Multi-Target Analysis

Multi-target analysis allows you to apply different settings to each sample within a single analysis run. This is useful when analyzing samples with different viral targets, experimental conditions, or analysis parameters.

When to Use Multi-Target Analysis

Use multi-target analysis when you have:

• Multiple viral targets in the same batch
• Different primer sets for different samples
• Varying analysis parameters (coverage thresholds, mismatch rates)
• Mixed experimental protocols

Samplesheet Format

Multi-target analysis requires a samplesheet in Excel (.xlsx), CSV (.csv), or TSV (.tsv) format. You can download an example Excel spreadsheet here.

Required Columns

Mandatory Columns

The following columns are required and analysis will fail without them:

• Sample: Must match FASTQ filenames exactly
• Virus: Viral target name (triggers presets if available)
• Reference: Path to reference FASTA file

Optional Columns

All other columns are optional and will use default values if not specified:

Column	Purpose	Example Values
Primers	Primer file path	/path/to/primers.fasta or NONE
Features	GFF file path	/path/to/features.gff or NONE
Match-ref	Enable reference matching	TRUE or FALSE
Segmented	Segmented virus analysis	TRUE or FALSE
min-coverage	Minimum coverage threshold	30, 50, etc.
primer-mismatch-rate	Primer mismatch tolerance	0.1, 0.15, etc.

Example Samplesheet

Sample	Virus	Match-ref	Segmented	Primers	Reference	Features	min-coverage	primer-mismatch-rate
Sample_1	SARS-CoV-2	FALSE	FALSE	/path/to/sars_cov_2_primers.fasta	/path/to/sars_cov_2_reference.fasta	/path/to/sars_cov_2.gff	50	0.1
Sample_2	Measles	FALSE	FALSE	/path/to/Measles_primers.fasta	/path/to/measles_reference.fasta	/path/to/measles.gff	30	0.15
Sample_3	Influenza_A	TRUE	TRUE	/path/to/Influenza-A_primers.fasta	/path/to/Influenza-A_reference.fasta	NONE	30	0.1
Sample_4	HPV	TRUE	FALSE	NONE	/path/to/HPV_reference.fasta	NONE	10	0.1

Important Notes

Sample Naming

Exact Filename Matching Required

The Sample column must correspond exactly to your FASTQ filenames in the input directory. ViroConstrictor will alert you if names don't match to prevent unexpected results.

Viral Target Names

• The Virus column serves as the viral target name for analysis
• Names trigger analysis presets if ViroConstrictor recognizes them
• If no preset exists, default settings are applied
• See presets documentation for available presets
• Use --disable-presets flag to force default settings for all samples

Running Multi-Target Analysis

Basic Command

Multi-Target Analysis Command

viroconstrictor \
    --input /path/to/FASTQ-files \
    --output /path/to/output-folder \
    --samplesheet /path/to/samplesheet.xlsx \
    --platform nanopore \
    --amplicon-type end-to-end

Combining Samplesheet with Command-Line Parameters

You can combine samplesheet settings with run-wide command-line parameters. This is useful when most parameters are the same across samples, but a few need to be customized per sample.

Mixed Parameter Approach

# Common platform and amplicon type for all samples
# Individual coverage thresholds specified in samplesheet
viroconstrictor \
    --input /path/to/FASTQ-files \
    --output /path/to/output-folder \
    --samplesheet /path/to/samplesheet.xlsx \
    --platform illumina \
    --amplicon-type end-to-end \
    --primer-mismatch-rate 0.15

Parameter priority: Samplesheet values override command-line parameters for individual samples.