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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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completoAbstract (sommario):
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.
Testo completo
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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.
Testo completoAbstract (sommario):
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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Babiker, Ahmed, Heath L. Bradley, Victoria D. Stittleburg, Jessica M. Ingersoll, Autum Key, Colleen S. Kraft, Jesse J. Waggoner e Anne Piantadosi. "Metagenomic Sequencing To Detect Respiratory Viruses in Persons under Investigation for COVID-19". Journal of Clinical Microbiology 59, n. 1 (16 ottobre 2020): e02142-20. http://dx.doi.org/10.1128/jcm.02142-20.
Testo completoAbstract (sommario):
ABSTRACTBroad testing for respiratory viruses among persons under investigation (PUIs) for SARS-CoV-2 has been performed inconsistently, limiting our understanding of alternative viral infections and coinfections in these patients. RNA metagenomic next-generation sequencing (mNGS) offers an agnostic tool for the detection of both SARS-CoV-2 and other RNA respiratory viruses in PUIs. Here, we used RNA mNGS to assess the frequencies of alternative viral infections in SARS-CoV-2 RT-PCR-negative PUIs (n = 30) and viral coinfections in SARS-CoV-2 RT-PCR-positive PUIs (n = 45). mNGS identified all viruses detected by routine clinical testing (influenza A [n = 3], human metapneumovirus [n = 2], and human coronavirus OC43 [n = 2], and human coronavirus HKU1 [n = 1]). mNGS also identified both coinfections (1, 2.2%) and alternative viral infections (4, 13.3%) that were not detected by routine clinical workup (respiratory syncytial virus [n = 3], human metapneumovirus [n = 1], and human coronavirus NL63 [n = 1]). Among SARS-CoV-2 RT-PCR-positive PUIs, lower cycle threshold (CT) values correlated with greater SARS-CoV-2 read recovery by mNGS (R2, 0.65; P < 0.001). Our results suggest that current broad-spectrum molecular testing algorithms identify most respiratory viral infections among SARS-CoV-2 PUIs, when available and implemented consistently.
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De Salazar, A., A. Fuentes-López, L. Viñuela, P. Camacho-Martinez, N. Chueca, L. Merino, J. Perez-Florido et al. "SARS-CoV-2 genome sequencing in Andalusia, methodology and study of variants". ACTUALIDAD MEDICA 106, n. 106(814) (gennaio 2022): 291–300. http://dx.doi.org/10.15568/am.2021.814.rev03.
Testo completoAbstract (sommario):
The incorporation of genomic sequencing techniques through next-generation sequencing has revolutionized clinical microbiology, innovating and improving the clinical diagnosis of infectious diseases. Today, whole genome sequencing in infectious diseases has many applications in virology, bacteriology, antibiotic resistance, epidemiology, and public health. With the appearance of SARS-CoV-2, the importance of the analysis and study of genetic sequences has been underlined. Since the initial identification of SARS-CoV-2, to date, more than 414,575 complete genomic sequences have been shared worldwide through public access databases. The ability to monitor viral evolution in near real time has a direct impact on the public health response to the COVID-19 pandemic. This paper presents the importance of genomic sequencing in microbiology, infectious diseases, epidemiology and public health, and describes how SARS-CoV-2 sequencing has been implemented in Andalusia, and what the main results are to date.
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Chen, Zhiyuan, Andrew S. Azman, Xinhua Chen, Junyi Zou, Yuyang Tian, Ruijia Sun, Xiangyanyu Xu et al. "Global landscape of SARS-CoV-2 genomic surveillance and data sharing". Nature Genetics 54, n. 4 (28 marzo 2022): 499–507. http://dx.doi.org/10.1038/s41588-022-01033-y.
Testo completoAbstract (sommario):
AbstractGenomic surveillance has shaped our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. We performed a global landscape analysis on SARS-CoV-2 genomic surveillance and genomic data using a collection of country-specific data. Here, we characterize increasing circulation of the Alpha variant in early 2021, subsequently replaced by the Delta variant around May 2021. SARS-CoV-2 genomic surveillance and sequencing availability varied markedly across countries, with 45 countries performing a high level of routine genomic surveillance and 96 countries with a high availability of SARS-CoV-2 sequencing. We also observed a marked heterogeneity of sequencing percentage, sequencing technologies, turnaround time and completeness of released metadata across regions and income groups. A total of 37% of countries with explicit reporting on variants shared less than half of their sequences of variants of concern (VOCs) in public repositories. Our findings indicate an urgent need to increase timely and full sharing of sequences, the standardization of metadata files and support for countries with limited sequencing and bioinformatics capacity.
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Pembaur, Anton, Erwan Sallard, Patrick Philipp Weil, Jennifer Ortelt, Parviz Ahmad-Nejad e Jan Postberg. "Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology". Microorganisms 9, n. 12 (16 dicembre 2021): 2598. http://dx.doi.org/10.3390/microorganisms9122598.
Testo completoAbstract (sommario):
The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.
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Shin, Yeun-Kyung, Oh-Kyu Kwon, Jinhwa Heo, Jinju Nah, Hae-Eun Kang, Yunhee Kang, In Jun Song e Ho-Kyung Sung. "Whole Genome Sequencing of SARS-CoV-2 in Cats and Dogs in South Korea in 2021". Veterinary Sciences 10, n. 1 (23 dicembre 2022): 6. http://dx.doi.org/10.3390/vetsci10010006.
