ASENext

Overview

ASENext is a Nextflow pipeline for allele-specific expression (ASE) analysis using STAR-WASP for alignment, UMI-tools for deduplication, and phaser for haplotype phasing and ASE detection.

Features

• STAR alignment with WASP mode for allele-specific mapping
• UMI-based deduplication
• Chromosome-specific analysis with configurable chromosome selection
• Beagle phasing integration
• Phaser-based allele-specific expression analysis
• Comprehensive QC with FastQC and MultiQC reporting

Requirements

• Nextflow (>=21.10.3)
• Singularity or Docker
• Reference genome and annotation files
• Beagle reference panel and genetic map (for phasing)

Usage

Basic usage

nextflow run ASENext --input samples.csv --outdir results --chromosome chr11

Input format

The pipeline requires a CSV file with the following columns:

sample,fastq_1,fastq_2,vcf
SAMPLE1,/path/to/sample1_R1.fastq.gz,/path/to/sample1_R2.fastq.gz,/path/to/sample1.vcf.gz

Parameters

Required parameters

• --input: Path to input CSV file
• --outdir: Path to output directory

Reference genome parameters

• --genome: Name of iGenomes reference (e.g., ‘GRCh38’)
• --fasta: Path to reference genome FASTA file (if not using --genome)
• --gtf: Path to GTF annotation file (if not using --genome)
• --star_index: Path to STAR index directory (if not using --genome)
• --gene_features: Path to BED file with gene features for phaser_gene_ae

Chromosome and phasing parameters

• --chromosome: Chromosome to analyze (default: ‘chr11’)
• --beagle_ref: Path to Beagle reference panel VCF
• --beagle_map: Path to Beagle genetic map file

UMI parameters

• --umi_separator: UMI separator character in read IDs (default: ’:‘)

Pipeline steps

1. Input validation and VCF preparation
2. FastQC for raw reads
3. STAR alignment with WASP mode
4. Filtering of WASP-passing reads
5. UMI-based deduplication
6. Sorting and indexing of BAM files
7. Chromosome extraction from VCF
8. Beagle phasing
9. Phaser for haplotype-level expression
10. Phaser_gene_ae for gene-level ASE
11. Extraction of ASE genes
12. MultiQC report generation

Output

The pipeline organizes outputs by sample name in the specified output directory:

• fastqc/: FastQC reports
• star/: STAR alignment results
• wasp/: WASP-filtered BAM files
• umi/: UMI-deduplicated BAM files
• beagle/: Phased VCF files
• phaser/: Phaser results
• ase/: Allele-specific expression results
• multiqc/: MultiQC report

Credits

• Pipeline framework: nf-core
• Tools: STAR, UMI-tools, samtools, bcftools, Beagle, phaser

Contributing

ASENext is under active development and we welcome contributions! If you find a bug, have an idea to improve it, or want to help implement new features (like better sex-chromosome support), feel free to open an issue or submit a pull request.

Let’s build this together.