Respiratory virus oligo panel

Sequencing for a first set of samples was performed at the Microbial Genome Sequencing Center (Pittsburgh, PA) in three independent sequencing runs. A Maxima double-stranded cDNA RT kit (Thermo Fisher) was used to generate cDNA. An Illumina Flex for Enrichment kit paired with an Illumina Respiratory Virus Oligo Panel (Illumina, Inc.) was used to enrich for respiratory virus cDNA with 15 PCR cycles in the final step. The libraries were then sequenced on a NextSeq 550 to yield on average 119 Mbp of 2 × 75 bp paired-end sequencing reads. For a second set of samples (see Table S1), rRNA depletion was performed, and oligonucleotide capture enriched and unenriched sequencing strategies were compared. The rRNA depletion was done using RiboZero Plus supplemented with a comprehensive “Gut Microbiome” probe set. Libraries were prepared using the Illumina RNA Prep with Enrichment (L) Tagmentation protocol. The rRNA-depleted samples were amplified for 20 cycles. Enrichment was performed using the Illumina Respiratory Virus Oligo Panel.

Purified RNA was prepared for sequencing using the Illumina RNA Prep with Enrichment (Illumina, 20040536) workflow, following the instructions for the Respiratory Virus Oligo Panel (Illumina, 20044311). The resulting libraries were run on a MiSeq instrument, with at least 1.5 million reads per sample. Reads were aligned to the SARS-CoV-2 MA10 genome using STAR (v2.7.9). Output BAM files were input into Geneious Prime, and variant calling was performed with a minimum of 100 reads per base and a quality score of 25, and a cutoff of 0.02 frequency variance. Variants in the nsp12 gene were averaged, then graphed along the length of the gene using a custom R script.

Automated nucleic acid extraction was performed as described previously (20 (link), 21 (link)) using the chemagic 360 (PerkinElmer) following the manufacturer’s protocol. Whole genome sequencing and analysis were performed as previously described (23 (link)).
For a subset of samples, 25 ng of RNA, previously extracted using QIAGEN’s Viral RNA mini kit, was processed following the Illumina RNA Prep with Enrichment (L) Tagmentation protocol with Illumina Respiratory Virus Oligo Panel for single-plex enrichment. Libraries were sequenced on the Illumina MiSeq (2 × 76 bp) or iSeq (2 × 151 bp) platform. FASTQ files were analyzed in Illumina’s BaseSpace using the DRAGEN Pathogen Detection application to generate consensus files. The pangolin web-based application, Phylogenetic Assignment of named Global Outbreak LINeages (PANGOLIN) (https://pangolin.cog-uk.io/) was used to identify the SARS-CoV2 lineages from these consensus sequences. Nextclade (https://clades.nextstrain.org/) was used for clade assignment, sequence quality check, and phylogenetic tree construction.