Testo completoAbstract (sommario):
SARS-CoV-2 infections have caused unprecedented damage worldwide by affecting humans and various animals. The first reported animal infection was observed in a pet dog in Hong Kong in March 2020. 36 countries reported 692 SARS-CoV-2 infections in 25 different animal species by 31 August 2022. Most outbreaks were caused by contact with SARS-CoV-2 infected humans. In South Korea, the first SARS-CoV-2 infection in an animal was reported in a cat in February 2021. As of 31 December 2021, 74 dogs and 42 cats have been confirmed to have SARS-CoV-2 in South Korea. Here, we identified various SARS-CoV-2 genomic lineages in SARS-CoV-2 confirmed cats and dogs. Among the 40 animal samples sequenced for lineage identification, a total of eight Pango lineages (B.1.1.7 (Alpha variant), B.1.429 (Epsilon variant), B.1.470, B.1.497, B.1.619.1, B.1.620, AY.69 (Delta variant), and AY.122.5 (Delta variant)) were identified. The dominant lineages were AY.69 (Delta variant; 37.5%), B.1.497 (35.0%), and B.1.619.1 (12.5%). This study provides the first reported cases of six lineages (B.1.470, B.1.497, B.1.620, B.1.619.1, AY.69 (Delta variant)), and AY.122.5 (Delta variant) in cats and dogs. Our results emphasize the importance of monitoring SARS-CoV-2 in pets because they are dynamic hosts of variant Pango lineages of SARS-CoV-2.
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Bahouq, Hanane, Madiha Bahouq e Abdelmajid Soulaymani. "Overview of genomic surveillance related to Severe Acute Respiratory Syndrom Coronavirus 2 (SARS- CoV-2)". E3S Web of Conferences 319 (2021): 01043. http://dx.doi.org/10.1051/e3sconf/202131901043.
Testo completoAbstract (sommario):
Since the start of the Severe Acute Respiratory Syndrom Coronavirus 2 (SARS-CoV-2) pandemic, several thousand of variants circulated and others are emerging. Therefore, genomic surveillance is crucial, which aims to detect the emergence of new variants, in particular Variants of Concern (VOC) and to assess the impact of priority mutations on the transmissibility and lethality of the virus, the performance of viral diagnostic methods and vaccine efficiency. An overview of available papers was performed to understand conduct, tools and utility of genomic sequencing and surveillance related to Covid-19 disease. We also report the experience of Morocco in this filed through available data. A national SARS-Cov-2 genomic consortium has been established in order to continuously inform the health authorities of the genetic evolution of circulating strains in Morocco. Genomic sequencing shows that Moroccan genomes spread did not show a predominant SARS-CoV-2 lineage. Genomes are dispersed across the evolutionary tree of SARS-CoV-2 and held between 4 and 16 mutations. As the pandemic ongoing, continuous genomic surveillance and regular sequencing are fundamental to understand the spread of SARS-CoV-2, to rapidly identify potential global transmission networks and to consolidate response strategies especially targeted Covid-19 vaccination.
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Harilal, Divinlal, Sathishkumar Ramaswamy, Tom Loney, Hanan Al Suwaidi, Hamda Khansaheb, Abdulmajeed Alkhaja, Rupa Varghese et al. "SARS-CoV-2 Whole Genome Amplification and Sequencing for Effective Population-Based Surveillance and Control of Viral Transmission". Clinical Chemistry 66, n. 11 (28 ottobre 2020): 1450–58. http://dx.doi.org/10.1093/clinchem/hvaa187.
Testo completoAbstract (sommario):
Abstract Background With the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing COVID-19 pandemic. Unlike RT-qPCR, SARS-CoV-2 whole genome sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. However, the practical and cost considerations of cWGS should be addressed before it is widely implemented. Methods We performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. To track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled SARS-CoV-2 genomes to publicly available sequences. Results We found considerable improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of SARS-CoV-2. With enrichment, more than 99% of the sequencing reads mapped to the viral genome, compared to an average of 0.63% without enrichment. Consequently, an increase in genome coverage was obtained using substantially less sequencing data, enabling higher scalability and sizable cost reductions. We also demonstrated how SARS-CoV-2 genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains. Conclusions SARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic.
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Alessandrini, Federica, Sara Caucci, Valerio Onofri, Filomena Melchionda, Adriano Tagliabracci, Patrizia Bagnarelli, Laura Di Sante, Chiara Turchi e Stefano Menzo. "Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing". Genes 11, n. 8 (12 agosto 2020): 929. http://dx.doi.org/10.3390/genes11080929.
Testo completoAbstract (sommario):
Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer’s instructions.
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Bhoyar, Rahul C., Abhinav Jain, Paras Sehgal, Mohit Kumar Divakar, Disha Sharma, Mohamed Imran, Bani Jolly et al. "High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing". PLOS ONE 16, n. 2 (17 febbraio 2021): e0247115. http://dx.doi.org/10.1371/journal.pone.0247115.
Testo completoAbstract (sommario):
The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.
