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

(A) Random hexamer-adapters were used in multiplex PCR to amplify 4,500 copies of plasmids in the background of 3,300 haploid human gDNA molecules. The resulting libraries were sequenced at an average of 0.6 million total reads. Of the total bases recovered, 16% were on SARS-CoV-2 S and N genes. (B) 80% of S and N genes, and 78% of the human mitochondrial chromosome were amplified with >= 1X coverage, while only 0.08% of the human chromosomes were. (C) On average, S and N genes were covered at 2,346X, mitochondria at 77X, and human chromosomes at 20X. (D) Human exons were relatively overamplified about 4-fold higher compared to their actual ratio within the genome. (E) Gaps and long regions of absence of amplification were observed for human chromosomes. An example shown here is chromosome Y. Small gaps were additionally found in the enlarged cluster of amplification. The long absent region (red double arrow) overlapped with the repetitive regions on Y chromosome. (F) Representation of the recovered regions in S and N genes and the human mitochondrial chromosome. The coverage was from 1,000-to 10,000-fold for S and N genes, and 30-to 500-fold for the mitochondrial chromosome. (G) The length of the majority of chromosomal amplification products are clustered around 100bp, while the amplified regions for the S and N genes, as well as the mitochondrial chromosome, were significantly longer.