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The copyright holder for this preprint (which was not peer-reviewed) is the .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 4 . qSanger detects as little as 20 GCE without RNA purification. ( A ) Representative Sanger sequencing traces of negative control virus (left) and SARS-CoV-2 sequence containing virus (right). Even when the signal is too low to detect mixed bases, the 3' offset caused by the deletion in the spike-in compared to the genomic sequence identifies positive samples. The sequencing peaks identified in the inset correspond to spike-in sequence offset by 4 bp (see paired arrows). (B) Twenty samples each of negative control virus and SARS-CoV-2 sequence containing virus were directly added to RT-PCR master mix with 100 spike-in molecules and Sanger sequencing was performed. All samples that successfully sequenced were accurately identified (one negative sample was undetermined due to sequencing failure). author/funder. All rights reserved. No reuse allowed without permission.