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Zouaki, Amal, Hakima Kabbaj, Ghizlane El Amin, Mouna Ouadghiri, Bouchra Belefquih, Azeddine Ibrahimi e Myriam Seffar. "Evaluation of the MAScIR SARS-CoV-2 M Kit 2.0 on the SARS-CoV-2 Infection". Advances in Virology 2023 (7 febbraio 2023): 1–13. http://dx.doi.org/10.1155/2023/9313666.
Testo completoAbstract (sommario):
SARS-CoV-2 is a major public health problem worldwide. Since its emergence, several diagnostic kits have been developed to ensure rapid patient management. The aim of our study is to check the performance of the new Moroccan SARS-CoV-2 detection kit: MAScIR SARS-CoV-2 M 2.0. The following parameters were studied: repeatability, reproducibility, analytical specificity, analytical sensitivity, and comparison with the GeneFinder™ COVID-19 Plus RealAmp Kit. In addition, an external quality evaluation comprising five specimens was carried out as part of an international program for the external quality evaluation of sublaboratories of the WHO and the Laboratory Office of the National Institute of Hygiene of Morocco. The results of all parameters studied showed an analytical performance that complied with the requirements of the method verification/validation protocol adopted by the Central Laboratory of Virology and met the recommendations of COFRAC (French Accreditation Committee). During the current study, the sequencing of some randomly selected positive samples was performed, among which the carriers of the Alpha variant, the Delta variant, and the Omicron variant were detected. These results allowed us to deduce that this kit was valid for detecting these three variants.
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Petrillo, Mauro, Maddalena Querci, Carlo Brogna, Jessica Ponti, Simone Cristoni, Peter V. Markov, Andrea Valsesia et al. "Evidence of SARS-CoV-2 bacteriophage potential in human gut microbiota". F1000Research 11 (9 marzo 2022): 292. http://dx.doi.org/10.12688/f1000research.109236.1.
Testo completoAbstract (sommario):
Background: In previous studies we have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth, and it is influenced by the administration of specific antibiotics. These observations are compatible with a ‘bacteriophage-like’ behaviour of SARS-CoV-2. Methods: We have further elaborated on these unusual findings and here we present the results of three different supplementary experiments: (1) an electron-microscope analysis of samples of bacteria obtained from a faecal sample of a subject positive to SARS-CoV-2; (2) mass spectrometric analysis of these cultures to assess the eventual de novo synthesis of SARS-CoV-2 spike protein; (3) sequencing of SARS-CoV-2 collected from plaques obtained from two different gut microbial bacteria inoculated with supernatant from faecal microbiota of an individual positive to SARS-CoV-2. Results: Immuno-labelling with Anti-SARS-CoV-2 nucleocapsid protein antibody confirmed presence of SARS-CoV-2 both outside and inside bacteria. De novo synthesis of SARS-CoV-2 spike protein was observed, as evidence that SARS-CoV-2 RNA is translated in the bacterial cultures. In addition, phage-like plaques were spotted on faecal bacteria cultures after inoculation with supernatant from faecal microbiota of an individual positive to SARS-CoV-2. Bioinformatic analyses on the reads obtained by sequencing RNA extracted from the plaques revealed nucleic acid polymorphisms, suggesting different replication environment in the two bacterial cultures. Conclusions: Based on these results we conclude that, in addition to its well-documented interactions with eukaryotic cells, SARS-CoV-2 may act as a bacteriophage when interacting with at least two bacterial species known to be present in the human microbiota. If the hypothesis proposed, i.e., that under certain conditions SARS-CoV-2 may multiply at the expense of human gut bacteria, is further substantiated, it would drastically change the model of acting and infecting of SARS-CoV-2, and most likely that of other human pathogenic viruses.
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Unselt, Desiree, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims e Jennifer Mason. "Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel". Cancer Research 82, n. 12_Supplement (15 giugno 2022): 437. http://dx.doi.org/10.1158/1538-7445.am2022-437.
Testo completoAbstract (sommario):
Abstract Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs. Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples. Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples. Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment. Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.
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Khateeb, Dina, Tslil Gabrieli, Bar Sofer, Adi Hattar, Sapir Cordela, Abigael Chaouat, Ilia Spivak et al. "SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection". PLOS Pathogens 18, n. 1 (12 gennaio 2022): e1010242. http://dx.doi.org/10.1371/journal.ppat.1010242.
Testo completoAbstract (sommario):
In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants.
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Bačenková, Darina, Marianna Trebuňová, Tatiana Špakovská, Marek Schnitzer, Lucia Bednarčíková e Jozef Živčák. "Comparison of Selected Characteristics of SARS-CoV-2, SARS-CoV, and HCoV-NL63". Applied Sciences 11, n. 4 (7 febbraio 2021): 1497. http://dx.doi.org/10.3390/app11041497.
Testo completoAbstract (sommario):
The global pandemic known as coronavirus disease 2019 (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This review article presents the taxonomy of SARS-CoV-2 coronaviruses, which have been classified as the seventh known human pathogenic coronavirus. The etiology of COVID-19 is also briefly discussed. Selected characteristics of SARS-CoV-2, SARS-CoV, and HCoV-NL63 are compared in the article. The angiotensin converting enzyme-2 (ACE-2) has been identified as the receptor for the SARS-CoV-2 viral entry. ACE2 is well-known as a counter-regulator of the renin-angiotensin system (RAAS) and plays a key role in the cardiovascular system. In the therapy of patients with COVID-19, there has been a concern about the use of RAAS inhibitors. As a result, it is hypothesized that ACE inhibitors do not directly affect ACE2 activity in clinical use. Coronaviruses are zoonotic RNA viruses. Identification of the primary causative agent of the SARS-CoV-2 is essential. Sequencing showed that the genome of the Bat CoVRaTG13 virus found in bats matches the genome of up to (96.2%) of SARS-CoV-2 virus. Sufficient knowledge of the molecular and biological mechanisms along with reliable information related to SARS-CoV-2 gives hope for a quick solution to epidemiological questions and therapeutic processes.
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Eichmeier, Ales, Tomas Kiss, Maria Kocanova, Eliska Hakalova, Milan Spetik, Jana Cechova e Boris Tichy. "Conserved MicroRNAs in Human Nasopharynx Tissue Samples from Swabs Are Differentially Expressed in Response to SARS-CoV-2". Genes 13, n. 2 (14 febbraio 2022): 348. http://dx.doi.org/10.3390/genes13020348.
Testo completoAbstract (sommario):
The use of high-throughput small RNA sequencing is well established as a technique to unveil the miRNAs in various tissues. The miRNA profiles are different between infected and non-infected tissues. We compare the SARS-CoV-2 positive and SARS-CoV-2 negative RNA samples extracted from human nasopharynx tissue samples to show different miRNA profiles. We explored differentially expressed miRNAs in response to SARS-CoV-2 in the RNA extracted from nasopharynx tissues of 10 SARS-CoV-2-positive and 10 SARS-CoV-2-negative patients. miRNAs were identified by small RNA sequencing, and the expression levels of selected miRNAs were validated by real-time RT-PCR. We identified 943 conserved miRNAs, likely generated through posttranscriptional modifications. The identified miRNAs were expressed in both RNA groups, NegS and PosS: miR-148a, miR-21, miR-34c, miR-34b, and miR-342. The most differentially expressed miRNA was miR-21, which is likely closely linked to the presence of SARS-CoV-2 in nasopharynx tissues. Our results contribute to further understanding the role of miRNAs in SARS-CoV-2 pathogenesis, which may be crucial for understanding disease symptom development in humans.
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Pećar, Dino, Ivana Čeko, Lana Salihefendić e Rijad Konjhodžić. "Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing". Genetics & Applications 5, n. 2 (27 dicembre 2021): 24. http://dx.doi.org/10.31383/ga.vol5iss2pp24-30.
Testo completoAbstract (sommario):
Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.
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Kockelbergh, Hannah, Shelley Evans, Tong Deng, Ella Clyne, Anna Kyriakidou, Andreas Economou, Kim Ngan Luu Hoang et al. "Utility of Bulk T-Cell Receptor Repertoire Sequencing Analysis in Understanding Immune Responses to COVID-19". Diagnostics 12, n. 5 (13 maggio 2022): 1222. http://dx.doi.org/10.3390/diagnostics12051222.
Testo completoAbstract (sommario):
Measuring immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), can rely on antibodies, reactive T cells and other factors, with T-cell-mediated responses appearing to have greater sensitivity and longevity. Because each T cell carries an essentially unique nucleic acid sequence for its T-cell receptor (TCR), we can interrogate sequence data derived from DNA or RNA to assess aspects of the immune response. This review deals with the utility of bulk, rather than single-cell, sequencing of TCR repertoires, considering the importance of study design, in terms of cohort selection, laboratory methods and analysis. The advances in understanding SARS-CoV-2 immunity that have resulted from bulk TCR repertoire sequencing are also be discussed. The complexity of sequencing data obtained by bulk repertoire sequencing makes analysis challenging, but simple descriptive analyses, clonal analysis, searches for specific sequences associated with immune responses to SARS-CoV-2, motif-based analyses, and machine learning approaches have all been applied. TCR repertoire sequencing has demonstrated early expansion followed by contraction of SARS-CoV-2-specific clonotypes, during active infection. Maintenance of TCR repertoire diversity, including the maintenance of diversity of anti-SARS-CoV-2 response, predicts a favourable outcome. TCR repertoire narrowing in severe COVID-19 is most likely a consequence of COVID-19-associated lymphopenia. It has been possible to follow clonotypic sequences longitudinally, which has been particularly valuable for clonotypes known to be associated with SARS-CoV-2 peptide/MHC tetramer binding or with SARS-CoV-2 peptide-induced cytokine responses. Closely related clonotypes to these previously identified sequences have been shown to respond with similar kinetics during infection. A possible superantigen-like effect of the SARS-CoV-2 spike protein has been identified, by means of observing V-segment skewing in patients with severe COVID-19, together with structural modelling. Such a superantigen-like activity, which is apparently absent from other coronaviruses, may be the basis of multisystem inflammatory syndrome and cytokine storms in COVID-19. Bulk TCR repertoire sequencing has proven to be a useful and cost-effective approach to understanding interactions between SARS-CoV-2 and the human host, with the potential to inform the design of therapeutics and vaccines, as well as to provide invaluable pathogenetic and epidemiological insights.
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Salles, Tiago Souza, Andrea Cony Cavalcanti, Fábio Burack da Costa, Vanessa Zaquieu Dias, Leandro Magalhães de Souza, Marcelo Damião Ferreira de Meneses, José Antônio Suzano da Silva et al. "Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing". PLOS ONE 17, n. 1 (20 gennaio 2022): e0262170. http://dx.doi.org/10.1371/journal.pone.0262170.
Testo completoAbstract (sommario):
The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.
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Parker, Matthew D., Benjamin B. Lindsey, Shay Leary, Silvana Gaudieri, Abha Chopra, Matthew Wyles, Adrienn Angyal et al. "Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data". Genome Research 31, n. 4 (15 marzo 2021): 645–58. http://dx.doi.org/10.1101/gr.268110.120.
Testo completo
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González-Recio, Oscar, Mónica Gutiérrez-Rivas, Ramón Peiró-Pastor, Pilar Aguilera-Sepúlveda, Cristina Cano-Gómez, Miguel Ángel Jiménez-Clavero e Jovita Fernández-Pinero. "Sequencing of SARS-CoV-2 genome using different nanopore chemistries". Applied Microbiology and Biotechnology 105, n. 8 (aprile 2021): 3225–34. http://dx.doi.org/10.1007/s00253-021-11250-w.
Testo completo
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Gladkikh, Anna, Ekaterina Klyuchnikova, Polina Pavlova, Valeriya Sbarzaglia, Nadezhda Tsyganova, Margarita Popova, Tatiana Arbuzova et al. "Comparative Analysis of Library Preparation Approaches for SARS-CoV-2 Genome Sequencing on the Illumina MiSeq Platform". International Journal of Molecular Sciences 24, n. 3 (25 gennaio 2023): 2374. http://dx.doi.org/10.3390/ijms24032374.
Testo completoAbstract (sommario):
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for over two years of the COVID-19 pandemic and a global health emergency. Genomic surveillance plays a key role in overcoming the ongoing COVID-19 pandemic despite its relative successive waves and the continuous emergence of new variants. Many technological approaches are currently applied for the whole genome sequencing (WGS) of SARS-CoV-2. They differ in key stages of the process, and they feature some differences in genomic coverage, sequencing depth, and in the accuracy of variant-calling options. In this study, three different protocols for SARS-CoV-2 WGS library construction are compared: an amplicon-based protocol with a commercial primer panel; an amplicon-based protocol with a custom panel; and a hybridization capture protocol. Specific differences in sequencing depth and genomic coverage as well as differences in SNP number were found. The custom panel showed suitable results and a predictable output applicable for the epidemiological surveillance of SARS-CoV-2 variants.
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Deminco, Felice, Sara N. Vaz, Daniele S. Santana, Celia Pedroso, Jean Tadeu, Andreas Stoecker, Sueli M. Vieira, Eduardo Netto e Carlos Brites. "A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants". Diagnostics 12, n. 11 (27 ottobre 2022): 2609. http://dx.doi.org/10.3390/diagnostics12112609.
Testo completoAbstract (sommario):
Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.
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Alasia, Datonye Dennis, Omosivie Maduka, Kennedy Wariso, Temitayo Awopeju e Faith Emuh. "SARS-CoV-2 recurrence and probable reinfection: outcome of a descriptive surveillance in a Nigerian tertiary hospital". International Journal of Research in Medical Sciences 9, n. 6 (27 maggio 2021): 1498. http://dx.doi.org/10.18203/2320-6012.ijrms20211912.
Testo completoAbstract (sommario):
Background: The reports of SARS-CoV-2 reinfection have increased. This stimulates the need for surveillance in diverse populations to establish the extent of reinfections and the challenges to diagnosis.Methods: A retrospective descriptive survey aimed at identifying probable SARS-CoV-2 reinfections using established criteria and proposed definitions was performed at a tertiary hospital in South-South, Nigeria.Results: The study found two cases for evaluation of reinfection. One case was identified as probable reinfection, pending the outcome of gene sequencing, while the second case was categorized as recurrence. The limited access to routine genetic sequencing for confirmation of reinfection was identified as a key challenge.Conclusions: Probable SARS-CoV-2 reinfections occur in Nigeria. Systematic surveillance of SARS-CoV-2 testing at the state and country-level is advocated to have a more accurate estimate of the burden of reinfections in the country. Access to genetic sequencing should be scaled up in Nigeria.
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Umair, Massab, Aamer Ikram, Muhammad Salman, Adnan Khurshid, Masroor Alam, Nazish Badar, Rana Suleman et al. "Whole-genome sequencing of SARS-CoV-2 reveals the detection of G614 variant in Pakistan". PLOS ONE 16, n. 3 (23 marzo 2021): e0248371. http://dx.doi.org/10.1371/journal.pone.0248371.
Testo completoAbstract (sommario):
Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.
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Peddu, Vikas, Ryan C. Shean, Hong Xie, Lasata Shrestha, Garrett A. Perchetti, Samuel S. Minot, Pavitra Roychoudhury et al. "Metagenomic Analysis Reveals Clinical SARS-CoV-2 Infection and Bacterial or Viral Superinfection and Colonization". Clinical Chemistry 66, n. 7 (12 giugno 2020): 966–72. http://dx.doi.org/10.1093/clinchem/hvaa106.
