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 Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries.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.

Six reference-quality genomes reveal evolution of bat adaptations.

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.

A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

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.

Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Investigating the cause of a 2021 winter wave of COVID-19 in a border region in eastern Germany: a mixed-methods study, August to November 2021.

It is so far unclear how the COVID-19 winter waves started and what should be done to prevent possible future waves. In this study, we deciphered the dynamic course of a winter wave in 2021 in Saxony, a state in Eastern Germany neighbouring the Czech Republic and Poland. The study was carried out through the integration of multiple virus genomic epidemiology approaches to track transmission chains, identify emerging variants and investigate dynamic changes in transmission clusters. For identified local variants of interest, functional evaluations were performed. Multiple long-lasting community transmission clusters have been identified acting as driving force for the winter wave 2021. Analysis of the dynamic courses of two representative clusters indicated a similar transmission pattern. However, the transmission cluster caused by a locally occurring new Delta variant AY.36.1 showed a distinct transmission pattern, and functional analyses revealed a replication advantage of it. This study indicated that long-lasting community transmission clusters starting since early autumn caused by imported or locally occurring variants all contributed to the development of the 2021 winter wave. The information we achieved might help future pandemic prevention.

The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Expanded olfactory system in ray-finned fishes capable of terrestrial exploration.

BACKGROUND: Smell abilities differ greatly among vertebrate species due to distinct sensory needs, with exceptional variability reported in the number of olfactory genes and the size of the odour-processing regions of the brain. However, key environmental factors shaping genomic and phenotypic changes linked to the olfactory system remain difficult to identify at macroevolutionary scales. Here, we investigate the association between diverse ecological traits and the number of olfactory chemoreceptors in approximately two hundred ray-finned fishes. RESULTS: We found independent expansions producing large gene repertoires in several lineages of nocturnal amphibious fishes, generally able to perform active terrestrial exploration. We reinforced this finding with on-purpose genomic and transcriptomic analysis of Channallabes apus, a catfish species from a clade with chemosensory-based aerial orientation. Furthermore, we also detected an augmented information-processing capacity in the olfactory bulb of nocturnal amphibious fishes by estimating the number of cells contained in this brain region in twenty-four actinopterygian species. CONCLUSIONS: Overall, we report a convergent genomic and phenotypic magnification of the olfactory system in nocturnal amphibious fishes. This finding suggests the possibility of an analogous evolutionary event in fish-like tetrapod ancestors during the first steps of the water-to-land transition, favouring terrestrial adaptation through enhanced aerial orientation.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany in 2020: insights into the cause of the second wave.

In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.

CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈ 90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish.

BACKGROUND: Vertebrate mitochondrial genomes are optimized for fast replication and low cost of RNA expression. Accordingly, they are devoid of introns, are transcribed as polycistrons and contain very little intergenic sequences. Usually, vertebrate mitochondrial genomes measure between 16.5 and 17 kilobases (kb). RESULTS: During genome sequencing projects for two novel vertebrate models, the invasive round goby and the sand goby, we found that the sand goby genome is exceptionally small (16.4 kb), while the mitochondrial genome of the round goby is much larger than expected for a vertebrate. It is 19 kb in size and is thus one of the largest fish and even vertebrate mitochondrial genomes known to date. The expansion is attributable to a sequence insertion downstream of the putative transcriptional start site. This insertion carries traces of repeats from the control region, but is mostly novel. To get more information about this phenomenon, we gathered all available mitochondrial genomes of Gobiidae and of nine gobioid species, performed phylogenetic analyses, analysed gene arrangements, and compared gobiid mitochondrial genome sizes, ecological information and other species characteristics with respect to the mitochondrial phylogeny. This allowed us amongst others to identify a unique arrangement of tRNAs among Ponto-Caspian gobies. CONCLUSIONS: Our results indicate that the round goby mitochondrial genome may contain novel features. Since mitochondrial genome organisation is tightly linked to energy metabolism, these features may be linked to its invasion success. Also, the unique tRNA arrangement among Ponto-Caspian gobies may be helpful in studying the evolution of this highly adaptive and invasive species group. Finally, we find that the phylogeny of gobiids can be further refined by the use of longer stretches of linked DNA sequence.

A haplotype-resolved reference genome of a long-distance migratory bat, Pipistrellus nathusii (Keyserling & Blasius, 1839).

We present a complete, chromosome-scale reference genome for the long-distance migratory bat Pipistrellus nathusii. The genome encompasses both haplotypic sets of autosomes and the separation of both sex chromosomes by utilizing highly accurate long-reads and preserving long-range phasing information through the use of three-dimensional chromatin conformation capture sequencing (Hi-C). This genome, accompanied by a comprehensive protein-coding sequence annotation, provides a valuable genomic resource for future investigations into the genomic bases of long-distance migratory flight in bats as well as uncovering the genetic architecture, population structure and evolutionary history of Pipistrellus nathusii. The reference-quality genome presented here gives a fundamental resource to further our understanding of bat genetics and evolution, adding to the growing number of high-quality genetic resources in this field. Here, we demonstrate its use in the phylogenetic reconstruction of the order Chiroptera, and in particular, we present the resources to allow detailed investigations into the genetic drivers and adaptations related to long-distance migration.

The genome sequence of Tadarida brasiliensis I. Geoffroy Saint-Hilaire, 1824 [Molossidae; Tadarida].

We present a genome assembly from an individual male Tadarida brasiliensis (The Brazilian free-tailed bat; Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.28 Gb in span. The majority of the assembly is scaffolded into 25 chromosomal pseudomolecules, with the X and Y sex chromosomes assembled.

A genome resource for the marine annelid Platynereis dumerilii.

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii and of the closely related P. massiliensis and P. megalops, to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ~1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 580 miRNA loci. We further explore the high genetic variation (~3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.