Real-time selective sequencing using nanopore technology

The Oxford Nanopore Technologies MinION sequencer enables the selection of specific DNA molecules for sequencing by reversing the driving voltage across individual nanopores. To directly select molecules for sequencing, we used dynamic time warping to match reads to reference sequences. We demonstrate our open-source Read Until software in real-time selective sequencing of regions within small genomes, individual amplicon enrichment and normalization of an amplicon set

AS AVENTURAS DE DOG MENDONÇA E PIZZA BOY: UMA LEITURA

Na trilogia As Aventuras de Dog Mendonça e Pizza Boy (2010; 2011; 2013) e sua prequela (2012), Filipe Melo e Juan Cavia povoam Portugal, e em particular Lisboa, com vários corpos fantásticos: gárgulas, vampiros, lobisomens, zombies, demónios e outros seres habitam o universo lusitano naquela que é uma obra com claras referências cinematográficas aos mestres do cinema de horror e outros. Ao mesmo tempo, esta homenagem apresenta também uma reflexão sobre o corpo fantástico, a monstruosidade e o Outro. Centrando-se essencialmente nas figuras sobrenaturais desta obra, este ensaio aborda os corpos fantásticos presentes nesta BD, em particular o caso de Dog Mendonça, o lobisomem, e de Pazuul, o demónio em corpo de criança. Desta forma, o presente estudo tem um duplo objetivo: por um lado, explorar as influências fantásticas de As Aventuras de Dog Mendonça e Pizza Boy e, por outro, estabelecer uma leitura do corpo fantástico a partir da análise de duas personagens que circulam entre dois mundos numa mesma cidade, o mundo sobrenatural e o mundo humano

Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism 937ACC>937GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

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

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved]

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work

Nevrological manifestations in patiens with West Nile fever

Гарячка Західного Нілу (ГЗН) є найпоширенішим зооантропонозним трансмісивним «комариним» захворюванням у групі природно осередкових інфекцій. Вірус ГЗН належить до роду Flavivirus родини Flaviviridae, поширений на усіх континентах за винятком Антарктиди. В Європі основним видом комарів, які передають вірус ГЗН людям, є Culex pipiens. Інфікування описане також при вертикальній передачі від матері до дитини та парентеральним шляхом. За філогенетичними властивостями вірусу розрізняють кілька генетичних груп – генотипів, деякі з яких містять підгрупи, що обумовлює нерівномірний територіальний розподіл та тяжкість клінічної маніфестації ГЗН