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1.
Front Cell Infect Microbiol ; 12: 819829, 2022.
Article in English | MEDLINE | ID: covidwho-2198682

ABSTRACT

Zika virus (ZIKV) emerged as a serious public health problem since the first major outbreak in 2007. Current ZIKV diagnostic methods can successfully identify known ZIKV but are impossible to track the origin of viruses and pathogens other than known ZIKV strains. We planned to determine the ability of Whole Genome Sequencing (WGS) in clinical epidemiology by evaluating whether it can successfully detect the origin of ZIKV in a suspected case of laboratory-acquired infection (LAI). ZIKV found in the patient sample was sequenced with nanopore sequencing technology, followed by the production of the phylogenetic tree, based on the alignment of 38 known ZIKV strains with the consensus sequence. The closest viral strain with the consensus sequence was the strain used in the laboratory, with a percent identity of 99.27%. We think WGS showed its time-effectiveness and ability to detect the difference between strains to the level of a single base. Additionally, to determine the global number of LAIs, a literature review of articles published in the last 10 years was performed, and 53 reports of 338 LAIs were found. The lack of a universal reporting system was worrisome, as in the majority of cases (81.1%), the exposure route was unknown.


Subject(s)
Nanopores , Vaccines , Zika Virus Infection , Zika Virus , Humans , Phylogeny , Whole Genome Sequencing , Zika Virus/genetics , Zika Virus Infection/epidemiology
2.
Front Cell Infect Microbiol ; 12: 960065, 2022.
Article in English | MEDLINE | ID: covidwho-2141708

ABSTRACT

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Genotype , Whole Genome Sequencing
3.
Am J Clin Pathol ; 157(5): 731-741, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-2114225

ABSTRACT

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern associated with immune escape is important to safeguard vaccination efficacy. We describe the potential of delayed N gene amplification in the Allplex SARS-CoV-2 Assay (Seegene) for screening of the B.1.351 (20H/501.V2, variant of concern 2 [VOC.V2], South African SARS-CoV-2 variant) lineage. METHODS: In a study cohort of 397 consecutive polymerase chain reaction-positive samples genotyped by whole-genome sequencing, amplification curves of E/N/S-RdRP targets indicated delayedN vs E gene amplification characteristic of B.1.351. Logistic regression was used to calculate a VOC.V2 probability score that was evaluated as a separate screening test in an independent validation cohort vs sequencing. RESULTS: B.1.351 showed a proportionally delayed amplification of the  N vs E gene. In logistic regression, only N and E gene cycle thresholds independently contributed to B.1.351 prediction, allowing calculation of a VOC.V2 probability score with an area under the curve of 0.94. At an optimal dichotomous cutoff point of 0.12, the VOC.V2 probability score achieved 98.7% sensitivity at 79.9% specificity, resulting in a negative predictive value (NPV) of 99.6% and a positive predictive value of 54.6%. The probability of B.1.351 increased with an increasing VOC.V2 probability score, achieving a likelihood ratio of 12.01 above 0.5. A near-maximal NPV was confirmed in 153 consecutive validation samples. CONCLUSIONS: Delayed N vs E gene amplification in the Allplex SARS-CoV-2 Assay can be used for fast and highly sensitive screening of B.1.351.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Probability , SARS-CoV-2/genetics , Whole Genome Sequencing
5.
J Mol Diagn ; 24(9): 963-976, 2022 09.
Article in English | MEDLINE | ID: covidwho-2083089

ABSTRACT

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Genome, Viral , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
6.
JAMA ; 328(15): 1523-1533, 2022 10 18.
Article in English | MEDLINE | ID: covidwho-2074838

ABSTRACT

Importance: Data on the epidemiology of mild to moderately severe COVID-19 are needed to inform public health guidance. Objective: To evaluate associations between 2 or 3 doses of mRNA COVID-19 vaccine and attenuation of symptoms and viral RNA load across SARS-CoV-2 viral lineages. Design, Setting, and Participants: A prospective cohort study of essential and frontline workers in Arizona, Florida, Minnesota, Oregon, Texas, and Utah with COVID-19 infection confirmed by reverse transcriptase-polymerase chain reaction testing and lineage classified by whole genome sequencing of specimens self-collected weekly and at COVID-19 illness symptom onset. This analysis was conducted among 1199 participants with SARS-CoV-2 from December 14, 2020, to April 19, 2022, with follow-up until May 9, 2022, reported. Exposures: SARS-CoV-2 lineage (origin strain, Delta variant, Omicron variant) and COVID-19 vaccination status. Main Outcomes and Measures: Clinical outcomes included presence of symptoms, specific symptoms (including fever or chills), illness duration, and medical care seeking. Virologic outcomes included viral load by quantitative reverse transcriptase-polymerase chain reaction testing along with viral viability. Results: Among 1199 participants with COVID-19 infection (714 [59.5%] women; median age, 41 years), 14.0% were infected with the origin strain, 24.0% with the Delta variant, and 62.0% with the Omicron variant. Participants vaccinated with the second vaccine dose 14 to 149 days before Delta infection were significantly less likely to be symptomatic compared with unvaccinated participants (21/27 [77.8%] vs 74/77 [96.1%]; OR, 0.13 [95% CI, 0-0.6]) and, when symptomatic, those vaccinated with the third dose 7 to 149 days before infection were significantly less likely to report fever or chills (5/13 [38.5%] vs 62/73 [84.9%]; OR, 0.07 [95% CI, 0.0-0.3]) and reported significantly fewer days of symptoms (10.2 vs 16.4; difference, -6.1 [95% CI, -11.8 to -0.4] days). Among those with Omicron infection, the risk of symptomatic infection did not differ significantly for the 2-dose vaccination status vs unvaccinated status and was significantly higher for the 3-dose recipients vs those who were unvaccinated (327/370 [88.4%] vs 85/107 [79.4%]; OR, 2.0 [95% CI, 1.1-3.5]). Among symptomatic Omicron infections, those vaccinated with the third dose 7 to 149 days before infection compared with those who were unvaccinated were significantly less likely to report fever or chills (160/311 [51.5%] vs 64/81 [79.0%]; OR, 0.25 [95% CI, 0.1-0.5]) or seek medical care (45/308 [14.6%] vs 20/81 [24.7%]; OR, 0.45 [95% CI, 0.2-0.9]). Participants with Delta and Omicron infections who received the second dose 14 to 149 days before infection had a significantly lower mean viral load compared with unvaccinated participants (3 vs 4.1 log10 copies/µL; difference, -1.0 [95% CI, -1.7 to -0.2] for Delta and 2.8 vs 3.5 log10 copies/µL, difference, -1.0 [95% CI, -1.7 to -0.3] for Omicron). Conclusions and Relevance: In a cohort of US essential and frontline workers with SARS-CoV-2 infections, recent vaccination with 2 or 3 mRNA vaccine doses less than 150 days before infection with Delta or Omicron variants, compared with being unvaccinated, was associated with attenuated symptoms, duration of illness, medical care seeking, or viral load for some comparisons, although the precision and statistical significance of specific estimates varied.


Subject(s)
COVID-19 Vaccines , COVID-19 , Vaccination , Viral Load , Adult , Female , Humans , Male , COVID-19/diagnosis , COVID-19/genetics , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/therapeutic use , Prospective Studies , RNA, Viral/analysis , RNA, Viral/genetics , RNA-Directed DNA Polymerase , SARS-CoV-2/genetics , Vaccination/statistics & numerical data , United States/epidemiology , Viral Load/drug effects , Viral Load/genetics , Viral Load/statistics & numerical data , Whole Genome Sequencing , Asymptomatic Infections/epidemiology , Asymptomatic Infections/therapy , Time Factors , Patient Acceptance of Health Care/statistics & numerical data
7.
Int J Mol Sci ; 23(17)2022 Aug 30.
Article in English | MEDLINE | ID: covidwho-2023749

ABSTRACT

Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and virulence potential of a single microbiological isolate during the diagnostic process. In contrast, metagenomic sequencing allows the analysis of DNA segments from multiple microorganisms within a community, either using an amplicon- or shotgun-based approach. However, WGS and shotgun metagenomic data are rarely combined, although such an approach may generate additive or synergistic information, critical for, e.g., patient management, infection control, and pathogen surveillance. To produce a combined workflow with actionable outputs, we need to understand the pre-to-post analytical process of both technologies. This will require specific databases storing interlinked sequencing and metadata, and also involves customized bioinformatic analytical pipelines. This review article will provide an overview of the critical steps and potential clinical application of combining WGS and metagenomics together for microbiological diagnosis.


Subject(s)
Metagenome , Metagenomics , Computational Biology , Humans , Whole Genome Sequencing , Workflow
8.
BMC Infect Dis ; 22(1): 713, 2022 Aug 29.
Article in English | MEDLINE | ID: covidwho-2021249

ABSTRACT

BACKGROUND: Variant of concern (VOC) SARS-CoV-2 alpha variant (B.1.1.7) was the dominant strain in the Netherlands between March 2021-June 2021. We describe three primary school outbreaks due to the alpha variant using whole genome sequencing with evidence of large-scale transmission among children, teachers and their household contacts. METHOD: All outbreaks described were investigated by the South Limburg Public Health Service, the Netherlands. A case was defined as an individual with a real-time polymerase chain reaction test or antigen test positive for SARS-CoV-2. Whole genome sequencing was performed on random samples from at least one child and one teacher of each affected class. RESULTS: Peak attack rates in classes were 53%, 33% and 39%, respectively. Specific genotypes were identified for each school across a majority of affected classes. Attack rates were high among staff members, likely to promote staff-to-children transmission. Cases in some classes were limited to children, indicating child-to-child transmission. At 39%, the secondary attack rate (SAR) in household contacts of infected children was remarkably high, similar to SAR in household contacts of staff members (42%). SAR of household contacts of asymptomatic children was only 9%. CONCLUSION: Our findings suggest increased transmissibility of the alpha variant in children compared to preceding non-VOC variants, consistent with a substantial rise in the incidence of cases observed in primary schools and children aged 5-12 since the alpha variant became dominant in March 2021. Lack of mandatory masking, insufficient ventilation and lack of physical distancing also probably contributed to the school outbreaks. The rise of the delta variant (B.1.617.2) since July 2021 which is estimated to be 55% more transmissible than the alpha variant, provides additional urgency to adequate infection prevention in school settings.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , Disease Outbreaks , Humans , Netherlands/epidemiology , SARS-CoV-2/genetics , Schools , Whole Genome Sequencing
9.
Int J Infect Dis ; 124: 104-106, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2007750

ABSTRACT

We reported herein a simultaneous co-identification with Omicron (B.1.1.529) and Delta (21A/478K.V1) SARS-CoV-2 variants, confirmed by whole genome sequencing in an 83-year-old French patient.


Subject(s)
COVID-19 , Coinfection , Humans , Aged, 80 and over , SARS-CoV-2/genetics , Genome, Viral , Sequence Analysis, DNA , COVID-19/diagnosis , Whole Genome Sequencing
10.
Sci Total Environ ; 851(Pt 2): 158266, 2022 Dec 10.
Article in English | MEDLINE | ID: covidwho-1996548

ABSTRACT

After a limited first wave of community transmission in March 2020 and until 2022, Western Australia was largely free of COVID-19, with cases restricted to hotel quarantine, commercial vessels, and small, infrequent community clusters. Despite the low case load setting, sequencing of wastewater samples from large municipal treatment plants produced SARS-CoV-2 genomes with coverage up to 99.7 % and depth to 4000×, which was sufficient to link wastewater sequences to those of active cases in the catchment at the time. This study demonstrates that ≤5 positive individuals can be enough to produce high genomic coverage (>90 %) assemblies even in catchments of up to a quarter of a million people. Genomic analysis of wastewater contemporaneous with clinical cases can also be used to rule out transmission between cases in different catchments, when their SARS-CoV-2 genomes have distinguishing nucleotide polymorphisms. These findings reveal a greater potential of wastewater WGS to inform outbreak management and disease surveillance than previously recognized.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Waste Water , COVID-19/epidemiology , Whole Genome Sequencing , Nucleotides
11.
Nat Biotechnol ; 40(7): 1035-1041, 2022 07.
Article in English | MEDLINE | ID: covidwho-1978002

ABSTRACT

Whole-genome sequencing (WGS) can identify variants that cause genetic disease, but the time required for sequencing and analysis has been a barrier to its use in acutely ill patients. In the present study, we develop an approach for ultra-rapid nanopore WGS that combines an optimized sample preparation protocol, distributing sequencing over 48 flow cells, near real-time base calling and alignment, accelerated variant calling and fast variant filtration for efficient manual review. Application to two example clinical cases identified a candidate variant in <8 h from sample preparation to variant identification. We show that this framework provides accurate variant calls and efficient prioritization, and accelerates diagnostic clinical genome sequencing twofold compared with previous approaches.