Testo completoAbstract (sommario):
Abstract Background More than 2 months separated the initial description of SARS-CoV-2 and discovery of its widespread dissemination in the United States. Despite this lengthy interval, implementation of specific quantitative reverse transcription (qRT)-PCR-based SARS-CoV-2 tests in the US has been slow, and testing is still not widely available. Metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence. Methods To evaluate metagenomic approaches in the context of the current SARS-CoV-2 epidemic, laboratory-confirmed positive and negative samples from Seattle, WA were evaluated by metagenomic sequencing, with comparison to a 2019 reference genomic database created before the emergence of SARS-CoV-2. Results Within 36 h our results showed clear identification of a novel human Betacoronavirus, closely related to known Betacoronaviruses of bats, in laboratory-proven cases of SARS-CoV-2. A subset of samples also showed superinfection or colonization with human parainfluenza virus 3 or Moraxella species, highlighting the need to test directly for SARS-CoV-2 as opposed to ruling out an infection using a viral respiratory panel. Samples negative for SARS-CoV-2 by RT-PCR were also negative by metagenomic analysis, and positive for Rhinovirus A and C. Unlike targeted SARS-CoV-2 qRT-PCR testing, metagenomic analysis of these SARS-CoV-2 negative samples identified candidate etiological agents for the patients’ respiratory symptoms. Conclusion Taken together, these results demonstrate the value of metagenomic analysis in the monitoring and response to this and future viral pandemics.
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Hernandez, Sarah, Phuong-Vi Nguyen, Taz Azmain, Anne Piantadosi e Jesse J. Waggoner. "SARS-CoV-2 genotyping and sequencing following a simple and economical RNA extraction and storage protocol". PLOS ONE 18, n. 1 (19 gennaio 2023): e0280577. http://dx.doi.org/10.1371/journal.pone.0280577.
Testo completoAbstract (sommario):
Since the beginning of the SARS-CoV-2 pandemic, supply chain shortages have caused major disruptions in sourcing the materials needed for laboratory-based molecular assays. With increasing demand for molecular testing, these disruptions have limited testing capacity and hindered efforts to mitigate spread of the virus and new variants. Here we evaluate an economical and reliable protocol for the extraction and short-term ambient temperature storage of SARS-CoV-2 RNA. Additional objectives of the study were to evaluate RNA from this protocol for 1) detection of single nucleotide polymorphisms (SNPs) in the spike gene and 2) whole genome sequencing of SARS-CoV-2. The RNAES protocol was evaluated with residual nasopharyngeal (NP) samples collected from Emory Healthcare and Emory Student Health services. All RNAES extractions were performed in duplicate and once with a commercial extraction robot for comparison. Following extraction, eluates were immediately tested by rRT-PCR. SARS-CoV-2 RNA was successfully detected in 56/60 (93.3%) RNAES replicates, and Ct values corresponded with comparator results. Upon testing in spike SNP assays, three genotypes were identified, and all variant calls were consistent with those previously obtained after commercial extraction. Additionally, the SARS-RNAES protocol yield eluate pure enough for downstream whole genome sequencing, and results were consistent with SARS-CoV-2 whole genome sequencing of eluates matched for Ct value. With reproducible results across a range of virus concentrations, the SARS-RNAES protocol could help increase SARS-CoV-2 diagnostic testing and monitoring for emerging variants in resource-constrained communities.
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Babiker, Ahmed, Heath L. Bradley, Victoria D. Stittleburg, Autum Key, Colleen Kraft, Jesse Waggoner e Anne Piantadosi. "64. Metagenomic Sequencing to Identify Alternative Infections and Co-infections in Persons Under Investigation for covid-19". Open Forum Infectious Diseases 7, Supplement_1 (1 ottobre 2020): S163—S164. http://dx.doi.org/10.1093/ofid/ofaa439.374.
