Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers

Abstract

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 the ~30,000 nucleotide SARS-CoV-2 genome that use 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). This study uses seven long-range PCR primers with an amplicon size of ~4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

Figure 1:

Comparison of ARTIC, Midnight, and Long-Range PCR primers. Long-range PCR primers used in this study include seven primer pairs to sequence the whole genome of SARS-CoV-2. The entire S-gene is sequenced by just one long-range primer. The horizontal dotted lines represent the viral genome segments amplified by each primer set.

Figure 2:

IGV plot showing seven different amplicons mapped to the SARS-CoV-2 reference genome for four samples with low CT values. A) Samples with CT values 11.6, 15.1,14.3, and 18.3 from top to bottom, respectively. B) IGV plot for two samples (zoomed for the sample with CT values of 11.6 and 18.3 from top to bottom, respectively. The scale [0–36678] for the top and [0–48783], respectively, represents the range of the total number of the quality filtered reads that mapped to each amplicon region. The details of the reads mapped to different amplicon regions for four samples sequenced are summarized in Table 3 and Figure 3.

Figure 3:

Histogram showing the number of reads mapped to each amplicon in the primer scheme. The ‘align_trim’ report file from the ARTIC pipeline was used in the ONTdeCIPHER tool. If the alignment length between the read and reference is <0.95% of the amplicon length, the read is discarded from the coverage plot. Amplicons are marked as dropped out if the total number of reads assigned to an amplicon is below 50. (X-axis: Seven amplicon regions tiled across the whole genome of SARS-CoV-2, amplifying seven regions along the viral genome. Y-axis: Total number of sequencing reads mapped to each amplicon region).

Figure 4:

Bar chart showing the total number of samples passing quality. A 96-well plate with samples of different CT values was sequenced using Long-range and Midnight primers for comparison. Long-range primers and midnight primers work to accurately assign lineages and generate good-quality genomes for GenBank.

Figure 5:

Bar chart showing samples sequenced with Midnight and Long-range primers with different CT values that passed quality. X-axis: CT value range, Y-axis Number of genomes passing quality.

Figure 6:

Plot of genome coverage and CT values for the genomes that passed quality when sequenced using long-range primers. Long-range primers are effective in sequencing samples with CT values less than 20 to get at least 99 % genome coverage.

Figure 7:

Diagrammatic representation of Oxford Nanopore Sequencing of SARS-CoV-2 using long-range PCR primers. (Figures made using BioRender.com)