High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples

Pater, Adrian A; Bosmeny, Michael S.; Parasrampuria, Mansi; Eddington, Seth B.; Ovington, Katy N; Barkau, Christopher L.; White, Adam A; Metz, Paige E; Sylvain, Rourke J; Chilamkurthy, Ramadevi; Yinusa, Abadat O; Benzinger, Scott W.; Hebert, Madison M; Gagnon, Keith T.

doi:10.1101/2021.02.09.430478

Cited by 21 publications

(22 citation statements)

References 19 publications

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“…Further reduction in turnaround time and costs can also be achieved, as exemplified by a combination use of primer sets that generate 1,200 bp amplicons and the ONT Rapid Barcoding Kit ( Freed et al, 2020 ). Moreover, protocols amenable to high throughput to accelerate sequencing efforts are also available ( Baker et al, 2021 ; Pater et al, 2021 ). The ongoing improvement in efficiency of cost and time is likely to increase the adoption of Nanopore sequencing of SARS-CoV-2 genomes in many more countries, thereby contributing to a better worldwide genomic surveillance of SARS-CoV-2.…”

Section: Discussionmentioning

confidence: 99%

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia

Prasetyoputri

Dharmayanthi

Iryanto

et al. 2022

PeerJ

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Background Indonesia is one of the Southeast Asian countries with high case numbers of COVID-19 with up to 4.2 million confirmed cases by 29 October 2021. Understanding the genome of SARS-CoV-2 is crucial for delivering public health intervention as certain variants may have different attributes that can potentially affect their transmissibility, as well as the performance of diagnostics, vaccines, and therapeutics. Objectives We aimed to investigate the dynamics of circulating SARS-CoV-2 variants over a 15-month period in Bogor and its surrounding areas in correlation with the first and second wave of COVID-19 in Indonesia. Methods Nasopharyngeal and oropharyngeal swab samples collected from suspected patients from Bogor, Jakarta and Tangerang were confirmed for SARS-CoV-2 infection with RT-PCR. RNA samples of those confirmed patients were subjected to whole genome sequencing using the ARTIC Network protocol and sequencer platform from Oxford Nanopore Technologies (ONT). Results We successfully identified 16 lineages and six clades out of 202 samples (male n = 116, female n = 86). Genome analysis revealed that Indonesian lineage B.1.466.2 dominated during the first wave (n = 48, 23.8%) while Delta variants (AY.23, AY.24, AY.39, AY.42, AY.43 dan AY.79) were dominant during the second wave (n = 53, 26.2%) following the highest number of confirmed cases in Indonesia. In the spike protein gene, S_D614G and S_P681R changes were dominant in both B.1.466.2 and Delta variants, while N439K was only observed in B.1.466.2 (n = 44) and B.1.470 (n = 1). Additionally, the S_T19R, S_E156G, S_F157del, S_R158del, S_L452R, S_T478K, S_D950N and S_V1264L changes were only detected in Delta variants, consistent with those changes being characteristic of Delta variants in general. Conclusions We demonstrated a shift in SARS-CoV-2 variants from the first wave of COVID-19 to Delta variants in the second wave, during which the number of confirmed cases surpassed those in the first wave of COVID-19 pandemic. Higher proportion of unique mutations detected in Delta variants compared to the first wave variants indicated potential mutational effects on viral transmissibility that correlated with a higher incidence of confirmed cases. Genomic surveillance of circulating variants, especially those with higher transmissibility, should be continuously conducted to rapidly inform decision making and support outbreak preparedness, prevention, and public health response.

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Section: Discussionmentioning

confidence: 99%

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia

Prasetyoputri

Dharmayanthi

Iryanto

et al. 2022

PeerJ

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“…The metadata included collection date, location, the origin of samples, passage history, and sequencing technology. Further analysis for possible missing metadata, FASTA sequences, a certain information, and frameshift mutations was undertaken following the result of GISAID quality control checking after submission 70 . In total, 12 genomes were submitted to GISAID 50 from NICCRAT-LIPI Team.…”

Section: Methodsmentioning

confidence: 99%

Genome Profiling of SARS-CoV-2 in Indonesia, ASEAN, and the Neighbouring East Asian Countries: Features, Challenges, and Achievements

Cahyani¹,

Putro

Ridwanuloh

et al. 2021

Preprint

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A year after the World Health Organisation declared COVID-19 as a pandemic, much has been learned with respect to SARS-CoV-2 epidemiology, vaccine production and disease treatment. Whole-genome sequencing (WGS) has played a significant role in contributing to our understanding of the epidemiology and biology of this virus. In this paper, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asia and the impact of technological development, access to resources, and demography of individual countries on its uptake. Facilitated by the Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative, we showcased a bilateral collaboration between the University of Nottingham and the Indonesian Institute of Sciences (LIPI/Lembaga Ilmu Pengetahuan Indonesia) to establish WGS of SARS-CoV-2 using Oxford Nanopore Technology® in Indonesia. Analyses of SARS-CoV-2 genomes deposited on GISAID from Southeast and East Asian countries reveals the importance of collecting clinical and demographic metadata and the importance of open access and data sharing. Lineage and phylogenetic analyses per 1 June 2021 found that: 1) B.1.466.2 variants were the most predominant in Indonesia, with mutations in the spike protein including D614G at 100%, N439K at 99.1%, and P681R at 69.7% frequency, 2) The variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) were first detected in Indonesia in January 2021, 2) B.1.470 was first detected in Indonesia and spread to the neighbouring regions, and 3) The highest rate of virus transmissions between Indonesia and the rest of the world appears to be through interactions with Singapore and Japan, two neighbouring countries with high degree of access and travels to and from Indonesia. Overall, we conclude that WGS of SARS-CoV-2 using Oxford Nanopore Technology® platforms fits well with the Indonesian context and can catalyse the increase of sequencing rates in the country.

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“…These amplicon dropouts are not news to the ARTIC Network, which already produced some work in order to fix this issue giving rise to the V3 primer scheme ( Tyson et al, 2020 ). However, a study still reported #74 amplicon dropout with the V3 primer scheme, as in our study, and fixed the problem by adjusting concentration of its primer set ( Pater et al, 2021 ). Other panels generating longer amplicons, designed for devices compatible with long-read sequencing such as the MinION, could also be considered.…”

Section: Discussionmentioning

confidence: 73%

SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters

Barbé

Schaeffer²,

Besnard³

et al. 2022

Front. Microbiol.

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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.

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High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples

Cited by 21 publications

References 19 publications

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia

Genome Profiling of SARS-CoV-2 in Indonesia, ASEAN, and the Neighbouring East Asian Countries: Features, Challenges, and Achievements

SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters

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