Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2

Changwoo Park; Kwan Woo Kim; Dongju Park; Zohaib ul Hassan; Edmond Changkyun Park; Chang Seop Lee; MD Tazikur Rahman; Hana Yi; Seil Kim

doi:10.3389/fmicb.2022.876085

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2

Changwoo Park, Kwan Woo Kim, Dongju Park, Zohaib ul Hassan, Edmond Changkyun Park, Chang Seop Lee, MD Tazikur Rahman, Hana Yi, Seil Kim

School of Biosystem and Biomedical Science

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Original language	English
Article number	876085
Journal	Frontiers in Microbiology
Volume	13
DOIs	https://doi.org/10.3389/fmicb.2022.876085
Publication status	Published - 2022 Aug 17

Bibliographical note

Funding Information:
This work was supported by the National Research Council of Science and Technology grant by the Ministry of Science and ICT (grant no. CRC-16-01-KRICT). This work was also supported by the “Establishment of measurement standards for microbiology,” grant number GP2021-0003-08, “Development of Rapid Diagnosis of Rift Valley Fever Virus based on Whole Genome Analysis,” grant number HI20C0558, and “Development of Rift valley fever virus RNA reference materials,” grant number 20015205 funded by the Korea Research Institute of Standards and Science, the Ministry of Health and Welfare, and Ministry of Trade, Industry and Energy, respectively.

Publisher Copyright:
Copyright © 2022 Park, Kim, Park, Hassan, Park, Lee, Rahman, Yi and Kim.

Keywords

MinION
SARS-CoV-2
amplicon-based genome sequencing
long amplicon
reverse transcription digital droplet PCR

ASJC Scopus subject areas

Microbiology
Microbiology (medical)

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10.3389/fmicb.2022.876085

Cite this

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title = "Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2",

abstract = "As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.",

keywords = "MinION, SARS-CoV-2, amplicon-based genome sequencing, long amplicon, reverse transcription digital droplet PCR",

author = "Changwoo Park and Kim, {Kwan Woo} and Dongju Park and Hassan, {Zohaib ul} and Park, {Edmond Changkyun} and Lee, {Chang Seop} and Rahman, {MD Tazikur} and Hana Yi and Seil Kim",

note = "Funding Information: This work was supported by the National Research Council of Science and Technology grant by the Ministry of Science and ICT (grant no. CRC-16-01-KRICT). This work was also supported by the “Establishment of measurement standards for microbiology,” grant number GP2021-0003-08, “Development of Rapid Diagnosis of Rift Valley Fever Virus based on Whole Genome Analysis,” grant number HI20C0558, and “Development of Rift valley fever virus RNA reference materials,” grant number 20015205 funded by the Korea Research Institute of Standards and Science, the Ministry of Health and Welfare, and Ministry of Trade, Industry and Energy, respectively. Publisher Copyright: Copyright {\textcopyright} 2022 Park, Kim, Park, Hassan, Park, Lee, Rahman, Yi and Kim.",

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AU - Lee, Chang Seop

AU - Rahman, MD Tazikur

AU - Yi, Hana

AU - Kim, Seil

N1 - Funding Information: This work was supported by the National Research Council of Science and Technology grant by the Ministry of Science and ICT (grant no. CRC-16-01-KRICT). This work was also supported by the “Establishment of measurement standards for microbiology,” grant number GP2021-0003-08, “Development of Rapid Diagnosis of Rift Valley Fever Virus based on Whole Genome Analysis,” grant number HI20C0558, and “Development of Rift valley fever virus RNA reference materials,” grant number 20015205 funded by the Korea Research Institute of Standards and Science, the Ministry of Health and Welfare, and Ministry of Trade, Industry and Energy, respectively. Publisher Copyright: Copyright © 2022 Park, Kim, Park, Hassan, Park, Lee, Rahman, Yi and Kim.

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N2 - As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

AB - As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

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VL - 13

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

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Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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