Nextflow pipeline for quality filtering, mapping, and AID-motif variant detection in Oxford Nanopore amplicon sequencing data. Specifically designed for immunoglobulin rearrangements to detect somatic hypermutation patterns, particularly C>T and G>A transitions associated with Activation-Induced Deaminase (AID) activity.

1. Reference Preparation: Converts AB1 files to FASTA format
2. Quality Filtering: Filters reads by quality and length (fastp)
3. Read Mapping: Aligns reads to reference sequences (minimap2)
4. Variant Calling: Pileup-based variant detection with dual frequency filters
   • Requires minimum read depth and allele frequency
   • Alt dominance filter: alternative allele must dominate among non-reference bases
   • Tracks variants in individual reads for per-sequence analysis
5. AID Motif Annotation: Annotates variants with AID motif patterns (WRCY, WA, RCG) and spot types

CSV sample sheet with columns: sample, fastq_file, reference_ab1 (optional), reference_fa (optional). Each sample requires either AB1 or FASTA reference. If there are multiple FASTQ files per sample, provide a reference for only one of them.

By default, results are stored in the results/ directory with the following structure:

• BAM files: Sorted and indexed alignment files (results/bam/)
• QC reports: fastp quality control reports in JSON and HTML formats (results/fastp/)
• Variant tables:
  • Per-position variants with AID motif annotations (results/variants/*_variants.csv)
  • Per-sequence variant summaries with C>T and G>A counts (results/variants/*_variants_per_seq.csv)

• Nextflow (>=24.04). See Nextflow installation guide for instructions.
• Docker or Apptainer/Singularity (for containerized execution). See Docker quick start guide or Apptainer quick start guide for installation instructions.

You need to provide at least a sample sheet CSV file via --samples_file, where you specify the paths to your FASTQ files and reference files. In the example_input/ folder you can find an example samples.csv file and example data.

To run the pipeline, use one of the following commands (adjust paths as needed):

# With Docker
nextflow run main.nf -profile docker --samples_file example_input/samples.csv

# With Apptainer/Singularity
nextflow run main.nf -profile apptainer --samples_file example_input/samples.csv

The pipeline can be configured via command-line parameters. The following parameters are available: