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1.
BMC Genomics ; 23(1): 319, 2022 Apr 22.
Article in English | MEDLINE | ID: covidwho-1799119

ABSTRACT

BACKGROUND: Over 4 million SARS-CoV-2 genomes have been sequenced globally in the past 2 years. This has been crucial in elucidating transmission chains within communities, the development of new diagnostic methods, vaccines, and antivirals. Although several sequencing technologies have been employed, Illumina and Oxford Nanopore remain the two most commonly used platforms. The sequence quality between these two platforms warrants a comparison of the genomes produced by the two technologies. Here, we compared the SARS-CoV-2 consensus genomes obtained from the Oxford Nanopore Technology GridION and the Illumina MiSeq for 28 sequencing runs. RESULTS: Our results show that the MiSeq had a significantly higher number of consensus genomes classified by Nextclade as good and mediocre compared to the GridION. The MiSeq also had a significantly higher genome coverage and mutation counts than the GridION. CONCLUSION: Due to the low genome coverage, high number of indels, and sensitivity to SARS-CoV-2 viral load noted with the GridION when compared to MiSeq, we can conclude that the MiSeq is more favourable for SARS-CoV-2 genomic surveillance, as successful genomic surveillance is dependent on high quality, near-whole consensus genomes.


Subject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Humans , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
2.
PLoS One ; 17(4): e0265220, 2022.
Article in English | MEDLINE | ID: covidwho-1775443

ABSTRACT

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies' (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
3.
PLoS One ; 17(2): e0263794, 2022.
Article in English | MEDLINE | ID: covidwho-1674022

ABSTRACT

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.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Culture Media/analysis , High-Throughput Nucleotide Sequencing/methods , SARS-CoV-2/genetics , Specimen Handling/methods , Whole Genome Sequencing/methods , COVID-19/genetics , COVID-19/virology , Culture Media/metabolism , Humans , SARS-CoV-2/isolation & purification
4.
PLoS One ; 17(1): e0261014, 2022.
Article in English | MEDLINE | ID: covidwho-1622333

ABSTRACT

High viral transmission in the COVID-19 pandemic has enabled SARS-CoV-2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.


Subject(s)
Genome, Viral/genetics , Genomics/methods , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , COVID-19/virology , Humans , RNA, Viral/genetics , Sequence Analysis/methods
5.
Genes (Basel) ; 12(11)2021 11 22.
Article in English | MEDLINE | ID: covidwho-1533885

ABSTRACT

Host genomic information, specifically genomic variations, may characterize susceptibility to disease and identify people with a higher risk of harm, leading to better targeting of care and vaccination. Italy was the epicentre for the spread of COVID-19 in Europe, the first country to go into a national lockdown and has one of the highest COVID-19 associated mortality rates. Qatar, on the other hand has a very low mortality rate. In this study, we compared whole-genome sequencing data of 14398 adults and Qatari-national to 925 Italian individuals. We also included in the comparison whole-exome sequence data from 189 Italian laboratory-confirmed COVID-19 cases. We focused our study on a curated list of 3619 candidate genes involved in innate immunity and host-pathogen interaction. Two population-gene metric scores, the Delta Singleton-Cohort variant score (DSC) and Sum Singleton-Cohort variant score (SSC), were applied to estimate the presence of selective constraints in the Qatari population and in the Italian cohorts. Results based on DSC and SSC metrics demonstrated a different selective pressure on three genes (MUC5AC, ABCA7, FLNA) between Qatari and Italian populations. This study highlighted the genetic differences between Qatari and Italian populations and identified a subset of genes involved in innate immunity and host-pathogen interaction.


Subject(s)
COVID-19/genetics , Genetic Predisposition to Disease/genetics , Host Microbial Interactions/genetics , Adult , Alleles , COVID-19/epidemiology , Communicable Disease Control , Disease Susceptibility/metabolism , Exome/genetics , Female , Gene Frequency/genetics , Genetic Predisposition to Disease/epidemiology , Genetics, Population , Genomics/methods , Humans , Immunity, Innate/immunology , Italy/epidemiology , Male , Qatar/epidemiology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Whole Exome Sequencing/methods , Whole Genome Sequencing/methods
6.
Genes (Basel) ; 12(11)2021 11 16.
Article in English | MEDLINE | ID: covidwho-1523931

ABSTRACT

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the "bridging variant" between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.


