Search

Bats carry viruses that can cause severe disease in other mammals. Asymptomatic infections in bats suggest limited tissue-damaging inflammation and immunopathology. To investigate the genomic basis of disease resistance, the Bat1K project generated reference-quality genomes of ten bat species. A systematic analysis showed that signatures of selection in immune genes are more prevalent in bats compared with other mammals. We found an excess of immune gene adaptations in the ancestral Chiroptera and many descending bat lineages, highlighting viral entry and detection factors, and regulators of antiviral and inflammatory responses. ISG15, an antiviral gene contributing to hyperinflammation during COVID-19, exhibits a deletion of a cysteine, required for homodimer formation, in rhinolophid and hipposiderid bats. Cellular infection experiments showed enhanced intracellular protein conjugation of bat ISG15 and lack of secretion into extracellular space, where human ISG15 stimulates inflammation. Our work highlights molecular mechanisms contributing to viral tolerance and disease resistance in bats.

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. This work was completed in part with resources provided by the University of Massachusetts' Green High Performance Computing Cluster (GHPCC). Funding was provided by the University of Massachusetts Amherst, NSF-IOS (grant #1904439 to L.M.K.), NOAA-Fisheries, National Research Council postdoctoral fellowship program to L.M.K., VGP, Rockefeller University, to E.D.J., HHMI to E.D.J., the Sanger Institute, Max-Planck-Gesellschaft, and grant contributions from Tom Gilbert, Paul Flicek, R.W.M., Karen A. Bjorndal, Alan B. Bolten, Ed Braun, N.J.G., T.M.-B., and A.F.S. We acknowledge CONICYT-DAAD for scholarship support to T.C.-V., the São Paulo Research Foundation to E.K.S.R.–FAPESP (grant #2020/10372-6). BeGenDiv is partially funded by the German Federal Ministry of Education and Research (BMbF, Förderkennzeichen 033W034A). The work of F.T.-N. and P.M. was supported by the Intramural Research Program of the National Library of Medicine, NIH. The work of M.P. was partially funded through the Federal Ministry of Education and Research (grant 01IS18026C). H.P. was supported by a Formació de Personal Investigador fellowship from Generalitat de Catalunya (FI_B100131). M.K. was supported by “la Caixa” Foundation (ID 100010434; code LCF/BQ/PR19/11700002), the Vienna Science and Technology Fund (WWTF), and the City of Vienna (VRG20-001). Funding for green turtle resequencing was provided by a Welsh Government Sêr Cymru II and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 663830-BU115 and the Sea Turtle Conservancy, Florida Sea Turtle Grants Program (17-033R).

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.

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 species1,2,3,4. 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., We thank them for their permission to publish. A.R., S.K., B.P.W. and A.M.P. were supported by the Intramural Research Program of the NHGRI, NIH (1ZIAHG200398). A.R. was also supported by the Korea Health Technology R&D Project through KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI17C2098). S.A.M., I.B. and R.D. were supported by Wellcome Trust grant WT207492; W.C., M. Smith, Z.N., Y.S., J.C., S. Pelan, J.T., A.T., J.W. and Kerstin Howe by WT206194; L.H., F.M., Kevin Howe and P. Flicek by WT108749/Z/15/Z, WT218328/B/19/Z and the European Molecular Biology Laboratory. O.F. and E.D.J. were supported by Howard Hughes Medical Institute and Rockefeller University start-up funds for this project. J.D. and H.A.L. were supported by the Robert and Rosabel Osborne Endowment. M.U.-S. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (750747). F.T.-N., J. Hoffman, P. Masterson and K.C. were supported by the Intramural Research Program of the NLM, NIH. C.L., B.J.K., J. Kim and H.K. were supported by the Marine Biotechnology Program of KIMST, funded by the Ministry of Ocean and Fisheries, Republic of Korea (20180430). M.C. was supported by Sloan Research Fellowship (FG-2020-12932). S.C.V. was funded by a Max Planck Research Group award from the Max Planck Society, and a Human Frontiers Science Program (HFSP) Research grant (RGP0058/2016). T.M.L., W.E.J. and the Canada lynx genome were funded by the Maine Department of Inland Fisheries & Wildlife (F11AF01099), including when W.E.J. held a National Research Council Research Associateship Award at the Walter Reed Army Institute of Research (WRAIR). C.B. was supported by the NSF (1457541 and 1456612). D.B. was funded by The University of Queensland (HFSP - RGP0030/2015). D.I. was supported by Science Exchange Inc. (Palo Alto, CA). H.W.D. was supported by NSF grants (OPP-0132032 ICEFISH 2004 Cruise, PLR-1444167 and OPP-1955368) and the Marine Science Center at Northeastern University (416). G.J.P.N. and the thorny skate genome were funded by Lenfest Ocean Program (30884). M.P. was funded by the German Federal Ministry of Education and Research (01IS18026C). M. Malinsky was supported by an EMBO fellowship (ALTF 456-2016). The following authors’ contributions were supported by the NIH: S. Selvaraj (R44HG008118); C.V.M., S.R.F., P.V.L. (R21 DC014432/DC/NIDCD); K.D.M. (R01GM130691); H.C. (5U41HG002371-19); M.D. (U41HG007234); and B.P. (R01HG010485). D.G. was supported by the National Key Research and Development Program of China (2017YFC1201201, 2018YFC0910504 and 2017YFC0907503). F.O.A. was supported by Al-Gannas Qatari Society and The Cultural Village Foundation-Katara, Doha, State of Qatar and Monash University Malaysia. C.T. was supported by The Rockefeller University. M. Hiller was supported by the LOEWE-Centre for Translational Biodiversity Genomics (TBG) funded by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK). H.C. was supported by the NHGRI (5U41HG002371-19). R.H.S.K. was funded by the Max Planck Society with computational resources at the bwUniCluster and BinAC funded by the Ministry of Science, Research and the Arts Baden-Württemberg and the Universities of the State of Baden-Württemberg, Germany (bwHPC-C5). B.V. was supported by the Biomedical Research Council of A*STAR, Singapore. T.M.-B. was funded by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (864203), MINECO/FEDER, UE (BFU2017-86471-P), Unidad de Excelencia María de Maeztu, AEI (CEX2018-000792-M), a Howard Hughes International Early Career award, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C.T. was supported by the European Research Council (ERC-2012-StG311000) and an Irish Research Council Laureate Award. M.T.P.G. was supported by an ERC Consolidator Award 681396-Extinction Genomics, and a Danish National Research Foundation Center Grant (DNRF143). T.W. was supported by the NSF (1458652). J. M. Graves was supported by the Australian Research Council (CEO561477). E.W.M. was partially supported by the German Federal Ministry of Education and Research (01IS18026C). Complementary sequencing support for the Anna’s hummingbird and several genomes was provided by Pacific Biosciences, Bionano Genomics, Dovetail Genomics, Arima Genomics, Phase Genomics, 10X Genomics, NRGene, Oxford Nanopore Technologies, Illumina, and DNAnexus. All other sequencing and assembly were conducted at the Rockefeller University, Sanger Institute, and Max Planck Institute Dresden genome labs. Part of this work used the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov). We acknowledge funding from the Wellcome Trust (108749/Z/15/Z) and the European Molecular Biology Laboratory., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2018-000792-M).

Since the ancestors of modern humans separated from those of Neandertals, around one hundred amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in the three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in the mouse, three substitutions in two of these proteins, KIF18a and KNL1, cause a prolongation of metaphase and a reduction in chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause a reduction in metaphase length and an increase in chromosome segregation errors in human brain organoids. Our data also show that, in these aspects, Neandertals were more similar to chimpanzees than to modern humans. Thus, the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neandertals.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis1when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways1,2,3that 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 generaPanagrolaimusandPlectus4. Here, we present precise radiocarbon dating indicating that thePanagrolaimusindividuals 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 namedPanagrolaimus n. sp. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis inPanagrolaimus n. sp. and inC. elegansis 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 thatC. elegansdauer 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution plays a significant role in shaping the dynamics of the coronavirus disease 2019 pandemic. To monitor the evolution of SARS-CoV-2 variants, through international collaborations, we performed genomic epidemiology analyses on a weekly basis with SARS-CoV-2 samples collected from a border region between Germany, Poland, and the Czech Republic in a global background. For identified virus mutant variants, active viruses were isolated and functional evaluations were performed to test their replication fitness and neutralization sensitivity against vaccine-elicited serum neutralizing antibodies. Thereby we identified a new B.1.1.7 sub-lineage carrying additional mutations of nucleoprotein G204P and open-reading-frame-8 K68stop. Of note, this B.1.1.7 sub-lineage is the predominant B.1.1.7 variant in several European countries such as Czech Republic, Austria, and Slovakia. The earliest samples belonging to this sub-lineage were detected in November 2020 in a few countries in the European continent, but not in the UK. We have also detected its further evolution with extra spike mutations D138Y and A701V, which are signature mutations shared with the Gamma and Beta variants, respectively. Antibody neutralization assay of virus variant isolations has revealed that the variant with extra spike mutations is 3.2-fold less sensitive to vaccine-elicited antibodies as compared to the other B.1.1.7 variants tested, indicating potential for immune evasion, but it also exhibited reduced replication fitness, suggesting lower transmissibility. The wide spread of this B.1.1.7 sub-lineage was related to the pandemic waves in early 2021 in various European countries. These findings about the emergence, spread, evolution, infection, and transmission abilities of this B.1.1.7 sub-lineage add to our understanding about the pandemic development in Europe and highlight the importance of international collaboration on virus mutant surveillance.

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard. Our analysis of these three independent limbless lineages revealed that signatures of shared sequence or transcription factor binding site divergence in individual limb regulatory elements are generally rare. Instead, shared divergence occurs more often at the level of signaling pathways, involving different regulatory elements associated with the same limb genes (such as Hand2 or Hox) and/or patterning mechanisms (such as Shh signaling). Interestingly, although snakes are known to have mutations in the Shh ZRS limb enhancer, this enhancer lacks relevant mutations in limbless lizards. Thus, different mechanisms could contribute to limb loss, and there are likely multiple evolutionary paths to limblessness in reptiles. ispartof: CELL REPORTS vol:38 issue:3 ispartof: location:United States status: published

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.

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

SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with many new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August to October 2020, indicating the major cause of the second wave was the introduction of new variants.

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease. This paper has an associated First Person interview with the co-first authors of the article.

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.

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., Author summary Parkinson’s disease is the second most common degenerative disorder of the brain. Mutations of the LRRK2 gene are the most recurrent genetic cause of pathology, and are thought to result in a more active LRRK2 protein, a large enzyme whose biological function is unclear. Therefore, LRRK2 inhibitors are regarded as promising therapeutics. However, mouse models do not reproduce human pathology unless they also lack LRRK1, and there is evidence of dominant negative effects of LRRK2 mutations. Therefore, the characterization of reliable LRRK2 knockout models might provide insights. In our study, we used the zebrafish as a tractable model to study both early developmental and adult phenotypes resulting from the loss of the entire endogenous lrrk2 gene. We found that mutant embryos displayed subtle brain phenotypes, including reduction of catecholaminergic neurons, the main target of human disease, that spontaneously resolved with development, and a late-onset and progressive increase of dopamine and serotonin degradation consistent with increased MAO enzyme activity. Our results suggest that similar defects might occur in the pre-symptomatic stage of the disease in humans, and warrant further evaluation of LRRK2 inhibition in a therapeutic perspective.

Defects of filaggrin (FLG) compromise epidermal barrier function and represent an important known genetic risk factor for atopic dermatitis (AD), but also for systemic atopy, including allergic sensitization and asthma. The flaky tail mouse model, widely used to address mechanisms of atopy induction by barrier-defective skin, harbors two mutations that affect the skin barrier, the mutation Flgft, resulting in near-complete loss of FLG expression, and the matted mutation inactivating transmembrane protein 79 (Tmem79). Upon separation of the two mutant loci, which are closely linked on chromosome 3, mice defective only for Tmem79 featured pronounced dermatitis and systemic atopy. Upon extensive backcross to BALB/c, also Flgft/ft mice (assumed to be wild type for Tmem79), developed AD-like dermatitis and reproduced the human ‘atopic march’, with high IgE levels and spontaneous asthma, suggesting a key role for functional Flg in protection from atopy also in mice. In contrast, BALB/c mice congenic for a targeted Flg knock out mutation did not develop skin inflammation or atopy. To resolve this discrepancy, we generated Flg-deficient mice on a pure BALB/c background by inactivating the Flg gene in BALB/c embryos. These animals feature an ichthyosis phenotype, but do not develop spontaneous dermatitis or systemic atopy. We sequenced the genome of the atopic Flgft BALB/c congenics and discovered that they were unexpectedly homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that Flg-deficiency does not cause atopy in mice. This finding is in line with lack of atopic disease in a fraction of Ichthyosis vulgaris patients carrying two FLG null alleles. However, absence of FLG may promote and modulate dermatitis caused by other genetic barrier defects, as skin inflammation in Tmem79ma/maFlgft/ft BALB/c congenics is qualitatively different compared to Tmem79ma/ma mice.

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. As part of the Vertebrate Genomes Project (VGP) we have developed 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 led to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We have observed 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 have identified errors, missing sequences, and incomplete genes in those references, particularly in repeat regions. Our assemblies have also identified novel gene region duplications, shedding new light on mitochondrial genome evolution and organization.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for 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 the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). 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 error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

Defects in the retina or the anterior segment of the eye lead to compromised vision and affect millions of people. Understanding how these ocular structures develop and are maintained is therefore of paramount importance. The maintenance of proper vision depends, among other factors, on the function of genes controlling apico-basal polarity. In fact, mutations in polarity genes are linked to retinal degeneration in several species, including human. Here we describe a novel zebrafish crb2b allele (crb2be40), which specifically affects the crb2b long isoform. crb2be40 mutants are viable and display normal ocular development. However, old crb2be40 mutant fish develop multiple defects in structures of the anterior segment, which includes the cornea, the iris and the lens. Phenotypes are characterised by smaller pupils due to expansion of the iris and tissues of the iridocorneal angle, an increased number of corneal stromal keratocytes, an abnormal corneal endothelium and an expanded lens capsule. These findings illustrate a novel role for crb2b in the maintenance of the anterior segment and hence add an important function to this polarity regulator, which may be conserved in other vertebrates including humans., Summary: Loss of the apico-basal polarity regulator Crb2b-lf does not affect retinal development but leads to ocular anterior segment defects in old zebrafish.

Loss of FLG causes ichthyosis vulgaris. Reduced FLG expression compromises epidermal barrier function and is associated with atopic dermatitis, allergy, and asthma. The flaky tail mouse harbors two mutations that affect the skin barrier, Flgft, resulting in hypomorphic FLG expression, and Tmem79ma, inactivating TMEM79. Mice defective only for TMEM79 featured dermatitis and systemic atopy, but also Flgft/ft BALB/c congenic mice developed eczema, high IgE, and spontaneous asthma, suggesting that FLG protects from atopy. In contrast, a targeted Flg-knockout mutation backcrossed to BALB/c did not result in dermatitis or atopy. To resolve this discrepancy, we generated FLG-deficient mice on pure BALB/c background by inactivating Flg in BALB/c embryos. These mice feature an ichthyosis phenotype, barrier defect, and facilitated percutaneous sensitization. However, they do not develop dermatitis or atopy. Whole-genome sequencing of the atopic Flgft BALB/c congenics revealed that they were homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that FLG deficiency does not cause atopy in mice, in line with lack of atopic disease in a fraction of patients with ichthyosis vulgaris carrying two Flg null alleles. However, the absence of FLG likely promotes and modulates dermatitis caused by other genetic barrier defects.

The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5'- to 3'-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory - European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

Background Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation. Results Here we present a high-quality genome assembly of the bat hawkmoth Hyles vespertilio, the first reference genome of a member of the Hyles genus. We generated 51× Pacific Biosciences long reads with an average read length of 8.9 kb. Pacific Biosciences reads longer than 4 kb were assembled into contigs, resulting in a 651.4-Mb assembly consisting of 530 contigs with an N50 value of 7.5 Mb. The circular mitochondrial contig has a length of 15,303 bp. The H. vespertilio genome is very repeat-rich and exhibits a higher repeat content (50.3%) than other Bombycoidea species such as Bombyx mori (45.7%) and Manduca sexta (27.5%). We developed a comprehensive gene annotation workflow to obtain consensus gene models from different evidence including gene projections, protein homology, transcriptome data, and ab initio predictions. The resulting gene annotation is highly complete with 94.5% of BUSCO genes being completely present, which is higher than the BUSCO completeness of the B. mori (92.2%) and M. sexta (90%) annotations. Conclusions Our gene annotation strategy has general applicability to other genomes, and the H. vespertilio genome provides a valuable molecular resource to study a range of questions in this genus, including phylogeny, incomplete lineage sorting, speciation, and hybridization. A genome browser displaying the genome, alignments, and annotations is available at https://genome-public.pks.mpg.de/cgi-bin/hgTracks?db=HLhylVes1.

