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1.
J Med Microbiol ; 70(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34165424

RESUMO

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019.


Assuntos
Macrolídeos/farmacologia , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/epidemiologia , Criança , Farmacorresistência Bacteriana/genética , Técnicas de Genotipagem/métodos , Humanos , Japão/epidemiologia , Mutação , Mycoplasma pneumoniae/classificação , Mycoplasma pneumoniae/efeitos dos fármacos , Nasofaringe/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/microbiologia , RNA Ribossômico 23S/genética
2.
Methods Mol Biol ; 2277: 143-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080150

RESUMO

Mice missing the Complex I subunit NADH:Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) of the electron transport chain are a leading model of the severe mitochondrial disease Leigh syndrome. These mice have enabled a better understanding of mitochondrial dysfunction in human disease, as well as in the discovery of interventions that can potentially suppress mitochondrial disease manifestations. In addition, increasing evidence suggests significant overlap between interventions that increase survival in NDUFS4 knockout mice and that extend life span during normative aging. This chapter discusses the practical aspects of handling and studying these mice, which can be challenging due to their severe disease phenotype. Common procedures such as breeding, genotyping, weaning, or treating these transgenic mice are also discussed.


Assuntos
Envelhecimento/genética , Ração Animal , Complexo I de Transporte de Elétrons/genética , Camundongos Knockout , Envelhecimento/fisiologia , Animais , Feminino , Técnicas de Genotipagem , Humanos , Doença de Leigh/genética , Masculino , Doenças Mitocondriais/genética , Sirolimo/farmacologia
3.
Medicine (Baltimore) ; 100(19): e25762, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34106605

RESUMO

ABSTRACT: The aim of this study was to explore the association of rs1836724 single-nucleotide polymorphism (SNP) of ERBB4 with risk and prognosis of non-small cell lung cancer (NSCLC) in the Chinese Han population.The genotype of rs1836724 SNP of ERBB4 from 258 patients with NSCLC and 200 noncancer controls were detected the TaqMan-MGB probes real-time fluorescence polymerase chain reaction. The distribution of genotype and alleles between the 2 groups was compared, and the association between clinicopathological characteristic and rs1836724 SNP was analyzed. Prognosis and influencing factors were analyzed by Kaplan-Meier and Cox regression analysis.There were significant differences in the genotype and allele distribution of ERBB4 rs1836724 between the NSCLC group and control group (P < .05). And CC genotype of rs1836724 was associated with increased risk of NSCLC in the Chinese Han population. Rs1836724 SNP was associated with TNM stage and lymph nodal metastasis (P = .001, P = .007). The median follow-up was 29 months, and the progression-free survival and overall survival of 258 NSCLC patients were 27.91% and 31.39%, respectively. Patients with GG genotype of rs1836724 had poor progression-free survival and overall survival. Rs1836724 SNP was an independent prognostic marker of NSCLC patients, CC genotype had a high risk of poor prognosis (odds ratio = 1.587, 95% confidence interval: 1.079-2.335, P = .019).In Chinese Han populations, rs1836724 SNP of ERBB4 may contribute toward the increased risk and poor prognosis of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Receptor ErbB-4/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/etnologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Estudos de Casos e Controles , China/epidemiologia , Feminino , Seguimentos , Genótipo , Técnicas de Genotipagem , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/etnologia , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Análise de Sobrevida
4.
Ann Saudi Med ; 41(3): 141-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085542

RESUMO

BACKGROUND: Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism may play a role in the pathogenesis of coronavirus-19 disease (COVID-19). OBJECTIVES: Investigate the relationship between ACE I/D polymorphism and the clinical severity of COVID-19. DESIGN: Prospective cohort study. SETTING: Tertiary care hospital. PATIENTS AND METHODS: The study included COVID-19 patients with asymptomatic, mild, and severe disease with clinical data and whole blood samples collected from 1 April 2020 to 1 July 2020. ACE I/D genotypes were determined by polymerase chain reaction and agarose gel electrophoresis. MAIN OUTCOME MEASURE: ACE DD, DI and II genotypes frequencies. SAMPLE SIZE: 90 cases, 30 in each disease severity group. RESULTS: Age and the frequency of general comorbidity increased significantly from the asymptomatic disease group to the severe disease group. Advanced age, diabetes mellitus and presence of ischemic heart disease were independent risk factors for severe COVID-19 [OR and 95 % CI: 1.052 (1.021-1.083), 5.204 (1.006-26.892) and 5.922 (1.109-31.633), respectively]. The ACE II genotype was the dominant genotype (50%) in asymptomatic patients, while the DD genotype was the dominant genotype (63.3 %) in severe disease. The ACE II geno-type was protective against severe COVID-19 [OR and 95% CI: .323 (.112-.929)]. All nine patients (8.9%) who died had severe disease. CONCLUSIONS: The clinical severity of COVID-19 infection may be associated with the ACE I/D polymorphism. LIMITATIONS: Small sample size and single center. CONFLICT OF INTEREST: None.


