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1.
Gigascience ; 8(5)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31089679

RESUMO

BACKGROUND: Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition. FINDINGS: We sequenced 2 commercially available mock communities containing 10 microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Both communities and the 10 individual species isolates were also sequenced with Illumina technology. We generated 14 and 16 gigabase pairs from 2 GridION flowcells and 150 and 153 gigabase pairs from 2 PromethION flowcells for the evenly distributed and log-distributed communities, respectively. Read length N50 ranged between 5.3 and 5.4 kilobase pairs over the 4 sequencing runs. Basecalls and corresponding signal data are made available (4.2 TB in total). Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning. CONCLUSIONS: We present ultra-deep, long-read nanopore datasets from a well-defined mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.

2.
Genome Biol ; 20(1): 8, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621750

RESUMO

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate our multiplexed amplicon approach, PrimalSeq, to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.


Assuntos
Genômica/métodos , Vírus do Nilo Ocidental/genética , Zika virus/genética , Variação Genética , Análise de Sequência de RNA
3.
Euro Surveill ; 23(12)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29589579

RESUMO

On 11 May 2015, the Dubréka prefecture, Guinea, reported nine laboratory-confirmed cases of Ebola virus disease (EVD). None could be epidemiologically linked to cases previously reported in the prefecture. We describe the epidemiological and molecular investigations of this event. We used the Dubréka EVD registers and the Ebola treatment centre's (ETC) records to characterise chains of transmission. Real-time field Ebola virus sequencing was employed to support epidemiological results. An epidemiological cluster of 32 cases was found, of which 27 were laboratory confirmed, 24 were isolated and 20 died. Real-time viral sequencing on 12 cases demonstrated SL3 lineage viruses with sequences differing by one to three nt inside a single phylogenetic cluster. For isolated cases, the average time between symptom onset and ETC referral was 2.8 days (interquartile range (IQR): 1-4). The average time between sample collection and molecular results' availability was 3 days (IQR: 2-5). In an area with scarce resources, the genetic characterisation supported the outbreak investigations in real time, linking cases where epidemiological investigation was limited and reassuring that the responsible strain was already circulating in Guinea. We recommend coupling thorough epidemiological and genomic investigations to control EVD clusters.

4.
Genome Announc ; 6(5)2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29437107

RESUMO

The genome sequence of the human pathogen Corynebacterium diphtheriae bv. mitis strain ISS 3319 was determined and closed in this study. The genome is estimated to have 2,404,936 bp encoding 2,257 proteins. This strain also possesses a plasmid of 1,960 bp.

5.
Nature ; 546(7658): 401-405, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28538723

RESUMO

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.


Assuntos
Infecção por Zika virus/epidemiologia , Infecção por Zika virus/virologia , Zika virus/genética , Aedes/virologia , Animais , Região do Caribe/epidemiologia , Surtos de Doenças/estatística & dados numéricos , Feminino , Florida/epidemiologia , Genoma Viral/genética , Humanos , Incidência , Epidemiologia Molecular , Mosquitos Vetores/virologia , Zika virus/isolamento & purificação , Infecção por Zika virus/transmissão
6.
Nat Protoc ; 12(6): 1261-1276, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28538739

RESUMO

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.


Assuntos
Vírus de DNA/genética , Genoma Viral , Reação em Cadeia da Polimerase Multiplex/métodos , Vírus de RNA/genética , Análise de Sequência de DNA/métodos , Epidemiologia Molecular/métodos
7.
Nature ; 544(7650): 309-315, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28405027

RESUMO

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.


Assuntos
Ebolavirus/genética , Ebolavirus/fisiologia , Genoma Viral/genética , Doença pelo Vírus Ebola/transmissão , Doença pelo Vírus Ebola/virologia , Clima , Surtos de Doenças/estatística & dados numéricos , Ebolavirus/isolamento & purificação , Geografia , Doença pelo Vírus Ebola/epidemiologia , Humanos , Internacionalidade , Modelos Lineares , Epidemiologia Molecular , Filogenia , Viagem/legislação & jurisprudência , Viagem/estatística & dados numéricos
8.
J Cardiothorac Vasc Anesth ; 31(2): 497-504, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28216204

