome Trust Sanger Institute. The manual annotation is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group and is released at regular intervals onto the Vertebrate Genome Annotation (Vega) database ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk) and may be viewed as a DAS source in Ensembl ("http://www.ensembl.org/Danio_rerio":http://www.ensembl.org/Danio_rerio). Our annotation is compiled in close collaboration with the Zebrafish Information Network (ZFIN) ("http://zfin.org/":http://zfin.org/), which has enabled us to provide an accurate, dynamic and distinct resource for the zebrafish community as a whole.Annotation is based on the reference genome sequence, which is derived from a minimal tile path assembly composed of clones that have been mapped, sequenced and meticulously finished to a sequence accuracy of over 99.9% per 100Kb. We expect to have 90% of the zebrafish genome to a finished standard by the end of 2009. Our approach to annotation uses two strategies. Firstly, the generation and annotation of gene lists comprising of cDNA (8995 in total) found in ZFIN that maps to our current reference assembly. And, secondly, by using clone by clone annotation, where we have annotated over 3200 genes, 1100 transcripts and 130 pseudogenes across 11 linkage groups and 3530 clones. As well as our on-going genome annotation we also welcome external annotation requests for specific genes and regions, which already include the annotation of 93 genes associated with human obesity and the scheduled annotation of the Major Histocompatability Complex, which will utilise reference sequence taken from libraries of a double haploid fish and complement our previous work on the human and mouse MHC already published. External requests and any feedback, questions or requests can be sent to zfish-help [at] sanger.ac.uk.

We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated “bystander” genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

Nature 496, 498–503 (2013); doi:10.1038/nature12111 In this Letter, five authors were inadvertently omitted: Sharmin Begum and Christine Lloyd from the Wellcome Trust Sanger Institute, and Christa Lanz, Gunter Raddatz and Stephan C. Schuster from the Max Planck Institute for Developmental Biology. David Elliot was incorrectly listed as David Eliot, Beverley Mortimore was incorrectly listed as Beverly Mortimer, and James D.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes., New reference genomes of the two extant monotreme lineages (platypus and echidna) reveal the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution.