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 Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries.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

Large-scale genome sampling reveals unique immunity and metabolic adaptations in bats.

Comprising more than 1,400 species, bats possess adaptations unique among mammals including powered flight, unexpected longevity, and extraordinary immunity. Some of the molecular mechanisms underlying these unique adaptations includes DNA repair, metabolism and immunity. However, analyses have been limited to a few divergent lineages, reducing the scope of inferences on gene family evolution across the Order Chiroptera. We conducted an exhaustive comparative genomic study of 37 bat species, one generated in this study, encompassing a large number of lineages, with a particular emphasis on multi-gene family evolution across immune and metabolic genes. In agreement with previous analyses, we found lineage-specific expansions of the APOBEC3 and MHC-I gene families, and loss of the proinflammatory PYHIN gene family. We inferred more than 1,000 gene losses unique to bats, including genes involved in the regulation of inflammasome pathways such as epithelial defence receptors, the natural killer gene complex and the interferon-gamma induced pathway. Gene set enrichment analyses revealed genes lost in bats are involved in defence response against pathogen-associated molecular patterns and damage-associated molecular patterns. Gene family evolution and selection analyses indicate bats have evolved fundamental functional differences compared to other mammals in both innate and adaptive immune system, with the potential to enhance antiviral immune response while dampening inflammatory signalling. In addition, metabolic genes have experienced repeated expansions related to convergent shifts to plant-based diets. Our analyses support the hypothesis that, in tandem with flight, ancestral bats had evolved a unique set of immune adaptations whose functional implications remain to be explored.

A landmark environmental law looks ahead.

In late December 1973, the United States enacted what some would come to call "the pitbull of environmental laws." In the 50 years since, the formidable regulatory teeth of the Endangered Species Act (ESA) have been credited with considerable successes, obliging agencies to draw upon the best available science to protect species and habitats. Yet human pressures continue to push the planet toward extinctions on a massive scale. With that prospect looming, and with scientific understanding ever changing, Science invited experts to discuss how the ESA has evolved and what its future might hold. -Brad Wible.

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.

BACKGROUND: 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. RESULTS: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. CONCLUSION: 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, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

The effects of model androgen 5α-dihydrotestosterone on mummichog (Fundulus heteroclitus) reproduction under different salinities.

Endocrine disrupting substances (EDSs) have the potential to disturb sensitive hormone pathways, particularly those involved in development and reproduction. Both fresh and estuarine water bodies receive inputs of EDSs from a variety of sources, including sewage effluent, industrial effluent and agricultural runoff. Based on current literature, freshwater species appear to respond to lower levels of EDSs than estuarine or marine species. Therefore, effects elicited by EDSs in freshwater teleosts may not be an accurate representation of how EDSs affect teleosts in estuarine and marine environments. To address this potential difference, a short-term reproductive bioassay was conducted under conditions of low and high salinity using mummichog (Fundulus heteroclitus), a euryhaline species that is native to the east coast of North America. The goals of this study were to determine the response of mummichog when exposed to an androgenic EDS and whether salinity affected the response. A model androgen, 5α-dihydrotestosterone (DHT), was selected for this experiment. Impacts on reproduction were evaluated at multiple biological levels, including physiological (sex steroid levels), organismal (gonad size and gonad morphology), and functional (egg production) endpoints. Under conditions of high salinity, egg production was significantly reduced at all exposure concentrations. Under conditions of low salinity, there were no significant differences based on DHT treatment; however, egg production in all treatment groups including the control were significantly reduced relative to the high salinity control group. Other reproductive endpoints, such as sex steroid production, showed stronger correlation to fecundity in females than males. This study demonstrates that mummichog fecundity is sensitive to androgenic endocrine disruption while also underscoring the importance of how changes in salinity, an environmental variable, can impact reproduction.