Performance of Long-Read Single-Molecule Real-Time Sequencing for SARS-CoV-2 Genotyping in Clinical Samples.

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Continuous genetic evolution of SARS-CoV-2 necessitates advanced whole genome sequencing for accurate virus typing.
• Various sequencing techniques have been developed to meet the demands of SARS-CoV-2 variant surveillance.

Purpose of the Study:

• To evaluate the performance and accuracy of PacBio single-molecule real-time (SMRT) sequencing for SARS-CoV-2 typing.
• To compare SMRT sequencing with short-read Illumina sequencing for SARS-CoV-2 variant identification.

Main Methods:

• PacBio SMRT sequencing was used to analyze 1646 clinical samples collected between January 2023 and June 2024.
• Reproducibility was assessed using two internal quality controls with high median read depths (1154X and 1059X).
• Performance was compared against Illumina sequencing using 271 samples from the same period.

Main Results:

• SMRT sequencing demonstrated an overall sensitivity of 83.6%, correlated with viral load.
• Illumina sequencing achieved a higher overall sensitivity of 90.8% over the same period.
• SMRT sequencing proved more efficient in identifying distinct lineages during co-infections due to long fragment amplification, despite minor lineage discrepancies attributed to rapid Pango taxonomy updates.

Conclusions:

• Both PacBio SMRT and Illumina sequencing provide accurate SARS-CoV-2 typing for epidemiological and clinical studies.
• SMRT sequencing offers advantages in resolving co-infections, complementing the higher sensitivity of Illumina sequencing.
• Ongoing evaluation of sequencing technologies is crucial for effective SARS-CoV-2 surveillance amidst viral evolution.