NGS-based ACE2 Amplicon Sequencing

The library for DNA sequencing using NGS was generated using the Fluidigm Access Array microfluidic chip as previously described^{38 (link)}. The confirmed target-specific ACE2 primers were then tagged with Fluidigm specific tag sequences and used for targeted next-generation sequencing^{38 (link)}. The prepared amplicon libraries were purified using AMPure XP beads (Beckman Coulter, USA) and quantified using a High Sensitivity DNA assay kit on BioAnalyzer (Agilent, USA). The libraries were further diluted to 1 pg for direct input into the emulsion polymerase chain reaction (PCR) with Ion SphereTM particles using the Ion Template OT2 kit (Ion OneTouch™ instrument). Enrichment of the clonal beads was carried out using the Ion OneTouch™ ES system following the manufacturer’s instructions (Thermo Fischer, USA). The pooled libraries were then sequenced using the Ion 520™ Chip on the Ion S5 XL Semiconductor sequencer, following the manufacturer’s instructions (Thermo Fisher).