Nasal and nasopharyngeal swabs were received at the Nevada State Public Health Lab (NSPHL) or Southern Nevada Public Health Lab (SNPHL) for SARS-CoV-2 diagnostic testing, following a RNA extraction using either a QIAamp Viral RNA Mini Kit (QIAGEN) or Mag-Bind Viral DNA/RNA kit (Omega Biotek). Specimens were tested for the presence of coronaviral RNA using FDA-approved kits that employed RT-PCR to detect SARS-COV-2 RNA. RNA samples from SARS-CoV-2 positive deidentified specimens were used for our studies in accordance with the guidelines of the Institutional Review Board (IRB) of the University of Nevada, Reno, which determined this study to be EXEMPT FROM IRB REVIEW according to federal regulations and university policy.

A set of 200 coronavirus-positive specimens were selected for genome sequencing. Specimens were treated with DNase I (QIAGEN) for 30 min at room temperature and concentrated using RNeasy Minelute spin columns (QIAGEN) based on the manufacturer-supplied protocol. These concentrated samples were converted into Illumina-compatible sequencing libraries with a QIAseq FX Single Cell RNA Library kit (QIAGEN). RNA samples were annealed to a 1:12.5 dilution of QIAseq FastSelect – HMR probes (QIAGEN) to reduce subsequent amplification of human ribosomal RNA. After treatment to remove trace DNA from the samples, a reverse transcription reaction was carried out using random hexamers to generate cDNA. This cDNA was ligated to one another, followed by isothermal linear amplification. Amplified DNA (1 μg) was enzymatically sheared to an average insert size of 300 bp, and Illumina-compatible dual-indexed sequencing adapters were ligated to the ends. Next, about 300 ng of adapter-ligated sample was amplified with 6 cycles of PCR with KAPA HiFi HotStart polymerase (Roche Sequencing Solutions). Enrichment of library molecules containing SARS-CoV-2 sequence was conducted with a myBaits kit and coronavirus-specific biotinylated probes (Arbor Biosciences). Each enrichment used 500 ng of PCR-amplified DNA, was carried out based on manufacturer instructions at a hybridization temperature of 65°C for 16 h, and was completed with 8–16 cycles of PCR using KAPA HiFi HotStart polymerase. Samples were sequenced using an Illumina NextSeq 500 mid-output (2 × 75) kit. The generated FASTQ files were analyzed as described below. The sequences are available at GISAID: hCoV-19/USA/NV-NSPHL-A (0004–0210)/2020.

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