UC Santa Cruz

Recently, wastewater surveillance of viral populations, especially SARS-CoV-2, has become a popular choice to actively monitor community spread of disease. While current methods like digital and quantitative PCR are popular choices for wastewater surveillance, their multiplexing capabilities are limited to the number of color channels available. Additionally, the use of probes requires prior knowledge of a target sequence making identification of an unknown agent impossible. The ability to identify, monitor, and track the spread of viral outbreaks in early stages are essential to preventing the spread of disease and potential pandemics. In this paper, we define a method to identify and quantify viral populations from wastewater samples on the MinION Nanopore. The MinION Nanopore, a Next Generation Sequencing device, is able to sequence nucleic acids without prior knowledge of its sequence and as a result, can distinguish between all unique species in a sample. Furthermore, by determining the number of molecules of a specific species and calculating its starting concentration from mass and read length information, we are able to quantify every unique sequence in a sample.Two nanopore experiments were run to test this model. In the first experiment, we quantified several gene blocks of a known starting concentration to test the precision and accuracy of the device. In the second experiment, we quantified viral RNA from wastewater and aligned the reads to the NCBI viral database to determine the identity of each molecule. Quantification of this run was directly compared to digital PCR results for SCV2 and Pepper Mild Mottle Virus amplification. Although we are only comparing the results of two viruses, depending on the results of this experiment, theoretically, this device has the potential to quantify every single unique organism or pathogen present in a sample.