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 Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries.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

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 species 1-4 . To address this issue, the international Genome 10K (G10K) consortium 5,6 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.

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Supplementary feeding is often a key tool in the intensive management of captive and threatened species. Although it can increase such parameters as breeding frequency and individual survival, supplementary feeding may produce undesirable side effects that increase overall extinction risk. Recent attempts to increase breeding frequency and success in the kakapo Strigops habroptilus using supplementary feeding inadvertently resulted in highly male-biased chick sex ratios. Here, we describe how the inclusion of sex allocation theory has remedied this conservation dilemma. Our study is the first to manipulate chick sex ratios in an endangered species by altering maternal condition and highlights the importance of incorporating evolutionary theory into modern conservation practice.

Telomere dynamics are intensively studied in human ageing research and epidemiology, with many correlations reported between telomere length and age-related diseases, cancer and death. While telomere length is influenced by environmental factors there is also good evidence for a strong heritable component. In human, the mode of telomere length inheritance appears to be paternal and telomere length differs between sexes, with females having longer telomeres than males. Genetic factors, e.g. sex chromosomal inactivation, and non-genetic factors, e.g. antioxidant properties of oestrogen, have been suggested as possible explanations for these sex-specific telomere inheritance and telomere length differences. To test the influence of sex chromosomes on telomere length, we investigated inheritance and sex-specificity of telomere length in a bird species, the kakapo (Strigops habroptilus), in which females are the heterogametic sex (ZW) and males are the homogametic (ZZ) sex. We found that, contrary to findings in humans, telomere length was maternally inherited and also longer in males. These results argue against an effect of sex hormones on telomere length and suggest that factors associated with heterogamy may play a role in telomere inheritance and sex-specific differences in telomere length.

bThe critically endangered New Zealand parrot, the kakapo, is subject to an intensive management regime aiming to maintain bird health and boost population size. Newly hatched kakapo chicks are subjected to human intervention and are frequently placed in captivity throughout their formative months. Hand rearing greatly reduces mortality among juveniles, but the potential long-term impact on the kakapo gut microbiota is uncertain. To track development of the kakapo gut microbiota, fecal samples from healthy, prefledged juvenile kakapos, as well as from unrelated adults, were analyzed by using 16S rRNA gene amplicon pyrosequencing. Following the original sampling, juvenile kakapos underwent a period of captivity, so further sampling during and after captivity aimed to elucidate the impact of captivity on the juvenile gut microbiota. Variation in the fecal microbiota over a year was also investigated, with resampling of the original juvenile population. Amplicon pyrosequencing revealed a juvenile fecal microbiota enriched with particular lactic acid bacteria compared to the microbiota of adults, although the overall community structure did not differ significantly among kakapos of different ages. The abundance of key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity, although the importance of these factors could not be proven unequivocally within the bounds of this study. Finally, the microbial community structure of juvenile and adult kakapos changed over time, reinforcing the need for continual monitoring of the microbiota as part of regular health screening.

Decreased genome-wide heterozygosity of inbred individuals can result in reduced survival and reproductive fitness (i.e. inbreeding depression). However, showing such heterozygosity-fitness correlations in endangered species, especially those that are already genetically impoverished, has proven to be difficult. New Zealand's kakapo (Strigops habroptilus) is a critically endangered, flightless parrot that now survives only on islands free of introduced predators. The recent population bottleneck of 51 individuals, lek mating system (where only females care for eggs and young) and the fact that all but one of the population's founders came from the same insular population, render them particularly susceptible to inbreeding depression. The present study uses 25 microsatellite loci to derive estimates of relatedness to investigate heterozygosity-fitness correlations in kakapo. After accounting for the effects of co-variables, there was no evidence that male heterozygosity affected variation in egg fertility, but there was evidence that female heterozygosity affected fecundity (i.e. clutch size) and hatching success; that is, more homozygous females lay smaller clutches and had lower hatching success. The present study detected significant heterozygosity-fitness correlations and demonstrates that inbreeding is a contributing factor towards relatively low hatching success, which in turn reduces population growth in this highly endangered, flightless parrot.

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