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Letteratura scientifica selezionata sul tema "SARS-CoV-2 sequencing"

Autore: Grafiati

Pubblicato: 14 marzo 2023

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Articoli di riviste sul tema "SARS-CoV-2 sequencing"

Nazario-Toole, Ashley, Holly M. Nguyen, Hui Xia, Dianne N. Frankel, John W. Kieffer e Thomas F. Gibbons. "Sequencing SARS-CoV-2 from antigen tests". PLOS ONE 17, n. 2 (8 febbraio 2022): e0263794. http://dx.doi.org/10.1371/journal.pone.0263794.

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Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.

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Mugnier, Nathalie, Aurélien Griffon, Bruno Simon, Maxence Rambaud, Hadrien Regue, Antonin Bal, Gregory Destras et al. "Evaluation of EPISEQ SARS-CoV-2 and a Fully Integrated Application to Identify SARS-CoV-2 Variants from Several Next-Generation Sequencing Approaches". Viruses 14, n. 8 (29 luglio 2022): 1674. http://dx.doi.org/10.3390/v14081674.

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Whole-genome sequencing has become an essential tool for real-time genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide. The handling of raw next-generation sequencing (NGS) data is a major challenge for sequencing laboratories. We developed an easy-to-use web-based application (EPISEQ SARS-CoV-2) to analyse SARS-CoV-2 NGS data generated on common sequencing platforms using a variety of commercially available reagents. This application performs in one click a quality check, a reference-based genome assembly, and the analysis of the generated consensus sequence as to coverage of the reference genome, mutation screening and variant identification according to the up-to-date Nextstrain clade and Pango lineage. In this study, we validated the EPISEQ SARS-CoV-2 pipeline against a reference pipeline and compared the performance of NGS data generated by different sequencing protocols using EPISEQ SARS-CoV-2. We showed a strong agreement in SARS-CoV-2 clade and lineage identification (>99%) and in spike mutation detection (>99%) between EPISEQ SARS-CoV-2 and the reference pipeline. The comparison of several sequencing approaches using EPISEQ SARS-CoV-2 revealed 100% concordance in clade and lineage classification. It also uncovered reagent-related sequencing issues with a potential impact on SARS-CoV-2 mutation reporting. Altogether, EPISEQ SARS-CoV-2 allows an easy, rapid and reliable analysis of raw NGS data to support the sequencing efforts of laboratories with limited bioinformatics capacity and those willing to accelerate genomic surveillance of SARS-CoV-2.

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Turakhia, Yatish, Nicola De Maio, Bryan Thornlow, Landen Gozashti, Robert Lanfear, Conor R. Walker, Angie S. Hinrichs et al. "Stability of SARS-CoV-2 phylogenies". PLOS Genetics 16, n. 11 (18 novembre 2020): e1009175. http://dx.doi.org/10.1371/journal.pgen.1009175.

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The SARS-CoV-2 pandemic has led to unprecedented, nearly real-time genetic tracing due to the rapid community sequencing response. Researchers immediately leveraged these data to infer the evolutionary relationships among viral samples and to study key biological questions, including whether host viral genome editing and recombination are features of SARS-CoV-2 evolution. This global sequencing effort is inherently decentralized and must rely on data collected by many labs using a wide variety of molecular and bioinformatic techniques. There is thus a strong possibility that systematic errors associated with lab—or protocol—specific practices affect some sequences in the repositories. We find that some recurrent mutations in reported SARS-CoV-2 genome sequences have been observed predominantly or exclusively by single labs, co-localize with commonly used primer binding sites and are more likely to affect the protein-coding sequences than other similarly recurrent mutations. We show that their inclusion can affect phylogenetic inference on scales relevant to local lineage tracing, and make it appear as though there has been an excess of recurrent mutation or recombination among viral lineages. We suggest how samples can be screened and problematic variants removed, and we plan to regularly inform the scientific community with our updated results as more SARS-CoV-2 genome sequences are shared (https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473 and https://virological.org/t/masking-strategies-for-sars-cov-2-alignments/480). We also develop tools for comparing and visualizing differences among very large phylogenies and we show that consistent clade- and tree-based comparisons can be made between phylogenies produced by different groups. These will facilitate evolutionary inferences and comparisons among phylogenies produced for a wide array of purposes. Building on the SARS-CoV-2 Genome Browser at UCSC, we present a toolkit to compare, analyze and combine SARS-CoV-2 phylogenies, find and remove potential sequencing errors and establish a widely shared, stable clade structure for a more accurate scientific inference and discourse.

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Sekulic, Miroslav, Holly Harper, Behtash G. Nezami, Daniel L. Shen, Simona Pichler Sekulic, Aaron T. Koeth, Clifford V. Harding, Hannah Gilmore e Navid Sadri. "Molecular Detection of SARS-CoV-2 Infection in FFPE Samples and Histopathologic Findings in Fatal SARS-CoV-2 Cases". American Journal of Clinical Pathology 154, n. 2 (26 maggio 2020): 190–200. http://dx.doi.org/10.1093/ajcp/aqaa091.

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Abstract Objectives To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. Methods Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. Results Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. Conclusions SARS-CoV-2 testing and viral sequencing can be performed from FFPE tissue. Detection and sequencing of SARS-CoV-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus’s pathophysiologic effects on human health.