Testo completoAbstract (sommario):
Abstract Background Broad testing for respiratory viruses among persons under investigation (PUI) for SARS-CoV-2 is performed inconsistently, limiting our understanding of alternative infections and co-infections in these patients. Here, we used unbiased metagenomic next-generation sequencing (mNGS) to assess the frequencies of 1) alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs and 2) viral co-infections in SARS-CoV-2 RT-PCR positive PUIs. Methods A convenience sample set was selected from PUIs who were tested for SARS-CoV-2 in the Emory Healthcare system during the first 2 months of the pandemic from 02/26-04/23/20. Laboratory results were extracted by chart review; Flu/RSV and multiplex respiratory pathogen PCRs had been performed at the discretion of treating physicians. Excess nasopharyngeal swab samples were retrieved within 72 hours of collection and underwent RNA extraction and SARS-CoV-2 testing by triplex RT-PCR. mNGS was performed by DNAse treatment, random primer cDNA synthesis, Nextera XT tagmentation, and high-depth Illumina sequencing. Reads underwent taxonomic classification by KrakenUniq, as implemented in viral-ngs. Results 53 PUIs were included, 30 negative and 23 positive for SARS-CoV-2 by RT-PCR. Among SARS-CoV-2 negative PUIs, 28 (93%) underwent clinical testing for alternative infections, and 8 (29%) tested positive for another respiratory virus. In all cases, mNGS identified the same virus (Table 1). In another 3 PUIs, mNGS identified two viruses that were not tested for and one that was missed by routine testing. No SARS-CoV-2 was detected by mNGS among RT-PCR negative PUIs. Among SARS-CoV-2 RT-PCR positive PUIs, 18 (69%) underwent clinical testing for co-infections, and none were detected. mNGS did not identify any viral co-infections but did detect SARS-CoV-2 in all 23 PUIs. Table 1: Molecular and Metagenomic Testing of Persons Under Investigation Conclusion Unbiased mNGS offers the powerful opportunity to streamline testing for PUIs by assessing for SARS-CoV-2 and alternative infections simultaneously; this technique can also be used to identify co-infections, but none were observed in our study population. Interestingly, many PUIs had no infection identified on routine testing or mNGS, which may reflect inadequate sampling, rapid virus clearance, or a non-viral process. Disclosures All Authors: No reported disclosures
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Rueca, Martina, Emanuela Giombini, Francesco Messina, Barbara Bartolini, Antonino Di Caro, Maria Rosaria Capobianchi e Cesare EM Gruber. "The Easy-to-Use SARS-CoV-2 Assembler for Genome Sequencing: Development Study". JMIR Bioinformatics and Biotechnology 3, n. 1 (14 marzo 2022): e31536. http://dx.doi.org/10.2196/31536.
Testo completoAbstract (sommario):
Background Early sequencing and quick analysis of the SARS-CoV-2 genome have contributed to the understanding of the dynamics of COVID-19 epidemics and in designing countermeasures at a global level. Objective Amplicon-based next-generation sequencing (NGS) methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession as well as harboring multiple deletions and amino acid–changing mutations. Methods To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here, we propose an easy-to-use SARS-CoV-2 genome assembler: the Easy-to-use SARS-CoV-2 Assembler (ESCA) pipeline. Results Our results have shown that ESCA could perform high-quality genome assembly from Ion Torrent and Illumina raw data and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility of comparing assembled genomes of multisample runs through an easy table format. Conclusions In conclusion, ESCA automatically furnished a variant table output file, fundamental to rapidly recognizing variants of interest. Our pipeline could be a useful method for obtaining a complete, rapid, and accurate analysis even with minimal knowledge in bioinformatics.
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Lim, Ho Jae, Min Young Park, Hye Soo Jung, Youngjin Kwon, Inhee Kim, Dong Kwan Kim, Nae Yu et al. "Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations". PLOS ONE 16, n. 12 (14 dicembre 2021): e0260850. http://dx.doi.org/10.1371/journal.pone.0260850.
Testo completoAbstract (sommario):
Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.
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Loconsole, Daniela, Anna Sallustio, Marisa Accogli, Francesca Centrone, Daniele Casulli, Antonino Madaro, Ersilia Tedeschi, Antonio Parisi e Maria Chironna. "Symptomatic SARS-CoV-2 Reinfection in a Healthy Healthcare Worker in Italy Confirmed by Whole-Genome Sequencing". Viruses 13, n. 5 (12 maggio 2021): 899. http://dx.doi.org/10.3390/v13050899.
Testo completoAbstract (sommario):
This study describes a case of SARS-CoV-2 reinfection confirmed by whole-genome sequencing in a healthy physician who had been working in a COVID-19 hospital in Italy since the beginning of the pandemic. Nasopharyngeal swabs were obtained from the patient at each presentation as part of routine surveillance. Nucleic acid amplification testing was performed on the two samples to confirm SARS-CoV-2 infection, and serological tests were used to detect SARS-CoV-2 IgG antibodies. Comparative genome analysis with whole-genome sequencing was performed on nasopharyngeal swabs collected during the two episodes of COVID-19. The first COVID-19 episode was in March 2020, and the second was in January 2021. Both SARS-CoV-2 infections presented with mild symptoms, and seroconversion for SARS-CoV-2 IgG was documented. Genomic analysis showed that the viral genome from the first infection belonged to the lineage B.1.1.74, while that from the second infection to the lineage B.1.177. Epidemiological, clinical, serological, and genomic analyses confirmed that the second episode of SARS-CoV-2 infection in the healthcare worker met the qualifications for “best evidence” for reinfection. Further studies are urgently needed to assess the frequency of such a worrisome occurrence, particularly in the light of the recent diffusion of SARS-CoV-2 variants of concern.
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So, Min-Kyung, Sholhui Park, Kyunghoon Lee, Soo-Kyung Kim, Hae-Sun Chung e Miae Lee. "Variant Prediction by Analyzing RdRp/S Gene Double or Low Amplification Pattern in Allplex SARS-CoV-2 Assay". Diagnostics 11, n. 10 (8 ottobre 2021): 1854. http://dx.doi.org/10.3390/diagnostics11101854.
