scholarly journals Low Represented Mutation Clustering in SARS-CoV-2 B.1.1.7 Sublineage Group with Synonymous Mutations in the E Gene

Diagnostics ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 2286
Author(s):  
Paolo Giuseppe Bonacci ◽  
Dalida Angela Bivona ◽  
Dafne Bongiorno ◽  
Stefano Stracquadanio ◽  
Mariacristina Massimino ◽  
...  
Keyword(s):  
Specific Area ◽  
Epidemiological Surveillance ◽  
N Gene ◽  
Synonymous Mutation ◽  
Sequencing Data ◽  
Gold Standard Method ◽  
E Gene ◽  
Synonymous Mutations ◽  
Frequent Mutations ◽  
Global Threat

Starting in 2019, the COVID-19 pandemic is a global threat that is difficult to monitor. SARS-CoV-2 is known to undergo frequent mutations, including SNPs and deletions, which seem to be transmitted together, forming clusters that define specific lineages. Reverse-Transcription quantitative PCR (RT-qPCR) has been used for SARS-CoV-2 diagnosis and is still considered the gold standard method. Our Eukaryotic Host Pathogens Interaction (EHPI) laboratory received six SARS-CoV-2-positive samples from a Sicilian private analysis laboratory, four of which showed a dropout of the E gene. Our sequencing data revealed the presence of a synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene of all four samples showing the dropout in RT-qPCR. Interestingly, these samples also harbored three other mutations (S137L—Orf1ab; N439K—S gene; A156S—N gene), which had a very low diffusion rate worldwide. This combination suggested that these mutations may be linked to each other and more common in a specific area than in the rest of the world. Thus, we decided to analyze the 103 sequences in our internal database in order to confirm or disprove our “mutation cluster hypothesis”. Within our database, one sample showed the synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene. This work underlines the importance of territorial epidemiological surveillance by means of NGS and the sequencing of samples with clinical and or technical particularities, e.g., post-vaccine infections or RT-qPCR amplification failures, to allow for the early identification of these SNPs. This approach may be an effective method to detect new mutational clusters and thus to predict new emerging SARS-CoV-2 lineages before they spread globally.

scholarly journals SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification

Micromachines ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 197
Author(s):  
Meiqing Liu ◽  
Haoran Li ◽  
Yanwei Jia ◽  
Pui-In Mak ◽  
Rui P. Martins
Keyword(s):  
Signal Amplification ◽  
Incubation Temperature ◽  
Dna Probes ◽  
Detection Sensitivity ◽  
N Gene ◽  
Rna Detection ◽  
Gold Standard Method ◽  
Complementary Method ◽  
Amplification Method ◽  
Virus Rna

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

scholarly journals Outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked to raw drinking milk resolved by rapid application of advanced pathogen characterisation methods, England, August to October 2017

2019 ◽  
Vol 24 (16) ◽  
Author(s):  
Juli Treacy ◽  
Claire Jenkins ◽  
Karthik Paranthaman ◽  
Frieda Jorgensen ◽  
Doris Mueller-Doblies ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Control Measures ◽  
Public Access ◽  
Epidemiological Surveillance ◽  
Effective Control ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
Drinking Milk

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred on the Isle of Wight between August and October 2017. Of the seven cases linked to the outbreak, five were identified through the statutory notification process and two were identified through national surveillance of whole genome sequencing data. Enhanced surveillance questionnaires established a common link to a farm, and link to the likely food vehicle, raw drinking milk (RDM). Microbiological investigations, including PCR, identified the presence of STEC O157:H7 in samples of RDM. Analysis of core genome single nucleotide polymorphism (SNP) data of STEC O157:H7 from human stool specimens, animal faecal samples and RDM demonstrated a one SNP difference between isolates, and therefore close genetic relatedness. Control measures that were put in place included suspension of sales and recall of RDM, as well as restrictions on public access to parts of the farm. Successful integration of traditional epidemiological surveillance and advanced laboratory methods for the detection and characterisation of STEC O157:H7 from human, animal and environmental samples enabled prompt identification of the outbreak vehicle and provided evidence to support the outbreak control team’s decision-making, leading to implementation of effective control measures in a timely manner.

scholarly journals Syntool: A Novel Region-Based Intolerance Score to Single Nucleotide Substitution for Synonymous Mutations Predictions Based on 123,136 Individuals

2017 ◽  
Vol 2017 ◽  
pp. 1-5
Author(s):  
Tongda Zhang ◽  
Yiran Wu ◽  
Zhangzhang Lan ◽  
Quan Shi ◽  
Ying Yang ◽  
...  
Keyword(s):  
Human Disease ◽  
Complex Traits ◽  
Nucleotide Substitution ◽  
Disease Risk ◽  
Substantial Contribution ◽  
Synonymous Mutation ◽  
Single Nucleotide Substitution ◽  
Single Nucleotide ◽  
Synonymous Mutations ◽  
Single Nucleotide Change

Background. Synonymous mutation is the single nucleotide change that does not cause an amino acid change but can affect the rate and efficiency of translation. So recent increase in our knowledge has revealed a substantial contribution of synonymous mutations to human disease risk and other complex traits. Nevertheless, there are still rarely synonymous mutation prediction methods. Methods. Nonsynonymous and synonymous coding SNPs show similar likelihood and effect size of human disease association. Here we defined synonymous and missense variation as single nucleotide substitution variation. And then we evaluated the intolerance of genic transcripts to single nucleotide substitution variation based on gnomAD 123136 individuals. After regressing all variations on common variations, we defined residuals of regression model as every genomics region intolerance scores. Results. We constructed a total of 24799 nonoverlapped region-based intolerance score by their intolerance to single nucleotide substitution variation (Syntool). The results show that Syntool score can discriminate synonymous disease causing mutations in Human Gene Mutation Database (HGMD Professional) and ClinVar database much better than others. Taken together, this study provides a novel prediction system for synonymous mutations, called Syntool, which could be helpful in identifying candidate synonymous disease causing mutations.

scholarly journals Detecting somatic mutations in genomic sequences by means of Kolmogorov–Arnold analysis

2015 ◽  
Vol 2 (8) ◽  
pp. 150143 ◽  
Author(s):  
V. G. Gurzadyan ◽  
H. Yan ◽  
G. Vlahovic ◽  
A. Kashin ◽  
P. Killela ◽  
...  
Keyword(s):  
Clinical Diagnostics ◽  
Genomic Research ◽  
Genomic Sequences ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Cancer Genome Sequencing ◽  
Frequent Mutations ◽  
Using Data ◽  
First Time ◽  
Generation Sequencing

The Kolmogorov–Arnold stochasticity parameter technique is applied for the first time to the study of cancer genome sequencing, to reveal mutations. Using data generated by next-generation sequencing technologies, we have analysed the exome sequences of brain tumour patients with matched tumour and normal blood. We show that mutations contained in sequencing data can be revealed using this technique, thus providing a new methodology for determining subsequences of given length containing mutations, i.e. its value differs from those of subsequences without mutations. A potential application for this technique involves simplifying the procedure of finding segments with mutations, speeding up genomic research and accelerating its implementation in clinical diagnostics. Moreover, the prediction of a mutation associated with a family of frequent mutations in numerous types of cancers based purely on the value of the Kolmogorov function indicates that this applied marker may recognize genomic sequences that are in extremely low abundance and can be used in revealing new types of mutations.

scholarly journals A novel point mutation in the N gene of SARS-CoV-2 may affect the detection of the virus by RT-qPCR

2021 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Sathyavathi Sundararaju ◽  
Chidambaram Manickam ◽  
Faheem Mirza ◽  
Hamad Al-Hail ◽  
...  
Keyword(s):  
Viral Genome ◽  
Large Scale ◽  
Target Genes ◽  
Cytidine Deaminase ◽  
World Health ◽  
N Gene ◽  
Single Nucleotide ◽  
E Gene ◽  
Reverse Transcription Pcr ◽  
Health Organization

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, laboratory testing to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription PCR (RT-qPCR) has played a central role in mitigating the spread of the virus (1). Soon after the viral genome sequences were available, several RT-qPCR assays were developed and made available by World Health Organization (WHO) for public use (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). The primer and probe sequences for these assays were chosen from multiple target genes within the viral genome such as the E gene, RdRp gene, ORF1ab and N gene. Many commercial and laboratory-developed assays were developed for SARS-CoV-2 detection based on these primer and probe sequences. The large-scale sustained person-to-person transmission of SARS-CoV-2 has led to many mutational events, some of which may affect the sensitivity and specificity of available PCR assays (2). Recently, mutations in the E gene (C26340T) and N gene (C29200T) were reported affecting the detection of target genes by two commercial assays in 8 and 1 patients, respectively. Interestingly, both mutations are of C>T type, a common single nucleotide polymorphism (SNP) that may be associated with strong host cell mRNA editing mechanisms known as APOBEC cytidine deaminase (3, 4). Another study found a G to U substitution in position 29140 that affected the sensitivity of detection of N gene-based assays (5). Here we report a novel N gene mutation (C29200A) seen in 3 patients, which affected the detection of SARS-CoV-2 N gene by a commercial assay.

scholarly journals Measuring the distribution of fitness effects in somatic evolution by combining clonal dynamics with dN/dS ratios

2019 ◽  
Author(s):  
Marc J Williams ◽  
Luiz Zapata ◽  
Benjamin Werner ◽  
Chris Barnes ◽  
Andrea Sottoriva ◽  
...  
Keyword(s):  
Evolutionary Dynamics ◽  
Quantitative Model ◽  
Driver Mutations ◽  
Cancer Evolution ◽  
Sequencing Data ◽  
Synonymous Mutations ◽  
Fitness Effects ◽  
Somatic Evolution ◽  
Normal Oesophagus

AbstractThe distribution of fitness effects (DFE) defines how new mutations spread through an evolving population. The ratio of non-synonymous to synonymous mutations (dN/dS) has become a popular method to detect selection in somatic cells, however the link, in somatic evolution, between dN/dS values and fitness coefficients is missing. Here we present a quantitative model of somatic evolutionary dynamics that yields the selective coefficients from individual driver mutations from dN/dS estimates, and then measure the DFE for somatic mutant clones in ostensibly normal oesophagus and skin. We reveal a broad distribution of fitness effects, with the largest fitness increases found for TP53 and NOTCH1 mutants (proliferative bias 1-5%). Accurate measurement of the per-gene DFE in cancer evolution is precluded by the quality of currently available sequencing data. This study provides the theoretical link between dN/dS values and selective coefficients in somatic evolution, and reveals the DFE for mutations in human tissues.

scholarly journals Witnessing Evolution of SARS-CoV-2 through Comparative Phylogenomics: The Proximate Origin is Guangdong, not Wuhan

2020 ◽  
Author(s):  
Özgül Doğan ◽  
Ertan Mahir Korkmaz ◽  
Mahir Budak ◽  
Battal Çıplak ◽  
Hasan Hüseyin Başıbüyük
Keyword(s):  
Exponential Growth ◽  
Viral Genome ◽  
Doubling Time ◽  
Rna Viruses ◽  
Amino Acid Sequences ◽  
Viral Population ◽  
N Gene ◽  
Pandemic Strain ◽  
Synonymous Mutations ◽  
S Gene

A new form of coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) is currently causing a pandemic. A six-month evolutionary history of SARS-CoV-2 is witnessed by characterising the total genome of 821 samples using comparative phylogenomic approaches. Our analyses produced striking inclusive results that may guide scientists/professionals for the past/future of pandemic. Phylogenetic and time estimation analyses suggest the proximate origin of pandemic strain as Guangdong and the origin time as first half of September 2019, not Wuhan and December 2019, respectively. The viral genome experienced a substitution rate similar to other RNA viruses, but it is particularly high in some of the peptides encoding sequences such as leader protein, E gene, orf8, orf10, nsp10, N gene, S gene and M gene and nsp4, while low in nsp11, orf7a, 3C-like proteinase, nsp9, nsp8 and endoRNase. Most strikingly, the divergence rate of amino acid sequences is high proportional to nucleotide divergence. Additionally, specific non-synonymous mutations in nsp3 and nsp6 evolved under positive selection. The exponential growth rate (r), doubling time (Td) and R0 were estimated to be 47.43 per year, 5.39 days and 2.72, respectively. Comparison of synapomorphies distinguishing the SARS-CoV-2 and the candidate ancestor bat coronavirus indicates that mutation pattern in nsp3 and S gene enabled the new strain to invade human and become a pandemic strain. We arrive at the following main conclusions: (i) six months evolution of viral genome is nearly neutral, (ii) origin of pandemic is not Wuhan and predates formal reports, (iii) although viral population is ongoing an exponential growth, the doubling time is evolving towards shortening, and (iv) divergence rate of total genome is similar to other RNA viruses, but it is prominently high in some genes while low in some others and evolution in these genes should be closely monitored as their protein products intervening to pathogenicity, virulence and immune response.

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 Global and Local Mutations in Bangladeshi SARS-CoV-2 Genomes

2020 ◽  
Author(s):  
Md. Mahbub Hasan ◽  
Rasel Das ◽  
Md. Rasheduzzaman ◽  
Md Hamed Hussain ◽  
Nazmul Hasan Muzahid ◽  
...  
Keyword(s):  
Virus Disease ◽  
Phylogenetic Analyses ◽  
Missense Mutations ◽  
Synonymous Mutations ◽  
Global Trend ◽  
Frequent Mutations ◽  
Bangladeshi Population ◽  
Virus Genomes ◽  
Global And Local ◽  
Unique Amino Acid

AbstractCorona Virus Disease-2019 (COVID-19) warrants comprehensive investigations of publicly available Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) genomes to gain new insight about their epidemiology, mutations and pathogenesis. Nearly 0.4 million mutations were identified so far in ∼60,000 SARS-CoV-2 genomic sequences. In this study, we compared 207 of SARS-CoV-2 genomes reported from different parts of Bangladesh and their comparison with 467 globally reported sequences to understand the origin of viruses, possible patterns of mutations, availability of unique mutations, and their apparent impact on pathogenicity of the virus in victims of Bangladeshi population. Phylogenetic analyses indicates that in Bangladesh, SARS-CoV-2 viruses might arrived through infected travelers from European countries, and the GR clade was found as predominant in this region. We found 2602 mutations including 1602 missense mutations, 612 synonymous mutations, 36 insertions and deletions with 352 other mutations types. In line with the global trend, D614G mutation in spike glycoprotein was predominantly high (95.6%) in Bangladeshi isolates. Interestingly, we found the average number of mutations in ORF1ab, S, ORF3a, M and N of genomes, having nucleotide shift at G614 (n=459), were significantly higher (p≤0.001) than those having mutation at D614 (n=215). Previously reported frequent mutations such as P4715L, D614G, R203K, G204R and I300F were also prevalent in Bangladeshi isolates. Additionally, 87 unique amino acid changes were revealed and were categorized as originating from different cities of Bangladesh. The analyses would increase our understanding of variations in virus genomes circulating in Bangladesh and elsewhere and help develop novel therapeutic targets against SARS-CoV-2.

scholarly journals Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Jonathan Hon-Kwan Chen ◽  
Cyril Chik-Yan Yip ◽  
Jasper Fuk-Woo Chan ◽  
Rosana Wing-Shan Poon ◽  
Kelvin Kai-Wang To ◽  
...  
Keyword(s):  
Hong Kong ◽  
High Throughput ◽  
Clinical Performance ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Gene Target ◽  
E Gene

ABSTRACT In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

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