A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

Devon Chandler-Brown; Anna M. Bueno; Oguzhan Atay; David S. Tsao

The copyright holder for this preprint (which was not peer-reviewed) is the . Figure 3 . qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules. author/funder. All rights reserved. No reuse allowed without permission.