Direct comparison of RT-ddPCR and targeted amplicon sequencing for SARS-CoV-2 mutation monitoring in wastewater

Over the course of the COVID-19 pandemic, variants of SARS-CoV-2 have emerged that are more contagious and more likely to cause breakthrough infections. Targeted amplicon sequencing approach is a gold standard for identification and analysis of variants. However, when applied to environmental sample...

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Published in:The Science of the total environment 2022-08, Vol.833, p.155059-155059, Article 155059
Main Authors: Lou, Esther G., Sapoval, Nicolae, McCall, Camille, Bauhs, Lauren, Carlson-Stadler, Russell, Kalvapalle, Prashant, Lai, Yanlai, Palmer, Kyle, Penn, Ryker, Rich, Whitney, Wolken, Madeline, Brown, Pamela, Ensor, Katherine B., Hopkins, Loren, Treangen, Todd J., Stadler, Lauren B.
Format: Article
Language:English
Subjects:
ARTIC
COVID-19
Humans
Mutation
Mutations
Pandemics
RT-ddPCR
SARS-CoV-2
SARS-CoV-2 - genetics
Variants of concern (VoC)
Wastewater
Wastewater-based epidemiology (WBE)
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Summary:Over the course of the COVID-19 pandemic, variants of SARS-CoV-2 have emerged that are more contagious and more likely to cause breakthrough infections. Targeted amplicon sequencing approach is a gold standard for identification and analysis of variants. However, when applied to environmental samples such as wastewater, it remains unclear how sensitive this method is for detecting variant-associated mutations in environmental samples. Here we directly compare a targeted amplicon sequencing approach (using ARTIC v3; hereafter referred to as sequencing) with RT-ddPCR quantification for the detection of five mutations that are characteristic of variants of concern (VoCs) in wastewater samples. In total, 547 wastewater samples were analyzed using both methods in parallel. When we observed positive mutation detections by RT-ddPCR, 42.6% of the detection events were missed by sequencing, due to negative detection or the limited read coverage at the mutation position. Further, when sequencing reported negative or depth-limited mutation detections, 26.7% of those events were instead positive detections by RT-ddPCR, highlighting the relatively poor sensitivity of sequencing. No or weak associations were observed between quantitative measurements of target mutations determined by RT-ddPCR and sequencing. These findings caution the use of quantitative measurements of SARS-CoV-2 variants in wastewater samples determined solely based on sequencing. [Display omitted] •547 wastewater samples were analyzed using RT-ddPCR and ARTIC-based targeted amplicon sequencing in parallel•RT-ddPCR is more sensitive than ARTIC-based amplicon sequencing for detection and quantification on five mutations•Neither AF nor reads containing mutations via sequencing can be directly used to estimate the mutation concentration•Sequencing detections were positively influenced by sequencing depth, SARS-CoV-2 concentration, and mutation concentration•RT-ddPCR should be applied for quantitative analysis and is preferable for early variant detection if targets are known
ISSN:0048-9697
1879-1026
DOI:10.1016/j.scitotenv.2022.155059