nf-core/longraredisease

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Introduction

nf-core/longraredisease is a comprehensive Nextflow pipeline designed for rare disease diagnostics using Oxford Nanopore long-read sequencing data. The pipeline integrates multiple state-of-the-art tools for variant discovery, including:

• Structural Variants (SVs): Sniffles, CuteSV, SVIM with SURVIVOR merging
• Single Nucleotide Variants (SNVs): Clair3 and DeepVariant
• Copy Number Variants (CNVs): Spectre and HiFiCNV
• Short Tandem Repeats (STRs): Straglr
• Methylation: Modkit
• Phasing: LongPhase with haplotagging
• Quality Control: NanoPlot, mosdepth, and MultiQC

The pipeline supports singleton analysis as well as family-based (trio) analyses with variant annotation and phenotype-driven prioritization.

Samplesheet input

You will need to create a samplesheet with information about the samples you would like to analyse before running the pipeline. Use this parameter to specify its location:

--input '[path to samplesheet file]'

The samplesheet is a comma-separated file (CSV) with a header row and the following columns:

Samplesheet format

Input file types

The file_path column can contain:

1. Directory path: A directory containing multiple files of the specified input_type
   • For fastq input: directory with .fastq.gz or .fq.gz files
   • For ubam input: directory with unaligned .bam files
2. Single file path: Path to a single file
   • For fastq input: single .fastq.gz or .fq.gz file
   • For bam or ubam input: single .bam file

The input_type is specified as a pipeline parameter (see Workflow Options).

Example samplesheets

Single sample (FASTQ input)

sample,file_path,hpo_terms,sex,phenotype,family_id,maternal_id,paternal_id
sample1,/path/to/sample1.fastq.gz,HP:0002721;HP:0002110,1,2,,,

Trio analysis

sample,file_path,hpo_terms,sex,phenotype,family_id,maternal_id,paternal_id
proband,/path/to/proband.fastq.gz,HP:0002721;HP:0001263,1,2,family1,mother,father
mother,/path/to/mother.fastq.gz,,2,1,family1,,
father,/path/to/father.fastq.gz,,1,1,family1,,

Multiple families

sample,file_path,hpo_terms,sex,phenotype,family_id,maternal_id,paternal_id
fam1_child,/path/to/fam1_child/,HP:0001263,1,2,family1,fam1_mom,fam1_dad
fam1_mom,/path/to/fam1_mom/,,,family1,,
fam1_dad,/path/to/fam1_dad/,,,family1,,
fam2_child,/path/to/fam2_child.fastq.gz,HP:0002110,2,2,family2,fam2_mom,fam2_dad
fam2_mom,/path/to/fam2_mom.fastq.gz,,,family2,,
fam2_dad,/path/to/fam2_dad.fastq.gz,,,family2,,

Example samplesheets are provided in the assets/ directory:

• samplesheet_test_fastq.csv
• samplesheet_test_ubam.csv

Reference genome

The pipeline requires a reference genome in FASTA format. You can provide this using:

--fasta '/path/to/reference.fasta'

The pipeline supports common reference genomes (GRCh37/hg19, GRCh38/hg38). Ensure the reference genome is indexed (.fai file) or the pipeline will create the index automatically.

Workflow options

Input type

Specify the type of input files using the --input_type parameter:

--input_type 'fastq'   # FASTQ files (default: ubam)
--input_type 'ubam'    # Unaligned BAM files
--input_type 'bam'     # Aligned BAM files

Sequencing platform

Specify the sequencing platform:

--sequencing_platform 'ont'     # Oxford Nanopore (default)
--sequencing_platform 'hifi'    # PacBio HiFi
--sequencing_platform 'pacbio'  # PacBio CLR

Alignment options

By default, the pipeline uses Minimap2 for alignment. You can customize the alignment model or use Winnowmap instead:

--minimap2_model 'map-ont'     # Minimap2 preset (default: auto-detected)
--use_winnowmap true           # Use Winnowmap instead of Minimap2
--winnowmap_model 'map-ont'    # Winnowmap preset
--winnowmap_kmers '/path/to/repetitive_k15.txt'  # Repetitive k-mer file

Analysis modules

Enable or disable specific analysis modules:

--skip_snv false             # Enable SNV calling (default: true)
--skip_sv false              # Enable SV calling (default: true)
--skip_cnv false             # Enable CNV calling (default: true)
--skip_str false             # Enable STR analysis (default: true)
--skip_methylation false     # Enable methylation calling (default: true)
--skip_phasing false         # Enable phasing (default: true)

Trio analysis

Enable family-based analysis for samples with pedigree information:

--trio_analysis true         # Enable trio analysis (default: false)
--haplotag_bam true          # Haplotag BAM files with phase information (default: true)

nextflow run nf-core/longraredisease
–input ./samplesheet.csv
–outdir ./results
–fasta /path/to/reference.fasta
–input_type fastq
-profile docker

This will launch the pipeline with the `docker` configuration profile. See below for more information about profiles.


### Example commands

#### Basic singleton analysis

```bash
nextflow run nf-core/longraredisease \
    --input samplesheet.csv \
    --outdir results \
    --fasta GRCh38.fasta \
    --input_type fastq \
    -profile docker

Trio analysis with all modules enabled

nextflow run nf-core/longraredisease \
    --input samplesheet_trio.csv \
    --outdir results_trio \
    --fasta GRCh38.fasta \
    --input_type ubam \
    --trio_analysis true \
    --haplotag_bam true \
    -profile docker

SV-focused analysis

nextflow run nf-core/longraredisease \
    --input samplesheet.csv \
    --outdir results_sv \
    --fasta GRCh38.fasta \
    --skip_snv true \
    --skip_cnv true \
    --skip_str true \
    --skip_methylation true \
    --skip_phasing true \
    -profile singularity

Analysis with target regions

nextflow run nf-core/longraredisease \
    --input samplesheet.csv \
    --outdir results_targeted \
    --fasta GRCh38.fasta \
    --filter_targets true \
    --targets_bed exome_targets.bed \
    -profile docker

nextflow run nf-core/nanoraredx -profile docker -params-file params.yaml

with:

```yaml title="params.yaml"
input: './samplesheet.csv'
outdir: './results/'
genome: 'GRCh37'
<...>

You can also generate such YAML/JSON files via nf-core/launch.

Updating the pipeline

When you run the above command, Nextflow automatically pulls the pipeline code from GitHub and stores it as a cached version. When running the pipeline after this, it will always use the cached version if available - even if the pipeline has been updated since. To make sure that you’re running the latest version of the pipeline, make sure that you regularly update the cached version of the pipeline:

nextflow pull nf-core/longraredisease

Reproducibility

It is a good idea to specify the pipeline version when running the pipeline on your data. This ensures that a specific version of the pipeline code and software are used when you run your pipeline. If you keep using the same tag, you’ll be running the same version of the pipeline, even if there have been changes to the code since.

First, go to the nf-core/longraredisease releases page and find the latest pipeline version - numeric only (eg. 1.0.0). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.0.0. Of course, you can switch to another version by changing the number after the -r flag.