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1.
J Transl Med ; 18(1): 362, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32967693

RESUMO

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Variação Genética , Pneumonia Viral/virologia , Substituição de Aminoácidos , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , Genoma Viral , Humanos , Itália/epidemiologia , Mutação , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Polimorfismo de Nucleotídeo Único , Pesquisa Médica Translacional , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Cultura de Vírus/métodos , Replicação Viral/genética , Replicação Viral/fisiologia , Sequenciamento Completo do Genoma
2.
Nat Commun ; 11(1): 4589, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917887

RESUMO

Mandibuloacral dysplasia syndromes are mainly due to recessive LMNA or ZMPSTE24 mutations, with cardinal nuclear morphological abnormalities and dysfunction. We report five homozygous null mutations in MTX2, encoding Metaxin-2 (MTX2), an outer mitochondrial membrane protein, in patients presenting with a severe laminopathy-like mandibuloacral dysplasia characterized by growth retardation, bone resorption, arterial calcification, renal glomerulosclerosis and severe hypertension. Loss of MTX2 in patients' primary fibroblasts leads to loss of Metaxin-1 (MTX1) and mitochondrial dysfunction, including network fragmentation and oxidative phosphorylation impairment. Furthermore, patients' fibroblasts are resistant to induced apoptosis, leading to increased cell senescence and mitophagy and reduced proliferation. Interestingly, secondary nuclear morphological defects are observed in both MTX2-mutant fibroblasts and mtx-2-depleted C. elegans. We thus report the identification of a severe premature aging syndrome revealing an unsuspected link between mitochondrial composition and function and nuclear morphology, establishing a pathophysiological link with premature aging laminopathies and likely explaining common clinical features.


Assuntos
Acro-Osteólise/metabolismo , Predisposição Genética para Doença/genética , Lipodistrofia/metabolismo , Mandíbula/anormalidades , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Acro-Osteólise/diagnóstico por imagem , Acro-Osteólise/genética , Acro-Osteólise/patologia , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Animais , Apoptose , Caenorhabditis elegans , Proliferação de Células , Criança , Regulação para Baixo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Genótipo , Homozigoto , Humanos , Lipodistrofia/diagnóstico por imagem , Lipodistrofia/genética , Lipodistrofia/patologia , Masculino , Mandíbula/diagnóstico por imagem , Proteínas de Membrana/genética , Metaloendopeptidases , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Mutação , Fenótipo , Pele , Sequenciamento Completo do Genoma
3.
BMC Infect Dis ; 20(1): 682, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32942989

RESUMO

BACKGROUND: Enterobacter cloacae species is responsible for nosocomial outbreaks in vulnerable patients in neonatal intensive care units (NICU). The environment can constitute the reservoir and source of infection in NICUs. Herein we report the impact of preventive measures implemented after an Enterobacter cloacae outbreak inside a NICU. METHODS: This retrospective study was conducted in one level 3 NICU in Lyon, France, over a 6 year-period (2012-2018). After an outbreak of Enterobacter cloacae infections in hospitalized neonates in 2013, several measures were implemented including intensive biocleaning and education of medical staff. Clinical and microbiological characteristics of infected patients and evolution of colonization/infection with Enterobacter spp. in this NICU were retrieved. Moreover, whole genome sequencing was performed on 6 outbreak strains. RESULTS: Enterobacter spp. was isolated in 469 patients and 30 patients developed an infection including 2 meningitis and 12 fatal cases. Preventive measures and education of medical staff were not associated with a significant decrease in patient colonisation but led to a persistent decreased use of cephalosporin in the NICU. Infection strains were genetically diverse, supporting the hypothesis of multiple hygiene defects rather than the diffusion of a single clone. CONCLUSIONS: Grouped cases of infections inside one setting are not necessarily related to a single-clone outbreak and could reveal other environmental and organisational problematics. The fight against implementation and transmission of Enterobacter spp. in NICUs remains a major challenge.


Assuntos
Enterobacter cloacae/patogenicidade , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/prevenção & controle , Controle de Infecções/métodos , Surtos de Doenças/prevenção & controle , Enterobacter cloacae/genética , Enterobacter cloacae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Fezes/microbiologia , Feminino , França , Humanos , Higiene , Recém-Nascido , Unidades de Terapia Intensiva Neonatal/estatística & dados numéricos , Masculino , Sepse Neonatal/epidemiologia , Sepse Neonatal/microbiologia , Estudos Retrospectivos , Sequenciamento Completo do Genoma
4.
Nat Commun ; 11(1): 4748, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958763

RESUMO

The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.


Assuntos
Genoma Humano/genética , Mutação , Neoplasias/genética , Composição de Bases , DNA Intergênico , Bases de Dados Genéticas , Exoma/genética , Éxons , Humanos , Estudos Retrospectivos , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
5.
Nat Commun ; 11(1): 4719, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948767

RESUMO

A small number of de novo assembled human genomes have been reported to date, and few have been complemented with population-based genetic variation, which is particularly important for North Africa, a region underrepresented in current genome-wide references. Here, we combine long- and short-read whole-genome sequencing data with recent assembly approaches into a de novo assembly of an Egyptian genome. The assembly demonstrates well-balanced quality metrics and is complemented with variant phasing via linked reads into haploblocks, which we associate with gene expression changes in blood. To construct an Egyptian genome reference, we identify genome-wide genetic variation within a cohort of 110 Egyptian individuals. We show that differences in allele frequencies and linkage disequilibrium between Egyptians and Europeans may compromise the transferability of European ancestry-based genetic disease risk and polygenic scores, substantiating the need for multi-ethnic genome references. Thus, the Egyptian genome reference will be a valuable resource for precision medicine.


Assuntos
Grupos Étnicos/genética , Genética Populacional , Genômica , Egito , Frequência do Gene , Variação Genética , Genoma Humano , Humanos , Desequilíbrio de Ligação , Masculino , Medicina de Precisão , Sequenciamento Completo do Genoma
6.
Antimicrob Resist Infect Control ; 9(1): 154, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32962759

RESUMO

BACKGROUND: Currently, hospitals have been forced to divert substantial resources to cope with the ongoing coronavirus disease 2019 (COVID-19) pandemic. It is unclear if this situation will affect long-standing infection prevention practices and impact on healthcare associated infections. Here, we report a nosocomial cluster of vancomycin-resistant enterococci (VRE) that occurred on a COVID-19 dedicated intensive care unit (ICU) despite intensified contact precautions during the current pandemic. Whole genome sequence-based typing (WGS) was used to investigate genetic relatedness of VRE isolates collected from COVID-19 and non-COVID-19 patients during the outbreak and to compare them to environmental VRE samples. METHODS: Five VRE isolated from patients (three clinical and two screening samples) as well as 11 VRE and six vancomycin susceptible Enterococcus faecium (E. faecium) samples from environmental sites underwent WGS during the outbreak investigation. Isolate relatedness was determined using core genome multilocus sequence typing (cgMLST). RESULTS: WGS revealed two genotypic distinct VRE clusters with genetically closely related patient and environmental isolates. The cluster was terminated by enhanced infection control bundle strategies. CONCLUSIONS: Our results illustrate the importance of continued adherence to infection prevention and control measures during the COVID-19 pandemic to prevent VRE transmission and healthcare associated infections.


Assuntos
Coinfecção/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/epidemiologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Betacoronavirus , Coinfecção/microbiologia , Infecções por Coronavirus/patologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Surtos de Doenças , Genoma Bacteriano/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Controle de Infecções , Unidades de Terapia Intensiva , Tipagem de Sequências Multilocus , Pandemias , Pneumonia Viral/patologia , Prevenção Primária , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/isolamento & purificação , Sequenciamento Completo do Genoma
7.
Sci Rep ; 10(1): 15643, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973171

RESUMO

As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses, for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/prevenção & controle , Proteínas do Nucleocapsídeo/genética , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/imunologia , Sequência de Bases , Infecções por Coronavirus/imunologia , Genoma Viral/genética , Humanos , Proteínas do Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Produtos Inativados/imunologia , Sequenciamento Completo do Genoma
8.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
9.
MMWR Morb Mortal Wkly Rep ; 69(37): 1288-1295, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32966272

RESUMO

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), can spread rapidly in high-risk congregate settings such as skilled nursing facilities (SNFs) (1). In Minnesota, SNF-associated cases accounted for 3,950 (8%) of 48,711 COVID-19 cases reported through July 21, 2020; 35% of SNF-associated cases involved health care personnel (HCP*), including six deaths. Facility-wide, serial testing in SNFs has been used to identify residents with asymptomatic and presymptomatic SARS-CoV-2 infection to inform mitigation efforts, including cohorting of residents with positive test results and exclusion of infected HCP from the workplace (2,3). During April-June 2020, the Minnesota Department of Health (MDH), with CDC assistance, conducted weekly serial testing at two SNFs experiencing COVID-19 outbreaks. Among 259 tested residents, and 341 tested HCP, 64% and 33%, respectively, had positive reverse transcription-polymerase chain reaction (RT-PCR) SARS-CoV-2 test results. Continued SARS-CoV-2 transmission was potentially facilitated by lapses in infection prevention and control (IPC) practices, up to 12-day delays in receiving HCP test results (53%) at one facility, and incomplete HCP participation (71%). Genetic sequencing demonstrated that SARS-CoV-2 viral genomes from HCP and resident specimens were clustered by facility, suggesting facility-based transmission. Residents and HCP working in SNFs are at risk for infection with SARS-CoV-2. As part of comprehensive COVID-19 preparation and response, including early identification of cases, SNFs should conduct serial testing of residents and HCP, maximize HCP testing participation, ensure availability of personal protective equipment (PPE), and enhance IPC practices† (4-5).


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Instituições de Cuidados Especializados de Enfermagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Feminino , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Pandemias , Medição de Risco , Sequenciamento Completo do Genoma , Adulto Jovem
10.
Virol J ; 17(1): 138, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32928234

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed significant threats to international health. The genetic traits as well as evolutionary processes in this novel coronavirus are not fully characterized, and their roles in viral pathogenesis are yet largely unknown. To get a better picture of the codon architecture of this newly emerging coronavirus, in this study we perform bioinformatic analysis, based on publicly available nucleotide sequences of SARS-CoV-2 along with those of other members of human coronaviruses as well as non-human coronaviruses in different hosts, to take a snapshot of the genome-wide codon usage pattern of SARS-CoV-2 and uncover that all over-represented codons end with A/U and this newly emerging coronavirus has a relatively low codon usage bias, which is shaped by both mutation pressure and natural selection. Additionally, there is slight variation in the codon usage pattern among the SARS-CoV-2 isolates from different geo-locations. Furthermore, the overall codon usage pattern of SARS-CoV-2 is generally similar to that of its phylogenetic relatives among non-human betacoronaviruses such as RaTG13. Taken together, we comprehensively analyze the characteristics of codon usage pattern in SARS-CoV-2 via bioinformatic approaches. The information from this research may not only be helpful to get new insights into the evolution of SARS-CoV-2, but also have potential value for developing coronavirus vaccines.


Assuntos
Betacoronavirus/genética , Uso do Códon , Infecções por Coronavirus/virologia , Genoma Viral , Pneumonia Viral/virologia , Animais , Sequência de Bases , Análise por Conglomerados , Códon , Biologia Computacional , Evolução Molecular , Humanos , Mutação , Pandemias , Filogenia , Seleção Genética , Proteínas Virais/genética , Sequenciamento Completo do Genoma
11.
J Infect ; 81(4): 621-624, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32735893

RESUMO

BACKGROUND: Care homes have been disproportionately affected by the COVID-19 pandemic and continue to suffer large outbreaks even when community infection rates are declining, thus representing important pockets of transmission. We assessed occupational risk factors for SARS-CoV-2 infection among staff in six care homes experiencing a COVID-19 outbreak during the peak of the pandemic in London, England. METHODS: Care home staff were tested for SARS-COV-2 infection by RT-PCR and asked to report any symptoms, their contact with residents and if they worked in different care homes. Whole genome sequencing (WGS) was performed on RT-PCR positive samples. RESULTS: In total, 53 (21%) of 254 staff were SARS-CoV-2 positive but only 12/53 (23%) were symptomatic. Among staff working in a single care home, SARS-CoV-2 positivity was 15% (2/13), 16% (7/45) and 18% (30/169) in those reporting no, occasional and regular contact with residents. In contrast, staff working across different care homes (14/27, 52%) had a 3.0-fold (95% CI, 1.9-4.8; P<0.001) higher risk of SARS-CoV-2 positivity than staff working in single care homes (39/227, 17%). WGS identified SARS-CoV-2 clusters involving staff only, including some that included staff working across different care homes. CONCLUSIONS: SARS-CoV-2 positivity was significantly higher among staff working across different care homes than those who were working in the same care home. We found local clusters of SARS-CoV-2 infection between staff only, including those with minimal resident contact. Infection control should be extended for all contact, including those between staff, whilst on care home premises.


Assuntos
Infecções por Coronavirus/epidemiologia , Instituição de Longa Permanência para Idosos/estatística & dados numéricos , Corpo Clínico/estatística & dados numéricos , Casas de Saúde/estatística & dados numéricos , Exposição Ocupacional/efeitos adversos , Pneumonia Viral/epidemiologia , Betacoronavirus/genética , Infecções por Coronavirus/transmissão , Inglaterra/epidemiologia , Genoma Viral/genética , Humanos , Controle de Infecções/métodos , Londres/epidemiologia , Pandemias , Pneumonia Viral/transmissão , Sequenciamento Completo do Genoma
12.
PLoS One ; 15(8): e0237087, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813723

RESUMO

Water buffalo (Bubalus bubalis) is an important source of meat and milk in countries with relatively warm weather. Compared to the cattle genome, a little has been done to reveal its genome structure and genomic traits. This is due to the complications stemming from the large genome size, the complexity of the genome, and the high repetitive content. In this paper, we introduce a high-quality draft assembly of the Egyptian water buffalo genome. The Egyptian breed is used as a dual purpose animal (milk/meat). It is distinguished by its adaptability to the local environment, quality of feed changes, as well as its high resistance to diseases. The genome assembly of the Egyptian water buffalo has been achieved using a reference-based assembly workflow. Our workflow significantly reduced the computational complexity of the assembly process, and improved the assembly quality by integrating different public resources. We also compared our assembly to the currently available draft assemblies of water buffalo breeds. A total of 21,128 genes were identified in the produced assembly. A list of milk virgin-related genes; milk pregnancy-related genes; milk lactation-related genes; milk involution-related genes; and milk mastitis-related genes were identified in the assembly. Our results will significantly contribute to a better understanding of the genetics of the Egyptian water buffalo which will eventually support the ongoing breeding efforts and facilitate the future discovery of genes responsible for complex processes of dairy, meat production and disease resistance among other significant traits.


Assuntos
Búfalos/genética , Genoma , Animais , Anotação de Sequência Molecular , Sequenciamento Completo do Genoma
13.
Nat Commun ; 11(1): 3883, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753598

RESUMO

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Regiões Promotoras Genéticas/genética , RNA-Seq , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
14.
PLoS Pathog ; 16(8): e1008779, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853289

RESUMO

The pandemic spread of African swine fever virus (ASFV) genotype II (GTII) has led to a global crisis. Since the circulating strains are almost identical, time and money have been mis-invested in whole-genome sequencing the last years. New methods, harmonised protocols for sample selection, sequencing, and bioinformatics are therefore urgently needed.


Assuntos
Vírus da Febre Suína Africana/classificação , Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , Genes Virais/genética , Variação Genética , Genoma Viral , Sequenciamento Completo do Genoma/métodos , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/isolamento & purificação , Animais , Biologia Computacional/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Filogenia , Controle de Qualidade , Suínos
15.
BMC Bioinformatics ; 21(1): 360, 2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807073

RESUMO

BACKGROUND: Discovering single nucleotide polymorphisms (SNPs) from agriculture crop genome sequences has been a widely used strategy for developing genetic markers for several applications including marker-assisted breeding, population diversity studies for eco-geographical adaption, genotyping crop germplasm collections, and others. Accurately detecting SNPs from large polyploid crop genomes such as wheat is crucial and challenging. A few variant calling methods have been previously developed but they show a low concordance between their variant calls. A gold standard of variant sets generated from one human individual sample was established for variant calling tool evaluations, however hitherto no gold standard of crop variant set is available for wheat use. The intent of this study was to evaluate seven SNP variant calling tools (FreeBayes, GATK, Platypus, Samtools/mpileup, SNVer, VarScan, VarDict) with the two most popular mapping tools (BWA-mem and Bowtie2) on wheat whole exome capture (WEC) re-sequencing data from allohexaploid wheat. RESULTS: We found the BWA-mem mapping tool had both a higher mapping rate and a higher accuracy rate than Bowtie2. With the same mapping quality (MQ) cutoff, BWA-mem detected more variant bases in mapping reads than Bowtie2. The reads preprocessed with quality trimming or duplicate removal did not significantly affect the final mapping performance in terms of mapped reads. Based on the concordance and receiver operating characteristic (ROC), the Samtools/mpileup variant calling tool with BWA-mem mapping of raw sequence reads outperformed other tests followed by FreeBayes and GATK in terms of specificity and sensitivity. VarDict and VarScan were the poorest performing variant calling tools with the wheat WEC sequence data. CONCLUSION: The BWA-mem and Samtools/mpileup pipeline, with no need to preprocess the raw read data before mapping onto the reference genome, was ascertained the optimum for SNP calling for the complex wheat genome re-sequencing. These results also provide useful guidelines for reliable variant identification from deep sequencing of other large polyploid crop genomes.


Assuntos
Genoma de Planta , Triticum/genética , Sequenciamento Completo do Genoma/métodos , Área Sob a Curva , Humanos , Polimorfismo de Nucleotídeo Único , Poliploidia , Análise de Componente Principal , Curva ROC , Software
16.
Genes (Basel) ; 11(8)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32824573

RESUMO

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Sequenciamento Completo do Genoma/métodos , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Pandemias , Pneumonia Viral/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma/normas
17.
Methods Mol Biol ; 2203: 67-74, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833204

RESUMO

This chapter reports the high-throughput sequencing protocol for sequencing Coronaviruses and other positive strand viruses to produce a dataset of significant depth of coverage. The protocol describes sequencing of infectious bronchitis virus propagated in embryonated eggs and harvested in the allantoic fluid. The protocol is composed of three main steps-enrichment of the allantoic fluid using ultracentrifugation, extraction of total RNA from allantoic fluid, and library preparation from total RNA to DNA sequencing libraries. The workflow will be suitable for all coronaviruses using high-throughput sequencing platforms.


Assuntos
Coronavirus/genética , Sequenciamento Completo do Genoma/métodos , Animais , Membrana Corioalantoide/virologia , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/isolamento & purificação , Fluxo de Trabalho
18.
Nat Commun ; 11(1): 4072, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792663

RESUMO

Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool.


Assuntos
Citidina/metabolismo , Endonucleases/metabolismo , Sequenciamento Completo do Genoma/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citidina/genética , DNA/genética , DNA/metabolismo , Endonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Genoma Humano/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Guia/genética
19.
EBioMedicine ; 59: 102960, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32853988

RESUMO

BACKGROUND: Some COVID-19 cases test positive again for SARS-CoV-2 RNA following negative test results and discharge, raising questions about the meaning of virus detection. Better characterization of re-positive cases is urgently needed. METHODS: Clinical data were obtained through Guangdong's COVID-19 surveillance network. Neutralization antibody titre was determined using microneutralization assays. Potential infectivity of clinical samples was evaluated by cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. FINDINGS: Among 619 discharged COVID-19 cases, 87 re-tested as SARS-CoV-2 positive in circumstances of social isolation. All re-positive cases had mild or moderate symptoms at initial diagnosis and were younger on average (median, 28). Re-positive cases (n = 59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n = 218) tested here. No infectious strain could be obtained by culture and no full-length viral genomes could be sequenced from re-positive cases. INTERPRETATION: Re-positive SARS-CoV-2 cases do not appear to be caused by active reinfection and were identified in ~14% of discharged cases. A robust NAb response and potential virus genome degradation were detected in almost all re-positive cases, suggesting a substantially lower transmission risk, especially through respiratory routes.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Betacoronavirus/isolamento & purificação , Criança , Pré-Escolar , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Pandemias , Alta do Paciente , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Sequenciamento Completo do Genoma , Adulto Jovem
20.
PLoS One ; 15(8): e0236285, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841250

RESUMO

Characterizing meiotic recombination rates across the genomes of nonhuman primates is important for understanding the genetics of primate populations, performing genetic analyses of phenotypic variation and reconstructing the evolution of human recombination. Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primates in biomedical research. We constructed a high-resolution genetic map of the rhesus genome based on whole genome sequence data from Indian-origin rhesus macaques. The genetic markers used were approximately 18 million SNPs, with marker density 6.93 per kb across the autosomes. We report that the genome-wide recombination rate in rhesus macaques is significantly lower than rates observed in apes or humans, while the distribution of recombination across the macaque genome is more uniform. These observations provide new comparative information regarding the evolution of recombination in primates.


Assuntos
Evolução Molecular , Macaca mulatta/genética , Meiose/genética , Recombinação Genética , Animais , Mapeamento Cromossômico , Marcadores Genéticos , Variação Genética , Genoma , Humanos , Polimorfismo de Nucleotídeo Único , Especificidade da Espécie , Sequenciamento Completo do Genoma
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