Subject(s)
Nanopore Sequencing , Nanopores , Chromosome Mapping , High-Throughput Nucleotide Sequencing/methods , Humans , Whole Genome Sequencing/methods
13.
Virulence ; 13(1): 1242-1251, 2022 12.
Article in English | MEDLINE | ID: covidwho-1956537

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have been emerging. However, knowledge of temporal and spatial dynamics of SARS-CoV-2 is limited. This study characterized SARS-CoV-2 evolution in immunosuppressed patients with long-term SARS-CoV-2 shedding for 73-250 days, without specific treatment. We conducted whole-genome sequencing of 27 serial samples, including 26 serial samples collected from various anatomic sites of two patients and the first positive sample from patient 2's mother. We analysed the intrahost temporal dynamics and genomic diversity of the viral population within different sample types. Intrahost variants emerging during infection showed diversity between individual hosts. Remarkably, N501Y, P681R, and E484K, key substitutions within spike protein, emerged in vivo during infection and became the dominant population. P681R, which had not yet been detected in the publicly available genome in Korea, appeared within patient 1 during infection. Mutually exclusive substitutions at residues R346 (R346S and R346I) and E484 (E484K and E484A) of spike protein and continuous turnover of these substitutions occurred. Unique genetic changes were observed in urine samples. A household transmission from patient 2 to his mother, at least 38 days after the diagnosis, was characterized. Viruses may differently mutate and adjust to the host selective pressure, which could enable the virus to replicate efficiently for fitness in each host. Intrahost variants could be candidate variants likely to spread to the population eventually. Our findings may provide new insights into the dynamics of SARS-CoV-2 in response to interactions between the virus and host.


Subject(s)
COVID-19 , Immunocompromised Host , SARS-CoV-2 , Virus Shedding , COVID-19/transmission , Humans , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Whole Genome Sequencing
14.
BMC Genomics ; 23(1): 406, 2022 May 30.
Article in English | MEDLINE | ID: covidwho-1951057

ABSTRACT

BACKGROUND: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. METHODS: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. RESULTS: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. CONCLUSIONS: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.


Subject(s)
COVID-19 , Influenza A Virus, H1N1 Subtype , RNA Viruses , Genome, Viral , Humans , SARS-CoV-2/genetics , Whole Genome Sequencing
15.
PLoS One ; 17(2): e0263678, 2022.
Article in English | MEDLINE | ID: covidwho-1910525

ABSTRACT

SARS-CoV-2 has spread throughout the world since its discovery in China, and Malaysia is no exception. WGS has been a crucial approach in studying the evolution and genetic diversity of SARS-CoV-2 in the ongoing pandemic. Despite considerable number of SARS-CoV-2 genome sequences have been submitted to GISAID and NCBI databases, there is still scarcity of data from Malaysia. This study aims to report new Malaysian lineages of the virus, responsible for the sustained spikes in COVID-19 cases during the third wave of the pandemic. Patients with nasopharyngeal and/or oropharyngeal swabs confirmed COVID-19 positive by real-time RT-PCR with CT value < 25 were chosen for WGS. The selected SARS-CoV-2 isolates were then sequenced, characterized and analyzed along with 986 sequences of the dominant lineages of D614G variants currently circulating throughout Malaysia. The prevalence of clade GH and G formed strong ground for the presence of two Malaysian lineages of AU.2 and B.1.524 that has caused sustained spikes of cases in the country. Statistical analysis on the association of gender and age group with Malaysian lineages revealed a significant association (p <0.05). Phylogenetic analysis revealed dispersion of 41 lineages, of these, 22 lineages are still active. Mutational analysis showed presence of unique G1223C missense mutation in transmembrane domain of the spike protein. For better understanding of the SARS-CoV-2 evolution in Malaysia especially with reference to the reported lineages, large scale studies based on WGS are warranted.