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Xiaoli, Lingzi, Jill V. Hagey, Daniel J. Park, Christopher A. Gulvik, Erin L. Young, Nabil-Fareed Alikhan, Adrian Lawsin et al. "Benchmark datasets for SARS-CoV-2 surveillance bioinformatics". PeerJ 10 (5 settembre 2022): e13821. http://dx.doi.org/10.7717/peerj.13821.

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Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has spread globally and is being surveilled with an international genome sequencing effort. Surveillance consists of sample acquisition, library preparation, and whole genome sequencing. This has necessitated a classification scheme detailing Variants of Concern (VOC) and Variants of Interest (VOI), and the rapid expansion of bioinformatics tools for sequence analysis. These bioinformatic tools are means for major actionable results: maintaining quality assurance and checks, defining population structure, performing genomic epidemiology, and inferring lineage to allow reliable and actionable identification and classification. Additionally, the pandemic has required public health laboratories to reach high throughput proficiency in sequencing library preparation and downstream data analysis rapidly. However, both processes can be limited by a lack of a standardized sequence dataset. Methods We identified six SARS-CoV-2 sequence datasets from recent publications, public databases and internal resources. In addition, we created a method to mine public databases to identify representative genomes for these datasets. Using this novel method, we identified several genomes as either VOI/VOC representatives or non-VOI/VOC representatives. To describe each dataset, we utilized a previously published datasets format, which describes accession information and whole dataset information. Additionally, a script from the same publication has been enhanced to download and verify all data from this study. Results The benchmark datasets focus on the two most widely used sequencing platforms: long read sequencing data from the Oxford Nanopore Technologies platform and short read sequencing data from the Illumina platform. There are six datasets: three were derived from recent publications; two were derived from data mining public databases to answer common questions not covered by published datasets; one unique dataset representing common sequence failures was obtained by rigorously scrutinizing data that did not pass quality checks. The dataset summary table, data mining script and quality control (QC) values for all sequence data are publicly available on GitHub: https://github.com/CDCgov/datasets-sars-cov-2. Discussion The datasets presented here were generated to help public health laboratories build sequencing and bioinformatics capacity, benchmark different workflows and pipelines, and calibrate QC thresholds to ensure sequencing quality. Together, improvements in these areas support accurate and timely outbreak investigation and surveillance, providing actionable data for pandemic management. Furthermore, these publicly available and standardized benchmark data will facilitate the development and adjudication of new pipelines.

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Mavzyutov, A. R., R. R. Garafutdinov, E. Yu Khalikova, R. R. Gazizov, An Kh Baymiev, Yu M. Nikonorov, I. V. Maksimov, B. R. Kuluev, Al Kh Baymiev e A. V. Chemeris. "The enigmas of the new coronavirus SARS-CoV-2". Biomics 13, n. 1 (2021): 75–99. http://dx.doi.org/10.31301/2221-6197.bmcs.2021-7.

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The emergence of the new SARS-CoV-2 coronavirus has given rise to many enigmas, to which there are no answers yet. However, the degree of threat to humanity, due to the fact that by the beginning of February 2021, more than 100 million people were ill in the world, of which 2 million died, led to the fact that the efforts of many researchers were aimed at combating this disease, including massive sequencing of the complete genomes of SARS-CoV-2, as this is necessary for diagnostics and prediction of the epidemiological situation, including in the long term. Currently, a fairly high level of conservativeness of the SARS-CoV-2 genome is shown, but there is also a significant variability of intra-host viral RNA, confirming the concept of the existence of quasispecies for RNA-containing viruses. As of February 2021, the complete genomes of almost half a million coronavirus isolates have been sequenced worldwide, and a number of nomenclatures have been proposed to streamline their analysis, including the convenient dynamic nomenclature Pango lineage. Variations of SARS-CoV-2 genomes in the form of consensus SNPs (Single Nucleotide Polymorphism) and intra-host iSNVs (intra-host Single Nucleotide Variant) were demonstrated. Taking into account iSNV and minor mutations, about 85% of the 29.9 thousand nucleotides viral genome were changed at least once, but only a very few of them turned into major mutations due to certain features that ensure the predominant distribution of such strains. The example of the S-protein gene, taking into account iSNV, minor and major mutations, shows its significant variability, which is detected when sequencing hundreds of thousands of SARS-CoV-2 genomes. On the basis of the analysis of 400 complete SARS-CoV-2 genomes isolated on the territory of the Russian Federation during 2020, the dynamics of the circulation of individual strains with acquired major mutations, the representation of which is slightly different from the changes in the SARS-CoV-2 genome in the rest world, is estimated. The possibility of long-term persistence of the new coronavirus in the human body is note, while the reservoirs for the latent existence of SARS-CoV-2, in contrast, for example, to the herpes simple virus, remain unknown. There is no consensus on the possibility of reactivation of SARS-CoV-2 or reinfection. The latter is theoretically possible in cases where SARS-CoV-2 strains belonging to other genetic lineages and clades are found in the body of the "re-infected". This, however, does not exclude the possibility of mutating the virus within a single host. Despite significant progress in monitoring the spread of SARS-CoV-2, many questions remain, but as knowledge of the biology of the new coronavirus accumulates, they will also be answered.

