OVERVIEW Data collected by Inqaba Biotec using HiFiViral SARS-CoV-2 Run of 39 samples suspected of being Omicron plus 3 Delta variant samples PROTOCOL https://www.pacb.com/wp-content/uploads/Procedure-Checklist-%E2%80%93-PacBio-HiFiViral-High-Throughput-Multiplexing-for-Full-Viral-Genome-Sequencing-of-SARS-CoV-2.pdf SMRTLINK DOCUMENTATION https://www.pacb.com/wp-content/uploads/SMRT_Link_User_Guide_v10.2.pdf METHODS Samples 25 Human swabs stored in Bosphore liquid collection media RNA isolation Three methods used: All samples (25) were collected using Bosphore liquid collection media https://www.launchdiagnostics.com/product-list/bosphore-ex-tract-dry-swab-rna-solution/ Batch A: samples processed directly from the Bosphore media. Upon collection, samples were immediately vortexed and incubated at 95*C for 8 minutes to release the viral material Batch B: samples from the Bosphore media were cleaned with Zymo RNA Clean and Concentrator before RT PCR, cDNA amplification and barcoding https://www.zymoresearch.com/collections/rna-clean-concentrator-kits-rcc Batch C: Viral RNA was extracted from Bosphore media using Zymo Quick-Viral RNA kit before RT PCR, cDNA amplification and barcoding https://www.zymoresearch.com/collections/quick-rna-viral-kits Viral Enrichment HiFiViral SARS-CoV-2 Kit Library prep SMRTbell® Express Template Prep Kit 2.0 + Enzyme Clean Up Kit 2.0 Sequencing Sequel IIe System, Sequel II Binding Kit 2.1, Sequel II Sequencing Plate 2.0, 8hr movie with no pre-extension Software ICS v10.1, SMRT® Link v10.2 DATA FILES m64187e_211217_130958.hifi_reads.bam HiFi reads (unprocessed) samples.hifi_reads.fastq.zip Processed HiFi reads: demultiplexed, trimmed, and QCed (96 samples) samples.mapped.bam.zip Processed HiFi reads mapped to Wuhan reference NC.045512.2 (96 samples) samples.consensus.fasta.zip Consensus sequences (96 samples) samples.consensus_mapped.bam.zip Consensus sequences (split at interior Ns) and mapped to Wuhan reference NC.045512.2 (96 samples) samples.vcf.zip Variant calls relative to Wuhan reference NC.045512.2 (96 samples) samples.raw.vcf.zip Variant calls relative to Wuhan reference NC.045512.2 before filtering on allele frequency (96 samples) sample_summary.csv Table of results by sample all_samples_coverage.png Summary of coverage across all samples samples.coverage.png.zip Coverage plot for each sample (96 samples) InqabaOmicronSamples.csv Table of results plus sample info (Ct, clade assignment, RNA extraction method, etc) SMRTLINK FILES InqabaOmicronSamples_PlateQC.csv Input file specifying barcode IDs, sample name, plate ID, and well coordinate (required for SMRTlink plateQC report) InqabaOmicronSamples_Barcoded_Sample.csv Input file specifying barcode IDs and sample names (required for Demultiplex Barcodes) PROBE AND BARCODE FILES HiFiViral_Barcoded_M13_Primer_Plate.fasta Barcoded M13 Primer Sequences