Testo completoAbstract (sommario):
The spread of delta variants (B.1.671.2) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a severe global threat. Multiplex real-time PCR is a common method for confirming SARS-CoV-2 infection, however, additional tests, such as whole genomic sequencing, are required to reveal the presence or type of viral mutation. Moreover, applying whole genomic sequencing to all SARS-CoV-2 positive samples is challenging due to time and cost constraints. Here, we report that the double or low amplification curve observed during RNA-dependent RNA polymerase (RdRp) gene/S gene amplification in the Allplex SARS-CoV-2 Assay is related to delta/alpha variants. We analyzed the RdRp/S gene amplification curve using 94 samples confirmed as SARS-CoV-2 infection by the Allplex SARS-CoV-2 Assay from January to August, 2021. These positive samples identified variant types using the Novaplex SARS-CoV-2 Variants I and IV Assays. Overall, 17 samples showing a double curve and 11 samples showing a low amplification pattern were associated with alpha-/delta-type strains with variants in the P681 region. The double or low curve shown in the RdRp gene amplification curve had 100% sensitivity and 100% specificity for diagnosing delta/alpha variants. During the SARS-CoV-2 virus diagnostic RT-PCR test using the Allplex SARS-CoV-2 Assay, we could consider the presence of delta/alpha variants in the samples with double or low amplification curve of the RdRp/S gene channel. This PCR amplification curve abnormality enables rapid and cost-effective variant type prediction during SARS-CoV-2 diagnostic testing in clinical laboratories.
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Vacca, Davide, Antonino Fiannaca, Fabio Tramuto, Valeria Cancila, Laura La Paglia, Walter Mazzucco, Alessandro Gulino et al. "Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs". Life 12, n. 1 (4 gennaio 2022): 69. http://dx.doi.org/10.3390/life12010069.
Testo completoAbstract (sommario):
In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.
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Gregory, Devon A., Monica Trujillo, Clayton Rushford, Anna Flury, Sherin Kannoly, Kaung Myat San, Dustin T. Lyfoung et al. "Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing". PLOS Pathogens 18, n. 10 (14 ottobre 2022): e1010636. http://dx.doi.org/10.1371/journal.ppat.1010636.
Testo completoAbstract (sommario):
Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.
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Wiegand, Tanner, Artem Nemudryi, Anna Nemudraia, Aidan McVey, Agusta Little, David N. Taylor, Seth T. Walk e Blake Wiedenheft. "The Rise and Fall of SARS-CoV-2 Variants and Ongoing Diversification of Omicron". Viruses 14, n. 9 (10 settembre 2022): 2009. http://dx.doi.org/10.3390/v14092009.
Testo completoAbstract (sommario):
In late December of 2019, high-throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we provide a short retrospective analysis of SARS-CoV-2 variants by analyzing a subset (n = 97,437) of all publicly available SARS-CoV-2 genomes (n = ~11.9 million) that were randomly selected but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron (BA.1) and Omicron sub-lineages (BA.2–BA.5) are significantly elevated compared to previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike and nucleocapsid proteins, while 24 other viral proteins—including those involved in SARS-CoV-2 replication—are generally conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered.
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Al-Ghazawy, Ola. "Egypt’s SARS-CoV-2 sequencing challenges". Nature Middle East, 7 maggio 2021. http://dx.doi.org/10.1038/nmiddleeast.2021.43.
Testo completo
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Alpert, Tara, Chantal B. F. Vogels, Mallery I. Breban, Mary E. Petrone, Anne L. Wyllie, Nathan D. Grubaugh e Joseph R. Fauver. "Sequencing SARS-CoV-2 Genomes from Saliva". Virus Evolution, 3 gennaio 2022. http://dx.doi.org/10.1093/ve/veab098.
Testo completoAbstract (sommario):
Abstract Genomic sequencing is crucial to understanding the epidemiology and evolution of SARS-CoV-2. Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal swabs, as input into whole genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays, however saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from nasopharyngeal swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.
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Rachiglio, Anna Maria, Luca De Sabato, Cristin Roma, Michele Cennamo, Mariano Fiorenza, Daniela Terracciano, Raffaella Pasquale et al. "SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system". Journal of Translational Medicine 19, n. 1 (5 giugno 2021). http://dx.doi.org/10.1186/s12967-021-02912-4.
Testo completoAbstract (sommario):
Abstract Background Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus. Methods RNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March–April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software. Results The complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes. Conclusions The Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.
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Bull, Rowena A., Thiruni N. Adikari, James M. Ferguson, Jillian M. Hammond, Igor Stevanovski, Alicia G. Beukers, Zin Naing et al. "Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis". Nature Communications 11, n. 1 (dicembre 2020). http://dx.doi.org/10.1038/s41467-020-20075-6.
Testo completoAbstract (sommario):
AbstractViral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.
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Park, Changwoo, Kwan Woo Kim, Dongju Park, Zohaib ul Hassan, Edmond Changkyun Park, Chang-Seop Lee, MD Tazikur Rahman, Hana Yi e Seil Kim. "Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2". Frontiers in Microbiology 13 (17 agosto 2022). http://dx.doi.org/10.3389/fmicb.2022.876085.
Testo completoAbstract (sommario):
As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.
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Brito, Anderson F., Elizaveta Semenova, Gytis Dudas, Gabriel W. Hassler, Chaney C. Kalinich, Moritz U. G. Kraemer, Joses Ho et al. "Global disparities in SARS-CoV-2 genomic surveillance". Nature Communications 13, n. 1 (16 novembre 2022). http://dx.doi.org/10.1038/s41467-022-33713-y.
Testo completoAbstract (sommario):
AbstractGenomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity.