The landscape of genomic structural variation in Indigenous Australians.

Indigenous Australians harbour rich and unique genomic diversity. However, Aboriginal and Torres Strait Islander ancestries are historically under-represented in genomics research and almost completely missing from reference datasets1-3. Addressing this representation gap is critical, both to advance our understanding of global human genomic diversity and as a prerequisite for ensuring equitable outcomes in genomic medicine. Here we apply population-scale whole-genome long-read sequencing4 to profile genomic structural variation across four remote Indigenous communities. We uncover an abundance of large insertion-deletion variants (20-49 bp; n = 136,797), structural variants (50 b-50 kb; n = 159,912) and regions of variable copy number (>50 kb; n = 156). The majority of variants are composed of tandem repeat or interspersed mobile element sequences (up to 90%) and have not been previously annotated (up to 62%). A large fraction of structural variants appear to be exclusive to Indigenous Australians (12% lower-bound estimate) and most of these are found in only a single community, underscoring the need for broad and deep sampling to achieve a comprehensive catalogue of genomic structural variation across the Australian continent. Finally, we explore short tandem repeats throughout the genome to characterize allelic diversity at 50 known disease loci5, uncover hundreds of novel repeat expansion sites within protein-coding genes, and identify unique patterns of diversity and constraint among short tandem repeat sequences. Our study sheds new light on the dimensions and dynamics of genomic structural variation within and beyond Australia.

Indigenous Australian genomes show deep structure and rich novel variation.

The Indigenous peoples of Australia have a rich linguistic and cultural history. How this relates to genetic diversity remains largely unknown because of their limited engagement with genomic studies. Here we analyse the genomes of 159 individuals from four remote Indigenous communities, including people who speak a language (Tiwi) not from the most widespread family (Pama-Nyungan). This large collection of Indigenous Australian genomes was made possible by careful community engagement and consultation. We observe exceptionally strong population structure across Australia, driven by divergence times between communities of 26,000-35,000 years ago and long-term low but stable effective population sizes. This demographic history, including early divergence from Papua New Guinean (47,000 years ago) and Eurasian groups1, has generated the highest proportion of previously undescribed genetic variation seen outside Africa and the most extended homozygosity compared with global samples. A substantial proportion of this variation is not observed in global reference panels or clinical datasets, and variation with predicted functional consequence is more likely to be homozygous than in other populations, with consequent implications for medical genomics2. Our results show that Indigenous Australians are not a single homogeneous genetic group and their genetic relationship with the peoples of New Guinea is not uniform. These patterns imply that the full breadth of Indigenous Australian genetic diversity remains uncharacterized, potentially limiting genomic medicine and equitable healthcare for Indigenous Australians.

Uncovering the Genetic and Molecular Features of Huntington's Disease in Northern Colombia.

Huntington's disease (HD) is a genetic disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene. Juan de Acosta, Atlántico, a city located on the Caribbean coast of Colombia, is home to the world's second-largest HD pedigree. Here, we include 291 descendants of this pedigree with at least one family member with HD. Blood samples were collected, and genomic DNA was extracted. We quantified the HTT CAG expansion using an amplicon sequencing protocol. The genetic heterogeneity was measured as the ratio of the mosaicism allele's read peak and the slippage ratio of the allele's read peak from our sequence data. The statistical and bioinformatic analyses were performed with a significance threshold of p < 0.05. We found that the average HTT CAG repeat length in all participants was 21.91 (SD = 8.92). Of the 291 participants, 33 (11.3%, 18 females) had a positive molecular diagnosis for HD. Most affected individuals were adults, and the most common primary and secondary alleles were 17/7 (CAG/CCG) and 17/10 (CAG/CCG), respectively. The mosaicism increased with age in the participants with HD, while the slippage analyses revealed differences by the HD allele type only for the secondary allele. The slippage tended to increase with the HTT CAG repeat length in the participants with HD, but the increase was not statistically significant. This study analyzed the genetic and molecular features of 291 participants, including 33 with HD. We found that the mosaicism increased with age in the participants with HD, particularly for the secondary allele. The most common haplotype was 17/7_17/10. The slippage for the secondary allele varied by the HD allele type, but there was no significant difference in the slippage by sex. Our findings offer valuable insights into HD and could have implications for future research and clinical management.

Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Arctic introgression and chromatin regulation facilitated rapid Qinghai-Tibet Plateau colonization by an avian predator.

The Qinghai-Tibet Plateau (QTP), possesses a climate as cold as that of the Arctic, and also presents uniquely low oxygen concentrations and intense ultraviolet (UV) radiation. QTP animals have adapted to these extreme conditions, but whether they obtained genetic variations from the Arctic during cold adaptation, and how genomic mutations in non-coding regions regulate gene expression under hypoxia and intense UV environment, remain largely unknown. Here, we assemble a high-quality saker falcon genome and resequence populations across Eurasia. We identify female-biased hybridization with Arctic gyrfalcons in the last glacial maximum, that endowed eastern sakers with alleles conveying larger body size and changes in fat metabolism, predisposing their QTP cold adaptation. We discover that QTP hypoxia and UV adaptations mainly involve independent changes in non-coding genomic variants. Our study highlights key roles of gene flow from Arctic relatives during QTP hypothermia adaptation, and cis-regulatory elements during hypoxic response and UV protection.

The genomic landscape of blood groups in Indigenous Australians in remote communities.

BACKGROUND: Red blood cell (RBC) membrane-associated blood group systems are clinically significant. Alloimmunisation is a persistent risk associated with blood transfusion owing to the antigen polymorphisms among these RBC-associated blood groups. Next-generation sequencing (NGS) offers an opportunity to characterize the blood group variant profile of a given individual. Australia comprises a large multiethnic population where most blood donors are Caucasian and blood group variants remain poorly studied among Indigenous Australians. In this study, we focused on the Tiwi Islanders, who have lived in relative isolation for thousands of years. METHODS AND MATERIALS: We predicted the blood group phenotype profiles in the Tiwi (457) and 1000 Genomes Phase 3 (1KGP3-2504) cohort individuals using RBCeq (https://www.rbceq.org/). The predicted phenotype prevalence was compared with the previous literature report. RESULTS: We report, for the first time, comprehensive blood group profiles corresponding to the 35 known blood group systems among the Indigenous Tiwi islander population and identify possible novel antigen variants therein. Our results demonstrate that the genetic makeup of the Tiwi participants is distinct from that of other populations, with a low prevalence of LU (Au[a-b+]) and ABO (A2) and D+C+c+E+e- phenotype, an absence of Diego blood group variants, and a unique RHD (DIII type4) variant. CONCLUSION: Our results may contribute to the development of a database of predicted phenotype donors among the Tiwi population and aid in improving transfusion safety for the ~2800 Tiwi people and the ~800,000 other Indigenous Australians throughout the nation.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Balancing openness with Indigenous data sovereignty: An opportunity to leave no one behind in the journey to sequence all of life.

The field of genomics has benefited greatly from its "openness" approach to data sharing. However, with the increasing volume of sequence information being created and stored and the growing number of international genomics efforts, the equity of openness is under question. The United Nations Convention of Biodiversity aims to develop and adopt a standard policy on access and benefit-sharing for sequence information across signatory parties. This standardization will have profound implications on genomics research, requiring a new definition of open data sharing. The redefinition of openness is not unwarranted, as its limitations have unintentionally introduced barriers of engagement to some, including Indigenous Peoples. This commentary provides an insight into the key challenges of openness faced by the researchers who aspire to protect and conserve global biodiversity, including Indigenous flora and fauna, and presents immediate, practical solutions that, if implemented, will equip the genomics community with both the diversity and inclusivity required to respectfully protect global biodiversity.

Pharmacogenomic analysis of a genetically distinct Indigenous population.

Indigenous Australians face a disproportionately severe burden of chronic disease relative to other Australians, with elevated rates of morbidity and mortality. While genomics technologies are slowly gaining momentum in personalised treatments for many, a lack of pharmacogenomic research in Indigenous peoples could delay adoption. Appropriately implementing pharmacogenomics in clinical care necessitates an understanding of the frequencies of pharmacologically relevant genetic variants within Indigenous populations. We analysed whole-genome sequence data from 187 individuals from the Tiwi Islands and characterised the pharmacogenomic landscape of this population. Specifically, we compared variant profiles and allelic distributions of previously described pharmacologically significant genes and variants with other population groups. We identified 22 translationally relevant pharmacogenomic variants and 18 clinically actionable guidelines with implications for drug dosing and treatment of conditions including heart disease, diabetes and cancer. We specifically observed increased poor and intermediate metabolizer phenotypes in the CYP2C9 (PM:19%, IM:44%) and CYP2C19 (PM:18%, IM:44%) genes.

Genomic attributes differ between Vibrio parahaemolyticus environmental and clinical isolates including pathotypes.

Vibrio parahaemolyticus is a marine bacterium and causes opportunistic gastroenteritis in humans. Clinical strains of V. parahaemolyticus contain haemolysin and type III secretion systems (T3SS) that define their pathotype. A growing number of strains isolated recently from the environment have acquired these virulence genes constituting a pool of potential pathogens. This study used comparative genomics to identify genetic factors that delineate environmental and clinical V. parahaemolyticus population and understand the similarities and differences between the T3SS2 phylotypes. The comparative analysis revealed the presence of a cluster of genes belonging to bacterial cellulose synthesis (bcs) in isolates of environmental origin. This cluster, previously unreported in V. parahaemolyticus, exhibit significant similarity to that of Aliivibrio fischeri, and might dictate a potentially new mechanism of its environmental adaptation and persistence. The study also identified many genes predicted in silico to be T3SS effectors that are unique to T3SS2ß of tdh- trh+ and tdh+ trh+ pathotype and having no identifiable homologue in tdh+ trh- T3SS2α. Overall, these findings highlight the importance of understanding the genes and strategies V. parahaemolyticus utilize for the myriad interactions with its hosts, either marine invertebrates or humans.

Microchromosomes are building blocks of bird, reptile, and mammal chromosomes.

Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical.

A globally diverse reference alignment and panel for imputation of mitochondrial DNA variants.

BACKGROUND: Variation in mitochondrial DNA (mtDNA) identified by genotyping microarrays or by sequencing only the hypervariable regions of the genome may be insufficient to reliably assign mitochondrial genomes to phylogenetic lineages or haplogroups. This lack of resolution can limit functional and clinical interpretation of a substantial body of existing mtDNA data. To address this limitation, we developed and evaluated a large, curated reference alignment of complete mtDNA sequences as part of a pipeline for imputing missing mtDNA single nucleotide variants (mtSNVs). We call our reference alignment and pipeline MitoImpute. RESULTS: We aligned the sequences of 36,960 complete human mitochondrial genomes downloaded from GenBank, filtered and controlled for quality. These sequences were reformatted for use in imputation software, IMPUTE2. We assessed the imputation accuracy of MitoImpute by measuring haplogroup and genotype concordance in data from the 1000 Genomes Project and the Alzheimer's Disease Neuroimaging Initiative (ADNI). The mean improvement of haplogroup assignment in the 1000 Genomes samples was 42.7% (Matthew's correlation coefficient = 0.64). In the ADNI cohort, we imputed missing single nucleotide variants. CONCLUSION: These results show that our reference alignment and panel can be used to impute missing mtSNVs in existing data obtained from using microarrays, thereby broadening the scope of functional and clinical investigation of mtDNA. This improvement may be particularly useful in studies where participants have been recruited over time and mtDNA data obtained using different methods, enabling better integration of early data collected using less accurate methods with more recent sequence data.

PACIFIC: a lightweight deep-learning classifier of SARS-CoV-2 and co-infecting RNA viruses.

Viral co-infections occur in COVID-19 patients, potentially impacting disease progression and severity. However, there is currently no dedicated method to identify viral co-infections in patient RNA-seq data. We developed PACIFIC, a deep-learning algorithm that accurately detects SARS-CoV-2 and other common RNA respiratory viruses from RNA-seq data. Using in silico data, PACIFIC recovers the presence and relative concentrations of viruses with > 99% precision and recall. PACIFIC accurately detects SARS-CoV-2 and other viral infections in 63 independent in vitro cell culture and patient datasets. PACIFIC is an end-to-end tool that enables the systematic monitoring of viral infections in the current global pandemic.

MicroRNA dynamics during hibernation of the Australian central bearded dragon (Pogona vitticeps).

Hibernation is a physiological state employed by many animals that are exposed to limited food and adverse winter conditions. Controlling tissue-specific and organism wide changes in metabolism and cellular function requires precise regulation of gene expression, including by microRNAs (miRNAs). Here we profile miRNA expression in the central bearded dragon (Pogona vitticeps) using small RNA sequencing of brain, heart, and skeletal muscle from individuals in late hibernation and four days post-arousal. A total of 1295 miRNAs were identified in the central bearded dragon genome; 664 of which were novel to central bearded dragon. We identified differentially expressed miRNAs (DEmiRs) in all tissues and correlated mRNA expression with known and predicted target mRNAs. Functional analysis of DEmiR targets revealed an enrichment of differentially expressed mRNA targets involved in metabolic processes. However, we failed to reveal biologically relevant tissue-specific processes subjected to miRNA-mediated regulation in heart and skeletal muscle. In brain, neuroprotective pathways were identified as potential targets regulated by miRNAs. Our data suggests that miRNAs are necessary for modulating the shift in cellular metabolism during hibernation and regulating neuroprotection in the brain. This study is the first of its kind in a hibernating reptile and provides key insight into this ephemeral phenotype.

Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

Visual Opsin Diversity in Sharks and Rays.

The diversity of color vision systems found in extant vertebrates suggests that different evolutionary selection pressures have driven specializations in photoreceptor complement and visual pigment spectral tuning appropriate for an animal's behavior, habitat, and life history. Aquatic vertebrates in particular show high variability in chromatic vision and have become important models for understanding the role of color vision in prey detection, predator avoidance, and social interactions. In this study, we examined the capacity for chromatic vision in elasmobranch fishes, a group that have received relatively little attention to date. We used microspectrophotometry to measure the spectral absorbance of the visual pigments in the outer segments of individual photoreceptors from several ray and shark species, and we sequenced the opsin mRNAs obtained from the retinas of the same species, as well as from additional elasmobranch species. We reveal the phylogenetically widespread occurrence of dichromatic color vision in rays based on two cone opsins, RH2 and LWS. We also confirm that all shark species studied to date appear to be cone monochromats but report that in different species the single cone opsin may be of either the LWS or the RH2 class. From this, we infer that cone monochromacy in sharks has evolved independently on multiple occasions. Together with earlier discoveries in secondarily aquatic marine mammals, this suggests that cone-based color vision may be of little use for large marine predators, such as sharks, pinnipeds, and cetaceans.

Waking the sleeping dragon: gene expression profiling reveals adaptive strategies of the hibernating reptile Pogona vitticeps.

BACKGROUND: Hibernation is a physiological state exploited by many animals exposed to prolonged adverse environmental conditions associated with winter. Large changes in metabolism and cellular function occur, with many stress response pathways modulated to tolerate physiological challenges that might otherwise be lethal. Many studies have sought to elucidate the molecular mechanisms of mammalian hibernation, but detailed analyses are lacking in reptiles. Here we examine gene expression in the Australian central bearded dragon (Pogona vitticeps) using mRNA-seq and label-free quantitative mass spectrometry in matched brain, heart and skeletal muscle samples from animals at late hibernation, 2 days post-arousal and 2 months post-arousal. RESULTS: We identified differentially expressed genes in all tissues between hibernation and post-arousal time points; with 4264 differentially expressed genes in brain, 5340 differentially expressed genes in heart, and 5587 differentially expressed genes in skeletal muscle. Furthermore, we identified 2482 differentially expressed genes across all tissues. Proteomic analysis identified 743 proteins (58 differentially expressed) in brain, 535 (57 differentially expressed) in heart, and 337 (36 differentially expressed) in skeletal muscle. Tissue-specific analyses revealed enrichment of protective mechanisms in all tissues, including neuroprotective pathways in brain, cardiac hypertrophic processes in heart, and atrophy protective pathways in skeletal muscle. In all tissues stress response pathways were induced during hibernation, as well as evidence for gene expression regulation at transcription, translation and post-translation. CONCLUSIONS: These results reveal critical stress response pathways and protective mechanisms that allow for maintenance of both tissue-specific function, and survival during hibernation in the central bearded dragon. Furthermore, we provide evidence for multiple levels of gene expression regulation during hibernation, particularly enrichment of miRNA-mediated translational repression machinery; a process that would allow for rapid and energy efficient reactivation of translation from mature mRNA molecules at arousal. This study is the first molecular investigation of its kind in a hibernating reptile, and identifies strategies not yet observed in other hibernators to cope stress associated with this remarkable state of metabolic depression.

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as 'vulnerable' due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala's ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala's survival in the wild.

Evolution of the shut-off steps of vertebrate phototransduction.

Different isoforms of the genes involved in phototransduction are expressed in vertebrate rod and cone photoreceptors, providing a unique example of parallel evolution via gene duplication. In this study, we determine the molecular phylogeny of the proteins underlying the shut-off steps of phototransduction in the agnathan and jawed vertebrate lineages. For the G-protein receptor kinases (GRKs), the GRK1 and GRK7 divisions arose prior to the divergence of tunicates, with further expansion during the two rounds of whole-genome duplication (2R); subsequently, jawed and agnathan vertebrates retained different subsets of three isoforms of GRK. For the arrestins, gene expansion occurred during 2R. Importantly, both for GRKs and arrestins, the respective rod isoforms did not emerge until the second round of 2R, just prior to the separation of jawed and agnathan vertebrates. For the triplet of proteins mediating shut-off of the G-protein transducin, RGS9 diverged from RGS11, probably at the second round of 2R, whereas Gß5 and R9AP appear not to have undergone 2R expansion. Overall, our analysis provides a description of the duplications and losses of phototransduction shut-off genes that occurred during the transition from a chordate with only cone-like photoreceptors to an ancestral vertebrate with both cone- and rod-like photoreceptors.