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Petersen, J. M., e D. Jhala. "Sequencing for COVID-19 in the Pandemic Era: What Does it Mean?" American Journal of Clinical Pathology 156, Supplement_1 (1 ottobre 2021): S140—S141. http://dx.doi.org/10.1093/ajcp/aqab191.300.

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Abstract Introduction/Objective SARS-CoV-2 has been developing mutations over the course of the pandemic, leading to the rise of variants. The sequencing of these variants, however, has an unclear role for the medical center providing patient treatment. Methods/Case Report Patient specimens that were positive for the presence of SARS-CoV-2 with a cycle threshold <30 by reverse transcriptase polymerase chain reaction (RT-PCR) were sent for sequencing at the Veterans Health Administration Public Health Reference Laboratory (PHRL). Testing for SARS-CoV-2 was by RT-PCR was initially done by either the Abbott Alinity m SARS-CoV-2 assay (Chicago IL) or the Cepheid Xpert Xpress SARS-CoV- 2/Flu/RSV assay (Sunnyvale CA). All sent patient specimens had been selected by the clinical team for concern of the presence of a SARS-CoV-2 variant. Results (if a Case Study enter NA) There were a total of 8 patients (4 males and 4 females) that were sent for sequencing. The patient ages ranged from 38 to 80 years (average 58.8). The racial proportion of the 8 patients was 2 African Americans, 2 Caucasian Americans, and 4 unanswered. All were positive for SARS-CoV-2 by RT-PCR (4 Abbott assay and 4 Cepheid assay). Six of the sequenced samples showed the NextClade 20I/501Y.V1, Pango Lineage B.1.1.7, a variant first identified in the United Kingdom; four of these six patients had documentation of vaccination prior to the infection. One sequence was a NextClade 20C Pango Lineage B.1.526.1, a variant first identified in New York. The last sequence identified was a NextClade 20G, Pango Lineage B.1, a variant predominantly seen in the United States. Conclusion At the present time, sequencing of SARS-CoV-2 does not have a clear clinical role. However, from a public health and epidemiological point of view, sequencing has a role in SARS-CoV-2 variant tracing and detection. Vaccine protection against variant SARS-CoV-2 may not be complete as some infected patients had been vaccinated.

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Nasir, Jalees A., Robert A. Kozak, Patryk Aftanas, Amogelang R. Raphenya, Kendrick M. Smith, Finlay Maguire, Hassaan Maan et al. "A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture". Viruses 12, n. 8 (15 agosto 2020): 895. http://dx.doi.org/10.3390/v12080895.

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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.

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Crawford, Dana C., e Scott M. Williams. "Global variation in sequencing impedes SARS-CoV-2 surveillance". PLOS Genetics 17, n. 7 (15 luglio 2021): e1009620. http://dx.doi.org/10.1371/journal.pgen.1009620.

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Avetyan, Diana, Siras Hakobyan, Maria Nikoghosyan, Lilit Ghukasyan, Gisane Khachatryan, Tamara Sirunyan, Nelli Muradyan et al. "Molecular Analysis of SARS-CoV-2 Lineages in Armenia". Viruses 14, n. 5 (17 maggio 2022): 1074. http://dx.doi.org/10.3390/v14051074.

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The sequencing of SARS-CoV-2 provides essential information on viral evolution, transmission, and epidemiology. In this paper, we performed the whole-genome sequencing of SARS-CoV-2 using nanopore and Illumina sequencing to describe the circulation of the virus lineages in Armenia. The analysis of 145 full genomes identified six clades (19A, 20A, 20B, 20I, 21J, and 21K) and considerable intra-clade PANGO lineage diversity. Phylodynamic and transmission analysis allowed to attribute specific clades as well as infer their importation routes. Thus, the first two waves of positive case increase were caused by the 20B clade, the third peak caused by the 20I (Alpha), while the last two peaks were caused by the 21J (Delta) and 21K (Omicron) variants. The functional analyses of mutations in sequences largely affected epitopes associated with protective HLA loci and did not cause the loss of the signal in PCR tests targeting ORF1ab and N genes as confirmed by RT-PCR. We also compared the performance of nanopore and Illumina short-read sequencing and showed the utility of nanopore sequencing as an efficient and affordable alternative for large-scale molecular epidemiology research. Thus, our paper describes new data on the genomic diversity of SARS-CoV-2 variants in Armenia in the global context of the virus molecular genomic surveillance.

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Più fonti

Tesi sul tema "SARS-CoV-2 sequencing"

Venkatesan, Lavanya. "Identifying and Tracking the Evolution of Mutations in the SARS-CoV-2 Virus". Thesis, Virginia Tech, 2021. http://hdl.handle.net/10919/103939.

