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1.
PLoS One ; 15(8): e0237384, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764809

RESUMO

Dengue fever is one of the major public health problems in Lao PDR. Over the last decade, dengue virus (DENV) epidemics were characterized by a novel predominant serotype accompanied by at least two other serotypes. Since 2008, DENV-2 circulated at a low level in Lao PDR but its epidemiologic profile changed at the end of 2018. Indeed, the number of confirmed DENV-2 cases suddenly increased in October 2018 and DENV-2 became predominant at the country level in early 2019. We developed a Genotype Screening Protocol (GSP) to determine the origin(s) of the Lao DENV-2 and study their genetic polymorphism. With a good correlation with full envelope gene sequencing data, this molecular epidemiology tool evidence the co-circulation of two highly polymorphic DENV-2 genotypes, i.e. Asian I and Cosmopolitan genotypes, over the last five years, suggesting multiple introductions of DENV-2 in the country. GSP approach provides relevant first line information that may help countries with limited laboratory resources to reinforce their capabilities to DENV-2 and to follow the epidemics progresses and assess situations at the regional level.


Assuntos
Vírus da Dengue/genética , Técnicas de Genotipagem/métodos , Inquéritos e Questionários , Vírus da Dengue/isolamento & purificação , Vírus da Dengue/fisiologia , Humanos , Laos , Sorotipagem , Fatores de Tempo , População Urbana/estatística & dados numéricos
2.
PLoS One ; 15(8): e0236483, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853203

RESUMO

Takifugu rubripes is more expensive than other species of the genus because of its high protein content and special flavor. However, it is easily confused with imported T. chinensis and T. pseudommus because they have similar morphological characteristics. We identified single nucleotide polymorphism (SNP) markers of T. rubripes by genotyping-by-sequencing (GBS) and evaluated their ability to distinguish among T. rubripes, T. chinensis, and T. pseudommus. In all, 18 polymorphic SNPs were subjected to phylogenetic analyses of the three Takifugu species. Additionally, we subjected a second set of samples to Sanger sequencing to verify that the polymorphic SNPs could be used to evaluate the genetic variation among the three Takifugu species. A phylogenetic tree that included the analyzed sequence of set A, which is referred to as the reference sequence, and a validation sequence of set B with 18 SNPs were produced. Based on this phylogenetic tree and STRUCTURE analyses, T. rubripes, T. chinensis and T. pseudommus have low genetic variation and should be considered the same gene pool. Our findings suggest that further studies are needed to estimate the genetic association of the three Takifugu species.


Assuntos
Técnicas de Genotipagem/métodos , Filogenia , Análise de Sequência de DNA/métodos , Takifugu/genética , Animais , Genótipo , Polimorfismo de Nucleotídeo Único/genética , Takifugu/classificação , Transcriptoma/genética
3.
PLoS One ; 15(8): e0237111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750100

RESUMO

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.


Assuntos
Proteínas de Peixes/genética , Peixes/genética , Técnicas de Genotipagem/métodos , Polimorfismo Genético , Tubulina (Proteína)/genética , Animais
4.
PLoS One ; 15(6): e0233800, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497070

RESUMO

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).


Assuntos
Análise Custo-Benefício , Metilação de DNA/genética , Eucalyptus/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Folhas de Planta/genética , Análise de Sequência de DNA/métodos , Árvores/genética , Cromossomos de Plantas/genética , Enzimas de Restrição do DNA/genética , Elementos de DNA Transponíveis/genética , Genes de Plantas/genética , Técnicas de Genotipagem/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Projetos Piloto , Reprodutibilidade dos Testes , Mapeamento por Restrição , Análise de Sequência de DNA/economia , Sulfitos/farmacologia
5.
PLoS One ; 15(6): e0234518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525936

RESUMO

BACKGROUND/OBJECTIVE: Human papillomavirus (HPV) genotyping and cytology have been recommended for colposcopy triage, but it is unclear which combinations of high-risk HPV (hrHPV) types and cytology with various thresholds provide clinically useful information for the triage after primary HPV screening on self-collected samples. METHOD: Chinese Multi-site Screening Trial (CHIMUST) database focused on self-collected samples was reviewed using the results of Cobas4800 HPV assay. Absolute risks of each genotype for cervical intraepithelial neoplasia 2 or worse/ 3 or worse (CIN2+/CIN3+) were calculated. Triage of atypical squamous cells of undetermined significance (ASCUS) or worse cytology was used as the comparator, and diagnostic accuracy for paired comparisons between algorithms was obtained using McNemar's test. RESULTS: A total of 10, 498 women were included, the overall prevalence of hrHPV, HPV16, HPV18, and Other hrHPV genotypes were 13.7%, 2.4%, 0.8%, and 10.5%, respectively. HPV16-positive women had the highest absolute risk among various genotypes for CIN2+/CIN3+ whether in normal or abnormal cytology (ASCUS or worse) and among all age groups. When compared with the comparator, combining HPV16 positivity and/or high-grade squamous intraepithelial lesion (HSIL) or worse yielded higher specificity (97.7% vs. 97.0%, p<0.0001), similar sensitivity (90.7% vs. 96.3%, p = 0.256) for detection of CIN3+, and a decrease in colposcopy referral rate from 3.5% to 2.7%, similar results were found for CIN2+. Positivity for HPV16 and/or (ASCUS or worse), and positivity for (HPV16 and/or HPV18) and/or (ASCUS or worse) achieved favorable sensitivity compared with the comparator (80.6% and 81.3% vs. 70.1% respectively for CIN2+, p<0.0001; both 96.3% vs. 96.3% for CIN3+, p = 1.000), these algorithms would reduce the colposcopy referral rate to 5.0% and 5.6% respectively, compared with 13.7% of that for HPV alone. CONCLUSIONS: Triage of HPV-positive women on self-collected samples by combining HPV16 or HPV16/18 genotyping with different thresholds of cytology could provide tradeoffs in sensitivity for detecting cervical lesions and colposcopy referral rates, and tailor management in various circumstances of clinical practice.


Assuntos
Técnicas de Genotipagem/métodos , Programas de Rastreamento/métodos , Infecções por Papillomavirus/diagnóstico , Triagem/métodos , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Adulto , Biópsia/estatística & dados numéricos , Colo do Útero/citologia , Colo do Útero/patologia , Colo do Útero/virologia , Colposcopia/estatística & dados numéricos , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Encaminhamento e Consulta/estatística & dados numéricos , Medição de Risco/métodos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Triagem/estatística & dados numéricos , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia
6.
Genet Sel Evol ; 52(1): 31, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32527317

RESUMO

BACKGROUND: The traditional way to estimate variance components (VC) is based on the animal model using a pedigree-based relationship matrix (A) (A-AM). After genomic selection was introduced into breeding programs, it was anticipated that VC estimates from A-AM would be biased because the effect of selection based on genomic information is not captured. The single-step method (H-AM), which uses an H matrix as (co)variance matrix, can be used as an alternative to estimate VC. Here, we compared VC estimates from A-AM and H-AM and investigated the effect of genomic selection, genotyping strategy and genotyping proportion on the estimation of VC from the two methods, by analyzing a dataset from a commercial broiler line and a simulated dataset that mimicked the broiler population. RESULTS: VC estimates from H-AM were severely overestimated with a high proportion of selective genotyping, and overestimation increased as proportion of genotyping increased in the analysis of both commercial and simulated data. This bias in H-AM estimates arises when selective genotyping is used to construct the H-matrix, regardless of whether selective genotyping is applied or not in the selection process. For simulated populations under genomic selection, estimates of genetic variance from A-AM were also significantly overestimated when the effect of genomic selection was strong. Our results suggest that VC estimates from H-AM under random genotyping have the expected values. Predicted breeding values from H-AM were inflated when VC estimates were biased, and inflation differed between genotyped and ungenotyped animals, which can lead to suboptimal selection decisions. CONCLUSIONS: We conclude that VC estimates from H-AM are biased with selective genotyping, but are close to expected values with random genotyping.VC estimates from A-AM in populations under genomic selection are also biased but to a much lesser degree. Therefore, we recommend the use of H-AM with random genotyping to estimate VC for populations under genomic selection. Our results indicate that it is still possible to use selective genotyping in selection, but then VC estimation should avoid the use of genotypes from one side only of the distribution of phenotypes. Hence, a dual genotyping strategy may be needed to address both selection and VC estimation.


Assuntos
Cruzamento/métodos , Técnicas de Genotipagem/métodos , Seleção Genética/genética , Análise de Variância , Animais , Galinhas/genética , Simulação por Computador , Genoma/genética , Genômica/métodos , Genótipo , Modelos Animais , Modelos Genéticos , Linhagem , Fenótipo
7.
Genet Sel Evol ; 52(1): 32, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576143

RESUMO

BACKGROUND: Cattle international genetic evaluations allow the comparison of estimated breeding values (EBV) across different environments, i.e. countries. For international evaluations, across-country genetic correlations (rg) need to be estimated. However, lack of convergence of the estimated parameters and high standard errors of the rg are often experienced for beef cattle populations due to limited across-country genetic connections. Furthermore, using all available genetic connections to estimate rg is prohibitive due to computational constraints, thus sub-setting the data is necessary. Our objective was to investigate and compare the impact of strategies of data sub-setting on estimated across-country rg and their computational requirements. METHODS: Phenotype and pedigree information for age-adjusted weaning weight was available for ten European countries and 3,128,338 Limousin beef cattle males and females. Using a Monte Carlo based expectation-maximization restricted maximum likelihood (MC EM REML) methodology, we estimated across-country rg by using a multi-trait animal model where countries are modelled as different correlated traits. Values of rg were estimated using the full data and four different sub-setting strategies that aimed at selecting the most connected herds from the largest population. RESULTS: Using all available data, direct and maternal rg (standard errors in parentheses) were on average equal to 0.79 (0.14) and 0.71 (0.19), respectively. Direct-maternal within-country and between-country rg were on average equal to - 0.12 (0.09) and 0.00 (0.14), respectively. Data sub-setting scenarios gave similar results: on average, estimated rg were smaller compared to using all data for direct (0.02) and maternal (0.05) genetic effects. The largest differences were obtained for the direct-maternal within-country and between-country rg, which were, on average 0.13 and 0.12 smaller compared to values obtained by using all data. Standard errors always increased when reducing the data, by 0.02 to 0.06, on average. The proposed sub-setting strategies reduced the required computing time up to 22% compared to using all data. CONCLUSIONS: Estimating all 120 across-country rg that are required for beef cattle international evaluations, using a multi-trait MC EM REML approach, is feasible but involves long computing time. We propose four strategies to reduce computational requirements while keeping a multi-trait estimation approach. In all scenarios with data sub-setting, the estimated rg were consistently smaller (mainly for direct-maternal rg) and had larger standard errors.


Assuntos
Bovinos/genética , Técnicas de Genotipagem/métodos , Seleção Genética/genética , Algoritmos , Animais , Peso Corporal , Cruzamento , Europa (Continente) , Feminino , Genoma/genética , Genômica/métodos , Genótipo , Masculino , Modelos Genéticos , Método de Monte Carlo , Linhagem , Fenótipo , Carne Vermelha , Desmame
8.
Genet Sel Evol ; 52(1): 33, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591011

RESUMO

BACKGROUND: Natural and artificial directional selection in cosmopolitan and autochthonous pig breeds and wild boars have shaped their genomes and resulted in a reservoir of animal genetic diversity. Signatures of selection are the result of these selection events that have contributed to the adaptation of breeds to different environments and production systems. In this study, we analysed the genome variability of 19 European autochthonous pig breeds (Alentejana, Bísara, Majorcan Black, Basque, Gascon, Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda, Krskopolje pig, Black Slavonian, Turopolje, Moravka, Swallow-Bellied Mangalitsa, Schwäbisch-Hällisches Schwein, Lithuanian indigenous wattle and Lithuanian White old type) from nine countries, three European commercial breeds (Italian Large White, Italian Landrace and Italian Duroc), and European wild boars, by mining whole-genome sequencing data obtained by using a DNA-pool sequencing approach. Signatures of selection were identified by using a single-breed approach with two statistics [within-breed pooled heterozygosity (HP) and fixation index (FST)] and group-based FST approaches, which compare groups of breeds defined according to external traits and use/specialization/type. RESULTS: We detected more than 22 million single nucleotide polymorphisms (SNPs) across the 23 compared populations and identified 359 chromosome regions showing signatures of selection. These regions harbour genes that are already known or new genes that are under selection and relevant for the domestication process in this species, and that affect several morphological and physiological traits (e.g. coat colours and patterns, body size, number of vertebrae and teats, ear size and conformation, reproductive traits, growth and fat deposition traits). Wild boar related signatures of selection were detected across all the genome of several autochthonous breeds, which suggests that crossbreeding (accidental or deliberate) occurred with wild boars. CONCLUSIONS: Our findings provide a catalogue of genetic variants of many European pig populations and identify genome regions that can explain, at least in part, the phenotypic diversity of these genetic resources.


Assuntos
Técnicas de Genotipagem/métodos , Seleção Genética/genética , Suínos/genética , Aclimatação/genética , Adaptação Fisiológica/genética , Algoritmos , Animais , Cruzamento , Domesticação , Europa (Continente) , Feminino , Genoma/genética , Genômica/métodos , Genótipo , Masculino , Modelos Genéticos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Genoma/métodos
9.
PLoS One ; 15(6): e0234694, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555734

RESUMO

Invasive alien species (IAS) are among the main causes of global biodiversity loss. Invasive brown (Rattus norvegicus) and black (R. rattus) rats, in particular, are leading drivers of extinction on islands, especially in the case of seabirds where >50% of all extinctions have been attributed to rat predation. Eradication is the primary form of invasive rat management, yet this strategy has resulted in a ~10-38% failure rate on islands globally. Genetic tools can help inform IAS management, but such applications to date have been largely reactive, time-consuming, and costly. Here, we developed a Genotyping-in-Thousands by sequencing (GT-seq) panel for rapid species identification and population assignment of invasive brown and black rats (RapidRat) in Haida Gwaii, an archipelago comprising ~150 islands off the central coast of British Columbia, Canada. We constructed an optimized panel of 443 single nucleotide polymorphisms (SNPs) using previously generated double-digest restriction-site associated DNA (ddRAD) genotypic data (27,686 SNPs) from brown (n = 295) and black rats (n = 241) sampled throughout Haida Gwaii. The informativeness of this panel for identifying individuals to species and island of origin was validated relative to the ddRAD results; in all comparisons, admixture coefficients and population assignments estimated using RapidRat were consistent. To demonstrate application, 20 individuals from novel invasions of three islands (Agglomerate, Hotspring, Ramsay) were genotyped using RapidRat, all of which were confidently assigned (>98.5% probability) to Faraday and Murchison Islands as putative source populations. These results indicated that a previous eradication on Hotspring Island was conducted at an inappropriate geographic scale; future management should expand the eradication unit to include neighboring islands to prevent re-invasion. Overall, we demonstrated that RapidRat is an effective tool for managing invasive rat populations in Haida Gwaii and provided a clear framework for GT-seq panel development for informing biodiversity conservation in other systems.


Assuntos
Conservação dos Recursos Naturais/métodos , Técnicas de Genotipagem/métodos , Espécies Introduzidas , Análise de Sequência de DNA/métodos , Animais , Colúmbia Britânica , Ilhas , Polimorfismo de Nucleotídeo Único/genética , Ratos , Especificidade da Espécie
10.
Genomics ; 112(5): 3588-3596, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32353474

RESUMO

The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.


Assuntos
Betacoronavirus/genética , Genômica , Técnicas de Genotipagem/métodos , Mutação , Polimorfismo de Nucleotídeo Único , Infecções por Coronavirus , Evolução Molecular , Genoma Viral , Humanos , Pandemias , Pneumonia Viral , Alinhamento de Sequência
11.
PLoS One ; 15(5): e0229207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357171

RESUMO

As genotyping technologies continue to evolve, so have their throughput and multiplexing capabilities. In this study, we demonstrate a new PCR-based genotyping technology that multiplexes thousands of single nucleotide polymorphism (SNP) markers with high-throughput capabilities in a simple protocol using a two-step PCR approach. The bioinformatic pipeline is user friendly and yields results that are intuitive to interpret. This method was tested on two recombinant inbred line (RIL) populations that had previous genotyping data from the Illumina Infinium assay for Triticum aestivum L. and the two data sets were found to be 100% in agreement. The genotyping by multiplexed sequencing (GMS) protocol multiplexes 1,656 wheat SNP markers, 207 syntenic barley SNP markers, and 49 known informative markers, which generate a possible 2,433 data points (including homoeoalleles and paralogs). This genotyping approach has the flexibility of being sequenced on either the Ion Torrent or Illumina next generation sequencing (NGS) platforms. Products are the result of direct sequencing and are therefore more reliable than scatter plot analysis which is the output of other genotyping methods such as the Illumina Infinium assay, komeptitive allele specific PCR and other like technologies.


Assuntos
Genômica , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Triticum/genética , Alelos , Biologia Computacional , Genótipo , Polimorfismo de Nucleotídeo Único/genética , Triticum/classificação
12.
PLoS One ; 15(3): e0230445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176732

RESUMO

Single-nucleotide polymorphisms (SNPs) is associated with efficacy of specific drugs. Although there are several methods for SNP genotyping in clinical settings, alternative methods with lower cost, higher throughout and less complexity are still needed. In this study, we modified Kompetitive Allele Specific PCR to enable multiplex SNP genotyping by introducing additional fluorescent cassettes that specifically help to differentiate more amplification signals in a single reaction. This new format of assay achieved a limit of detection down to 310 copies/ reactions for simultaneous detection of 2 SNPs with only standard end-point PCR workflow for synthetic controls, and genotyped 117 clinical samples with results that were in 100% agreement with hospital reports. This study presented a simplified, cost-effective high-throughput SNP genotyping alternative for pharmacogenetic variants, and enabled easier access to pharmaceutical guidance when needed.


Assuntos
Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Farmacogenética/métodos , Polimorfismo de Nucleotídeo Único/genética , Alelos , Genótipo , Humanos
13.
Parasit Vectors ; 13(1): 122, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32143704

RESUMO

BACKGROUND: Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS: We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS: This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS: Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.


Assuntos
Cyclospora/genética , Cyclospora/isolamento & purificação , Ciclosporíase/diagnóstico , Genoma Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Tipagem Molecular/métodos , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , Cyclospora/classificação , Ciclosporíase/parasitologia , DNA de Protozoário/genética , Fezes/parasitologia , Técnicas de Genotipagem/métodos , Humanos , Oocistos/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
14.
Curr Protoc Mouse Biol ; 10(1): e69, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32159922

RESUMO

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Genotipagem/métodos , Modelos Animais , Animais , Técnicas de Genotipagem/instrumentação , Indicadores e Reagentes , Camundongos , Camundongos Transgênicos
15.
PLoS Genet ; 16(2): e1008576, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053607

RESUMO

Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.


Assuntos
Surtos de Doenças/prevenção & controle , Genoma de Protozoário/genética , Técnicas de Genotipagem/métodos , Malária Vivax/transmissão , Plasmodium vivax/genética , África Oriental , Ásia , Conjuntos de Dados como Assunto , Erradicação de Doenças/métodos , Marcadores Genéticos/genética , Genótipo , Geografia , Humanos , Malária Vivax/epidemiologia , Malária Vivax/parasitologia , Metadados , Repetições de Microssatélites/genética , Plasmodium vivax/isolamento & purificação , Polimorfismo de Nucleotídeo Único/genética , Valor Preditivo dos Testes , América do Sul , Doença Relacionada a Viagens , Reino Unido , Sequenciamento Completo do Genoma
16.
Muscle Nerve ; 61(6): 801-807, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32086834

RESUMO

BACKGROUND: Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed. METHODS: we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running. RESULTS: This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing. CONCLUSIONS: The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases.


Assuntos
Genótipo , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Especificidade da Espécie
17.
BMC Cancer ; 20(1): 81, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005181

RESUMO

BACKGROUND: Increased usage of genomic risk assessment assays suggests increased reliance on data provided by these assays to guide therapy decisions. The current study aimed to assess the change in treatment decision and physician confidence based on the 70-gene risk of recurrence signature (70-GS, MammaPrint) and the 80-gene molecular subtype signature (80-GS, BluePrint) in early stage breast cancer patients. METHODS: IMPACt, a prospective, case-only study, enrolled 452 patients between November 2015 and August 2017. The primary objective population included 358 patients with stage I-II, hormone receptor-positive, HER2-negative breast cancer. The recommended treatment plan and physician confidence were captured before and after receiving results for 70-GS and 80-GS. Treatment was started after obtaining results. The distribution of 70-GS High Risk (HR) and Low Risk (LR) patients was evaluated, in addition to the distribution of 80-GS compared to IHC status. RESULTS: The 70-GS classified 62.5% (n = 224/358) of patients as LR and 37.5% (n = 134/358) as HR. Treatment decisions were changed for 24.0% (n = 86/358) of patients after receiving 70-GS and 80-GS results. Of the LR patients initially prescribed CT, 71.0% (44/62) had CT removed from their treatment recommendation. Of the HR patients not initially prescribed CT, 65.1% (41/63) had CT added. After receiving 70-GS results, CT was included in 83.6% (n = 112/134) of 70-GS HR patient treatment plans, and 91.5% (n = 205/224) of 70-GS LR patient treatment plans did not include CT. For patients who disagreed with the treatment recommended by their physicians, most (94.1%, n = 16/17) elected not to receive CT when it was recommended. For patients whose physician-recommended treatment plan was discordant with 70-GS results, discordance was significantly associated with age and lymph node status. CONCLUSIONS: The IMPACt trial showed that treatment plans were 88.5% (n = 317/358) in agreement with 70-GS results, indicating that physicians make treatment decisions in clinical practice based on the 70-GS result. In clinically high risk, 70-GS Low Risk patients, there was a 60.0% reduction in treatment recommendations that include CT. Additionally, physicians reported having greater confidence in treatment decisions for their patients in 72% (n = 258/358) of cases after receiving 70-GS results. TRIAL REGISTRATION: "Measuring the Impact of MammaPrint on Adjuvant and Neoadjuvant Treatment in Breast Cancer Patients: A Prospective Registry" (NCT02670577) retrospectively registered on Jan 27, 2016.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Tomada de Decisão Clínica/métodos , Técnicas de Genotipagem/métodos , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Competência Clínica , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Medicina de Precisão , Estudos Prospectivos
18.
PLoS One ; 15(1): e0227294, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940389

RESUMO

BACKGROUND: Cell-free DNA detection is becoming a surrogate assay for tumor genotyping. Biological fluids often content a very low amount of cell-free tumor DNA and assays able to detect very low allele frequency mutant with a few quantities of DNA are required. We evaluated the ability of the fully-automated molecular diagnostics platform Idylla for the detection of KRAS, NRAS and BRAF hotspot mutations in plasma from patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: First, we evaluated the limit of detection of the system using two set of laboratory made samples that mimic mCRC patient plasma, then plasma samples from patients with mCRC were assessed using Idylla system and BEAMing digital PCR technology. RESULTS: Limits of detection of 0.1%, 0.4% and 0.01% for KRAS, NRAS and BRAF respectively have been reached. With our laboratory made samples, sensitivity up to 0.008% has been reached. Among 15 patients' samples tested for KRAS mutation, 2 discrepant results were found between Idylla and BEAMing dPCR. A 100% concordance between the two assays has been found for the detection of NRAS and BRAF mutations in plasma samples. CONCLUSIONS: The Idylla system does not reach as high sensitivity as assays like ddPCR but has an equivalent sensitivity to modified NGS technics with a lower cost and a lower time to results. These data allowed to consider the Idylla system in a routine laboratory workflow for KRAS, NRAS and BRAF mutations detection in plasma.


Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/isolamento & purificação , Neoplasias Colorretais/diagnóstico , Análise Mutacional de DNA/instrumentação , Técnicas de Genotipagem/instrumentação , Linhagem Celular Tumoral , DNA Tumoral Circulante/genética , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , GTP Fosfo-Hidrolases/genética , Frequência do Gene , Técnicas de Genotipagem/métodos , Humanos , Limite de Detecção , Proteínas de Membrana/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Sensibilidade e Especificidade
19.
BMC Plant Biol ; 20(1): 2, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898487

RESUMO

BACKGROUND: Apple (Malus x domestica Borkh.) is one of the most important fruit tree crops of temperate areas, with great economic and cultural value. Apple cultivars can be maintained for centuries in plant collections through grafting, and some are thought to date as far back as Roman times. Molecular markers provide a means to reconstruct pedigrees and thus shed light on the recent history of migration and trade of biological materials. The objective of the present study was to identify relationships within a set of over 1400 mostly old apple cultivars using whole-genome SNP data (~ 253 K SNPs) in order to reconstruct pedigrees. RESULTS: Using simple exclusion tests, based on counting the number of Mendelian errors, more than one thousand parent-offspring relations and 295 complete parent-offspring families were identified. Additionally, a grandparent couple was identified for the missing parental side of 26 parent-offspring pairings. Among the 407 parent-offspring relations without a second identified parent, 327 could be oriented because one of the individuals was an offspring in a complete family or by using historical data on parentage or date of recording. Parents of emblematic cultivars such as 'Ribston Pippin', 'White Transparent' and 'Braeburn' were identified. The overall pedigree combining all the identified relationships encompassed seven generations and revealed a major impact of two Renaissance cultivars of French and English origin, namely 'Reinette Franche' and 'Margil', and one North-Eastern Europe cultivar from the 1700s, 'Alexander'. On the contrary, several older cultivars, from the Middle Ages or the Roman times, had no, or only single, identifiable offspring in the set of studied accessions. Frequent crosses between cultivars originating from different European regions were identified, especially from the nineteenth century onwards. CONCLUSIONS: The availability of over 1400 apple genotypes, previously filtered for genetic uniqueness and providing a broad representation of European germplasm, has been instrumental for the success of this large pedigree reconstruction. It enlightens the history of empirical selection and recent breeding of apple cultivars in Europe and provides insights to speed-up future breeding and selection.


Assuntos
Genoma de Planta , Malus/genética , Polimorfismo de Nucleotídeo Único/genética , Cruzamento , Europa (Continente) , Genótipo , Técnicas de Genotipagem/métodos , Linhagem , Sequenciamento Completo do Genoma
20.
Food Chem ; 313: 126167, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31951885

RESUMO

Cows' milk may contain two types of ß-casein: A1 and A2. A1 digestion is associated with the release of ß-casomorphine-7 peptide, which can cause adverse gastrointestinal effects. Two methods - high-resolution melting (HRM) and rhAmp® SNP genotyping - were developed to identify the ß-casein gene (CSN2) A1 and A2 alleles directly in milk. DNA milk samples from 45 animals were examined and 10 samples were also sequenced to confirm the accuracy of the assays. The analytical sensitivities of both strategies for A1 allele identification were evaluated by testing decreasing dilutions of A1 allele DNA copies (500 - 5 copies) in the A2 sample. The limits of detection for A1 in A2 samples were 10% (100 copies) and 2% (10 copies) for HRM and rhAmp, respectively. Both techniques were specific, differentiating between A1 and A2 alleles. However, we recommend rhAmp genotyping testing over HRM because of its enhanced sensitivity for A1.


Assuntos
Alelos , Caseínas/genética , Análise de Alimentos/métodos , Técnicas de Genotipagem/métodos , Leite/fisiologia , Reação em Cadeia da Polimerase/métodos , Animais , Bovinos , Feminino , Limite de Detecção , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade
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