Starting an Analysis

Once ViroConstrictor is installed, you can use it to analyze your NGS FASTQ data. ViroConstrictor supports multiple analysis workflows depending on your data and requirements.

Before starting your analysis, ensure your input files are properly formatted according to the input formatting guide. ViroConstrictor supports various sequencing platforms and amplicon types - see the amplicons documentation for technical details about amplicon configurations.

You can run analyses in different ways:

• Single-target analysis: Single-target analysis with uniform parameters across all samples
• Multi-target analysis: Use samplesheets to apply different settings per sample (see samplesheet documentation)
• Multi-reference analysis: Let ViroConstrictor choose the best reference for each sample (see multi-reference guide)
• Preset configurations: Use predefined settings for common viral targets (see presets documentation)

For a quick start, see the quickstart which includes a basic command template.

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Overview of Command-Line Options

Below is a brief summary of the available command-line options and their meanings.

Command	Argument	Explanation
--input / -i	[input directory]	The folder containing your input FASTQ files. Both absolute and relative paths are supported.
--output / -o	[output directory]	The desired output folder for data and results. If the folder does not exist, it will be created.
--reference / -ref	[reference FASTA]	The input reference genome sequence in FASTA format.
--primers / -pr	[primer FASTA] / NONE	The primer sequences in FASTA format. Use NONE if your sequencing protocol does not use primers.
--features / -gff	[GFF file] / NONE	The GFF3 file containing genomic features (e.g., open reading frames) that match the given reference FASTA. Use NONE if you do not have a GFF3 file.
--platform	nanopore / illumina / iontorrent	The sequencing platform used to generate the dataset. The default is nanopore.
--amplicon-type / -at	end-to-end / end-to-mid / fragmented	The amplicon type that matches your sequencing experiment or protocol. Options are end-to-end, end-to-mid, or fragmented.
--min-coverage / -mc	Minimum coverage	The minimum coverage for the consensus sequence(s). The default is 30.
--primer-mismatch-rate/ -pmr	Fraction of maximum primer mismatches	The maximum percentage of mismatches allowed during the primer search. Only substitutions are counted (insertions and deletions are not considered). The default is 0.1 (10%). For example, if your primer is 30 nt long, up to 3 mismatches are allowed.
--target / --preset	Name of the viral target	A descriptive name for the viral target under analysis (e.g., "Measles", "Sars-CoV-2", "HPV16", or "Influenza_A"). This target will trigger the use of an analysis preset if one is available. If no preset is available, default settings will be used. See information regarding presets. Disable analysis presets using the --disable-presets flag.
--disable-presets / -dp	N/A	Disables the use of analysis presets so that default analysis settings are used for all samples or viral targets. The viral target must still be provided.
--match-ref / -mr	N/A	Enables the match-ref process for all samples. See additional information regarding the match reference process.
--segmented / -seg	N/A	Indicates that the samples to be analyzed are segmented rather than being based on a single reference genome. This setting applies only to the match-ref process. See details for segmented viruses.
--threads / -t	Number of threads	The number of local threads available for use. The default is the number of available threads on your system.
--dryrun	N/A	Runs the ViroConstrictor workflow without performing any actions. (default: False)
--skip-updates	N/A	Skips the check for a new version.
--version / -v	N/A	Displays the current version of ViroConstrictor and exits.
--help / -h	N/A	Displays the ViroConstrictor help document and exits.