Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

High heteroplasmy is associated with low mitochondrial copy number and selection against non-synonymous mutations in the snail Cepaea nemoralis.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Caecilian Genomes Reveal the Molecular Basis of Adaptation and Convergent Evolution of Limblessness in Snakes and Caecilians.

We present genome sequences for the caecilians Geotrypetes seraphini (3.8 Gb) and Microcaecilia unicolor (4.7 Gb), representatives of a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. More than 69% of both genomes are composed of repeats, with retrotransposons being the most abundant. We identify 1,150 orthogroups that are unique to caecilians and enriched for functions in olfaction and detection of chemical signals. There are 379 orthogroups with signatures of positive selection on caecilian lineages with roles in organ development and morphogenesis, sensory perception, and immunity amongst others. We discover that caecilian genomes are missing the zone of polarizing activity regulatorysequence (ZRS) enhancer of Sonic Hedgehog which is also mutated in snakes. In vivo deletions have shown ZRS is required for limb development in mice, thus, revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.

MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio high fidelity reads.

BACKGROUND:  PacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (> Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. RESULTS:  MitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (368 Metazoa and 6 Fungi species) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. CONCLUSIONS:  MitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on GitHub ( https://github.com/marcelauliano/MitoHiFi ). MitoHiFi is available with its dependencies as a Docker container on GitHub (ghcr.io/marcelauliano/mitohifi:master).

Whole genomes from the extinct Xerces Blue butterfly can help identify declining insect species.

The Xerces Blue (Glaucopsyche xerces) is considered to be the first butterfly to become extinct in historical times. It was notable for its chalky lavender wings with conspicuous white spots on the ventral wings. The last individuals were collected in their restricted habitat, in the dunes near the Presidio military base in San Francisco, in 1941. We sequenced the genomes of four 80- to 100-year-old Xerces Blue, and seven historical and one modern specimens of its closest relative, the Silvery Blue (Glaucopsyche lygdamus). We compared these to a novel annotated genome of the Green-Underside Blue (Glaucopsyche alexis). Phylogenetic relationships inferred from complete mitochondrial genomes indicate that Xerces Blue was a distinct species that diverged from the Silvery Blue lineage at least 850,000 years ago. Using nuclear genomes, both species experienced population growth during the Eemian interglacial period, but the Xerces Blue decreased to a very low effective population size subsequently, a trend opposite to that observed in the Silvery Blue. Runs of homozygosity and deleterious load in the former were significantly greater than in the later, suggesting a higher incidence of inbreeding. These signals of population decline observed in Xerces Blue could be used to identify and monitor other insects threatened by human activities, whose extinction patterns are still not well known.

Distinct patterns of genetic variation at low-recombining genomic regions represent haplotype structure.

Genomic regions sometimes show patterns of genetic variation distinct from the genome-wide population structure. Such deviations have often been interpreted to represent effects of selection. However, systematic investigation of whether and how non-selective factors, such as recombination rates, can affect distinct patterns has been limited. Here, we associate distinct patterns of genetic variation with reduced recombination rates in a songbird, the Eurasian blackcap (Sylvia atricapilla), using a new reference genome assembly, whole-genome resequenc- ing data and recombination maps. We find that distinct patterns of genetic variation reflect haplotype structure at genomic regions with different prevalence of reduced recombination rate across populations. At low-recombining regions shared in most populations, distinct patterns reflect conspicuous haplotypes segregating in multiple populations. At low-recombining regions found only in a few populations, distinct patterns represent variance among cryptic haplotypes within the low-recombining populations. With simulations, we confirm that these distinct patterns evolve neutrally by reduced recombination rate, on which the effects of selection can be overlaid. Our results highlight that distinct patterns of genetic variation can emerge through evolutionary reduction of local recombination rate. The recombination landscape as an evolvable trait therefore plays an important role determining the heterogeneous distribution of genetic variation along the genome.

Chromosomal reference genome sequences for the malaria mosquito, Anopheles coustani, Laveran, 1900.

We present genome assembly from individual female An. coustani (African malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae) from Lopé, Gabon. The genome sequence is 270 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled for both species. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Scalable, accessible, and reproducible reference genome assembly and evaluation in Galaxy.

Improvements in genome sequencing and assembly are enabling high-quality reference genomes for all species. However, the assembly process is still laborious, computationally and technically demanding, lacks standards for reproducibility, and is not readily scalable. Here we present the latest Vertebrate Genomes Project assembly pipeline and demonstrate that it delivers high-quality reference genomes at scale across a set of vertebrate species arising over the last ~500 million years. The pipeline is versatile and combines PacBio HiFi long-reads and Hi-C-based haplotype phasing in a new graph-based paradigm. Standardized quality control is performed automatically to troubleshoot assembly issues and assess biological complexities. We make the pipeline freely accessible through Galaxy, accommodating researchers even without local computational resources and enhanced reproducibility by democratizing the training and assembly process. We demonstrate the flexibility and reliability of the pipeline by assembling reference genomes for 51 vertebrate species from major taxonomic groups (fish, amphibians, reptiles, birds, and mammals).

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared to its predecessor. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. We jointly analyzed 163 short-read whole genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ~20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

A chromosome-level assembly supports genome-wide investigation of the DMRT gene family in the golden mussel (Limnoperna fortunei).

BACKGROUND: The golden mussel (Limnoperna fortunei) is a highly invasive species that causes environmental and socioeconomic losses in invaded areas. Reference genomes have proven to be a valuable resource for studying the biology of invasive species. While the current golden mussel genome has been useful for identifying new genes, its high fragmentation hinders some applications. FINDINGS: In this study, we provide the first chromosome-level reference genome for the golden mussel. The genome was built using PacBio HiFi, 10X, and Hi-C sequencing data. The final assembly contains 99.4% of its total length assembled to the 15 chromosomes of the species and a scaffold N50 of 97.05 Mb. A total of 34,862 protein-coding genes were predicted, of which 84.7% were functionally annotated. A significant (6.48%) proportion of the genome was found to be in a hemizygous state. Using the new genome, we have performed a genome-wide characterization of the Doublesex and Mab-3 related transcription factor gene family, which has been proposed as a target for population control strategies in other species. CONCLUSIONS: From the applied research perspective, a higher-quality genome will support genome editing with the aim of developing biotechnology-based solutions to control invasion. From the basic research perspective, the new genome is a high-quality reference for molecular evolutionary studies of Mytilida and other Lophotrochozoa, and it may be used as a reference for future resequencing studies to assess genomic variation among different golden mussel populations, unveiling potential routes of dispersion and helping to establish better control policies.

The genome sequence of the malaria mosquito, Anopheles funestus, Giles, 1900.

We present a genome assembly from an individual female Anopheles funestus (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae). The genome sequence is 251 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Genome assembly and annotation of the tambaqui (Colossoma macropomum): an emblematic fish of the Amazon River Basin.

Colossoma macropomum, known as "tambaqui", is the largest Characiformes fish in the Amazon River Basin and a leading species in Brazilian aquaculture and fisheries. Good quality meat and excellent adaptability to culture systems are some of its remarkable farming features. To support studies into the genetics and genomics of the tambaqui, we have produced the first high-quality genome for the species. We combined Illumina and PacBio sequencing technologies to generate a reference genome, assembled with 39× coverage of long reads and polished to a consensus quality value (QV) of 36 with 130× coverage of short reads. The genome was assembled into 1269 scaffolds (a total of 1,221,847,006 bases), with a scaffold N50 size of 40 Mb, where 93% of all assembled bases were placed in the largest 54 scaffolds corresponding to the diploid karyotype of the tambaqui. Furthermore, the NCBI Annotation Pipeline annotated genes, pseudogenes, and non-coding transcripts using the RefSeq database as evidence, guaranteeing a high-quality annotation. A Genome Data Viewer for the tambaqui was produced, which will benefit groups interested in exploring the unique genomic features of the species. The availability of a highly accurate genome assembly for tambaqui provides the foundation for the discovery of novel ecological and evolutionary insights, and is a helpful resource for aquaculture.

A Long-Term Conserved Satellite DNA That Remains Unexpanded in Several Genomes of Characiformes Fish Is Actively Transcribed.

Eukaryotic genomes contain large amounts of repetitive DNA sequences, such as tandemly repeated satellite DNAs (satDNAs). These sequences are highly dynamic and tend to be genus- or species-specific due to their particular evolutionary pathways, although there are few unusual cases of conserved satDNAs over long periods of time. Here, we used multiple approaches to reveal that an satDNA named CharSat01-52 originated in the last common ancestor of Characoidei fish, a superfamily within the Characiformes order, ∼140-78 Ma, whereas its nucleotide composition has remained considerably conserved in several taxa. We show that 14 distantly related species within Characoidei share the presence of this satDNA, which is highly amplified and clustered in subtelomeric regions in a single species (Characidium gomesi), while remained organized as small clusters in all the other species. Defying predictions of the molecular drive of satellite evolution, CharSat01-52 shows similar values of intra- and interspecific divergence. Although we did not provide evidence for a specific functional role of CharSat01-52, its transcriptional activity was demonstrated in different species. In addition, we identified short tandem arrays of CharSat01-52 embedded within single-molecule real-time long reads of Astyanax paranae (536 bp-3.1 kb) and A. mexicanus (501 bp-3.9 kb). Such arrays consisted of head-to-tail repeats and could be found interspersed with other sequences, inverted sequences, or neighbored by other satellites. Our results provide a detailed characterization of an old and conserved satDNA, challenging general predictions of satDNA evolution.

A high-quality genome and comparison of short- versus long-read transcriptome of the palaearctic duck Aythya fuligula (tufted duck).

BACKGROUND: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. FINDINGS: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. CONCLUSIONS: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long-read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

The genome sequence of the Norway rat, Rattus norvegicus Berkenhout 1769.

We present a genome assembly from an individual male Rattus norvegicus (the Norway rat; Chordata; Mammalia; Rodentia; Muridae). The genome sequence is 2.44 gigabases in span. The majority of the assembly is scaffolded into 20 chromosomal pseudomolecules, with both X and Y sex chromosomes assembled. This genome assembly, mRatBN7.2, represents the new reference genome for R. norvegicus and has been adopted by the Genome Reference Consortium.

The genome sequence of the European turtle dove, Streptopelia turtur Linnaeus 1758.

We present a genome assembly from an individual female Streptopelia turtur (the European turtle dove; Chordata; Aves; Columbidae). The genome sequence is 1.18 gigabases in span. The majority of the assembly is scaffolded into 35 chromosomal pseudomolecules, with the W and Z sex chromosomes assembled.

Complete vertebrate mitogenomes reveal widespread repeats and gene duplications.

BACKGROUND: Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. RESULTS: As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100-300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. CONCLUSIONS: Our results indicate that even in the "simple" case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone.