Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 904
Filtrar
1.
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
2.
Commun Biol ; 3(1): 538, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994472

RESUMO

The advent of portable nanopore sequencing devices has enabled DNA and RNA sequencing to be performed in the field or the clinic. However, advances in in situ genomics require parallel development of portable, offline solutions for the computational analysis of sequencing data. Here we introduce Genopo, a mobile toolkit for nanopore sequencing analysis. Genopo compacts popular bioinformatics tools to an Android application, enabling fully portable computation. To demonstrate its utility for in situ genome analysis, we use Genopo to determine the complete genome sequence of the human coronavirus SARS-CoV-2 in nine patient isolates sequenced on a nanopore device, with Genopo executing this workflow in less than 30 min per sample on a range of popular smartphones. We further show how Genopo can be used to profile DNA methylation in a human genome sample, illustrating a flexible, efficient architecture that is suitable to run many popular bioinformatics tools and accommodate small or large genomes. As the first ever smartphone application for nanopore sequencing analysis, Genopo enables the genomics community to harness this cheap, ubiquitous computational resource.


Assuntos
Betacoronavirus/genética , Biologia Computacional/métodos , Genoma Humano , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento Completo do Genoma/métodos , Betacoronavirus/patogenicidade , Telefone Celular/instrumentação , Biologia Computacional/instrumentação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Nanoporos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Sequenciamento Completo do Genoma/instrumentação
3.
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
4.
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
5.
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
6.
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
7.
Genes (Basel) ; 11(8)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32806776

RESUMO

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Betacoronavirus/patogenicidade , Chlorocebus aethiops , Primers do DNA/normas , Feminino , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase/normas , Células Vero , Sequenciamento Completo do Genoma/normas
8.
Viruses ; 12(8)2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824272

RESUMO

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , DNA Complementar/genética , Genoma Viral , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Sequenciamento Completo do Genoma/métodos
9.
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
10.
PLoS One ; 15(8): e0238245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845907

RESUMO

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.


Assuntos
Mapeamento Cromossômico/métodos , Limite de Detecção , Teste Pré-Natal não Invasivo/métodos , Sequenciamento Completo do Genoma/métodos , Ácidos Nucleicos Livres/análise , Deleção Cromossômica , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 1/genética , Síndrome do Miado do Gato/diagnóstico , Síndrome do Miado do Gato/genética , Síndrome de DiGeorge/diagnóstico , Síndrome de DiGeorge/genética , Feminino , Humanos , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Gravidez , Cuidado Pré-Natal , Síndrome de Wolf-Hirschhorn/diagnóstico , Síndrome de Wolf-Hirschhorn/genética
11.
Emerg Infect Dis ; 26(10): 2401-2405, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32610037

RESUMO

We describe validated protocols for generating high-quality, full-length severe acute respiratory syndrome coronavirus 2 genomes from primary samples. One protocol uses multiplex reverse transcription PCR, followed by MinION or MiSeq sequencing; the other uses singleplex, nested reverse transcription PCR and Sanger sequencing. These protocols enable sensitive virus sequencing in different laboratory environments.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/análise , Análise de Sequência de RNA/métodos , Sequenciamento Completo do Genoma/métodos , Reação em Cadeia da Polimerase Multiplex , Pandemias
12.
Nat Commun ; 11(1): 3375, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632155

RESUMO

Hybridization can drive speciation. We examine the hypothesis that Castanea henryi var. omeiensis is an evolutionary lineage that originated from hybridization between two near-sympatric diploid taxa, C. henryi var. henryi and C. mollissima. We produce a high-quality genome assembly for mollissima and characterize evolutionary relationships among related chestnut taxa. Our results show that C. henryi var. omeiensis has a mosaic genome but has accumulated divergence in all 12 chromosomes. We observe positive correlation between admixture proportions and recombination rates across the genome. Candidate barrier genomic regions, which isolate var. henryi and mollissima, are re-assorted in the hybrid lineage. We further find that the putative barrier segments concentrate in genomic regions with less recombination, suggesting that interaction between natural selection and recombination shapes the evolution of hybrid genomes during hybrid speciation. This study highlights that reassortment of parental barriers is an important mechanism in generating biodiversity.


Assuntos
Diploide , Fagaceae/genética , Genoma de Planta/genética , Hibridização Genética , Mosaicismo , Ploidias , Algoritmos , Evolução Molecular , Fagaceae/classificação , Especiação Genética , Modelos Genéticos , Recombinação Genética , Seleção Genética , Especificidade da Espécie , Sequenciamento Completo do Genoma/métodos
13.
ACS Infect Dis ; 6(8): 1998-2016, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32677821

RESUMO

Since late December 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-negative rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathology and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomography will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Infecções por Coronavirus/virologia , Efeito Citopatogênico Viral , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoquímica/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise Espectral Raman/métodos , Tomografia Computadorizada por Raios X/métodos , Sequenciamento Completo do Genoma/métodos
14.
PLoS One ; 15(7): e0235641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614888

RESUMO

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Salmonella enterica/genética , Sequenciamento Completo do Genoma/métodos , Antibacterianos/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Salmonella enteritidis/classificação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência
15.
Nat Genet ; 52(8): 778-789, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661416

RESUMO

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.


Assuntos
Metilação de DNA/genética , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genoma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Estudos Prospectivos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Exoma/métodos , Sequenciamento Completo do Genoma/métodos
16.
Genome Med ; 12(1): 57, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32605661

RESUMO

BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.


Assuntos
Betacoronavirus/genética , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento Completo do Genoma/métodos , Infecções por Coronavirus , Variação Genética/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Pneumonia Viral , RNA Viral/genética
17.
Euro Surveill ; 25(22)2020 Jun.
Artigo em Inglês | MEDLINE | ID: covidwho-525969

RESUMO

We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city's international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Genoma Viral/genética , Pandemias , Pneumonia Viral/diagnóstico , Sequenciamento Completo do Genoma/métodos , Betacoronavirus/isolamento & purificação , Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Alemanha/epidemiologia , Humanos , Pneumonia Viral/epidemiologia , DNA Polimerase Dirigida por RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Med Microbiol ; 69(7): 1013-1019, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32579102

RESUMO

Introduction. Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem globally, including in Indonesia. Whole-genome sequencing (WGS) analysis has rarely been used for the study of TB and MDR-TB in Indonesia.Aim. We evaluated the use of WGS for drug-susceptibility testing (DST) and to investigate the population structure of drug-resistant Mycobacterium tuberculosis in Java, Indonesia.Methodology. Thirty suspected MDR-TB isolates were subjected to MGIT 960 system (MGIT)-based DST and to WGS. Phylogenetic analysis was done using the WGS data. Results obtained using MGIT-based DST and WGS-based DST were compared.Results. Agreement between WGS and MGIT was 93.33 % for rifampicin, 83.33 % for isoniazid and 76.67 % for streptomycin but only 63.33 % for ethambutol. Moderate WGS-MGIT agreement was found for second-line drugs including amikacin, kanamycin and fluoroquinolone (73.33-76.67 %). MDR-TB was more common in isolates of the East Asian Lineage (63.3%). No evidence of clonal transmission of DR-TB was found among members of the tested population.Conclusion. Our study demonstrated the applicability of WGS for DST and molecular epidemiology of DR-TB in Java, Indonesia. We found no transmission of DR-TB in Indonesia.


Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Adulto , Antituberculosos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Genótipo , Humanos , Indonésia/epidemiologia , Canamicina/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Mutação , Fenótipo , Filogenia , Rifampina/farmacologia , Estreptomicina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Sequenciamento Completo do Genoma/métodos
19.
Arch Virol ; 165(8): 1915-1918, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32504395

RESUMO

We determined the complete genomic sequence of begonia flower breaking virus (BFBV), a novel putative member of the genus Potyvirus isolated from Begonia bowerae cv. 'Tiger' plants grown in Kunming. The genomic RNA comprises 9540 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and contains a typical open reading frame (ORF) of potyviruses. The ORF consists of 9219 nucleotides and encodes a 3073-amino-acid polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Sequence comparison indicated that BFBV shares 43.9-55.12% amino acid sequence identity with known potyviruses and that BFBV shares the highest amino acid sequence identity (55.12%) with beet mosaic virus. The results from the complete genomic sequence analysis further suggest that BFBV is a member of a novel species in the genus Potyvirus.


Assuntos
Begoniaceae/virologia , Flores/virologia , Genoma Viral/genética , Potyvirus/genética , Sequência de Aminoácidos , Genômica/métodos , Fases de Leitura Aberta/genética , Filogenia , Doenças das Plantas/virologia , RNA Viral/genética , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos
20.
Nat Commun ; 11(1): 2719, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483195

RESUMO

National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor resistance trends and disseminate these data to AMR stakeholders. Whole-genome sequencing (WGS) can support surveillance by pinpointing resistance mechanisms and uncovering transmission patterns. However, genomic surveillance is rare in low- and middle-income countries. Here, we implement WGS within the established Antimicrobial Resistance Surveillance Program of the Philippines via a binational collaboration. In parallel, we characterize bacterial populations of key bug-drug combinations via a retrospective sequencing survey. By linking the resistance phenotypes to genomic data, we reveal the interplay of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying the expansion of specific resistance phenotypes that coincide with the growing carbapenem resistance rates observed since 2010. Our results enhance our understanding of the drivers of carbapenem resistance in the Philippines, while also serving as the genetic background to contextualize ongoing local prospective surveillance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Genômica/métodos , Sequenciamento Completo do Genoma/métodos , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Humanos , Testes de Sensibilidade Microbiana/métodos , Filipinas/epidemiologia , Inquéritos e Questionários
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA