Dataset Information

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

ABSTRACT: We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The proposed protocol allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titer virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.

SUBMITTER: Kalendar R

PROVIDER: S-EPMC10293217 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

Kalendar Ruslan R Kairov Ulykbek U Karabayev Daniyar D Aitkulova Akbota A Tynyshtykbayeva Nuray N Daniyarov Asset A Otarbay Zhenis Z Rakhimova Saule S Akilzhanova Ainur A Sarbassov Dos D

Scientific reports 20230626 1

We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized ...[more]

PMID: 37365249

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Project description:Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from ∼5% to reach ∼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.

Project description:Severe acute respiratory syndrome (SARS-CoV-2) is responsible for the worldwide pandemic, COVID-19. The original viral whole-genome was sequenced by a high-throughput sequencing approach from the samples obtained from Wuhan, China. Real-time gene sequencing is the main parameter to manage viral outbreaks because it expands our understanding of virus proliferation, spread, and evolution. Whole-genome sequencing is critical for SARS-CoV-2 variant surveillance, the development of new vaccines and boosters, and the representation of epidemiological situations in the country. A significant increase in the number of COVID-19 cases confirmed in August 2021 in Kazakhstan facilitated a need to establish an effective and proficient system for further study of SARS-CoV-2 genetic variants and the development of future Kazakhstan's genomic surveillance program. The SARS-CoV-2 whole-genome was sequenced according to SARS-CoV-2 ARTIC protocol (EXP-MRT001) by Oxford Nanopore Technologies at the National Laboratory Astana, Kazakhstan to track viral variants circulating in the country. The 500 samples kindly provided by the Republican Diagnostic Center (UMC-NU) and private laboratory KDL "Olymp" were collected from individuals in Nur-Sultan city diagnosed with COVID-19 from August 2021 to May 2022 using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All samples had a cycle threshold (Ct) value below 20 with an average Ct value of 17.03. The overall average value of sequencing depth coverage for samples is 244X. 341 whole-genome sequences that passed quality control were deposited in the Global initiative on sharing all influenza data (GISAID). The BA.1.1 (n = 189), BA.1 (n = 15), BA.2 (n = 3), BA.1.15 (n = 1), BA.1.17.2 (n = 1) omicron lineages, AY.122 (n = 119), B.1.617.2 (n = 8), AY.111 (n = 2), AY.126 (n = 1), AY.4 (n = 1) delta lineages, one sample B.1.1.7 (n = 1) belongs to alpha lineage, and one sample B.1.637 (n = 1) belongs to small sublineage were detected in this study. This is the first study of SARS-CoV-2 whole-genome sequencing by the ONT approach in Kazakhstan, which can be expanded for the investigation of other emerging viral or bacterial infections on the country level.

Dataset Information

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

Publications

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

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