Bats account for ~20% of all extant mammal species and are considered exceptional given their extraordinary adaptations, including biosonar, true flight, extreme longevity, and unparalleled immune systems. To understand these adaptations, we generated reference-quality genomes of six species representing the key divergent lineages. We assembled these genomes with a novel pipeline incorporating state-of-the-art long-read and long-range sequencing and assembly techniques. The genomes were annotated using a maximal evidence approach, de novo predictions, protein/mRNA alignments, Iso-seq long read and RNA-seq short read transcripts, and gene projections from our new TOGA pipeline, retrieving virtually all (>99%) mammalian BUSCO genes. Phylogenetic analyses of 12,931 protein coding-genes and 10,857 conserved non-coding elements identified across 48 mammalian genomes helped to resolve bats’ closest extant relatives within Laurasiatheria, supporting a basal position for bats within Scrotifera. Genome-wide screens along the bat ancestral branch revealed (a) selection on hearing-involved genes (e.g LRP2, SERPINB6, TJP2), which suggest that laryngeal echolocation is a shared ancestral trait of bats; (b) selection (e.g INAVA, CXCL13, NPSR1) and loss of immunity related proteins (e.g. LRRC70, IL36G), including pro-inflammatory NF-kB signalling; and (c) expansion of the APOBEC family, associated with restricting viral infection, transposon activity and interferon signalling. We also identified unique integrated viruses, indicating that bats have a history of tolerating viral pathogens, lethal to other mammal species. Non-coding RNA analyses identified variant and novel microRNAs, revealing regulatory relationships that may contribute to phenotypic diversity in bats. Together, our reference-quality genomes, high-quality annotations, genome-wide screens and in-vitro tests revealed previously unknown genomic adaptations in bats that may explain their extraordinary traits.

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., Reference-quality genomes for six bat species shed light on the phylogenetic position of Chiroptera, and provide insight into the genetic underpinnings of the unique adaptations of this clade.

Since the beginning of global trade, hundreds of species have colonized territories outside of their native range. Some of these species proliferate at the expense of native ecosystems, i.e., have become invasive. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales. They also present promising case studies for ecological and evolutionary success in novel environments.We seize this unique opportunity to study genomic substrates for ecological success and adaptability to novel environments in a vertebrate. We report a highly contiguous long-read based genome assembly for the most successful temperate invasive fish, the benthic round goby (Neogobius melanostomus), and analyse gene families that may promote its impressive ecological success.Our approach provides 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 in epigenetic regulators, and the presence of 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 are gene expansions. This suggests that gene duplications may promote ecological flexibility, superior performance in novel environments, and underlie the impressive colonization success of the round goby. Gobiidae generally feature fascinating adaptations and are excellent colonizers. Further long-read genome approaches across the goby family may reveal whether the ability to conquer new habitats relates more generally to gene copy number expansions.

SummaryBovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery, but the molecular consequences that link defects in splicing factor genes to the aetiology of the disease remain to be elucidated. Mutations in PRPF31, one of the splicing factors, are linked to RP11. To get insight into the mechanisms by which mutations in this gene lead to retinal degeneration, we induced mutations in theDrosophilaorthologuePrp31. Flies heterozygous mutant forPrp31are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype.Prp31mutant flies show a high degree of phenotypic variability, similar as reported for human RP11 patients. Degeneration is associated with increased accumulation of rhodopsin 1, both in the rhabdomere and in the cell body. In fact, reducing rhodopsin levels by raising animals in a carotenoid-free medium not only suppressed rhodopsin accumulation, but also retinal degeneration. In addition, our results underscore the relevance of eye color mutations on phenotypic traits, in particular whilst studying a complex process such as retinal degeneration.Article SummaryRetinitis pigmentosa (RP) is a human disease affecting 1.6 million people worldwide. So far >50 genes have been identified that are causally related to RP. Mutations in the splicing factor PRPF31 are linked to RP11. We induced mutations in theDrosophilaorthologuePrp31and show that flies heterozygous forPrp31undergo light-dependent retinal degeneration. Degeneration is associated with increased accumulation of the light-sensitive molecule, rhodopsin 1. In fact, reducing rhodopsin levels by dietary intervention suppressed retinal degeneration. We believe that this model will help to better understand the aetiology of the human disease.