Subject(s)
COVID-19/epidemiology , COVID-19/transmission , SARS-CoV-2/genetics , Base Sequence/genetics , Evolution, Molecular , Humans , India/epidemiology , Mutation/genetics , Phylogeny , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Whole Genome Sequencing/methods
7.
J Infect Dev Ctries ; 15(10): 1376-1383, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1518653

ABSTRACT

INTRODUCTION: The first case of severe acute respiratory syndrome 2 (SARS-CoV-2) was imported to Pakistan in February 2020, since then 8,260 deaths have been witnessed. The virus has been constantly mutating and local transmission cases from different countries vary due to host dependent viral adaptation. Many distinct clusters of variant SARS-CoV-2 have been defined globally. In this study, the epidemiology of SARS-CoV-2 was studied and locally transmitted SARS-CoV-2 isolates from Karachi were sequenced to compared and identify any possible variants. METHODOLOGY: The real time PCR was performed on nasopharyngeal specimen to confirm SARS-CoV-2 with Orf 1ab and E gene as targets. The virus isolates were sequenced through oxford nanopore technology MinION platform. Isolates from the first and second wave of COVID-19 outbreak in Karachi were compared. RESULTS: The overall positivity rate for PCR was 26.24% with the highest number of positive cases in June. Approximately, 37.45% PCR positive subjects aged between 19-40 years. All the isolates belonged to GH clade and shared missense mutation D614G in spike protein linked to increased transmission rate worldwide. Another spike protein mutation A222V coexisted with D614G in the virus from the second wave of COVID-19. CONCLUSIONS: Based on the present findings it is suggested that the locally transmitted virus from Karachi varies from those reported from other parts of Pakistan. Slight variability was also observed between viruses from the first and second wave. Variability in any potential vaccine target may result in failed trials, therefore information on any local viral variants is always useful for effective vaccine design and/or selection.


Subject(s)
COVID-19/transmission , Genome, Viral , Nanopores , Nasopharynx/virology , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , Adult , COVID-19/epidemiology , COVID-19/virology , Female , Humans , Male , Middle Aged , Mutation , Pakistan , Phylogeny , Polymerase Chain Reaction , Whole Genome Sequencing/instrumentation , Young Adult
8.
Sci Rep ; 11(1): 21352, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493207

ABSTRACT

The outcome of SARS-CoV-2 infection is determined by multiple factors, including the viral, host genetics, age, and comorbidities. This study investigated the association between prognostic factors and disease outcomes of patients infected by SARS-CoV-2 with multiple S protein mutations. Fifty-one COVID-19 patients were recruited in this study. Whole-genome sequencing of 170 full-genomes of SARS-CoV-2 was conducted with the Illumina MiSeq sequencer. Most patients (47%) had mild symptoms of COVID-19 followed by moderate (19.6%), no symptoms (13.7%), severe (4%), and critical (2%). Mortality was found in 13.7% of the COVID-19 patients. There was a significant difference between the age of hospitalized patients (53.4 ± 18 years) and the age of non-hospitalized patients (34.6 ± 19) (p = 0.001). The patients' hospitalization was strongly associated with hypertension, diabetes, and anticoagulant and were strongly significant with the OR of 17 (95% CI 2-144; p = 0.001), 4.47 (95% CI 1.07-18.58; p = 0.039), and 27.97 (95% CI 1.54-507.13; p = 0.02), respectively; while the patients' mortality was significantly correlated with patients' age, anticoagulant, steroid, and diabetes, with OR of 8.44 (95% CI 1.5-47.49; p = 0.016), 46.8 (95% CI 4.63-472.77; p = 0.001), 15.75 (95% CI 2-123.86; p = 0.009), and 8.5 (95% CI 1.43-50.66; p = 0.019), respectively. This study found the clade: L (2%), GH (84.3%), GR (11.7%), and O (2%). Besides the D614G mutation, we found L5F (18.8%), V213A (18.8%), and S689R (8.3%). No significant association between multiple S protein mutations and the patients' hospitalization or mortality. Multivariate analysis revealed that hypertension and anticoagulant were the significant factors influencing the hospitalization and mortality of patients with COVID-19 with an OR of 17.06 (95% CI 2.02-144.36; p = 0.009) and 46.8 (95% CI 4.63-472.77; p = 0.001), respectively. Moreover, the multiple S protein mutations almost reached a strong association with patients' hospitalization (p = 0.07). We concluded that hypertension and anticoagulant therapy have a significant impact on COVID-19 outcomes. This study also suggests that multiple S protein mutations may impact the COVID-19 outcomes. This further emphasized the significance of monitoring SARS-CoV-2 variants through genomic surveillance, particularly those that may impact the COVID-19 outcomes.


