Abstract
ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low-frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole-genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low-frequency variants. Our analyses reveal that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina Respiratory Viral Oligo panel and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high-quality sequence data from low-viral-load samples for public health and genomic surveillance purposes.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern
Seven SARS-CoV-2-positive clinical specimens were cultured as representatives of different SARS-CoV-2 genomic clusters that were cocirculating in New South Wales (NSW) between February and April 2020 [3]
The genome of isolate 2 reverted to wild type at position C:26213; the single nucleotide polymorphisms (SNPs) C:26213:T detected in the original clinical specimen was still present as a low-frequency variant
Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high-quality sequence data from low-viral-load samples for public health and genomic surveillance purposes. A high level of variability exists between sequencing protocols in obtaining complete SARS-CoV-2 genomes, from clinical samples with low viral loads (as reflected by real-time PCR [RT-PCR] cycle threshold [CT] values), such as those collected from patients without symptoms, with mild disease, or late in the course of infection. The accuracy required to detect and call variants using different protocols has not been adequately validated All of these factors—sequencing method, reproducibility, and thresholds for variant calling—may affect the quality and impact of genomic surveillance and public health efforts to contain outbreaks
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