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SARS-CoV-2 is caused by a pathogenic and highly transmissible beta coronavirus leading to severe infections in immuno-compromised individuals. This study first evaluates the primers used in the Reverse Transcription Polymerase Chain Reaction (RT-PCR) to detect SARS-CoV-2 by understanding how mutations might affect the primer efficiency with the SARS-CoV-2 sequences. Mutations on the Spike protein of SARS-CoV-2 are the most important as the spike protein mediates the viral entry into host cells. This study tracks the course of mutations on the spike protein by focusing on the haplogroups of the sequences across the world. A comprehensive database linking three important, currently available databases is curated as part of this study to fill the gaps caused by sequencing errors. Further, this study exploits the data generated by the Illumina and Oxford Nanopore next generation sequencing methods to study the evolution of mutations in a single Septuagenarian patient over an infection period of 102 days using the gene analysis software Geneious Prime.
Master of Science
A novel corona virus named Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has taken down the entire world by causing Covid-19 pandemic. Initially detected in Wuhan, China, the virus has now made its way to more than 200 countries with a heavy death toll. Understanding the virus through mutation tracking and improving diagnostics and vaccine design have now become the top priority of researchers. Most of these researchers depend on quality viral sequence datasets to identify and track mutations. One aim of this study is to provide a comprehensive dataset linking the GISAID (Global Initiative on Sharing All Influenza Data), NCBI (National Center for Biological Information) and the SRA (Sequence Read Archive) sequences. The dataset can be used for genome analysis and mutation tracking which can provide important insights for vaccine design and in improving diagnostic assays. In addition, this study provides an analysis of viral mutations in in the genomic regions targeted by commonly used primers in the RT-PCR tests for SARS-CoV-2 that may affect the efficiency of detection. This study also uses the haplogroup information of people across the world to track the D614G mutation on the S gene of SARS-CoV-2 as it may be associated with increased transmissibility. To track the course of mutations in SARS-CoV-2, it is important to analyze the sequencing data provided by the Illumina and Oxford Nanopore next generation sequencing methods. We present a case study to investigate the course of SARS-CoV-2 mutations in a single septuagenarian patient over a period of 102days using the Sequence Read Archive (SRA) data generated by two Next Generation Sequencing methods and compare the advantages that one has over the other.

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GRIMALDI, ANTONIO. "AN IMPROVED GENOMIC SURVEILLANCE APPROACH TO DISSECT THE SARS-COV-2 PANDEMIC". Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/946458.

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Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the only approach to rapidly monitor and tackle emerging variants of concern (VOC) of the COVID-19 pandemic. Such scrutiny is crucial to limit the spread of VOC that might escape the immune protection conferred by vaccination strategies. It is also becoming clear now that efficient genomic surveillance would require monitoring the host gene expression to identify prognostic biomarkers of efficacy and disease progression. Here we applied an integrated workflow for RNA extracted from nasal swabs to obtain in parallel the entire genome of SARS-CoV-2 and host respiratory epithelium transcriptome, representing the majority of Italian processed genomic samples. In addition, we have matured and applied novel proof-of-principle approaches to prioritize possible gain-of-function mutations by leveraging patients' metadata and isolated patient-specific signatures of SARS-CoV-2 infection. The goals mentioned above have all been achieved in a cost-effective manner that does not require automation, in an effort to allow any lab with a benchtop sequencer and a limited budget to perform integrated genomic surveillance on premises.

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Martignano, Filippo, Salvatore Di Giorgio, Maria Gabriella Torcia, Giorgio Mattiuz, Silvestro Conticello, Stefania Crucitta, Alessandra Mingrino et al. "Sequencing-based approaches for the study of Lung-related diseases". Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1120728.

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My thesis is focused on sequencing-based methods for lung diseases monitoring. Modern sequencing techniques allow us to comprehensively characterize nucleic acids obtained from patient-derived biological material, with potential applications in both basic research and clinical practice. The thesis is divided in two sections: In Section 1: “Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2” I describe the presence of RNA editing events in SARS-CoV-2, by analysing publicly available second generation RNA sequencing data from infected patients: Emerging viral infections represent a threat to global health, and the recent outbreak of novel coronavirus disease 2019 caused by SARS-CoV-2 exemplifies the risks. RNA editing is a physiological mechanism mediated by two enzyme families: APOBEC and ADAR, which introduce A-to-I and C-to-U mutations in double strand and single strand RNA respectively. RNA editing typically involves endogenous RNAs but, if targeting viral RNA, it is potentially deleterious for virus’ viability itself, by generating premature stop codons and missense mutations in the viral genome. On the other hand, RNA editing could fuel virus evolution by increasing the basal mutational rate. I have downloaded publicly available Illumina transcriptomic data, from BALF samples of infected patients; using a combination of published tools (Reditools2, JACUSA) I have detected an enrichment of APOBEC and ADAR related mutation on SARS-CoV-2 RNA. A similar enrichment was observed in genomic RNA SARS-CoV-2, SARS and MERS sequences downloaded from GISAID and NCBI virus. The evidence of RNA editing on SARS-CoV-2 suggests that APOBEC and ADAR can interact with viral RNAs, probably with an anti-viral purpose. C-to-U changes leading to stop codons are overrepresented in the transcriptomic data but—as expected—disappear in the genomic dataset. This might point—again—to an antiviral role for these editing enzymes. Also, the proportion of ADAR-related mutation was unexpectedly lower in genomic RNA sequences, compared to transcriptomic data. It is possible that A-to-I editing is somehow restricting viral propagation, thus reducing the number of viral progeny showing evidence of these changes. In Section 2: “Analysis of copy number variations from cell-free DNA of lung cancer patients via Nanopore sequencing” I have developed a customized workflow to exploit Nanopore sequence for the analysis of plasmatic cell-free DNA: Cancer is an extremely dynamic disease: malignant cells are constantly under selective pressure and the evolutionary path of each tumor can take different directions due to such pressure. It is hence important to monitor cancer development at multiple timepoints to closely follow its evolution; unfortunately, the risks and invasiveness of conventional biopsy make it unsuitable for repeated sampling. A valid and non-invasive alternative to tissue sampling is represented by the analysis of cfDNA from liquid biopsy samples (plasma). CNVs are an important class of genetic alterations that can affect tumor aggressivity and resistance to treatment. To date, the only reliable approach to obtain a whole-genome CNV profile from plasmatic cfDNA is Illumina sequencing. However the need for expensive sequencers is often an obstacle for smaller laboratories. Oxford Nanopore Technologies has recently released MinION: a fast and extremely inexpensive third generation sequencer based on the Nanopore technology. However, this technology is not designed for low quality DNA such as cfDNA (very fragmented, low concentration). I have modified Nanopore standard protocols to make them compatible with the characteristics of cfDNA. The technique has been tested on plasma samples obtained from lung cancer patients, with the aim of detecting tumor-specific copy number variations. The approach has been subsequently validated by comparing it with the current standard technique (Illumina). Nanopore and Illumina results strongly correlate (R = 0.96 – 0.99, p << 0.001), with concordant log2ratio values in 97-99% of genome positions. Nanopore features (i.e. reduced costs) represent advantages over current sequencing technologies, and might drive the adoption of molecular karyotyping from liquid biopsies as a tool for cancer monitoring in clinical settings.

