Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, has necessitated rapid genomic surveillance.
• The ARTIC Network developed a multiplex PCR method for whole-genome amplification of SARS-CoV-2.
• The original ARTIC primer set showed amplification bias, particularly with low viral load samples.

Purpose of the Study:

• To identify the cause of coverage bias in the ARTIC Network's multiplex PCR protocol.
• To develop an improved primer set that minimizes amplification bias for SARS-CoV-2 genome sequencing.

Main Methods:

• Analysis of primer interactions and dimer formation within the ARTIC multiplex PCR primer set.
• In silico prediction of primers involved in significant primer-dimer interactions.
• Design and testing of a modified primer set (version N1, or NIID-1) with alternative primers.

Main Results:

• Primer dimer formation was identified as a major cause of amplicon coverage bias.
• Twelve alternative primers were designed to replace primers predicted to form dimers.
• The new NIID-1 primer set demonstrated improved overall genome coverage compared to ARTIC V1 and V3.

Conclusions:

• Primer dimer formation significantly impacts the accuracy of SARS-CoV-2 whole-genome amplification.
• The NIID-1 primer set offers enhanced performance for SARS-CoV-2 genome sequencing, especially for low viral load samples.
• This improved primer set can aid in more reliable genomic surveillance and research of SARS-CoV-2.