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

For both Seracare negative and positive control specimens, eight replicates each were performed on the cross-product of conditions for Luna vs OneTaq polymerases and direct VTM vs purified RNA, for 64 reactions total. 25ul of the Seracare specimen (corresponding to 125 GCE) was added to a 100ul total reaction volume. Additionally, 16 replicates of no-template controls were performed for each polymerase wherein nuclease free water was added to the reaction. All 32 NTC samples across all conditions were negative by qSanger assay and analysis (Fig. 3A) . Nearly all Seracare negative control samples were determined to be negative by qSanger; indeterminate results were obtained from 2 purified RNA Luna specimens and 1 purified OneTaq specimen. All Seracare positive controls were identified by qSanger except for a OneTaq direct VTM specimen (Fig. 3A ). Samples were classified as undetermined due to low chromatogram signal intensity (Signal to noise ratio < 500) or lack of sequence alignment. Possible reasons for undetermined chromatograms could be failure in RNA extraction, PCR amplification, or cycle sequencing. Since the majority of undetermined specimens used purified RNA, the possibility that the majority of assay failures were due to the RNA extraction process itself, perhaps by carryover of high salt buffers, should be considered.