Targeted accurate RNA consensus sequencing (tARC-seq) reveals mechanisms of replication error affecting SARS-CoV-2...

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• RNA viruses like SARS-CoV-2 rely on error-prone RNA-dependent RNA polymerases (RdRp) for replication.
• Understanding viral evolution necessitates precise monitoring of replication errors and de novo mutations.
• Existing methods struggle to detect rare mutations in low-input samples, limiting insights into viral dynamics.

Purpose of the Study:

• To introduce a novel targeted accurate RNA consensus sequencing (tARC-seq) method.
• To accurately quantify mutation frequency and types in SARS-CoV-2.
• To investigate viral mutation patterns in both cell culture and clinical samples.

Main Methods:

• Development and application of targeted accurate RNA consensus sequencing (tARC-seq).
• Analysis of de novo mutation rates and types in SARS-CoV-2.
• Identification of mutation hotspots and cold spots across the viral genome.

Main Results:

• An average of 2.68 × 10^-5 de novo errors per replication cycle was observed.
• A C>T mutation bias was detected, not solely explained by APOBEC editing.
• Mutation susceptibility correlated with GC content and transcription regulatory sites.
• tARC-seq identified template switching events, including insertions, deletions, and complex mutations.

Conclusions:

• tARC-seq provides high-precision mutation detection for RNA viruses.
• The study elucidates SARS-CoV-2 mutation patterns, evolutionary hotspots, and mechanisms of genetic diversity.
• Findings offer critical insights into the evolutionary trajectory of SARS-CoV-2.