For viral genomic sequencing, total RNA was extracted from nasopharyngeal swabs as described. 70 μL of extracted RNA was treated for 30 min at room temperature with Qiagen DNase I (Qiagen, Germantown, MD, USA) and then cleaned and concentrated with silica spin columns (Qiagen RNeasy MinElute; Qiagen) with a 12 μL water elution. A portion (7 μL) of this RNA was annealed to an rRNA inhibitor (Qiagen FastSelect rRNA HMR; Qiagen) and then reverse-transcribed (cDNA) using random hexamers. The synthesised DNA was strand-ligated and isothermally amplified into micrograms of DNA (Qiagen FX Single Cell RNA Library Kit; Qiagen). A portion (1 μg) of this amplified DNA was sheared and ligated to Illumina-compatible sequencing adapters, followed by six cycles of PCR amplification (KAPA HiFi HotStart Library Amplification Kit; Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA, USA) to enrich for library molecules with adapters at both ends. Next, these sequencing libraries were enriched for a sequence specific to SARS-CoV-2 using biotinylated oligonucleotide baits (myBaits Expert Virus, Arbor Biosciences, Ann Arbor, MI, USA). A further eight to 16 cycles of PCR were done after enrichment (98°C for 45 s, 98°C for 15 s, 60°C for 30 s, repeat for eight to 16 cycles, then 72°C for 60 s and 4°C to complete), and these SARS-CoV-2-enriched sequencing libraries were pooled and sequenced with an Illumina NextSeq 500 (Illumina, San Diego, CA USA) as paired-end 2 × 75 base pair reads using the NextSeq version 2.5 mid-output 150 cycle kit (Illumina).
