scholarly journals Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 123
Author(s):  
Nicolò Musso ◽  
Paolo Giuseppe Bonacci ◽  
Dafne Bongiorno ◽  
Stefano Stracquadanio ◽  
Dalida Angela Bivona ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Sanger Sequencing ◽  
N Gene ◽  
Whole Genome ◽  
S Gene ◽  
Rapid Spread ◽  
Alpha Beta ◽  
Alpha Variant ◽  
Commercial Kits ◽  
Gene Portion

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

scholarly journals Viral loads and profile of the patients infected with SARS-CoV-2 Delta, Alpha or R.1 variants in Tokyo

2021 ◽  
Author(s):  
Chihiro Tani-Sassa ◽  
Yumi Iwasaki ◽  
Naoya Ichimura ◽  
Katsutoshi Nagano ◽  
Yuna Takatsuki ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Whole Genome ◽  
Elderly Individuals ◽  
Viral Loads ◽  
Copy Numbers ◽  
Significant Difference ◽  
Rapid Spread ◽  
Alpha Variant

The rapid spread of the Delta variant of SARS-CoV-2 became a serious concern worldwide in summer 2021. We examined the copy number and variant types of all SARS-CoV-2-positive patients who visited our hospital from February to August 2021 using PCR tests. Whole genome sequencing was performed for some samples. The R.1 variant (B.1.1.316) was responsible for most infections in March, replacing the previous variant (B.1.1.214); the Alpha (B.1.1.7) variant caused most infections in April and May; and the Delta variant (B.1.617.2) was the most prevalent in July and August. There was no significant difference in copy numbers among the previous variant cases (n=29, median 3.0x104 copies/μL), R.1 variant cases (n=28, 2.1x105 copies/μL), Alpha variant cases (n=125, 4.1x105 copies/μL), and Delta variant cases (n=106, 2.4x105 copies/μL). Patients with Delta variant infection were significantly younger than those infected with R.1 and the previous variants, possibly because many elderly individuals in Tokyo were vaccinated between May and August. There was no significant difference in mortality among the four groups. Our results suggest that the increased infectivity of Delta variant may be caused by factors other than the higher viral loads. Clarifying these factors is important to control the spread of Delta variant infection.

scholarly journals Real-Time SARS-CoV-2 Genotyping by High-Throughput Multiplex PCR Reveals the Epidemiology of the Variants of Concern in Qatar

2021 ◽  
Author(s):  
Mohammad R. Hasan ◽  
Mahesh K. R. Kalikiri ◽  
Faheem Mirza ◽  
Sathyavathi Sundararaju ◽  
Anju Sharma ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Genome Sequencing ◽  
High Throughput ◽  
Sanger Sequencing ◽  
Transmission Dynamics ◽  
Whole Genome ◽  
Real Time Tracking ◽  
Alpha Beta ◽  
Pcr Genotyping

Complementing whole genome sequencing strategies with high-throughput multiplex RT-qPCR genotyping allows for more comprehensive and real-time tracking of SARS-CoV-2 variants of concern. During the second and third waves of COVID-19 in Qatar, PCR genotyping, combined with Sanger sequencing of un-typeable samples, was employed to describe the epidemiology of the Alpha, Beta and Delta variants. A total of 9792 nasopharyngeal PCR-positive samples collected between April-June 2021 were successfully genotyped, revealing the importation and transmission dynamics of these three variants in Qatar.

Phylogeny of 2019-nCoVs and SARS-like CoVs of Human, Bat and Pangolin Origin

2020 ◽  
Keyword(s):  
N Gene ◽  
Whole Genome ◽  
Human Origin ◽  
Gene Sequences ◽  
M Gene ◽  
S Gene ◽  
Shared Identity ◽  
Novel Coronavirus ◽  
S Genes

