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

Area of Science:

• Virology
• Genomics
• Epidemiology

Background:

• Whole Genome Sequencing (WGS) of SARS-CoV-2 is essential for tracking COVID-19 pandemic evolution and identifying Variants of Concern (VoCs).
• Current popular primer schemes, such as Midnight and ARTIC V4.1, amplify SARS-CoV-2 in smaller segments, which can lead to amplicon dropouts and coverage bias due to mutations and primer interactions.
• These issues result in low-quality genome assemblies with ambiguous regions ('N's), hindering accurate lineage assignment and the monitoring of critical mutations.

Approach:

• This study developed and utilized seven long-range PCR primers designed to tile across the entire SARS-CoV-2 genome.
• The primers generate larger amplicons, approximately 4500 bp in size, simplifying genome sequencing.
• A specific primer set was designed to cover the full-length S-gene, a critical region for viral characterization.

Key Points:

• The long-range PCR approach significantly reduces the likelihood of amplicon dropouts compared to shorter-fragment methods.
• Minimizing coverage bias leads to more complete and accurate SARS-CoV-2 genome sequences.
• This method facilitates more reliable lineage assignment and tracking of mutations in SARS-CoV-2 variants.

Conclusions:

• The use of a reduced set of long-range PCR primers offers a more robust method for sequencing SARS-CoV-2 genomes.
• This approach enhances genome quality, improving the accuracy of epidemiological surveillance and variant tracking.
• This strategy is crucial for effective monitoring of the COVID-19 pandemic and the emergence of new SARS-CoV-2 variants.