Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance
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Abstract
Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain performance for emerging virus lineages. We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 196 Delta lineage SARS-CoV-2 clinical specimens, and 71 mostly Omicron lineage samples with S gene target failure (SGTF), reflecting circulating lineages in the United Kingdom during December 2021. 96-plexed nanopore sequencing was used. For Delta lineage samples, ARTIC v4 recovered the greatest proportion of ≥90% complete genomes (81.1%; 159/193), followed by Midnight (71.5%; 138/193) and ARTIC v3 (34.1%; 14/41). Midnight protocol however yielded higher average genome recovery (mean 98.8%) than ARTIC v4 (98.1%) and ARTIC v3 (75.4%), resulting in less ambiguous final consensus assemblies overall. Explaining these observations were ARTIC v4’s superior genome recovery in low viral titre/high cycle threshold (Ct) samples and inferior performance in high titre/low Ct samples, where Midnight excelled. We evaluated Omicron sequencing performance using a revised Midnight primer mix alongside prototype ARTIC v4.1 primers, head-to-head with the existing commercially available Midnight and ARTIC v4 protocols. The revised protocols both improved considerably the recovery of Omicron genomes and exhibited similar overall performance to one another. Revised Midnight protocol recovered ≥90% complete genomes for 85.9% (61/71) of Omicron samples vs. 88.7% (63/71) for ARTIC v4.1. Approximate cost per sample for Midnight (£12) is lower than ARTIC (£16) while hands-on time is considerably lower for Midnight (∼7 hours) than ARTIC protocols (∼9.5 hours).
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SciScore for 10.1101/2021.12.28.21268461: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Sample processing and analysis was covered by ethical approval for the use of anonymised oro- or nasopharyngeal specimens from patients for the diagnosis of influenza and other respiratory pathogens, including SARS-CoV-2 (North West-Greater Manchester South Research Ethics Committee [REC], REC Ref:19/NW/0730). Sex as a biological variable not detected. Randomization Clinical negative controls were randomly included throughout each plate. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Depth of coverage and cov20 was calculated from primer-trimmed BAM files using Samtools (Li et al., 2009) depth, including aligned deletions in the … SciScore for 10.1101/2021.12.28.21268461: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Sample processing and analysis was covered by ethical approval for the use of anonymised oro- or nasopharyngeal specimens from patients for the diagnosis of influenza and other respiratory pathogens, including SARS-CoV-2 (North West-Greater Manchester South Research Ethics Committee [REC], REC Ref:19/NW/0730). Sex as a biological variable not detected. Randomization Clinical negative controls were randomly included throughout each plate. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Depth of coverage and cov20 was calculated from primer-trimmed BAM files using Samtools (Li et al., 2009) depth, including aligned deletions in the depth calculation. Samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Results from OddPub: Thank you for sharing your code.
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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