Ion Torrent S5 sequencing; SARS-CoV-2 genome sequencing from post-mortem FFPE lung tissues
The Ion AmpliSeq™ SARS-CoV-2 Research Panel
For library construction, 10ng of RNA (5ng and 1ng for testing robustness) were retro-transcribed   with the SuperScript™ VILO™ (ThermoFisher Scientific) in accordance with the manufacturer's instructions. The Ion AmpliSeq™ SARS-CoV-2 Research Panel (Ion AmpliSeq™, ThermoFisher Scientific) was used to manually prepare the libraries. The panel consists of two 5X primer pair pools that target 237 amplicons specific to the SARS-CoV-2 coronavirus and 5 human expression controls. The amplicon lengths range from 125 base pairs (bp) to 275bp and are designed to provide >99% coverage of the SARS-CoV-2 genome, covering from position 43 to position 29 842 (positions related to reference sequence3). Amplification condition was 98°C for two minutes for initial denaturation, followed by 20, 25 or 30 cycles (Supplemental Table 1) at 98°C for 15 seconds and 60°C for four minutes. Then, the amplicons were digested, barcoded, purified using AMPure XP Beads (Beckman Coulter, Brea, California, USA). The libraries were amplified by PCR and size selection was performed using AMPure XP Beads (Beckman Coulter). The Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef and the Ion Chef (ThermoFisher Scientific) were used for template preparation and chip loading. Sequencing was performed using the S5 Gene StudioTM instrument (ThermoFisher Scientific).