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PINZAUTI, DAVID. "Implementation of a flexible Oxford Nanopore sequencing platform for microbial genomics". Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1138520.

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Oxford Nanopore sequencing technology is slowly revolutionising the entire microbiology field. Its ease of use and cost effective approaches coupled with long reads sequencing represent an essential and powerful tool. In the present thesis I have implemented a sequencing platform based on the Oxford Nanopore technology, a flexible system suitable for both research and diagnostic fields. The first part of my work was dedicated to the optimization of a DNA extraction protocol capable of isolating high molecular weight (HMW) genomic DNA. In fact, Nanopore sequencing readouts are highly influenced by both the quality and the integrity of the genomic DNA. An enzymatic lysis based extraction protocol was optimized, recovering HMW DNA from two strains of Streptococcus mitis and generating multiple ultra long reads (i.e. >100 Kb in length), making it possible to achieve complete genome assemblies. As the extraction protocol was mainly optimized for Gram-positive bacteria, it is also suitable to lyse the thinner cell wall of Gram-negatives. Oxford Nanopore Whole Genome Sequencing (WGS) approaches have enabled the complete genome sequencing and assembly of 16 Enterococcus faecalis isolates from clinical dental samples. Sequencing data provided enough information enabling i) population studies, defining genomic clusters based on isolates homologies; ii) bacterial profiling, assessing antimicrobial resistance genes and virulence traits; iii) comparative analysis, identifying genomic rearrangements and homologies based on synteny blocks. Finally, the platform was used for the monitoring of the ongoing SARS-CoV-2 pandemic. We have proposed a 900 bp amplicon sequencing protocol, adapted from the ARTIC sequencing protocol (https://artic.network/), enabling a near-complete genome assembly of SARS-CoV-2 strains, helping in the detection of nucleotide changes and monitoring the circulating viral lineages. In conclusion, the Oxford Nanopore sequencing platform can bring several improvements in the microbiology field, allowing i) complete genome assembly, ii) rapid microbial profiling, and iii) helping in the monitoring of local or global outbreaks.

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Wang, Xuanting. "Identification of SARS-CoV-2 Polymerase and Exonuclease Inhibitors and Novel Methods for Single-Color Fluorescent DNA Sequencing by Synthesis". Thesis, 2021. https://doi.org/10.7916/d8-n6ah-nt76.

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This dissertation is divided into two main sections describing major portions of my Ph.D. research: (1) development of two enzymatic assays for identifying inhibitors of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and the associated proofreading exonuclease complexes, two key enzymatic activities of SARS-CoV-2, the virus responsible for the COVID-19 pandemic and (2) the design and implementation of four novel single-color fluorescent DNA sequencing by synthesis (SBS) methods, including the synthesis of many of the key nucleotide analogues required for these studies. In response to the COVID-19 pandemic, the first part of my research is focused on the discovery of potential therapeutics for combating coronavirus infections. Chapter 1 describes the identification of several polymerase and exonuclease inhibitors for SARS-CoV-2 using novel mass spectrometry-based molecular assays. SARS-CoV-2 has an exonuclease complex, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RdRp and the exonuclease could overcome this deficiency. Chapter 1 reports the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of identified exonuclease inhibitors, RNAs terminated with the active forms of the prodrugs like Sofosbuvir, Remdesivir and Favipiravir were largely protected from excision by the exonuclease, while in the absence of exonuclease inhibitors, there was rapid excision. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. Chapters 2-6 describe the single-color DNA SBS studies. Chapter 2 provides essential background on the structure of DNA, the DNA polymerase reaction, and several key DNA sequencing technologies, with an emphasis on the design of nucleotide analogues for the DNA SBS approach. Chapter 3 delineates a one-color fluorescent DNA SBS method based on a set of nucleotide reversible terminators (NRTs) comprising two orthogonal cleavable linkers, one fluorescent dye and one anchor. Chapter 4 describes a one-color hybrid DNA sequencing approach using a set of dideoxynucleotide analogues bearing two orthogonal cleavable linkers, one fluorophore and one anchor as well as a set of unlabeled NRTs. By introducing a pH responsive fluorophore into the design of nucleotide analogues, Chapter 5 demonstrates a novel type of single-color DNA SBS method using a set of NRTs comprising one pH-responsive fluorescent dye or one non-responsive fluorescent dye tethered with one cleavable linker. Chapter 6 presents another option for the single-color DNA sequencing technique using a set of deoxynucleotide analogues comprising the above pH responsive or non-responsive dyes tethered with a cleavable linker, along with a set of unlabeled NRTs. The one-color SBS approaches have the potential for higher sensitivity, miniaturization and cost effectiveness compared with four-color SBS methods. Finally, Chapter 7 summarizes the SARS-CoV-2 antiviral drug discovery and one-color sequencing techniques and discusses potential follow-up research on these projects.

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Capitoli di libri sul tema "SARS-CoV-2 sequencing"

Yan, Yan, e Qinxue Hu. "Molecular Epidemiology of SARS-CoV-2 by Sequencing". In Methods in Molecular Biology, 19–32. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2111-0_2.

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Knyazev, Sergey, Daniel Novikov, Mark Grinshpon, Harman Singh, Ram Ayyala, Varuni Sarwal, Roya Hosseini et al. "A Novel Network Representation of SARS-CoV-2 Sequencing Data". In Bioinformatics Research and Applications, 165–75. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-91415-8_15.

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Chourasia, Prakash, Sarwan Ali, Simone Ciccolella, Gianluca Della Vedova e Murray Patterson. "Clustering SARS-CoV-2 Variants from Raw High-Throughput Sequencing Reads Data". In Computational Advances in Bio and Medical Sciences, 133–48. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-17531-2_11.

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Speicher, David J., Jalees A. Nasir, Peng Zhou e Danielle E. Anderson. "Whole-Genome Sequencing of Pathogens in : A Target-Enrichment Approach for SARS-CoV-2". In Methods in Molecular Biology, 119–37. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1518-8_8.

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Corredor-Vargas, A. M., R. Torezani, G. Paneto e T. F. Bastos-Filho. "Importance of Sequencing the SARS-CoV-2 Genome Using the Nanopore Technique to Understand Its Origin, Evolution and Development of Possible Cures". In XXVII Brazilian Congress on Biomedical Engineering, 1341–44. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-70601-2_199.

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Kumar, Arbind, Aashish Sharma, Narendra Vijay Tirpude, Yogendra Padwad, Shaifali Sharma e Sanjay Kumar. "Perspective Chapter: Emerging SARS-CoV-2 Variants of Concern (VOCs) and Their Impact on Transmission Rate, Disease Severity and Breakthrough Infections". In Infectious Diseases. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.107844.

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SARS-CoV-2, like all RNA viruses, evolves over time, and genetic mutations have been linked to increased replication fitness and evolvability. SARS-CoV-2 spreads quickly between countries, resulting in new mutations. SARS-CoV-2 genome sequencing reveals that variants emerge through point mutations, insertions, and deletions. Concerns have been raised about the ability of currently approved vaccines to protect against emerging variants. Viral spike protein is a component of many approved vaccine candidates, and mutations in the S-protein may affect transmission dynamics and the risk of immune escape, resulting this pandemic last-longer in populations. Understanding the evolution of the SARS-CoV-2 virus, as well as its potential relationship with transmissibility, infectivity, and disease severity, may help us predict the consequences of future pandemics. SARS-CoV-2 genome studies have identified a few mutations that could potentially alter the transmissibility and pathogenicity of the SARS-CoV-2 virus. At the moment, it is worth mentioning that a few variants have increased the transmissibility of SARS-CoV-2. The Alpha, Beta, Gamma, Delta, Delta+, and omicron variants are designated as variants of concern (VOCs) by the World Health Organisation and have been linked with an increased risk to the community in terms of transmission, hospitalisation, and mortality. This chapter thoroughly discusses the impact of SARS-CoV-2 mutations, mainly VOCs, on public health by mining many published articles.

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Patil, Shashank M., Chandrashekar Srinivasa, Ramith Ramu, Shiva Prasad Kollur, Suhas Ramesh e Chandan Shivamallu. "SARS-CoV-2 genome sequencing and promising druggable targets". In Coronavirus Drug Discovery, 3–22. Elsevier, 2022. http://dx.doi.org/10.1016/b978-0-323-95578-2.00004-2.

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Paz, Mercedes, Pilar Moreno e Gonzalo Moratorio. "Perspective Chapter: Real-Time Genomic Surveillance for SARS-CoV-2 on Center Stage". In Infectious Diseases. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.107842.

