2.3 Next-generation sequencing (NGS)
RNA extracts from the cats (representing one real-time RT-PCR-positive
oropharyngeal and one fecal swab per animal; Figure 1) along with a
pharyngeal swab from the owner (obtained on D7) were further processed
for NGS-based SARS-CoV-2 whole genome sequencing. Amplification of the
complete viral genomes was performed via a commercially available
oligonucleotide panel (Ion AmpliSeq™ SARS-CoV-2 Research Panel, ID:
05280253; Thermo Fisher Scientific). The Ion AmpliSeq™ Library Kit Plus
was used for library preparation, according to the manufacturer’s
instructions. Briefly, the library preparation involved reverse
transcription using the SuperScript™ VILO™ cDNA Synthesis Kit (Thermo
Fisher Scientific), 16 cycles of PCR amplification, adapter ligation,
library purification using Agencourt™ AMPure™ XP (Beckman Coulter, Brea,
CA, USA), and library quantification using the Qubit™ Fluorometer dsDNA
HS Assay Kit. An ion 540™ Chip was prepared using the Ion Chef™
instrument, and NGS reactions were run on an Ion GeneStudio™ S5
semiconductor benchtop sequencer (Thermo Fisher Scientific).