Choosing the Right Workflow

WASP2 supports four major data types. Use this guide to find your workflow.

Data Type	Input	Goal	Start Here
Bulk RNA-seq	BAM + phased VCF	Allele-specific expression (ASE)	Bulk Workflow (RNA-seq / ATAC-seq)
Bulk ATAC-seq	BAM + phased VCF	Allele-specific chromatin accessibility	Bulk Workflow (RNA-seq / ATAC-seq)
scRNA-seq (10x)	Cell Ranger BAM + VCF + barcodes	Per-cell or per-cell-type ASE	Single-Cell Workflow (scRNA-seq / scATAC-seq)
scATAC-seq (10x)	Fragments/BAM + VCF + barcodes	Single-cell allelic imbalance in ATAC peaks	Single-Cell Workflow (scRNA-seq / scATAC-seq)

Decision Flowchart

Step 1: What sequencing assay did you run?

• RNA-seq → go to Step 2

• ATAC-seq → go to Step 3

Step 2: Bulk or single-cell RNA-seq?

• Bulk RNA-seq → Bulk Workflow (RNA-seq / ATAC-seq)

• 10x Chromium scRNA-seq → