Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2

Paden, Clinton R.; Tao, Ying; Queen, Krista; Zhang, Jing; Li, Yan; Uehara, Anna; Tong, Suxiang

doi:10.3201/eid2610.201800

Cited by 140 publications

(123 citation statements)

References 10 publications

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“…We observed that the RT-LAMP assay was less sensitive and informative than multiplex PCR-based NGS in our study and other literature (20)(21)(22)(23)(24). NGS is a robust tool for obtaining extensive genetic information, allowing LOD values as low as 10 copies/ml for SARS-CoV-2 and serving as a reference test for COVID-19, especially for those challenging samples with a low viral content (2,(22)(23)(24). However, several experimental issues, such as erroneous barcode sequencing, the production of primer dimers, and potential cross-contamination between runs, may complicate sequence-based analyses and impact the validity of NGS results (25,26).…”

Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 48%

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Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

mSphere

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals. IMPORTANCE We developed a visual and rapid reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 48%

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

mSphere

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“…The present study does have several limitations. First, the 4 methods were tested on a limited number of European samples, all affiliated to the lineage B1, according to the Pangolin classification (23); the present evaluation should be confirmed in larger studies including other lineages in order to be more representative of the global ecology of this emerging virus.As SARS-CoV-2 sequencing is an expanding market and other methods has been published(24)(25)(26) or are probably under development, the present study is not exhaustive. However, the approaches selected herein are representative of the methods mostly used during pandemics and are easy to implement in a diagnostic laboratory.…”

mentioning

confidence: 99%

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

Charre¹,

Ginevra²,

Sabatier³

et al. 2020

Preprint

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Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.

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“…To conserve resources, pools with up to five specimens were tested together, and individual specimens within positive or inconclusive pools were retested. Nucleic acid from RT‐PCR–positive specimens was then extracted and subjected to Oxford Nanopore MinION sequencing, and full genome sequences were generated using methods described previously 3 . Phylogenetic relations were inferred using the Nextstrain pipeline, 4 including the 36 positive SARS‐CoV‐2 sentinel specimens and selected full genome sequences available as of April 1, 2020, from the Global Initiative on Sharing All Influenza Data (GISAID) 5 .…”

Section: Characteristic Age Group All Ages <18 Years Wk Beginning Marmentioning

confidence: 99%

Detection and genetic characterization of community-based SARS-CoV-2 infections – New York City, March 2020

Bushman

Alroy

Greene

et al. 2020

American Journal of Transplantation

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Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2

Cited by 140 publications

References 10 publications

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

Detection and genetic characterization of community-based SARS-CoV-2 infections – New York City, March 2020

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