OVERVIEW                                                                                                                                                                                                                                                                         
    Example data for HiFiViral SARS-CoV-2 for Synthetic RNA Controls
    These control sequences are NOT FULL LENGTH and consist of non-overlapping 5kb fragments
    Controls were diluted to the following copy numbers: 6E6, 3E6, 1E6, 1E5, 1E4, 1E3, 100, 25  
    Samples with >= 10,000 copies have > 95% genome coverage
    Samples with >= 1000 copies have > 90% genome coverage    

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         	Twist Bioscience control RNAs: 14 (Alpha), 15 (Alpha), 16 (Beta), 17 (Gamma)
    Experimental Design 4 Controls X 8 input quantities X 3 replicates = 96 samples
    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
    m64012_210821_095651.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)
    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)

SMRTLINK FILES
    HiFiViral_SARS-CoV-2_TwistControls.PlateQC.csv		Input file specifying barcode IDs, sample name, plate ID, and well coordinate (required for SMRTlink plateQC report)
    HiFiViral_SARS-CoV-2_TwistControls.Barcoded_Samples.csv	Input file specifying barcode IDs and sample names (required for Demultiplex Barcodes)

PROBE AND BARCODE FILES
    HiFiViral_SARS-CoV-2_Enrichment_Probes.fasta		Probe Arm Sequences
    HiFiViral_Barcoded_M13_Primer_Plate.fasta			Barcoded M13 Primer Sequences

KNOWN ISSUES
    Multi-strain detection for samples with Ct >= 26 is not supported, false positives may be observed