Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance
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Abstract
Genome sequencing is pivotal to virus surveillance, revealing the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated frequent 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 96-plexed nanopore sequencing of 193 Delta-lineage SARS-CoV-2 clinical specimens with complete paired results and 71 S-gene target failure-selected specimens collected during emergence of Omicron BA.1. For Delta samples, ARTIC v4 recovered ≥90% of the genome in 82.4% (159/193) of specimens compared with 71.5% (138/193) for Midnight. ARTIC v4 was more robust in low-titre, high-Ct specimens, whereas Midnight achieved slightly greater completeness at lower Ct. In the S-gene target failure-selected cohort, revised Midnight v2 and ARTIC v4.1 primer schemes substantially improved genome completeness: ≥99% genome recovery was achieved in 74.6% (53/71) with Midnight v2 versus 18.3% (13/71) with Midnight, and 57.7% (41/71) with ARTIC v4.1 versus 0% (0/71) with ARTIC v4. Estimated consumable costs were lower for Midnight than ARTIC (£12 versus £16 per sample at Q1 2022 prices), and the interval from reverse transcription to flow cell loading was shorter (~7 versus ~9.5 hours). Midnight proved a practical alternative for rapid nanopore sequencing, with lower consumable costs and shorter preparation time, although ARTIC v4 was more robust in high-Ct Delta specimens.
Impact statement
Tiling amplicon whole genome sequencing enables fast and cost-effective virus sequencing, yet is adversely affected by mutations at primer binding sites. We evaluated ARTIC and Midnight nanopore sequencing protocols using SARS-CoV-2 clinical samples in terms of genome recovery, turnaround time and consumable costs. Our evaluation contributed to deployment of a nanopore sequencing service embedded in an NHS hospital microbiology laboratory supporting genomic surveillance and subsequently the RECOVERY platform trial.
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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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