Rna prep with enrichment

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.

Sequencing libraries were prepared by Illumina RNA Prep with Enrichment with 8.5 µL RNA input, according to the manufacturer’s protocol. Total RNA for all samples was below the recommended 10 ng. Briefly, RNA is synthesized to cDNA, followed by tagmentation library preparation. The indexed library is then hybridized with the custom probe panel, captured, and, finally, PCR amplified. Pre-enrichment controls were removed from the workflow after purification of the indexed library. Enriched sample libraries were normalized to 200 ng and pooled for three-plex hybridization overnight (16 hours) using the previously described pan-HAV probes (100 uM). Final sequencing libraries were normalized to 20 nM prior to pooling for sequencing on an Illumina NextSeq2000 using a P1 2 × 150 bp reagent kit.