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

We sequenced a library made with 4,500 copies of N and S gene-containing plasmids spiked into 10 ng of human gDNA, which roughly represents 3,300 haploid genomes. Even though the molar ratios of viral targets and human haploid genomes were comparable, N and S genes which encompass about 4kb of targets, were a negligible fraction of the 3 billion base pairs of a human genome. If every region of the human genome were amplified and sequenced at 0.6 million reads per sample, only one read of viral target would be recovered. In fact, our results showed that 16% of the recovered bases, or 13% of the recovered reads, were within the viral N and S genes ( Fig. 3A and Supplemental Table 2 ). 80% and 78% of SARS-CoV-2 and mitochondrial targets were covered, respectively (Fig. 3B) , and the base coverage was significantly higher than human targets (Fig. 3C) . In contrast, only 0.08% of regions in human chromosomes were amplified. Furthermore, the human exonic regions were preferentially amplified (Fig. 3D) . This suggested that the random hexamers deselected a large portion of the human genome, while favorably amplifying regions that were more "random" in base composition. Indeed, long gaps and absence of coverage in very large repetitive regions were observed in human chromosomes (Fig. 3E) . On the contrary, the gaps in SARS-CoV-2 and mitochondrial regions were significantly shorter (Fig. 3F) , whereas the amplified targets overlapped and were longer than human targets (Fig. 3G) .