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The course of the COVID-19 pandemic depends not only on how the SARS-CoV-2 virus mutates but on the actions taken to respond to it. Important public health decisions can only be taken if we know viral dynamics, viral variants distribution, and whether new variants are emerging that may be more transmissible or/and more virulent, displaying evasion to vaccines or antiviral treatments. This situation has put the use of different approaches, such as molecular techniques and real-time genomic sequencing, to support public health decision-making on center stage. To achieve this, robust programs based on: (i) diagnostic capacity; (ii) high-throughput sequencing technologies; and (iii) high-performance bioinformatic resources, need to be established. This chapter focuses on how SARS-CoV-2 evolved since its discovery and it summarizes the scientific efforts to obtain genomic data as the virus spread throughout the globe.

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Cristina Diaconu, Carmen, Ioana Madalina Pitica, Mihaela Chivu-Economescu, Laura Georgiana Necula, Anca Botezatu, Iulia Virginia Iancu, Ana Iulia Neagu et al. "SARS-CoV-2 Variant Surveillance in Genomic Medicine Era". In Infectious Diseases. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.107137.

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In the genomic medicine era, the emergence of SARS-CoV-2 was immediately followed by viral genome sequencing and world-wide sequences sharing. Almost in real-time, based on these sequences, resources were developed and applied around the world, such as molecular diagnostic tests, informed public health decisions, and vaccines. Molecular SARS-CoV-2 variant surveillance was a normal approach in this context yet, considering that the viral genome modification occurs commonly in viral replication process, the challenge is to identify the modifications that significantly affect virulence, transmissibility, reduced effectiveness of vaccines and therapeutics or failure of diagnostic tests. However, assessing the importance of the emergence of new mutations and linking them to epidemiological trend, is still a laborious process and faster phenotypic evaluation approaches, in conjunction with genomic data, are required in order to release timely and efficient control measures.

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Vlasova-St. Louis, Irina, Andrew Gorzalski e Mark Pandori. "Diagnostic Applications for RNA-Seq Technology and Transcriptome Analyses in Human Diseases Caused by RNA Viruses". In Applications of RNA-Seq in Biology and Medicine [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.99156.

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Human diseases caused by single-stranded, positive-sense RNA viruses, are among the deadliest of the 21st Century. In particular, there are two notable standouts: human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of these disease-causing viral transcripts, by next-generation RNA sequencing (RNA-Seq), represents the most immediate opportunity for advances in diagnostic, therapeutic, and preventive applicability in infectious diseases (e.g., AIDS and COVID-19). Moreover, RNA-Seq technologies add significant value to public health studies by first, providing real-time surveillance of known viral strains, and second, by the augmentation of epidemiological databases, construction of annotations and classifications of novel sequence variants. This chapter intends to recapitulate the current knowledge of HIV and SARS-CoV-2 transcriptome architecture, pathogenicity, and some features of the host immune response. Additionally, it provides an overview of recent advances in diagnostic sequencing methodologies and discusses the future challenges and prospects on the utilization of RNA-Seq technologies.

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Atti di convegni sul tema "SARS-CoV-2 sequencing"

"APPLICATIONS OF SARS-COV-2 SEQUENCING DATA". In 14th International Conference on Computer Graphics, Visualization, Computer Vision and Image Processing. IADIS Press, 2020. http://dx.doi.org/10.33965/bigdaci2020_202011c034.

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Benslimane, Fatiha M., Hebah Al Khatib, Dana Albatesh, Ola Al-Jamal, Sonia Boughattas, Asmaa A. Althani e Hadi M. Yassine. "Nanopore Sequencing SARS-CoV-2 Genome in Qatar". In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0289.

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Background: The current pandemic, COVID-19, is cause by an RNA Coronavirus that was recently identified as SARS-CoV-2. RNA viruses tend to have a high mutation rate; the rate is around a million times greater than that of their hosts. The mutagenic potential of the virus depends on many factors, including the fidelity of nucleic acid-replicating viral enzymes, such as SARSCoV-2 RNA dependent RNA polymerase (RdRp). The rate of mutation drives viral evolution and genome variability, consequently allowing viruses to escape the immunity of the host and develop resistance to drugs. Therefore, the characterization of SARS-CoV-2 variants might lead to implement better therapeutics treatments, vaccines design and identify new diagnostics approaches. Aim: The aim of this study was to establish a fast sequencing method to identify SARS-CoV-2 mutations in Qatar. This will help to assess if there are new viral variants that are spreading in country. Methods: RNA was isolated from samples collected from Qatar COVID-19 positive patients. The Artic Network V3 primer scheme and Oxford Nanopore ligation sequencing kit were used to prepare the sequencing libraries. Libraries were loaded on to R9.4.1 flow cells and ran on a GridION. Bioinformatics analysis was done following the Artic Network SARA-CoV-2 bioinformatics tools. Results: Genome coverage of sequenced samples was >80% and the depth was average at 200x. The coverage was highly dependable on sample viral load; samples of CT value lower than 30 resulted in better sequence coverage. The sequenced genomes were deposited in GISAID and were mainly clustering with genomes deposited from the UK. Sequences were compared to Illumina and sanger sequences and they showed compatible results. Conclusion: The use of ONT to sequence SARA-CoV-2 is a quick, affordable, and reliable technique to determine viral mutation. Using this technique, the first sequences from Qatar were deposited in to GISAID. Up to date, 700 genomes have been sequenced from Qatari samples.

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Yuan, Yingzhi. "SARS-CoV-2: next generation sequencing and analysis". In International Conference on Biomedical and Intelligent Systems (IC-BIS 2022), a cura di Ahmed El-Hashash. SPIE, 2022. http://dx.doi.org/10.1117/12.2660958.

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Kille, Bryce, Yunxi Liu, Nicolae Sapoval, Michael Nute, Lawrence Rauchwerger, Nancy Amato e Todd J. Treangen. "Accelerating SARS-CoV-2 low frequency variant calling on ultra deep sequencing datasets". In 2021 IEEE International Parallel and Distributed Processing Symposium Workshops (IPDPSW). IEEE, 2021. http://dx.doi.org/10.1109/ipdpsw52791.2021.00038.

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Hsu, Po-Kai, e Shimeng Yu. "In-Memory 3D NAND Flash Hyperdimensional Computing Engine for Energy-Efficient SARS-CoV-2 Genome Sequencing". In 2022 IEEE International Memory Workshop (IMW). IEEE, 2022. http://dx.doi.org/10.1109/imw52921.2022.9779291.

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Kaptelova, V. V., A. S. Speranskaya, A. E. Samoilov, A. V. Valdokhina, V. P. Bulanenko, E. V. Korneenko, O. Y. Shipulina e V. G. Akimkin. "MUTATIONS IN THE GENOMES OF SARS-COV-2 FROM CLINICAL SAMPLES OBTAINED IN LATE MARCH-EARLY APRIL FROM PATIENTS IN MOSCOW". In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-147.

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Many papers suggested that D614G mutation in the viral spike (S) protein SARS-CoV-2 can influence the ability of virus transmission. In recent work [1], it was shown D614G influences the rate of disease transmission only in combination with the P323L mutation in the viral polymerase. We have sequenced 28 full genomes of SARS-CoV-2, obtained from clinical material from patients of different ages. The analyzed isolates belong to clades B.1 (GH) and B1.1 (GR). Combinations of mutations P323L and D614G were found in all genomes. These differences can be explained by sampling: the samples for the sequencing of the whole genome were selected with high viral load, it can be related to the rate of viral replication in intra-host. That, in turn, can be dependent on the presence of P323L/D614G mutations in the virus genome.

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Al Khatib, Hebah A., Fatiha M. Benslimane, Israa El Bashir, Asmaa A. Al Thani e Hadi M. Yassine. "Within-Host Diversity of SARS-Cov-2 in COVID-19 Patients with Variable Disease Severities". In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0280.

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Background: The ongoing pandemic of SARS-COV-2 has already infected more than eight million people worldwide. The majority of COVID-19 patients are either asymptomatic or have mild symptoms. Yet, about 15% of the cases experience severe complications and require intensive care. Factors determining disease severity are not yet fully characterized. Aim: Here, we investigated the within-host virus diversity in COVID-19 patients with different clinical manifestations. Methods: We compared SARS-COV-2 genetic diversity in 19 mild and 27 severe cases. Viral RNA was extracted from nasopharyngeal samples and sequenced using Illumina MiSeq platform. This was followed by deep-sequencing analyses of SARS-CoV-2 genomes at both consensus and sub-consensus sequence levels. Results: Consensus sequences of all viruses were very similar, showing more than 99·8% sequence identity regardless of the disease severity. However, the sub-consensus analysis revealed significant differences in within-host diversity between mild and severe cases. Patients with severe symptoms exhibited a significantly (p-value 0.001) higher number of variants in coding and non-coding regions compared to mild cases. Analysis also revealed higher prevalence of some variants among severe cases. Most importantly, severe cases exhibited significantly higher within-host diversity (mean= 13) compared to mild cases (mean=6). Further, higher within-host diversity was observed in patients above the age of 60 compared to the younger age group. Conclusion: These observations provided evidence that within-host diversity might play a role in the development of severe disease outcomes in COVID19 patients; however, further investigations is required to elucidate this association.

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Derecichei, Iulian, e Govindaraja Atikukke. "Machine Learning Model to Track SARS-CoV-2 Viral Mutation Evolution and Speciation Using Next-generation Sequencing Data". In BCB '20: 11th ACM International Conference on Bioinformatics, Computational Biology and Health Informatics. New York, NY, USA: ACM, 2020. http://dx.doi.org/10.1145/3388440.3415991.

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Nouailles, Geraldine, Emanuel Wyler, Peter Pennitz, Dylan Postmus, Daria Vladimirova, Julia Kazmierski, Fabian Pott et al. "Single-cell-sequencing in SARS-COV-2-infected hamsters sheds light on endothelial cell involvement in COVID-19". In ERS International Congress 2021 abstracts. European Respiratory Society, 2021. http://dx.doi.org/10.1183/13993003.congress-2021.pa2355.

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Li, Chenyu, David N. Debruyne, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Bo Zhou, Utsav Pandey et al. "Abstract S08-01: Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing". In AACR Virtual Meeting: COVID-19 and Cancer; July 20-22, 2020. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1557-3265.covid-19-s08-01.

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Rapporti di organizzazioni sul tema "SARS-CoV-2 sequencing"

Gleasner, Cheryl, Kimberly Mcmurry, Julia Kelliher e Andrew Hatch. Illumina Sequencing for SARS-CoV-2 Training [Slides]. Office of Scientific and Technical Information (OSTI), maggio 2021. http://dx.doi.org/10.2172/1782612.

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