Investigating the Extent of Primer Dropout in SARS-CoV-2 Genome Sequences During the Early Circulation of Delta Variants

The SARS-CoV-2 Delta variant, corresponding to the Pangolin lineage B.1.617.2, was first detected in India in July 2020 and rapidly became dominant worldwide. The ARTIC v3 protocol for SARS-CoV-2 whole-genome sequencing, which relies on a large number of PCR primers, was among the first available early in the pandemic, but may be prone to coverage dropouts that result in incomplete genome sequences. A new set of primers (v4) was designed to circumvent this issue in June 2021. In this study, we investigated whether the sequencing community adopted the new sets of primers, especially in the context of the spread of the Delta lineage, in July 2021. Because information about protocols from individual laboratories is generally difficult to obtain, the aims of the study were to identify whether large under-sequenced regions were present in deposited Delta variant genome sequences (from April to August 2021), to investigate the extent of the coverage dropout among all the currently available Delta sequences in six countries, and to propose simple PCR primer modifications to sequence the missing region, especially for the first circulating Delta variants observed in 2021 in Switzerland. Candidate primers were tested on few clinical samples, highlighting the need to further pursue primer optimization and validation on a larger and diverse set of samples

The problem of a metal impurity in an oxide: ab-initio study of electronic and structural properties of Cd in Rutile TiO2

In this work we undertake the problem of a transition metal impurity in an oxide. We present an ab-initio study of the relaxations introduced in TiO2 when a Cd impurity replaces substitutionally a Ti atom. Using the Full-Potential Linearized-Augmented-Plane-Wave method we obtain relaxed structures for different charge states of the impurity and computed the electric-field gradients (EFGs) at the Cd site. We find that EFGs, and also relaxations, are dependent on the charge state of the impurity. This dependence is very remarkable in the case of the EFG and is explained analyzing the electronic structure of the studied system. We predict fairly anisotropic relaxations for the nearest oxygen neighbors of the Cd impurity. The experimental confirmation of this prediction and a brief report of these calculations have recently been presented [P.R.L. 89, 55503 (2002)]. Our results for relaxations and EFGs are in clear contradiction with previous studies of this system that assumed isotropic relaxations and point out that no simple model is viable to describe relaxations and the EFG at Cd in TiO2 even approximately.Comment: 11 pages, 8 figures, Revtex 4, published in Physical Review

Genome Sequences of Rare Human Enterovirus Genotypes Recovered from Clinical Respiratory Samples in Bern, Switzerland.

We report on genomic sequences of human enteroviruses (EVs) that were identified in respiratory samples in Bern, Switzerland, in 2018 and 2019. Besides providing sequences for coxsackievirus A2, echovirus 11, and echovirus 30, we determined the sequences of rare EV-D68 and EV-C105 genotypes circulating in Switzerland

NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A rapid, PCR-free method for whole genome sequencing of pathogens in clinical samples

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach, called NASCarD (Nanopore adaptive sampling with carrier DNA), which allows low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using clinical samples positive for SARS-CoV-2 during the Omicron wave, we demonstrate how the method leads to up to >100x coverage of the full genome sequences of the target organism as compared to standard shotgun metatranscriptomics approach. It provides complete and accurate genome sequence reconstruction within seven hours at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples

NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A Rapid, PCR-Free Method for SARS-CoV-2 Whole-Genome Sequencing in Clinical Samples.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples

Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains

Investigating the biological and technical origins of unknown bases in the S region of the SARS-CoV-2 Delta variant genome sequences

We are reporting on the observation of a large, under-sequenced region of the S gene of the SARS-CoV2 Delta variant genomes, identified in sequences originating from various sequencing centres worldwide (e.g. USA, India, England, Switzerland, France, Germany). This poorly sequenced region was identified from the early phases of the Delta variant spread and the phenomenon is still ongoing. As many commonly-used protocols rely on amplicon-based sequencing procedures, we investigated the likely origin of the issue. We established its biological origin as resulting from mutations in the viral genomes at primer binding sites. We designed and evaluated new PCR primers to circumvent this issue in order to complement the ARTIC v3 set, and validated their performance for the sequencing of circulating Delta variants

Hyperfine interaction studies with (181)Ta and (111)Cd probes in the compound Ti(2)Ag

By using the time-differential perturbed angular correlation technique, the electric field gradients (EFG) at (181)Hf/(181)Ta and (111)In/(111)Cd probe sites in the MoSi(2)-type compound Ti(2)Ag have been measured as a function of temperature in the range from 24 to 1073 K. Ab initio EFG calculations have been performed within the framework of density functional theory using the full-potential augmented plane wave + local orbitals method as implemented in the WIEN2k package. These calculations allowed assignments of the probe lattice sites. For Ta, a single well-defined EFG with very weak temperature dependence was established and attributed to the [4(e)4mm] Ti site. For (111)Cd probes, two of the three measured EFGs are well defined and correlated with substitutional lattice sites, i.e. both the [4(e)4mm] Ti site and the [2(a)4/mmm] Ag site.Deutscher Akademischer Austauschdienst (DAAD)DAADDFGDeutsche Forschungsgemeinschaf (DFG)FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CapesCnpqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Caracterização e identificação de esquemas Ponzi: casos Charles Ponzi, Bernard Madoff e Dona Branca

Mestrado em AuditoriaEsta investigação objetivou estudar um tipo de fraude financeira milenar, que se tem repetido em diferentes períodos e ocorrido em vários continentes, denominada por esquema Ponzi. Face à aparente simplicidade e ao elevado lucro gerado, este esquema atrai a curiosidade e o interesse mesmo de pessoas mais conservadoras. Numa fase inicial, a oportunidade de investimento só é acessível a um grupo restrito. Contudo, posteriormente, estas propostas começam a ser objeto de comentário e até de admiração pelo sucesso alcançado, alargando-se o universo de aderentes. No mundo globalizado, face há grande oferta no mercado de produtos financeiros, complexos e difíceis de entender, não se torna fácil resistir à tentação de investir neste tipo de produtos. Embora já existam instrumentos de alerta para estas fraudes, tem-se verificado que não são suficientes para permitir uma deteção e identificação atempada e impedir que sejam lesados muitos investidores, tanto de pequenas poupanças como de grandes investimentos, assim como para impossibilitar a afetação do sistema financeiro na área de atuação. Tendo em conta este cenário, uma das motivações que esteve na origem da presente investigação prende-se com a importância de se dispor de uma ferramenta que permita detetar, o mais cedo possível, este tipo de fraude de forma a minimizar os danos. Neste contexto, surge a pergunta de investigação: Como funciona um esquema Ponzi e de que forma é possível detetar e identificar, de forma atempada, esta fraude financeira? Pretendendo-se dar resposta à questão em causa, analisaram-se três casos de esquemas Ponzi, Charles Ponzi, Bernard Madoff e o caso português Dona Branca, tendo-se identificado a existência de caraterísticas e formas de funcionamento semelhantes, no desenvolvimento do esquema. O resultado do estudo possibilitou a criação de um modelo padrão da evolução dos esquemas Ponzi, denominado por «Modelo das cinco fases de um esquema Ponzi». Este modelo permite ao auditor e ao investigador identificar a fase do esquema em que se encontra. Alguns autores têm aconselhado a utilização das red flags como medida preventiva na deteção das fraudes financeiras. Este Modelo aliado às red flags constitui uma ferramenta mais robusta à identificação de fraudes, em momento oportuno. A aptidão desta técnica na deteção de padrões sintomáticos dos esquemas Ponzi poderá também ser do interesse de investidores e de reguladores financeiros, quando confrontados com propostas de investimento com rendibilidades atrativas mas irrealistas, face a oportunidades similares existentes no mercado.The following research had the objective of studying an ancient type of fraud that has been repeated time over time throughout history and spread over the five continents – The Ponzi Scheme. Because of it’s simplicity and the promised high profit margins this scheme attracts people from all specters of society even the more conservative ones. Usually it starts off with a very restricted number of individuals and as rumor of high gains spread out on a person to person basis, more people get attracted to the scheme. At each new entrant, the pyramid scheme gets fed on it’s basis, distributing the entrant money as earnings to the tiers above. This pyramid chain keeps feeding itself as new entrants keep coming into the scheme. But as new entrants start being scares, the high earnings stop being delivered and the as the base links break the pyramid eventually collapses. What is of a surprise is that in a globalized world, with the information age at our doorstep, these kind of schemes are still possible to happen. This is mainly due to the overwhelming amount of financial products, some of them of high complexity, difficult to understand and with low yield’s compared to these apparently simple and high return proposals. If one adds the economic cyclic downturns it becomes more clear to understand the appetite for these schemes. In light of all this it became apparent that a better tool that would allow for early detection of this type of fraud was necessary. As such the research question arises: How do Ponzi schemes work and how they can be detected and identified at an early stage? To answer this question, three well known and documented cases were analyzed: Charles Ponzi case, Bernard Madoff case and the Portuguese case of Dona Branca. The main common characteristics and ways of working between them were identified and analyzed. The resulting study gave way to the identification of a pattern for these types of scheme evolution, which in turn allowed to develop a model denominated «5 Step Ponzi Scheme Model». This model gives an auditor and/or investigator to identify the development stage at which the scheme is. Some authors have recommended the use of the so called red flags as a preventive measure for early detection. When we use these red flags in conjunction with the «5 steps model» this makes up a very robust model for identifying this type of fraud early. This tool is also applicable when regulators and financial investors are taking investment decisions at which point the model can come into play identifying if some high return proposals are realistic or a possible Ponzi scheme.info:eu-repo/semantics/publishedVersio