A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing
A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spr...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.910955/full |
| _version_ | 1818549392232153088 |
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| author | Maria T. Arévalo Maria T. Arévalo Mark A. Karavis Sarah E. Katoski Jacquelyn V. Harris Jessica M. Hill Samir V. Deshpande Pierce A. Roth Alvin T. Liem R. Cory Bernhards |
| author_facet | Maria T. Arévalo Maria T. Arévalo Mark A. Karavis Sarah E. Katoski Jacquelyn V. Harris Jessica M. Hill Samir V. Deshpande Pierce A. Roth Alvin T. Liem R. Cory Bernhards |
| author_sort | Maria T. Arévalo |
| collection | DOAJ |
| description | A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army’s Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology’s Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective. |
| first_indexed | 2024-12-12T08:32:45Z |
| format | Article |
| id | doaj.art-eab3da68d713481e9fae404c8d87b631 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T08:32:45Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-eab3da68d713481e9fae404c8d87b6312022-12-22T00:31:03ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-06-011310.3389/fmicb.2022.910955910955A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore SequencingMaria T. Arévalo0Maria T. Arévalo1Mark A. Karavis2Sarah E. Katoski3Jacquelyn V. Harris4Jessica M. Hill5Samir V. Deshpande6Pierce A. Roth7Alvin T. Liem8R. Cory Bernhards9Defense Threat Reduction Agency, Aberdeen Proving Ground, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesDCS Corporation, Belcamp, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesDCS Corporation, Belcamp, MD, United StatesDCS Corporation, Belcamp, MD, United StatesUnited States Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, United StatesA new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army’s Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology’s Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.https://www.frontiersin.org/articles/10.3389/fmicb.2022.910955/fullnanopore sequencingCOVID-19SARS-CoV-2whole genome sequencingwhole genome assembly |
| spellingShingle | Maria T. Arévalo Maria T. Arévalo Mark A. Karavis Sarah E. Katoski Jacquelyn V. Harris Jessica M. Hill Samir V. Deshpande Pierce A. Roth Alvin T. Liem R. Cory Bernhards A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing Frontiers in Microbiology nanopore sequencing COVID-19 SARS-CoV-2 whole genome sequencing whole genome assembly |
| title | A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing |
| title_full | A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing |
| title_fullStr | A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing |
| title_full_unstemmed | A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing |
| title_short | A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing |
| title_sort | rapid whole genome sequencing assay for detection and characterization of novel coronavirus sars cov 2 clinical specimens using nanopore sequencing |
| topic | nanopore sequencing COVID-19 SARS-CoV-2 whole genome sequencing whole genome assembly |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.910955/full |
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