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The copyright holder for this preprint (which was not peer-reviewed) is the .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 Quantitation for figure 2 was performed with the same quality check as above but quantifying the terminal 6 bases of reference and spike-in sequence for all primary sequences, regardless of whether genomic, spike-in, or mixed sequence dominates. All analysis was performed using custom scripts in R, employing the seqinr and tidyverse packages. Reverse-transcription (RT) and PCR amplification of a SARS-CoV-2 target region is accomplished by directly addition of Viral Transport Media (VTM) to a one-step RT-PCR master mix containing~200 copies of synthetic spike-in DNA. The SARS-CoV-2 target region and spike-in DNA are co-amplified on a standard thermal cycler, and the amplification products are Sanger sequenced. Custom data analysis of the resulting chromatogram is then used to determine whether the specimen is COVID-19 negative or positive. ( B ) Synthetic spike-in is designed with sequence homology to the SARS-CoV-2 target so that it co-amplifies with the SARS-CoV-2 target. A 4-base pair (bp) deletion in the spike-in design enables quantification of relative abundances of spike-in and SARS-CoV-2 DNA from a Sanger sequencing chromatogram. The depicted forward and reverse primers are not to scale. ( C ) Representative Sanger sequencing traces showing pure genomic sequence (top), pure spike-in sequence (middle), and sequencing from a mixture of genomic and spike-in sequences (bottom). The spike-in used has a 4-bp offset compared to wild type (wt), which means that when two sequences are present, the signal from each sequence can be used to estimate their relative abundances (see arrows for examples of paired bases).