High sensitivity detection of coronavirus SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Chenyu Li; David N. Debruyne; Julia Spencer; Vidushi Kapoor; Lily Y. Liu; Bo Zhou; Lucie Lee; Rounak Feigelman; Grayson Burdon; Jeffrey Liu; Alejandra Oliva; Adam Borcherding; Hongdong Tan; Alexander E. Urban; Guoying Liu; Zhitong Liu

The coverage was from 1000-to 10,000-fold for S and N genes, and 30-to 500-fold for the mitochondrial chromosome (Fig. 3F) . The coverage is roughly within a 10-fold range, as is observed in human chromosomes (Fig. 3E) . Therefore, increasing sequencing depth might not significantly improve the coverage. This 10-fold difference in coverage has been routinely observed with our multiplex PCR technology (Supplemental Table 3 and Supplemental Fig. 4) . We were able to detect 80% of the regions in S and N genes in libraries generated using 4,500 copies of plasmids, with an average base coverage of 5,000 for a total sequencing depth of 0.6 million reads. A few targets of 150 to 200 bp in length in S and N genes were preferentially amplified. Even when copy number went down to a few copies (3-14), these targets were still detected (Fig.4) . They represented 14% and 11% of the target regions when 14 and 2.8 copies of plasmids were amplified, respectively.