Subject(s)
COVID-19/mortality , Mutation , SARS-CoV-2/genetics , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics , Adolescent , Adult , Aged , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Comorbidity , Female , High-Throughput Nucleotide Sequencing/methods , Hospitalization , Humans , Indonesia/epidemiology , Male , Middle Aged , Phylogeny , Prognosis , Retrospective Studies , Risk Factors , Whole Genome Sequencing/methods , Young Adult
9.
Viruses ; 13(10)2021 10 08.
Article in English | MEDLINE | ID: covidwho-1463840

ABSTRACT

The emergence of variants of SARS-CoV-2 has created challenges for the testing infrastructure. Although large-scale genome sequencing of SARS-CoV-2 has facilitated hospital and public health responses, access to sequencing facilities globally is variable and turnaround times can be significant, so there is a requirement for rapid and cost-effective alternatives. Applying a polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) approach enables rapid (<4 h) identification of SARS-CoV-2 lineages from nucleic acid extracts, through the presence or absence of a panel of defined of genomic polymorphisms. For example, the B.1.1.7 lineage ("UK", "Alpha", or "Kent" variant) is characterised by 23 mutations compared to the reference strain, and the most biologically significant of these are found in the S gene. We have developed a SARS-CoV-2 typing assay focused on five positions in the S gene (HV69/70, N501, K417, E484 and P681). This configuration can identify a range of variants, including all the "Variants of Concern" currently designated by national and international public health bodies. The panel has been evaluated using a range of clinical isolates and standardised control materials at four UK hospitals and shows excellent concordance with the known lineage information derived from full sequence analysis. The assay has a turnaround time of about three hours for a set of up to 24 samples and has been utilised to identify emerging variants in a clinical setting.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Genome, Viral/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Polymorphism, Single Nucleotide/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Whole Genome Sequencing/methods
10.
PLoS One ; 16(10): e0258513, 2021.
Article in English | MEDLINE | ID: covidwho-1463324

ABSTRACT

INTRODUCTION: Since their emergence, SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.351 have spread worldwide. We estimated the risk of hospitalisation and admission to an intensive care unit (ICU) for infections with B.1.1.7 and B.1.351 in Norway, compared to infections with non-VOC. MATERIALS AND METHODS: Using linked individual-level data from national registries, we conducted a cohort study on laboratory-confirmed cases of SARS-CoV-2 in Norway diagnosed between 28 December 2020 and 2 May 2021. Variants were identified based on whole genome sequencing, partial sequencing by Sanger sequencing or PCR screening for selected targets. The outcome was hospitalisation or ICU admission. We calculated adjusted risk ratios (aRR) with 95% confidence intervals (CIs) using multivariable binomial regression to examine the association between SARS-CoV-2 variants B.1.1.7 and B.1.351 with i) hospital admission and ii) ICU admission compared to non-VOC. RESULTS: We included 23,169 cases of B.1.1.7, 548 B.1.351 and 4,584 non-VOC. Overall, 1,017 cases were hospitalised (3.6%) and 206 admitted to ICU (0.7%). B.1.1.7 was associated with a 1.9-fold increased risk of hospitalisation (aRR 95%CI 1.6-2.3) and a 1.8-fold increased risk of ICU admission (aRR 95%CI 1.2-2.8) compared to non-VOC. Among hospitalised cases, no difference was found in the risk of ICU admission between B.1.1.7 and non-VOC. B.1.351 was associated with a 2.4-fold increased risk of hospitalisation (aRR 95%CI 1.7-3.3) and a 2.7-fold increased risk of ICU admission (aRR 95%CI 1.2-6.5) compared to non-VOC. DISCUSSION: Our findings add to the growing evidence of a higher risk of severe disease among persons infected with B.1.1.7 or B.1.351. This highlights the importance of prevention and control measures to reduce transmission of these VOC in society, particularly ongoing vaccination programmes, and preparedness plans for hospital surge capacity.


Subject(s)
COVID-19/epidemiology , COVID-19/prevention & control , Critical Care/methods , Hospitalization , Patient Admission , Registries , SARS-CoV-2/genetics , Adolescent , Adult , Aged , COVID-19/virology , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Infant , Infant, Newborn , Intensive Care Units , Male , Middle Aged , Norway/epidemiology , Real-Time Polymerase Chain Reaction/methods , Risk , Whole Genome Sequencing/methods , Young Adult
11.
J Med Virol ; 94(1): 327-334, 2022 01.
Article in English | MEDLINE | ID: covidwho-1410052

ABSTRACT

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.


Subject(s)
Genome, Viral/genetics , Multiplex Polymerase Chain Reaction/methods , Nanopore Sequencing/methods , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Nasopharynx/virology , RNA, Viral/genetics , Sequence Analysis, RNA/methods
12.
Sci Rep ; 11(1): 18126, 2021 09 13.
Article in English | MEDLINE | ID: covidwho-1406407

ABSTRACT

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.


Subject(s)
COVID-19/diagnosis , Mutation, Missense , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , COVID-19/epidemiology , COVID-19/virology , Evolution, Molecular , Genome, Viral/genetics , India , Multiplex Polymerase Chain Reaction/methods , Pandemics/prevention & control , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/physiology , Whole Genome Sequencing/methods
13.
Cell ; 184(20): 5179-5188.e8, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1401294

ABSTRACT

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.


Subject(s)
COVID-19/epidemiology , COVID-19/transmission , Pandemics , Recombination, Genetic , SARS-CoV-2/genetics , Base Sequence/genetics , COVID-19/virology , Computational Biology/methods , Gene Frequency , Genome, Viral , Genotype , Humans , Mutation , Phylogeny , Polymorphism, Single Nucleotide , United Kingdom/epidemiology , Whole Genome Sequencing/methods
14.
Euro Surveill ; 25(13)2020 04.
Article in English | MEDLINE | ID: covidwho-1389098

ABSTRACT

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus/genetics , Genome, Viral/genetics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Severe Acute Respiratory Syndrome/diagnosis , Travel , Whole Genome Sequencing/methods , Betacoronavirus/isolation & purification , COVID-19 , China , Coronavirus/classification , Coronavirus/isolation & purification , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Germany , Humans , Italy , Molecular Epidemiology , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Point Mutation , RNA, Viral/isolation & purification , SARS-CoV-2 , Severe Acute Respiratory Syndrome/virology
15.
J Med Virol ; 93(12): 6833-6836, 2021 12.
Article in English | MEDLINE | ID: covidwho-1372746

ABSTRACT

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as B.1.1.7 and B.1.351, has become a crucial issue worldwide. Therefore, we began testing all patients with COVID-19 for the N501Y and E484K mutations by using polymerase chain reaction (PCR)-based methods. Nasopharyngeal swab samples from 108 patients who visited our hospital between February and April 2021 were analyzed. The samples were analyzed using reverse transcription-PCR with melting curve analysis to detect the N501Y and E484K mutations. A part of the samples was also subjected to whole-genome sequencing (WGS). Clinical parameters such as mortality and admission to the intensive care unit were analyzed to examine the association between increased disease severity and the E484K mutation. The ratio of cases showing the 501N + 484K mutation rapidly increased from 8% in February to 46% in March. WGS revealed that the viruses with 501N + 484K mutation are R.1 lineage variants. Evidence of increased disease severity related to the R.1 variants was not found. We found that the R.1 lineage variants rapidly prevailed in Tokyo in March 2021, which suggests the increased transmissibility of R.1 variants, while they showed no increased severity.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , SARS-CoV-2/genetics , Aged , Female , Humans , Male , Mutation/genetics , Spike Glycoprotein, Coronavirus/genetics , Tokyo/epidemiology , Whole Genome Sequencing/methods
16.
Genome Biol Evol ; 13(9)2021 09 01.
Article in English | MEDLINE | ID: covidwho-1371724