Subject(s)
COVID-19/virology , Genome, Viral , Mutation , Nasopharynx/virology , SARS-CoV-2/genetics , Humans , Malaysia , Spike Glycoprotein, Coronavirus/genetics , Whole Genome Sequencing
16.
Viruses ; 14(6)2022 06 10.
Article in English | MEDLINE | ID: covidwho-1911621

ABSTRACT

The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (>90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.


Subject(s)
Encephalitis Viruses, Tick-Borne , Encephalitis, Tick-Borne , Base Sequence , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis, Tick-Borne/diagnosis , Genes, Viral , Humans , Whole Genome Sequencing
17.
PLoS One ; 17(5): e0267847, 2022.
Article in English | MEDLINE | ID: covidwho-1910619

ABSTRACT

PURPOSE: Whole genome sequencing of SARS-CoV2 is important to find useful information about the viral lineages, variants of interests and variants of concern. As there are not enough data about the circulating SARS-CoV2 variants in Iran, we sequenced 54 SARS-CoV2 genomes during the 5 waves of pandemic in Iran. METHODS: After viral RNA extraction from clinical samples collected during the COVID-19 pandemic, next generation sequencing was performed using the Nextseq platform. The sequencing data were analyzed and compared with reference sequences. RESULTS: During the 1st wave, V and L clades were detected. The second wave was recognized by G, GH and GR clades. Circulating clades during the 3rd wave were GH and GR. In the fourth wave GRY (alpha variant), GK (delta variant) and one GH clade (beta variant) were detected. All viruses in the fifth wave were in clade GK (delta variant). There were different mutations in all parts of the genomes but Spike-D614G, NSP12-P323L, N-R203K and N-G204R were the most frequent mutants in these studied viruses. CONCLUSIONS: These findings display the significance of SARS-CoV2 monitoring to help on time detection of possible variants for pandemic control and vaccination plans.


Subject(s)
COVID-19 , Influenza, Human , COVID-19/epidemiology , Humans , Influenza, Human/epidemiology , Iran/epidemiology , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Whole Genome Sequencing
18.
Pathology ; 54(5): 615-622, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1907625

ABSTRACT

Extensive studies and analyses into the molecular features of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) have enhanced the surveillance and investigation of its clusters and transmission worldwide. The whole genome sequencing (WGS) approach is crucial in identifying the source of infection and transmission routes by monitoring the emergence of variants over time and through communities. Varying SARS-CoV-2 genomics capacity and capability levels have been established in public health laboratories across different Australian states and territories. Therefore, laboratories performing SARS-CoV-2 WGS for public health purposes are recommended to participate in an external proficiency testing program (PTP). This study describes the development of a SARS-CoV-2 WGS PTP. The PTP assessed the performance of laboratories while providing valuable insight into the current state of SARS-CoV-2 genomics in public health across Australia. Part 1 of the PTP contained eight simulated SARS-CoV-2 positive and negative specimens to assess laboratories' wet and dry laboratory capacity. Part 2 involved the analysis of a genomic dataset that consisted of a multi-FASTA file of 70 consensus genomes of SARS-CoV-2. Participating laboratories were required to (1) submit raw data for independent bioinformatics analysis, (2) analyse the data with their processes, and (3) answer relevant questions about the data. The performance of the laboratories was commendable, despite some variation in the reported results due to the different sequencing and bioinformatics approaches used by laboratories. The overall outcome is positive and demonstrates the critical role of the PTP in supporting the implementation and validation of SARS-CoV-2 WGS processes. The data derived from this PTP will contribute to the development of SARS-CoV-2 bioinformatic quality control (QC) and performance benchmarking for accreditation.


Subject(s)
COVID-19 , SARS-CoV-2 , Australia , COVID-19/diagnosis , Humans , Laboratory Proficiency Testing , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
19.
Sci Rep ; 12(1): 2054, 2022 02 08.
Article in English | MEDLINE | ID: covidwho-1900600

ABSTRACT

Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.


Subject(s)
COVID-19/genetics , Genome, Viral , Mutation , SARS-CoV-2/genetics , Whole Genome Sequencing , Humans
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