Assuntos
COVID-19/genética , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Índice de Gravidade de Doença , Adulto , Idoso , Sequência de Bases , COVID-19/diagnóstico , Feminino , Seguimentos , Marcadores Genéticos , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Estudos Prospectivos , Deleção de Sequência
5.
Arch Virol ; 166(8): 2249-2254, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33999261

RESUMO

Porcine parvovirus 1 (PPV1) is a major cause of reproductive failure in pigs. To date, six additional porcine parvoviruses (PPV2-PPV7) have been identified. In this study, we detected 11 PPV1 strains, five PPV3 strains, three PPV4 strains, six PPV5 strains, five PPV6 strains, and one PPV7 strain in Korean wild boars. PPV1, -3, and -5, and PPV6 from Korean wild boars harbor conserved motifs within the Ca2+ binding loop and the catalytic center of the PLA1 motif. Intra-species recombination among PPV7 strains was also identified. Genetic characterization revealed that PPV1 from Korean wild boars may be similar to virulent PPV strains.


Assuntos
DNA Viral/genética , Infecções por Parvoviridae/virologia , Parvovirus Suíno/classificação , Sus scrofa/virologia , Substituição de Aminoácidos , Animais , Feminino , Técnicas de Genotipagem , Masculino , Parvovirus Suíno/genética , Parvovirus Suíno/isolamento & purificação , Filogenia , República da Coreia , Suínos
6.
Nat Immunol ; 22(6): 781-793, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031617

RESUMO

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.


Assuntos
Memória Imunológica , Mycobacterium tuberculosis/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Progressão da Doença , Feminino , Seguimentos , Predisposição Genética para Doença , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Peru , RNA-Seq , Fatores Sexuais , Análise de Célula Única , Fatores Socioeconômicos , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/microbiologia , Adulto Jovem
7.
Eur J Protistol ; 79: 125799, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34044353

RESUMO

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.


Assuntos
Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da Espécie
8.
BMC Genomics ; 22(1): 378, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34030629

RESUMO

BACKGROUND: Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. "Next generation" high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose. RESULTS: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807-819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123-139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic. CONCLUSIONS: The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.


Assuntos
Técnicas de Genotipagem , Polimorfismo de Nucleotídeo Único , Animais , Custos e Análise de Custo , Genótipo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
9.
Mol Biol Rep ; 48(4): 3593-3604, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33973139

RESUMO

Variants in the epidermal growth factor receptor (EGFR) gene are recognized as predictors of therapy response and are correlated with progression-free and overall survival in non-small cell lung cancer (NSCLC) patients. Molecularly guided therapy needs precise and cost-effective molecular tests. This review focused primarily on screening or target methods for the EGFR variants detection with diagnostic and prognostic potential in the clinical research published papers. Concerning the inclusion and exclusion criteria, the search interval comprised available articles published from 2010 until 2020 in three electronic databases, ISI Web of Science, Pub Med, and Scopus. The analysis of eligible studies started with 5647 and obtained the final 987 full-text articles analyzed as clinical research. The regions comprised were Africa, America, Australia, Asia, Euro-Asia, Europe, or a consortium of different countries. All of the tested methods were applied prevalently in Asia. In clinical research, the polymerase chain reaction (PCR), followed by sequencing methods have been involved mostly over the years. The identified high-through output approaches evolved to improve the survival and quality of the NSCLC patient's life becoming more sensitive, specific, and cost-effective.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Testes Genéticos/métodos , Técnicas de Genotipagem/métodos , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Humanos , Polimorfismo Genético
10.
Nat Commun ; 12(1): 2981, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016985

RESUMO

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Recombinação Genética , Espermatócitos/metabolismo , Animais , Evolução Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos , Cromossomos/genética , Europa (Continente) , Fertilidade/genética , Técnicas de Genotipagem/métodos , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Análise do Sêmen , Espermatócitos/citologia
11.
Medicine (Baltimore) ; 100(18): e25203, 2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-33950918

RESUMO

ABSTRACT: Introduction: Hepatitis C virus (HCV) infection is a major public health issue. HCV genotype identification is clinically important to tailor the dosage and duration of treatment, and recombination in intra-patient populations of HCV may lead to the generation of escape mutants, as previously observed for other RNA viruses. Up to now, there is no study assessing HCV genotypes and subtypes in Heilongjiang Province, China.Methods: To determine genotype and phylogenetic analysis of HCV in Heilongjiang Province is crucial. In this study, we amplified 3 genome regions (5'UTR, E1, and NS5B) of 30 HCV patients in Heilongjiang Province, amplified products were analyzed by bioinformatics.Results: We found that 23 specimens had concordant subtypes in the 3 gene regions (2a and 1b), 7 HCV patients were considered the recombinants, the recombination pattern of the 7 HCV patients in the 5'UTR, E1, and NS5B region as followed: 1b/2a/1b, 2a/2a/1b, 1b/2a/2a, 1b/2a/1b, 1b/2a/1b, 1b/2a/1b, 2a/2a/1b.Conclusions: The findings in the present study showed that a higher recombination rate (23%) than other researches, and the recombination of 2a/1b in the 5'UTR, E1, and NS5B region was only found in the present study up to now.


Assuntos
Hepacivirus/genética , Hepatite C Crônica/virologia , Regiões 5' não Traduzidas/genética , Adulto , Idoso , China/epidemiologia , Biologia Computacional , DNA Viral/genética , Feminino , Técnicas de Genotipagem , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Adulto Jovem
13.
Genes (Basel) ; 12(3)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802939

RESUMO

Gotland sheep, a breed native to Gotland, Sweden (an island in the Baltic Sea), split from the Gute sheep breed approximately 100 years ago, and since, has probably been crossed with other breeds. This breed has recently gained popularity, due to its pelt quality. This study estimates the shared ancestors and identifies recent selection signatures in Gotland sheep using 600 K single nucleotide polymorphism (SNP) genotype data. Admixture analysis shows that the Gotland sheep is a distinct breed, but also has shared ancestral genomic components with Gute (~50%), Karakul (~30%), Romanov (~20%), and Fjällnäs (~10%) sheep breeds. Two complementary methods were applied to detect selection signatures: A Bayesian population differentiation FST and an integrated haplotype homozygosity score (iHS). Our results find that seven significant SNPs (q-value < 0.05) using the FST analysis and 55 significant SNPs (p-value < 0.0001) using the iHS analysis. Of the candidate genes that contain significant markers, or are in proximity to them, we identify several belongings to the keratin genes, RXFP2, ADCY1, ENOX1, USF2, COX7A1, ARHGAP28, CRYBB2, CAPNS1, FMO3, and GREB1. These genes are involved in wool quality, polled and horned phenotypes, fertility, twining rate, meat quality, and growth traits. In summary, our results provide shared founders of Gotland sheep and insight into genomic regions maintained under selection after the breed was formed. These results contribute to the detection of candidate genes and QTLs underlying economic traits in sheep.


Assuntos
Técnicas de Genotipagem/veterinária , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Carneiro Doméstico/classificação , Animais , Teorema de Bayes , Cruzamento , Efeito Fundador , Genótipo , Seleção Genética , Ovinos , Carneiro Doméstico/genética , Suécia
14.
Genes (Basel) ; 12(3)2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803820

RESUMO

There is a general and solid theoretical framework to explain how the interplay between natural selection and gene flow affects local adaptation. Yet, to what extent coexisting closely related species evolve collectively or show distinctive evolutionary responses remains a fundamental question. To address this, we studied the population genetic structure and morphological differentiation of sympatric three-spined and nine-spined stickleback. We conducted genotyping-by-sequencing and morphological trait characterisation using 24 individuals of each species from four lowland brackish water (LBW), four lowland freshwater (LFW) and three upland freshwater (UFW) sites in Belgium and the Netherlands. This combination of sites allowed us to contrast populations from isolated but environmentally similar locations (LFW vs. UFW), isolated but environmentally heterogeneous locations (LBW vs. UFW), and well-connected but environmentally heterogenous locations (LBW vs. LFW). Overall, both species showed comparable levels of genetic diversity and neutral genetic differentiation. However, for all three spatial scales, signatures of morphological and genomic adaptive divergence were substantially stronger among populations of the three-spined stickleback than among populations of the nine-spined stickleback. Furthermore, most outlier SNPs in the two species were associated with local freshwater sites. The few outlier SNPs that were associated with the split between brackish water and freshwater populations were located on one linkage group in three-spined stickleback and two linkage groups in nine-spined stickleback. We conclude that while both species show congruent evolutionary and genomic patterns of divergent selection, both species differ in the magnitude of their response to selection regardless of the geographical and environmental context.


Assuntos
Técnicas de Genotipagem/veterinária , Polimorfismo de Nucleotídeo Único , Smegmamorpha/classificação , Smegmamorpha/fisiologia , Adaptação Fisiológica , Animais , Bélgica , Fluxo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Países Baixos , Compostos Orgânicos , Análise de Sequência de DNA/veterinária , Smegmamorpha/genética
15.
Toxins (Basel) ; 13(5)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925199

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) can cause chronic lung infections in patients with Cystic Fibrosis (CF). One option for managing them is the use of linezolid. We hereby report the in-host emergence of linezolid resistance (LR) in MRSA in CF siblings via a population analysis. A collection of 171 MRSA strains from 68 samples were characterized by determining their linezolid Minimal Inhibitory Concentrations (MICs), analyzing the locus of staphylococcal protein A (spa) and whole genome sequencing. Courses of linezolid were retraced. Strains belonged to three spa types (t002, t045, t127) and two sequence types (ST1, ST5). Emergence of LR occurred under treatment, one year apart in both siblings, in the CC5-MRSA-I Geraldine clone harboring the toxic shock syndrome toxin-1-encoding gene. Resistance was related to a G2576T substitution present in a variable number of 23S rRNA gene copies. Susceptible and resistant strains were co-isolated within samples. Single Nucleotide Polymorphism-based analysis revealed complex colonizations by highly diversified, clonally related populations. LR remains rare in MRSA and there are very few longitudinal analyses documenting its emergence. Analyzing a large MRSA collection revealed new aspects of LR emergence: it emerges in specific subclonal lineages resulting from adaptive diversification of MRSA in the CF lung and this heterogeneity of intra-sample resistance may contribute to compromising antibiotic management.


Assuntos
Fibrose Cística/complicações , Linezolida/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Choque Séptico/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Adolescente , Criança , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Técnicas de Genotipagem , Humanos , Linezolida/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Choque Séptico/tratamento farmacológico , Irmãos , Infecções Estafilocócicas/microbiologia , Sequenciamento Completo do Genoma
16.
Clin Microbiol Infect ; 27(7): 1036.e1-1036.e8, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33813118

RESUMO

OBJECTIVES: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. CONCLUSIONS: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.


Assuntos
COVID-19/diagnóstico , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/normas , Artefatos , COVID-19/virologia , Genoma Viral , Técnicas de Genotipagem/métodos , Guias como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Viral , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma/métodos , Fluxo de Trabalho
17.
Curr Protoc ; 1(4): e100, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33826801

RESUMO

Genome editing technologies have revolutionized genetic studies in the life sciences community in recent years. The application of these technologies allows researchers to conveniently generate mutations in almost any gene of interest. This is very useful for species such as maize that have complex genomes and lack comprehensive mutant collections. With the improvement of genome editing tools and transformation methods, these technologies are also widely used to assist breeding research and implementation in maize. However, the detection and genotyping of genomic edits rely on low-throughput, high-cost methods, such as traditional agarose gel electrophoresis and Sanger sequencing. This article describes a method to barcode the target regions of genomic edits from many individuals by low-cost polymerase chain reaction (PCR) amplification. It also employs next-generation sequencing (NGS) to genotype the genome-edited plants at high throughput and low cost. This protocol can be used for initial screening of genomic edits as well as derived population genotyping on a small or large scale, at high efficiency and low cost. © 2021 Wiley Periodicals LLC. Basic Protocol 1: A fast genomic DNA preparation method from genome edited plants Basic Protocol 2: Barcoding the amplicons of edited regions from each individual by two rounds of PCR Basic Protocol 3: Bioinformatics analysis.


Assuntos
Edição de Genes , Melhoramento Vegetal , Genoma de Planta/genética , Genótipo , Técnicas de Genotipagem , Humanos
18.
Methods Mol Biol ; 2250: 75-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33900593

RESUMO

Miniature inverted-repeat transposable elements (MITEs) are a subset of short, non-autonomous class II transposable elements and also a major source of eukaryotic genomic variation. Therefore, genome-wide identification of MITE insertions can help to shed light on their copy number variation and genome insertion features. Here, we present a protocol for targeted MITE identification and genotyping by high-throughput sequencing. By introducing genome-wide detection of the rice mJing MITE as an example, we describe DNA extraction, DNA fragmentation, targeted DNA fragment enrichment, library construction for high-throughput sequencing, and sequence analysis.


Assuntos
Elementos de DNA Transponíveis , Sequências Repetidas Invertidas , Oryza/genética , Variações do Número de Cópias de DNA , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento Completo do Genoma
19.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810044

RESUMO

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genes de Plantas , Mutagênese , Oryza/genética , Proteínas de Plantas/genética , Característica Quantitativa Herdável , Sequência de Bases , Inibidores de Cisteína Proteinase , DNA Bacteriano/genética , Grão Comestível/genética , Grão Comestível/metabolismo , Regulação da Expressão Gênica de Plantas , Ordem dos Genes , Redes Reguladoras de Genes , Estudos de Associação Genética , Vetores Genéticos/genética , Genoma de Planta , Técnicas de Genotipagem , Mutação , Oryza/classificação , Oryza/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Proteômica , Transdução de Sinais
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