ABSTRACT

Discrete classification of SARS-CoV-2 viral genotypes can identify emerging strains and detect geographic spread, viral diversity, and transmission events. We developed a tool (GNU-based Virus IDentification [GNUVID]) that integrates whole-genome multilocus sequence typing and a supervised machine learning random forest-based classifier. We used GNUVID to assign sequence type (ST) profiles to all high-quality genomes available from GISAID. STs were clustered into clonal complexes (CCs) and then used to train a machine learning classifier. We used this tool to detect potential introduction and exportation events and to estimate effective viral diversity across locations and over time in 16 US states. GNUVID is a highly scalable tool for viral genotype classification (https://github.com/ahmedmagds/GNUVID) that can quickly classify hundreds of thousands of genomes in a way that is consistent with phylogeny. Our genotyping ST/CC analysis uncovered dynamic local changes in ST/CC prevalence and diversity with multiple replacement events in different states, an average of 20.6 putative introductions and 7.5 exportations for each state over the time period analyzed. We introduce the use of effective diversity metrics (Hill numbers) that can be used to estimate the impact of interventions (e.g., travel restrictions, vaccine uptake, mask mandates) on the variation in circulating viruses. Our classification tool uncovered multiple introduction and exportation events, as well as waves of expansion and replacement of SARS-CoV-2 genotypes in different states. GNUVID classification lends itself to measures of ecological diversity, and, with systematic genomic sampling, it could be used to track circulating viral diversity and identify emerging clones and hotspots.


Subject(s)
COVID-19/virology , Genome, Viral/genetics , SARS-CoV-2/genetics , Genomics/methods , Genotype , Humans , Machine Learning , Whole Genome Sequencing/methods
17.
Diagn Microbiol Infect Dis ; 101(4): 115521, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1363964

ABSTRACT

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Freezing , Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Whole Genome Sequencing/methods , COVID-19/virology , Cryopreservation , Genome, Viral , Humans , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/genetics
18.
Methods Mol Biol ; 2327: 119-137, 2021.
Article in English | MEDLINE | ID: covidwho-1363726

ABSTRACT

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.


Subject(s)
Genome, Viral , SARS-CoV-2/genetics , Saliva/virology , Whole Genome Sequencing/methods , Computational Biology , DNA, Complementary/genetics , Humans , Molecular Probes/genetics , Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Streptavidin , Whole Genome Sequencing/instrumentation
19.
Trop Biomed ; 38(3): 283-288, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1346923

ABSTRACT

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.


Subject(s)
Genome, Viral , High-Throughput Nucleotide Sequencing , Multiplex Polymerase Chain Reaction , SARS-CoV-2/genetics , Whole Genome Sequencing , Base Sequence , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Multiplex Polymerase Chain Reaction/methods , Whole Genome Sequencing/methods
20.
PLoS One ; 16(8): e0255691, 2021.
Article in English | MEDLINE | ID: covidwho-1344159

ABSTRACT

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is clinically essential, and is required also to monitor confirmed cases aiming to prevent further spread. Positive real-time PCR results at late time points following initial diagnosis may be clinically misleading as this methodology cannot account for the infection capabilities and the existence of whole genome sequences. In this study, 47 serial respiratory samples were tested by Allplex-nCoV test (Seegene), a triplex of three assays targeting the SARS-CoV-2 RdRP, E and N genes and subsequently assessed by next generation sequencing (NGS). COVID19 patients were tested at an early stage of the disease, when all these viral gene targets were positive, and at an advanced stage, when only the N gene target was positive in the Allplex-nCoV test. The corresponding NGS results showed the presence of complete viral genome copies at both early and advanced stages of the disease, although the total number of mapped sequences was lower in samples from advanced disease stages. We conclude that reduced viral transmission at this late disease stage may result from the low quantities of complete viral sequences and not solely from transcription favoring the N gene.


Subject(s)
COVID-19/genetics , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , Female , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Middle Aged , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity
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