Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology
The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerston...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-12-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/9/12/2598 |
| _version_ | 1797502290323570688 |
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| author | Anton Pembaur Erwan Sallard Patrick Philipp Weil Jennifer Ortelt Parviz Ahmad-Nejad Jan Postberg |
| author_facet | Anton Pembaur Erwan Sallard Patrick Philipp Weil Jennifer Ortelt Parviz Ahmad-Nejad Jan Postberg |
| author_sort | Anton Pembaur |
| collection | DOAJ |
| description | The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10<sup>6</sup> were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage. |
| first_indexed | 2024-03-10T03:30:58Z |
| format | Article |
| id | doaj.art-07c5c091d3ec4364b7cf29e4462072cb |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T03:30:58Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-07c5c091d3ec4364b7cf29e4462072cb2023-11-23T09:40:25ZengMDPI AGMicroorganisms2076-26072021-12-01912259810.3390/microorganisms9122598Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore TechnologyAnton Pembaur0Erwan Sallard1Patrick Philipp Weil2Jennifer Ortelt3Parviz Ahmad-Nejad4Jan Postberg5Clinical Molecular Genetics and Epigenetics, Centre for Biomedical Education & Research (ZBAF), Faculty of Health, Witten/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, GermanyInstitute of Virology and Microbiology, Centre for Biomedical Education & Research (ZBAF), Faculty of Health, Witten/Herdecke University, Stockumer Str. 10, 58453 Witten, GermanyClinical Molecular Genetics and Epigenetics, Centre for Biomedical Education & Research (ZBAF), Faculty of Health, Witten/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, GermanyInstitute of Medical Laboratory Diagnostics, Centre for Clinical & Translational Research (CCTR), HELIOS University Hospital Wuppertal, Witten/Herdecke University, Heusnerstr. 40, 42283 Wuppertal, GermanyInstitute of Medical Laboratory Diagnostics, Centre for Clinical & Translational Research (CCTR), HELIOS University Hospital Wuppertal, Witten/Herdecke University, Heusnerstr. 40, 42283 Wuppertal, GermanyClinical Molecular Genetics and Epigenetics, Centre for Biomedical Education & Research (ZBAF), Faculty of Health, Witten/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, GermanyThe scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10<sup>6</sup> were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.https://www.mdpi.com/2076-2607/9/12/2598(+)RNA genome sequencingCOVID-19 surveillancevariant-of-concern (VOC)variant-of-interest (VOI) |
| spellingShingle | Anton Pembaur Erwan Sallard Patrick Philipp Weil Jennifer Ortelt Parviz Ahmad-Nejad Jan Postberg Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology Microorganisms (+)RNA genome sequencing COVID-19 surveillance variant-of-concern (VOC) variant-of-interest (VOI) |
| title | Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology |
| title_full | Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology |
| title_fullStr | Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology |
| title_full_unstemmed | Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology |
| title_short | Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology |
| title_sort | simplified point of care full sars cov 2 genome sequencing using nanopore technology |
| topic | (+)RNA genome sequencing COVID-19 surveillance variant-of-concern (VOC) variant-of-interest (VOI) |
| url | https://www.mdpi.com/2076-2607/9/12/2598 |
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