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.

Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that 15 human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene, NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution.

International audience; Salamanders boast an illustrious history in biological research as the animal in which the Spemann organizer 1 and Sperry's chemoaffinity theory of axonal guidance 2 were discovered. Since 1768, when Spallanzani discovered tail and limb regeneration, researchers have probed this animal's remarkable regenerative capabilities with increasing molecular resolution. A. mexicanum (Fig. 1a) was first collected by von Humboldt, and has been cultivated in the laboratory since 1864 as a model for investigating phenomena such as nuclear reprogramming, the embryology of germ-cell induction, retinal neuron processing and regeneration 3. Owing to the ease with which A. mexicanum can be bred in the laboratory, a sophisticated molecular toolkit has been developed for this species, including germline transgenesis and CRISPR-mediated gene mutation as well as viral and other transfection methods. These tools have enabled discoveries such as the identification of the source cells of regeneration and molecular pathways that control regeneration 4,5. A full exploitation of the axolotl model, including understanding regeneration and why it is limited in other tetrapods, requires analysis of its genome regulation and evolution. However, efforts towards comprehensive assembly of salamander genomes have been challenging owing to their large genome sizes (14-120 Gb) and the large number of repetitive regions they contain; the 32-Gb axolotl genome is ten times the size of the human genome. Here we report the sequencing, assembly and analysis of the axolotl genome. A long-read assembler for large genomes Our aim was to generate a genome sequence assembly for the d/d axolotl strain (Fig. 1a), which is commonly used in laboratory regene ration studies owing to its compatibility with live imaging. Taking into consideration the expected challenge of assembling the complex 32-Gb genome 6 , we sequenced 110 million long reads (32× coverage, N50 read length 14.2 kb) using Pacific Biosciences (PacBio) instruments (Supplementary Information section 1) to avoid the read sampling bias that is often found when using other technologies and to span repeat-rich genomic regions that cause breaks in short-read assemblies (Fig. 1b, c). We developed an assembly algorithm (MARVEL) that integrates a two-phase read-correction procedure that keeps long PacBio reads intact for assembly (Supplementary Information section 2). MARVEL produced a contig assembly with an N50 of 218 kb. Next, we used 7× Illumina-based sequencing to correct sequence errors in 1% of the contig bases (Fig. 1b), which yielded a sequence accuracy of more than 99.2%. On the basis of the Illumina data, we estimated a heterozygosity of 0.47% (Supplementary Information section 2.2). To provide a scaffold for the contig assembly, we generated de novo optical maps using the Bionano Saphyr system (Supplementary Information section 2.3). The Bionano map resolved contig chimaeras, which were found in 1.7% of contigs, slightly reducing N50 contig length to 216 kb (Fig. 1d). The final hybrid assembly yielded an N50 scaffold length of 3 Mb. Compared to the short-read assembly of the 20-Gb spruce genome 7 or the 22-Gb loblolly pine genome 8 , which involved 12× long-read coverage, the axolotl assembly showed 56-and 29-fold improvements in contiguity, respectively (Table 1). To assess the completeness of the assembly (Supplementary Information section 4.1), we first determined the number of aligning non-exonic ultraconserved elements 9 (UCEs). We found that 194 (98.5%) of 197 non-exonic UCEs that are conserved across vertebrates align to the axolotl assembly. By comparison, 189 and 192 UCEs align to the Tibetan frog and Xenopus genomes, respectively, and 195 UCEs align to the coelacanth genome, indicating that the completeness of the axolotl genome assembly is comparable to or better than the two other amphibian genomes, which are smaller than 2.3 Gb 10. 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.