RESUMO

OBJECTIVE: To determine whether the ratio of peak systolic-to-nadir diastolic velocity (S/D ratio) measured using Doppler at the left ventricular assist device (LVAD) inflow and outflow cannulae is associated with pump thrombosis and to determine whether there is an absolute decrease in the diastolic cannula velocities in LVAD thrombosis. DESIGN: Retrospective chart review. SETTING: University hospital. PARTICIPANTS: Patients who underwent LVAD exchange. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Transesophageal echocardiograms were reviewed from all patients with the HeartMate II device (Thoratec Corporation, Pleasanton, CA) over a 6-year period and who underwent LVAD exchange for pump thrombosis. The following 3 time points were evaluated: (1) initial LVAD placement (prethrombosis), (2) thrombosis, and (3) exchanged LVAD placement (postthrombosis). Systolic and diastolic flow velocities were examined using pulse-wave spectral Doppler at the inflow and outflow cannulae, and the S/D ratio for each was determined. Statistical analysis was performed with SAS, version 9.4 (SAS Institute, Cary, NC), using 2-tailed tests and alpha = 0.05. Thirteen patients were included in the study. Significant differences were observed in S/D ratios among the 3 phases at both the inflow (p = 0.0234) and outflow (p = 0.0047) cannulae. Pairwise tests of the inflow cannulae showed that the mean S/D ratio at the time of thrombosis (mean ± standard deviation [SD], 4.29 ± 1.74) was significantly greater than the prethrombosis ratio (2.49 ± 0.65; p = 0.0069). Among outflow measurements, the mean S/D ratio at thrombosis (3.94 ± 1.34) was significantly higher than both the prethrombosis (2.63 ± 0.56; p = 0.0025) and postthrombosis (2.74 ± 0.83) (p = 0.0093) ratios. Decreases in diastolic velocities were not statistically significant at the inflow cannula. At the outflow cannula, there was a significant difference in diastolic velocity among the phases (p = 0.0233). Specifically, the postthrombosis diastolic measurements (41.50 ± 9.94) were significantly higher than both the prethrombosis (26.85 ± 10.13; p = 0.0140) and thrombosis (26.7 ± 15.35; p = 0.0151) values. CONCLUSIONS: An increased S/D ratio measured with Doppler at the LVAD inflow and outflow cannulas may be associated with pump thrombosis. Decreased diastolic cannula velocities were not observed in LVAD thrombosis.


Assuntos
Cânula , Ecocardiografia Transesofagiana , Coração Auxiliar/efeitos adversos , Trombose/diagnóstico por imagem , Trombose/etiologia , Idoso , Ecocardiografia Transesofagiana/tendências , Feminino , Coração Auxiliar/tendências , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombose/fisiopatologia
9.
Emerg Infect Dis ; 22(12): 2149-2152, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27869596

RESUMO

In October 2015, a new case of Ebola virus disease in Guinea was detected. Case investigation, serology, and whole-genome sequencing indicated possible transmission of the virus from an Ebola virus disease survivor to another person and then to the case-patient reported here. This transmission chain over 11 months suggests slow Ebola virus evolution.


Assuntos
Surtos de Doenças , Ebolavirus , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/transmissão , Criança , Ebolavirus/classificação , Ebolavirus/genética , Feminino , Guiné/epidemiologia , Doença pelo Vírus Ebola/história , Doença pelo Vírus Ebola/virologia , História do Século XXI , Humanos , Masculino , Filogenia , Vigilância da População , Estudos Soroepidemiológicos
10.
Nature ; 530(7589): 228-232, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26840485

RESUMO

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.


Assuntos
Ebolavirus/genética , Monitoramento Epidemiológico , Genoma Viral/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , Aeronaves , Surtos de Doenças/estatística & dados numéricos , Ebolavirus/classificação , Ebolavirus/patogenicidade , Guiné/epidemiologia , Humanos , Mutagênese/genética , Taxa de Mutação , Fatores de Tempo
11.
Am J Gastroenterol ; 110(12): 1718-29; quiz 1730, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26526081

RESUMO

OBJECTIVES: Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn's disease (CD). METHODS: Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics. RESULTS: Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN. CONCLUSIONS: Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.


Assuntos
Doença de Crohn/genética , Doença de Crohn/microbiologia , Nutrição Enteral , Fezes , Metagenoma , Microbiota , Adolescente , Criança , Doença de Crohn/sangue , Doença de Crohn/metabolismo , Fezes/química , Feminino , Humanos , Complexo Antígeno L1 Leucocitário/metabolismo , Modelos Lineares , Masculino , Metagenômica/métodos , Microbiota/genética , RNA Ribossômico 16S , Análise de Sequência de RNA
12.
Int J Antimicrob Agents ; 46(5): 572-5, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26364847

RESUMO

Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125 mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8 mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5 mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Minociclina/análogos & derivados , Proteínas Ribossômicas/genética , Deleção de Sequência , Substituição de Aminoácidos , Antibacterianos/uso terapêutico , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Testes de Sensibilidade Microbiana , Minociclina/farmacologia , Minociclina/uso terapêutico , Dados de Sequência Molecular , Análise de Sequência de DNA , Tigeciclina
13.
Nat Methods ; 12(8): 733-5, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26076426

RESUMO

We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.


Assuntos
Biologia Computacional/métodos , Escherichia coli K12/genética , Genoma Bacteriano , Nanoporos , Nanotecnologia/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Mapeamento de Sequências Contíguas/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reprodutibilidade dos Testes , Software
14.
Genome Biol ; 16: 114, 2015 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-26025440

RESUMO

BACKGROUND: Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. RESULTS: We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. CONCLUSIONS: Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices.


Assuntos
Infecção Hospitalar/epidemiologia , Surtos de Doenças , Infecções por Salmonella/epidemiologia , Salmonella enteritidis/genética , Análise de Sequência de DNA/métodos , Infecção Hospitalar/microbiologia , Hospitalização , Humanos , Infecções por Salmonella/microbiologia , Salmonella enteritidis/classificação , Salmonella enteritidis/isolamento & purificação
15.
Nat Commun ; 6: 6717, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25848958

RESUMO

Tuberculosis (TB) was once a major killer in Europe, but it is unclear how the strains and patterns of infection at 'peak TB' relate to what we see today. Here we describe 14 genome sequences of M. tuberculosis, representing 12 distinct genotypes, obtained from human remains from eighteenth-century Hungary using metagenomics. All our historic genotypes belong to M. tuberculosis Lineage 4. Bayesian phylogenetic dating, based on samples with well-documented dates, places the most recent common ancestor of this lineage in the late Roman period. We find that most bodies yielded more than one M. tuberculosis genotype and we document an intimate epidemiological link between infections in two long-dead individuals. Our results suggest that metagenomic approaches usefully inform detection and characterization of historical and contemporary infections.


Assuntos
Coinfecção/microbiologia , DNA Bacteriano/análise , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Adulto , Teorema de Bayes , Europa (Continente)/epidemiologia , Feminino , Genótipo , História do Século XVIII , Humanos , Hungria/epidemiologia , Masculino , Metagenômica , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , Tuberculose/epidemiologia , Tuberculose/história , Adulto Jovem
16.
Gigascience ; 4: 6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25695306

RESUMO

[This corrects the article DOI: 10.1186/2047-217X-3-22.].

17.
BMJ Open ; 4(11): e006278, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25371418

RESUMO

OBJECTIVES: Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. STUDY DESIGN: An observational prospective cohort study. SETTING: Burns care ward and critical care ward in the UK. PARTICIPANTS: Patients with >7% total burns by surface area were recruited into the study. METHODS: All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. RESULTS: WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. CONCLUSIONS: This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward.


Assuntos
Infecção Hospitalar/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Adulto , Feminino , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Hospitais , Humanos , Masculino , Estudos Prospectivos
18.
Gigascience ; 3: 22, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25386338

RESUMO

BACKGROUND: The MinION™ is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. The MinION™ measures the change in current resulting from DNA strands interacting with a charged protein nanopore. These measurements can then be used to deduce the underlying nucleotide sequence. FINDINGS: We present a read dataset from whole-genome shotgun sequencing of the model organism Escherichia coli K-12 substr. MG1655 generated on a MinION™ device during the early-access MinION™ Access Program (MAP). Sequencing runs of the MinION™ are presented, one generated using R7 chemistry (released in July 2014) and one using R7.3 (released in September 2014). CONCLUSIONS: Base-called sequence data are provided to demonstrate the nature of data produced by the MinION™ platform and to encourage the development of customised methods for alignment, consensus and variant calling, de novo assembly and scaffolding. FAST5 files containing event data within the HDF5 container format are provided to assist with the development of improved base-calling methods.

19.
Nat Methods ; 11(11): 1144-6, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25218180

RESUMO

Shotgun sequencing enables the reconstruction of genomes from complex microbial communities, but because assembly does not reconstruct entire genomes, it is necessary to bin genome fragments. Here we present CONCOCT, a new algorithm that combines sequence composition and coverage across multiple samples, to automatically cluster contigs into genomes. We demonstrate high recall and precision on artificial as well as real human gut metagenome data sets.


Assuntos
Mapeamento de Sequências Contíguas/métodos , Trato Gastrointestinal/microbiologia , Genoma Bacteriano/genética , Metagenoma/genética , Metagenômica/métodos , Microbiota/genética , Análise de Sequência de DNA/métodos , Software , Algoritmos , Bifidobacterium/genética , Escherichia coli K12/genética , Fezes/microbiologia , Humanos , Escherichia coli Shiga Toxigênica/genética
20.
Genome Announc ; 2(3)2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24812216

RESUMO

We report the draft genome assembly of Elizabethkingia meningoseptica strain 502. The sample was isolated from the wound of a repatriated military serviceperson who suffered major trauma from an improvised explosive device (IED), resulting in wounds with extensive environmental contamination. E. meningoseptica was isolated from wounds in both legs. The draft genome assembly has 21 contigs with a total size of 3,960,744 bases. The genome contains genes encoding 26 putative ß-lactamases.

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