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

We developed custom bioinformatic analyses to extract the relative abundance of SARS-CoV-2 and spike-in amplified products from Sanger chromatograms and automate analysis of qSanger chromatograms (see Methods). Briefly, peak amplitudes were assigned to either the spike-in or SARS-CoV-2 sequence at each base position, and a linear regression analysis was performed to determine the qSanger ratio between SARS-CoV-2 and spike-in trace intensities. qSanger ratios near 0 were recovered in the samples with 0 GCE, indicating the complete absence of SARS-CoV-2 RNA. (Fig. 1C ). Since all of the SARS-CoV-2 RNA at 10 GCE or qSanger ratios of 3% or greater, this provided further evidence that the limit of detection of qSanger COVID-19 is~10 GCE or fewer. Quantitative analysis of chromatogram peak heights was able to recover a qSanger ratio for even the 5000 GCE condition, and the qSanger ratios had excellent linearity over 10-5000 GCE (Fig. 2C) . Furthermore, qSanger ratios were in good agreement qPCR Ct values (Fig. 2D ).