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Introduction

longraredisease is a comprehensive Nextflow pipeline for Oxford Nanopore sequencing analysis, designed for rare disease research and diagnostics. It delivers high-confidence variant discovery by integrating multiple state-of-the-art tools. longraredisease performs multi-caller structural variant (SV) detection, single nucleotide variant (SNV) calling, copy number variant (CNV) analysis, short tandem repeat (STR) detection, and phasing analysis in a reproducible, modular workflow.

Pipeline Overview

• Structural Variants (SVs): Sniffles, CuteSV, SVIM, with SURVIVOR merging
• Single Nucleotide Variants (SNVs): Clair3, DeepVariant
• Copy Number Variants (CNVs): Spectre, QDNAseq
• Short Tandem Repeats (STRs): STRaglr
• Phasing: LongPhase
• Quality Control: Coverage analysis with mosdepth

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Requirements

Software:

• Nextflow (≥22.10.0)
• Docker or Singularity/Apptainer

Hardware: Will be updated later in the project

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Quick Start

1. Clone the Repository

git clone https://github.com/nourmahfel/nf-core-longraredisease.git
cd nf-core-longraredisease

2. Test Installation

nextflow run main.nf -profile test,docker

3. Run with Your Data

nextflow run main.nf     --bam_dir /path/to/bam/files      --outdir results     -profile docker

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Input Data Requirements

Parameter	Description	Format	Required
--bam_dir	Directory containing BAM files	Directory path	✅
--fasta_file	Reference genome FASTA	.fasta/.fa	✅
--outdir	Output directory	Directory path	✅
--str_bed_file	STR regions for analysis	.bed	✅
--bed_file	Target regions BED file	.bed	Optional
--chrom_sizes	Chromosome sizes file	.txt	Optional

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Configuration Parameters

Core Analysis Options

--snv true/false              # SNV calling (default: true)
--cnv true/false              # CNV calling (default: true)
--str true/false              # STR analysis (default: true)
--phase true/false            # Phasing analysis (default: true)
--phase_with_sv true/false    # Include SVs in phasing (default: true)

SV Calling Parameters

--filter_sv_calls true/false           # Apply coverage-based filtering (default: true)
--min_read_support auto/integer        # Minimum read support (default: auto)
--min_read_support_limit integer       # Minimum support limit (default: 3)
--merge_sv_calls true/false            # Merge calls from multiple callers (default: true)
--max_distance_breakpoints integer     # Max distance for merging (default: 1000)
--min_supporting_callers integer       # Min callers supporting variant (default: 2)
--min_sv_size integer                  # Minimum SV size (default: 30)

SNV Calling Parameters

--clair3_model string                  # Model name (default: r1041_e82_400bps_sup_v500)
--clair3_platform ont/pacbio           # Sequencing platform (default: ont)
--use_deepvariant true/false           # Run DeepVariant alongside Clair3 (default: true)

CNV Calling Parameters

--use_qdnaseq true/false               # Use QDNAseq instead of Spectre (default: false)
--genome_build hg38/hg19               # Genome build (default: hg38)
--qdnaseq_bin_size integer             # Bin size in kb (default: 1000)
--cutoff float                         # CNV calling cutoff (default: 0.5)
--spectre_fasta_file path              # Full genome FASTA for Spectre
--spectre_mosdepth path                # Mosdepth regions file
--spectre_snv_vcf path                 # SNV VCF for Spectre

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Usage Examples

Basic Run

nextflow run main.nf     --bam_dir /data/bam_files     --fasta_file /ref/genome.fasta     --outdir results     -profile docker

SV-Only Analysis

nextflow run main.nf     --bam_dir /data/bam_files     --fasta_file /ref/genome.fasta     --snv false     --cnv false     --str false     --phase false     --outdir sv_results     -profile docker

Targeted Analysis with BED File

nextflow run main.nf     --bam_dir /data/bam_files     --fasta_file /ref/genome.fasta     --bed_file /targets/exome.bed     --use_qdnaseq true     --outdir targeted_results     -profile docker

High-Sensitivity SV Calling

nextflow run main.nf     --bam_dir /data/bam_files     --fasta_file /ref/genome.fasta     --min_supporting_callers 1     --min_sv_size 20     --filter_sv_calls false     --outdir sensitive_sv     -profile docker

Custom Resource Limits

nextflow run main.nf     --bam_dir /data/bam_files     --fasta_file /ref/genome.fasta     --outdir results     -profile docker     --max_cpus 32     --max_memory 128.GB

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Output Structure

results/
├── minimap2/           # Aligned BAM files
├── mosdepth/           # Coverage analysis
├── sniffles/           # Sniffles SV calls
├── cutesv/             # CuteSV SV calls
├── svim/               # SVIM SV calls
├── survivor/           # Merged SV calls
├── clair3/             # Clair3 SNV calls
├── deepvariant/        # DeepVariant SNV calls (if enabled)
├── snv_combined/       # Combined SNV calls
├── longphase/          # Phasing results (if enabled)
├── spectre/            # Spectre CNV calls (if enabled)
├── runqdnaseq/         # QDNAseq CNV calls (if enabled)
├── straglr/            # STR analysis (if enabled)
└── pipeline_info/      # Execution reports

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Configuration Profiles

Available Profiles:

• test: Minimal test dataset
• docker: Use Docker containers
• singularity: Use Singularity containers

Custom Configuration

// custom.config
params {
    max_cpus = 16
    max_memory = '64.GB'
    outdir = '/scratch/results'
}

process {
    withName: 'CLAIR3' {
        cpus = 8
        memory = '32.GB'
    }
}

Run with:

nextflow run main.nf -c custom.config -profile docker

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Test Data

The pipeline includes test data for validation:

• Location: assets/test_data/
• Genome: Chromosome 22 subset
• Samples: Simulated nanopore data
• Runtime: ~10-15 minutes

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Performance Optimization

For Large Datasets

• Increase resource limits: --max_cpus 64 --max_memory 256.GB
• Use faster storage: --outdir /fast_storage/results
• Enable process caching: -resume

For Limited Resources

• Reduce parallel processes: --max_cpus 4 --max_memory 16.GB
• Disable resource-intensive analyses: --use_deepvariant false --cnv false

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Troubleshooting

Common Issues

• Out of Memory Errors: Increase memory limits, e.g. --max_memory 64.GB
• File Not Found Errors: Check file paths and permissions (ls -la /path/to/input/files)
• Container Issues: Try different container engine (-profile singularity)
• SURVIVOR Filename Collisions: Ensure BAM files have unique prefixes Check that filter_sv_calls is properly configured

Getting Help

• Check the .nextflow.log file for detailed error messages
• Use -resume to restart from the last successful step
• Enable debug mode:

nextflow run main.nf -profile test,docker --debug

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Citation

Citation paper to be added soon as writing one.

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Contributing

We welcome contributions! Please see our Contributing Guidelines for details.

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License

This project is licensed under the MIT License – see the LICENSE file for details.

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This pipeline integrates several tools for variant calling: Sniffles, CuteSV, SVIM, SURVIVOR, Clair3, DeepVariant, LongPhase, Spectre, STRaglr