Abstract
Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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COVID-19 / diagnosis*
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Humans
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Nanopore Sequencing / methods*
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Pandemics*
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SARS-CoV-2* / genetics
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SARS-CoV-2* / isolation & purification
Grants and funding
The research was supported by grants from the Slovak Research and Development Agency (
https://www.apvv.sk; APVV-18-0239 to JN, PP-COVID-20-0017 to BK), the Scientific Grant Agency (
https://www.minedu.sk/vedecka-grantova-agentura-msvvas-sr-a-sav-vega/; VEGA 1/0463/20 to BB, VEGA 1/0458/18 to TV, VEGA 1/0027/19 to JN, VEGA 1/0136/20 to MN), and the European Union’s Horizon 2020 research and innovation program (
https://ec.europa.eu/programmes/horizon2020/; EVA-GLOBAL project #871029 to BK and PANGAIA project #872539 to TV). The research was also supported in part by the Operation Program of Integrated Infrastructure (OPII) projects ITMS2014: 313011ATL7 and ITMS2014+: 313021X329 (Advancing University Capacity and Competence in Research, Development and Innovation), co-financed by the European Regional Development Fund (
https://ec.europa.eu/regional_policy/en/funding/erdf/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.