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

Area of Science:

• Genomics
• Virology
• Molecular Biology

Background:

• Whole Genome Sequencing (WGS) is vital for SARS-CoV-2 pandemic surveillance.
• Existing primer schemes like Midnight and ARTIC V4.1 can suffer from amplicon dropouts and coverage bias due to mutations and primer interactions.
• These issues lead to low-quality genomes, inaccurate lineage assignments, and challenges in tracking Variants of Concern (VoCs).

Purpose of the Study:

• To evaluate the efficacy of long-range PCR primers for SARS-CoV-2 whole genome sequencing.
• To compare the performance of long-range primers against established methods like Midnight primers.
• To assess the utility of long-range primers for accurate variant identification, particularly for the S-gene.

Main Methods:

• Utilized a set of seven long-range PCR primer pairs to generate approximately 4500 bp amplicons covering the entire SARS-CoV-2 genome.
• Employed Oxford Nanopore sequencing for clinical isolates.
• Sequenced 94 clinical isolates to compare long-range primers with Midnight primers.

Main Results:

• Long-range primers significantly reduce amplicon dropout and coverage bias compared to shorter amplicon methods.
• This approach enables high-quality whole genome sequencing of SARS-CoV-2.
• Primers flanking the S-gene provide reliable identification of SARS-CoV-2 variants.

Conclusions:

• Long-range PCR primers are effective for single-molecule sequencing of RNA viruses like SARS-CoV-2.
• This method enhances the surveillance of emerging variants by providing more accurate genomic data.
• The use of long-range primers improves the reliability of lineage assignment and tracking of mutations in VoCs.