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1.
Mol Ecol ; 31(11): 3035-3055, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35344635

RESUMO

Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species' capacity to adapt in the face of extreme selective pressures. Here, we employed exon capture and high-throughput next-generation sequencing to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range. An examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. The initial divergence of these clusters appears to have been concordant with the Mid-Brunhes Transition (~430 to 300 kya), a major climatic reorganisation that increased the amplitude of Pleistocene glacial-interglacial cycles. While signatures of polygenic selection and environmental adaptation were detected, strong evidence for repeated, climate-associated range contractions and demographic bottleneck events suggests that geographically isolated refugia may have played a more significant role in the survival of the koala through the Pleistocene glaciation than in situ adaptation. Consequently, the conservation of genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of vulnerable populations to accelerating anthropogenic threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g., guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.


Assuntos
Phascolarctidae , Animais , Austrália , Evolução Biológica , Ecossistema , Variação Genética/genética , Genômica , Phascolarctidae/genética
2.
Proc Biol Sci ; 288(1945): 20202244, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33622136

RESUMO

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala (Phascolarctos cinereus), and present the first whole-genome, multi-tissue marsupial 'methylome atlas'. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.


Assuntos
Phascolarctidae , Animais , Metilação de DNA , Epigênese Genética , Epigenoma , Feminino , Masculino , Phascolarctidae/genética , Cromossomo X/genética
3.
Proc Natl Acad Sci U S A ; 115(34): 8609-8614, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30082403

RESUMO

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.


Assuntos
Retrovirus Endógenos/genética , Phascolarctidae/genética , Recombinação Genética , Animais , Feminino , Masculino , New South Wales
4.
J Infect Dis ; 220(8): 1312-1324, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31253993

RESUMO

BACKGROUND: Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data. METHODS: In this study, we applied a diverse selection of presequencing enrichment methods targeting all major viral groups, to characterize the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing, we generated 710 datasets constituting 57 billion sequencing reads. RESULTS: Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveal a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained 1 or more viruses; however, no link between specific viruses and cancer types were found. CONCLUSIONS: Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , Neoplasias/virologia , Anelloviridae/genética , Anelloviridae/isolamento & purificação , Biópsia , Conjuntos de Dados como Assunto , Feminino , Herpesviridae/genética , Herpesviridae/isolamento & purificação , Humanos , Masculino , Neoplasias/patologia , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Parvovirus/genética , Parvovirus/isolamento & purificação
5.
Ecol Evol ; 14(8): e11700, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39091325

RESUMO

Genetic management is a critical component of threatened species conservation. Understanding spatial patterns of genetic diversity is essential for evaluating the resilience of fragmented populations to accelerating anthropogenic threats. Nowhere is this more relevant than on the Australian continent, which is experiencing an ongoing loss of biodiversity that exceeds any other developed nation. Using a proprietary genome complexity reduction-based method (DArTSeq), we generated a data set of 3239 high quality Single Nucleotide Polymorphisms (SNPs) to investigate spatial patterns and indices of genetic diversity in the koala (Phascolarctos cinereus), a highly specialised folivorous marsupial that is experiencing rapid and widespread population declines across much of its former range. Our findings demonstrate that current management divisions across the state of New South Wales (NSW) do not fully represent the distribution of genetic diversity among extant koala populations, and that care must be taken to ensure that translocation paradigms based on these frameworks do not inadvertently restrict gene flow between populations and regions that were historically interconnected. We also recommend that koala populations should be prioritised for conservation action based on the scale and severity of the threatening processes that they are currently faced with, rather than placing too much emphasis on their perceived value (e.g., as reservoirs of potentially adaptive alleles), as our data indicate that existing genetic variation in koalas is primarily partitioned among individual animals. As such, the extirpation of koalas from any part of their range represents a potentially critical reduction of genetic diversity for this iconic Australian species.

7.
Nat Commun ; 12(1): 1316, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637755

RESUMO

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.


Assuntos
Células Germinativas , Neoplasias/genética , Infecções por Retroviridae/genética , Retroviridae/genética , Animais , Retrovirus Endógenos , Evolução Molecular , Gammaretrovirus/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Neoplasias/virologia , Phascolarctidae/genética , Phascolarctidae/virologia , Proteínas Repressoras/genética , Infecções por Retroviridae/virologia , Proteína bcl-X/genética
8.
Mol Ecol Resour ; 19(2): 512-525, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30575257

RESUMO

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome-scale studies to characterize both model and non-model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome-wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site-associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms-enabling the exploration of diverse evolutionary and conservation questions.


Assuntos
Elementos de DNA Transponíveis , Genética Populacional/métodos , Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Animais , Cervos , Reação em Cadeia da Polimerase/métodos , Ratos , Lobos
9.
Nat Genet ; 50(8): 1102-1111, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29967444

RESUMO

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.


Assuntos
Adaptação Fisiológica/genética , Phascolarctidae/genética , Animais , Austrália , Infecções por Chlamydia/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Genoma , Anotação de Sequência Molecular/métodos , Phascolarctidae/metabolismo , Translocação Genética
10.
PeerJ ; 4: e1847, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27069793

RESUMO

Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small.

11.
Emerg Microbes Infect ; 5(8): e90, 2016 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-27530749

RESUMO

Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.


Assuntos
Reservatórios de Doenças , Fezes/virologia , Microbioma Gastrointestinal , Variação Genética , Genoma Viral , Infecções por Picornaviridae/epidemiologia , Picornaviridae/genética , Ratos/virologia , Sequência de Aminoácidos , Animais , Dinamarca/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala , Hong Kong/epidemiologia , Humanos , Malásia/epidemiologia , Metagenômica , Filogenia , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , Infecções por Picornaviridae/transmissão , RNA Viral , Proteínas Virais/química , Proteínas Virais/genética , Zoonoses
12.
Viruses ; 8(2)2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26907326

RESUMO

Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. We applied the workflow to 686 sequencing libraries from 252 cancer samples of different cancer and tissue types, 32 non-template controls, and 24 test samples. Recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. We provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. Unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified.


Assuntos
Neoplasias/virologia , Vírus/genética , Vírus/isolamento & purificação , Biologia Computacional , Sequência Conservada , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Viral/genética , Vírus/classificação
13.
Viruses ; 7(11): 6089-107, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26610552

RESUMO

Transcriptome analysis of polar bear (Ursus maritimus) tissues identified sequences with similarity to Porcine Endogenous Retroviruses (PERV). Based on these sequences, four proviral copies and 15 solo long terminal repeats (LTRs) of a newly described endogenous retrovirus were characterized from the polar bear draft genome sequence. Closely related sequences were identified by PCR analysis of brown bear (Ursus arctos) and black bear (Ursus americanus) but were absent in non-Ursinae bear species. The virus was therefore designated UrsusERV. Two distinct groups of LTRs were observed including a recombinant ERV that contained one LTR belonging to each group indicating that genomic invasions by at least two UrsusERV variants have recently occurred. Age estimates based on proviral LTR divergence and conservation of integration sites among ursids suggest the viral group is only a few million years old. The youngest provirus was polar bear specific, had intact open reading frames (ORFs) and could potentially encode functional proteins. Phylogenetic analyses of UrsusERV consensus protein sequences suggest that it is part of a pig, gibbon and koala retrovirus clade. The young age estimates and lineage specificity of the virus suggests UrsusERV is a recent cross species transmission from an unknown reservoir and places the viral group among the youngest of ERVs identified in mammals.


Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Ursidae/genética , Ursidae/virologia , Animais , Feminino , Masculino , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificação , Homologia de Sequência , Sequências Repetidas Terminais
14.
J Clin Virol ; 61(2): 189-95, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25129855

RESUMO

The emergence of encephalitis lethargica (EL), an acute-onset polioencephalitis of unknown etiology as an epidemic in the years 1917-1925 is still unexplainable today. Questioned by the first descriptor of EL himself, Constantin von Economo, there has been much debate shrouding a possible role of the "Spanish" H1N1 influenza A pandemic virus in the development of EL. Previous molecular studies employing conventional PCR for the detection of influenza A virus RNA in archived human brain samples from patients who died of acute EL were negative. However, the clinical and laboratory characteristics of EL and its epidemiology are consistent with an infectious disease, and recently a possible enterovirus cause was investigated. With the rapid development of high-throughput sequencing, new information about a possible viral etiology can be obtained if sufficient specimens for analysis were still available today. Here, we discuss the implications of these technologies for the investigation of a possible infectious cause of EL from archived material, as well as a prospectus for future work for acquiring viral nucleic acids from these sources.


Assuntos
Encéfalo/virologia , Encefalite por Arbovirus/complicações , Encefalite por Arbovirus/etiologia , Doença de Parkinson Pós-Encefalítica/etiologia , Vírus/isolamento & purificação , Encefalite por Arbovirus/história , Sequenciamento de Nucleotídeos em Larga Escala/métodos , História do Século XX , Humanos , Doença de Parkinson Pós-Encefalítica/história , Patologia Molecular/métodos
15.
Sci Rep ; 3: 2468, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24002378

RESUMO

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent "primer walking" combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.


Assuntos
Metagenômica , Vírus da Parainfluenza 4 Humana/genética , Infecções Respiratórias/virologia , Sequência de Bases , Variação Genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenômica/métodos , Dados de Sequência Molecular , Fases de Leitura Aberta , Vírus da Parainfluenza 4 Humana/classificação , Vírus da Parainfluenza 4 Humana/isolamento & purificação , Filogenia , Prevalência , Infecções Respiratórias/epidemiologia , Alinhamento de Sequência
16.
PLoS One ; 8(1): e50961, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23326310

RESUMO

The characterization of biomolecules from ancient samples can shed otherwise unobtainable insights into the past. Despite the fundamental role of transcriptomal change in evolution, the potential of ancient RNA remains unexploited - perhaps due to dogma associated with the fragility of RNA. We hypothesize that seeds offer a plausible refuge for long-term RNA survival, due to the fundamental role of RNA during seed germination. Using RNA-Seq on cDNA synthesized from nucleic acid extracts, we validate this hypothesis through demonstration of partial transcriptomal recovery from two sources of ancient maize kernels. The results suggest that ancient seed transcriptomics may offer a powerful new tool with which to study plant domestication.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Plantas/genética , Sementes/genética , Zea mays/genética , DNA Complementar/química , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação/genética , Sementes/crescimento & desenvolvimento , Fatores de Tempo , Transcriptoma , Zea mays/crescimento & desenvolvimento
17.
Methods Mol Biol ; 888: 109-18, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22665278

RESUMO

Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred to as "ancient DNA," using the Roche GS FLX 454 platform.


Assuntos
DNA/análise , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA , Animais , Bancos de Espécimes Biológicos , Mapeamento Cromossômico , DNA/química , Fragmentação do DNA , Extinção Biológica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mamutes/genética , Homem de Neandertal/genética , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , Ursidae/genética
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