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. 2024 Feb 14:15:1272972.
doi: 10.3389/fmicb.2024.1272972. eCollection 2024.

Genomic surveillance of SARS-CoV-2 using long-range PCR primers

Sangam Kandel  1 , Susanna L Hartzell  2 , Ashton K Ingold  2 , Grace A Turner  2 , Joshua L Kennedy  2   3   4 , David W Ussery  1
Affiliations

Genomic surveillance of SARS-CoV-2 using long-range PCR primers

Sangam Kandel et al. Front Microbiol. .

Abstract

Introduction: Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs).

Methods: In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell.

Results and discussion: Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.

Keywords: SARS-CoV-2; genomic epidemiology; long-range primers; nanopore; sequencing; surveillance.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Comparison of ARTIC, Midnight, and Long-Range PCR primers.
Figure 2
Figure 2
Diagrammatic representation of Oxford Nanopore Sequencing of SARS-CoV-2 using long-range PCR primers. (Figures made using BioRender.com).
Figure 3
Figure 3
IGV plot showing seven different amplicons mapped to the SARS-CoV-2 reference genome for four samples with low CT values.
Figure 4
Figure 4
Bar chart showing samples sequenced with Midnight and Long-range primers with different CT values that passed quality.
See this image and copyright information in PMC

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