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Updated: Aug 30, 2025

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing
Florence Nicot1, Pauline Trémeaux1, Justine Latour1
1Virology Laboratory, Toulouse University Hospital, Toulouse, France.
Abstract:
Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. Single-molecule real-time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole-genome sequencing (WGS) of SARS-CoV-2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3-99.5]) than with the amplicon protocol (99.3% [IQR: 69.9-99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS-CoV-2.
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