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Identification of Genomic Variants of SARS-CoV-2 Using Nanopore Sequencing.

ABSTRACT: Background and Objectives: SARS-CoV-2 is the first global threat and life-changing event of the twenty-first century. Although efficient treatments and vaccines have been developed, due to the virus's ability to mutate in key regions of the genome, whole viral genome sequencing is needed for efficient monitoring, evaluation of the spread, and even the adjustment of the molecular diagnostic assays. Materials and Methods: In this study, Nanopore and Ion Torrent sequencing technologies were used to detect the main SARS-CoV-2 circulating strains in Timis County, Romania, between February 2021 and May 2022. Results: We identified 22 virus lineages belonging to seven clades: 20A, 20I (Alpha, V1), 21B (Kappa), 21I (Delta), 21J (Delta), 21K (Omicron), and 21L (Omicron). Conclusions: Results obtained with both methods are comparable, and we confirm the utility of Nanopore sequencing in large-scale epidemiological surveillance due to the lower cost and reduced time for library preparation.

SUBMITTER: Capraru ID

PROVIDER: S-EPMC9788413 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Identification of Genomic Variants of SARS-CoV-2 Using Nanopore Sequencing.

Capraru Ionut Dragos ID Romanescu Mirabela M Anghel Flavia Medana FM Oancea Cristian C Marian Catalin C Sirbu Ioan Ovidiu IO Chis Aimee Rodica AR Ciordas Paula Diana PD

Medicina (Kaunas, Lithuania) 20221215 12

<i>Background and Objectives</i>: SARS-CoV-2 is the first global threat and life-changing event of the twenty-first century. Although efficient treatments and vaccines have been developed, due to the virus's ability to mutate in key regions of the genome, whole viral genome sequencing is needed for efficient monitoring, evaluation of the spread, and even the adjustment of the molecular diagnostic assays. <i>Materials and Methods</i>: In this study, Nanopore and Ion Torrent sequencing technologie ...[more]

PMID: 36557043

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Project description:The genomic analysis of SARS-CoV-2 has served as a crucial tool for generating invaluable data that fulfils both epidemiological and clinical necessities. Long-read sequencing technology (e.g., ONT) has been widely used, providing a real-time and faster response when necessitated. A novel nanopore-based long-read sequencing platform named QNome nanopore has been successfully used for bacterial genome sequencing and assembly; however, its performance in the SARS-CoV-2 genomic surveillance is still lacking. Synthetic SARS-CoV-2 controls and 120 nasopharyngeal swab (NPS) samples that tested positive by real-time polymerase chain reaction were sequenced on both QNome and MGI platforms in parallel. The analytical performance of QNome was compared to the short-read sequencing on MGI. For the synthetic SARS-CoV-2 controls, despite the increased error rates observed in QNome nanopore sequencing reads, accurate consensus-level sequence determination was achieved with an average mapping quality score of approximately 60 (i.e., a mapping accuracy of 99.9999%). For the NPS samples, the average genomic coverage was 89.35% on the QNome nanopore platform compared with 90.39% for MGI. In addition, fewer consensus genomes from QNome were determined to be good by Nextclade compare with MGI (p < 0.05). A total of 9004 mutations were identified using QNome sequencing, with substitutions being the most prevalent, in contrast, 10 997 mutations were detected on MGI (p < 0.05). Furthermore, 23 large deletions (i.e., deletions≥ 10 bp) were identified by QNome while 19/23 were supported by evidence from short-read sequencing. Phylogenetic analysis revealed that the Pango lineage of consensus genomes for SARS-CoV-2 sequenced by QNome concorded 83.04% with MGI. QNome nanopore sequencing, though challenged by read quality and accuracy compared to MGI, is overcoming these issues through bioinformatics and computational advances. The advantage of structure variation (SV) detection capabilities and real-time data analysis renders it a promising alternative nanopore platform for the surveillance of the SARS-CoV-2.

Project description:COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2's spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant (P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.

Project description:BackgroundThe Centers for Disease Control and Prevention contracted with laboratories to sequence the SARS-CoV-2 genome from positive samples across the United States to enable public health officials to investigate the impact of variants on disease severity as well as the effectiveness of vaccines and treatment. Herein we present the initial results correlating RT-PCR quality control metrics with sample collection and sequencing methods from full SARS-CoV-2 viral genomic sequencing of 24,441 positive patient samples between April and June 2021.MethodsRT-PCR confirmed (N Gene Ct value < 30) positive patient samples, with nucleic acid extracted from saliva, nasopharyngeal and oropharyngeal swabs were selected for viral whole genome SARS-CoV-2 sequencing. Sequencing was performed using Illumina COVIDSeq™ protocol on either the NextSeq550 or NovaSeq6000 systems. Informatic variant calling, and lineage analysis were performed using DRAGEN COVID Lineage applications on Illumina's Basespace cloud analytical system. All sequence data and variant calls were uploaded to NCBI and GISAID.ResultsAn association was observed between higher sequencing coverage, quality, and samples with a lower Ct value, with < 27 being optimal, across both sequencing platforms and sample collection methods. Both nasopharyngeal swabs and saliva samples were found to be optimal samples of choice for SARS-CoV-2 surveillance sequencing studies, both in terms of strain identification and sequencing depth of coverage, with NovaSeq 6000 providing higher coverage than the NextSeq 550. The most frequent variants identified were the B.1.617.2 Delta (India) and P.1 Gamma (Brazil) variants in the samples sequenced between April 2021 and June 2021. At the time of submission, the most common variant > 99% of positives sequenced was Omicron.ConclusionThese initial analyses highlight the importance of sequencing platform, sample collection methods, and RT-PCR Ct values in guiding surveillance efforts. These surveillance studies evaluating genetic changes of SARS-CoV-2 have been identified as critical by the CDC that can affect many aspects of public health including transmission, disease severity, diagnostics, therapeutics, and vaccines.

Dataset Information

Identification of Genomic Variants of SARS-CoV-2 Using Nanopore Sequencing.

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Identification of Genomic Variants of SARS-CoV-2 Using Nanopore Sequencing.

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