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The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.029199 doi: bioRxiv preprint Seracare positive control specimens (Fig. 3B ). Since each reaction contained 125 GCE of Sars-CoV-2 and 200 GCE of spike-in, we expected to measure a qSanger ratio of 0.625. The OneTaq results yielded a qSanger ratio consistently around 2-3.5. This discrepancy could be due to a slightly more efficient amplification of the SARS-CoV-2 sequence compared to spike-in sequence. Remarkably, Luna polymerase mix yielded a qSanger ratio of 0.74 +/-0.04 s.e.m. for VTM and a qSanger ratio of 0.97 +/-0.04 s.e.m. for purified RNA, which is very close to the expected ratio. The~20% difference is on par with typical imprecisions for pipetting and DNA quantification. The coefficient of variation (CV) for the VTM and RNA purified Luna assays were 12% and 16%, respectively. Notably, this is in good agreement with the imprecision associated with measuring~125 molecules at the Poisson limit. Since Luna exhibited better accuracy and precision compared to OneTaq, and the Luna direct VTM method resulted in correct calls for all NTC, Seracare positive, and negative specimens without any failed reactions, subsequent experiments were performed with Luna polymerase mix.