COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens

Figure 1

Workflow of the redundant tiling PCR. A schematic illustration of the redundant tiling PCR strategy for the whole-genome sequencing of SARS-CoV-2 is shown. Multiplex PCR was performed for three groups (orange, green, and blue amplicons) with no overlaps within each multiplex PCR. The primers used for this scheme are listed in Supplementary Table 1. After the first cDNA strand was synthesized via reverse transcription, it was subjected to tiling PCR. Using prior knowledge, consistently overrepresented amplicons were marked with biotin using biotinylated primers (red crossed marks) and subtracted from the multiplex PCR products via streptavidin-dependent subtraction. The subtracted multiplex PCR products were then used for NGS library preparation.