A novel coronavirus first broke out in Wuhan, China in December, 2019 has been declared a pandemic by WHO on March, 2020. This work aimed to search for probable ancestor of the virus, phylogeny of 2019-nCoVs and similar SL-CoVs based on the whole genome, M, N, ORF1ab, orf3a, and S gene sequences (n=84) obtained from GenBank using BLASTn software in the NCBI was done. Nucleotides of ORF3a and S-genes among 2019-nCoVs are identical, whereas its similar on the whole genome (99.9-100%), M-gene (99.7-100%), N-gene (99.9-100%) and ORF1ab-gene (99.7-100%). nCoVs are similar to bat CoV/RaTG13 on the whole genome (96.2%), M-gene (95.0%), N-gene (97%), ORF1ab-gene (95.3%), ORF3a-gene (99.1%) and S-gene (90.7%). Likewise, nCoVs exhibited homology to bat-CoVZXC21 on M-gene (93.2%), N-gene (91.5%), ORF1ab-gene (93.1%) and ORF3a-gene (94.4%). The emergent viruses shared identity to bat-CoVZC45 on N-gene (91.3%), ORF1ab-gene (92.8%) and ORF3a-gene (94.0%). In addition, pangolin-CoV/MP789 exhibited common sequences on M-gene (91.0%), N-gene (96.3%) and ORF3a-gene (93.3%) to nCoV. Furthermore, pangolin-CoV/MP789 is analogous to bat CoV/RaTG13 (91.3%) and bat-SL-CoVZXC21 (92.2%) on M-gene and to bat CoV/RaTG13 (94.8%) on N-gene. Nevertheless, nCoVs are distinct from the previously identified SL-CoVs of human origin. The present analysis indicates that nCoVs may have transmitted from bats, pangolin and/or unidentified hosts.

scholarly journals First detection and report of SARS-CoV-2 Spike protein N501Y mutations in Oklahoma USA

2021 ◽  
Author(s):  
Sai Narayanan ◽  
Girish Patil ◽  
Sunil More ◽  
Jeremiah Saliki ◽  
Anil Kaul ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Spike Protein ◽  
Whole Genome ◽  
S Gene ◽  
Pathogen Dynamics ◽  
Geographical Locations

AbstractWe describe the detection of SARS-CoV-2 (VOC)B.1.1.7 lineage in Oklahoma, USA. Various mutations in the S gene and ORF8 with similarity to the genome of B.1.1.7 lineage were detected in 4 of the 6 genomes sequenced and reported here. The sequences have been made available in GISAID. Presence of novel lineages indicate the need for frequent whole genome sequencing to better understand pathogen dynamics in different geographical locations.

scholarly journals SARS-CoV-2 Omicron Outbreak in a Dormitory in Saint-Petersburg, Russia

2022 ◽  
Author(s):  
Georgii A. Bazykin ◽  
Daria M. Danilenko ◽  
Andrey B. Komissarov ◽  
Nikita Yolshin ◽  
Olga V. Shneider ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Immune Evasion ◽  
Whole Genome ◽  
Saint Petersburg ◽  
Single Origin ◽  
Rate Of Spread ◽  
Rapid Spread ◽  
Community Transmission

Abstract The B.1.1.529 Omicron variant of SARS-CoV-2 is rapidly spreading, displacing the globally prevalent Delta variant. Before December 16, 2021, community transmission had already been observed in tens of countries globally. However, in Russia, all reported cases had been sporadic and associated with travel. Here, we report an Omicron outbreak at a students’ dormitory in Saint Petersburg, Russia. Out of the 462 sampled residents of the dormitory, 206 (44.6%) tested PCR positive, and 159 (77.1%) of these infections carried the S:ins214EPE insertion, indicating that they were of the Omicron strain. 104 (65%) of Omicron-positive patients have been vaccinated and/or reported previous covid-19. Whole genome sequencing confirmed that the outbreak is caused by the Omicron variant. Phylogenetic analysis showed that the outbreak has a single origin, and belongs to the S:346K sublineage of Omicron which may be characterized by an increased rate of spread, compared to other Omicron sublineages. The rapid spread of Omicron in a population with preexisting immunity to previous variants underlines its propensity for immune evasion.

scholarly journals From partial to whole genome imputation of SARS-CoV-2 for epidemiological surveillance

2021 ◽  
Author(s):  
Francisco M Ortuno ◽  
Carlos Loucera ◽  
Carlos S Casimiro-Soriguer ◽  
Jose A Lepe ◽  
Pedro Camacho Martinez ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Rate ◽  
Epidemiological Surveillance ◽  
Primary Data ◽  
Whole Genome ◽  
Sequencing Data ◽  
Wide Range ◽  
Commercial Kits ◽  
Almost All

The current SARS-CoV-2 pandemic has emphasized the utility of viral whole genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is increasingly producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and therefore useless, sequences. However, viral sequences evolve in the context of a complex phylogeny and therefore different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data. We developed impuSARS, an application that includes Minimac, the most widely used strategy for genomic data imputation and, taking advantage of the enormous amount of SARS-CoV-2 whole genome sequences available, a reference panel containing 239,301 sequences was built. The impuSARS application was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing) showing great fidelity when reconstructing the original sequences. The impuSARS application is also able to impute whole genomes from commercial kits covering less than 20% of the genome or only from the Spike protein with a precision of 0.96. It also recovers the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (< 20%). Imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. impuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole genome sequencing.

scholarly journals Evaluation of Molecular Methods for Identification of Salmonella Serovars

2016 ◽  
Vol 54 (8) ◽  
pp. 1992-1998 ◽  
Author(s):  
Catherine Yoshida ◽  
Simone Gurnik ◽  
Aaminah Ahmad ◽  
Travis Blimkie ◽  
Stephanie A. Murphy ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Molecular Methods ◽  
Outbreak Detection ◽  
Broad Host Range ◽  
Whole Genome ◽  
Content Type ◽  
Molecular Serotyping ◽  
Commercial Kits

Classification by serotyping is the essential first step in the characterization ofSalmonellaisolates and is important for surveillance, source tracking, and outbreak detection. To improve detection and reduce the burden of salmonellosis, several rapid and high-throughput molecularSalmonellaserotyping methods have been developed.The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAPSalmonellaserotyping assay), to theSalmonellagenoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically importantSalmonellaserovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host rangeSalmonellaserovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity ofSalmonellaserovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively.It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping ofSalmonella.

scholarly journals Arthrobacter woluwensis Bacteremia: A Clinical and Genomic Report

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 443
Author(s):  
Shu-Yuan Li ◽  
Chin-Chuan Kao ◽  
Yu-Cheng Hu ◽  
Chung-Hsu Lai ◽  
Yi-Ping Jiang ◽  
...  
Keyword(s):  
Case Report ◽  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Immunocompromised Host ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Commercial Kits

Arthrobacter woluwensis is a Gram-positive, aerobic Actinobacteria that is widely distributed in the environment worldwide. Little is known about A. woluwensis infection and it is commonly mis-identified by culturing with commercial kits. To date, only six cases of bacteremia caused by A. woluwensis have been reported in the literature. Herein, we report a case of Arthrobacter woluwensis bacteremia in an immunocompromised host. In this case report, the results of antimicrobial susceptibility testing showed that this clinical isolate of A. woluwensis is sensitive to vancomycin, teicoplanin, but resistant to penicillin, cephalosporin and ciprofloxacin. Additionally, whole genome sequencing analysis identified common subunits of the urease system.

scholarly journals Genomic diversity of SARS-CoV-2 in Pakistan during fourth wave of pandemic

2021 ◽  
Author(s):  
Massab Umair ◽  
Aamer Ikram ◽  
Zaira Rehman ◽  
Adnan Haider ◽  
Nazish Badar ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Mutation Profile ◽  
Alpha Variant ◽  
Fourth Wave

AbstractThe emergence of different variants of concern of SARS-CoV-2 has resulted in upsurges of COVID positive cases around the globe. Pakistan is also experiencing fourth wave of COVID-19 with increasing number of positive cases. In order to understand the genomic diversity of circulating SARS-CoV-2 strains during fourth wave of pandemic in Pakistan, the current study was designed. The samples from 89 COVID-19 positive patients were subjected to whole genome sequencing using GeneStudio S5. The results showed that 99% (n=88) of isolates belonged to delta variant and only one isolate belonged to alpha variant. Among delta variant cases 26.1% (n=23) isolates were showing B.1.617.2 while 74% of isolates showing AY.4 lineage. Islamabad was found to be the most affected city with 54% (n=48) of cases, followed by Karachi (28%, n=25), and Rawalpindi (10%, n=9). AY.4 has slight difference in mutation profile compared to B.1.617.2. E156del, G142D and V26I mutations in spike and T181I in NSP6 were present in B.1.617.2 but not in AY.4. Interestingly, A446V mutation in NSP4 has been only observed in AY.4. The current study highlights the circulation of primarily delta variant (B.1.617.2 and AY.4) during fourth wave of pandemic in Pakistan.

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