Tiled amplicon sequencing of SARS-CoV-2: RNA extracts from nasopharyngeal swabs OR VeroE6 cells
Automated extraction of total nucleic acid extraction from samples was carried out using the Roche MagNA pure 96 with the Roche MagNA Pure DNA and total NA kit. The superscript IV VILO Master Mix (Thermo Fisher) was used for reverse-transcription of RNA extracts. Prepared cDNA was then amplified separately using the Midnight ~1.2 kb amplicons tiling the SARS-CoV-2 genome using the Platinum SuperFi Green PCR Mastermix (Thermo Fisher) with 1.5 L of cDNA per reaction. PCR products were cleaned using AMPure XP beads (0.8X bead ratio), and quantified using PicoGreen dsDNA Assay (Thermo Fisher). All 14x amplicon products from a given sample were then pooled at equal abundance, and prepped for short-read sequencing using the Illumina DNA Prep Kit, according to the manufacturers protocol. Samples were multiplexed using Nextera DNA CD Indexes and sequenced using 150 bp paired-end sequencing on an Illumina MiSeq.