Crykey: Comprehensive Identification of SARS-CoV-2 Cryptic Mutations in Wastewater

We present Crykey, a computational tool for rapidly identifying cryptic mutations of SARS-CoV-2. Specifically, we identify co-occurring single nucleotide mutations on the same sequencing read, called linked-read mutations, that are rare or entirely missing in existing databases, and have the potential to represent novel cryptic lineages found in wastewater. While previous approaches exist for identifying cryptic linked-read mutations from specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and for tens of thousands of samples and with increased scrutiny, given their potential to represent either artifacts or hidden SARS-CoV-2 lineages. Crykey fills this gap by identifying rare linked-read mutations that pass stringent computational filters to limit the potential for artifacts. We evaluate the utility of Crykey on >3,000 wastewater and >22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over a three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations representing potential cryptic lineages in wastewater.