Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
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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.
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SciScore for 10.1101/2021.05.12.21256693: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Collection of samples and RNA preparation: Oropharyngeal swabs of patients with suspected COVID-19, collected between March 30, 2020 and March 19, 2021, were preserved in 2-3 ml of viral transport medium and delivered to the laboratory of the Biomedical Research Centre of the Slovak Academy of Sciences in Bratislava, Slovakia in frame of the routine RT-qPCR diagnostics for SARS-CoV-2. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Applied Biosystem, Foster City, California, USA) Applied Biosystemsuggested: (Applied Biosystems, RRID:SCR_005039)For … SciScore for 10.1101/2021.05.12.21256693: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Collection of samples and RNA preparation: Oropharyngeal swabs of patients with suspected COVID-19, collected between March 30, 2020 and March 19, 2021, were preserved in 2-3 ml of viral transport medium and delivered to the laboratory of the Biomedical Research Centre of the Slovak Academy of Sciences in Bratislava, Slovakia in frame of the routine RT-qPCR diagnostics for SARS-CoV-2. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Applied Biosystem, Foster City, California, USA) Applied Biosystemsuggested: (Applied Biosystems, RRID:SCR_005039)For batch UKBA-2, the final sequences were produced by first combining sequencing reads from both standard and Flongle runs with the same primer set and running the Artic pipeline. Articsuggested: NoneTo detect sub-genomic RNAs, reads were aligned to transcripts downloaded from the UCSC genome browser by minimap2, and classified as sub-genomic, if the alignment to a sub-genomic RNA has at least 5 matches more than the best alignment to the reference genome. UCSC genome browsersuggested: (UCSC Genome Browser, RRID:SCR_005780)Read coverage was computed using genomecov tool from BEDTools (43) with options -bga -split. BEDToolssuggested: (BEDTools, RRID:SCR_006646)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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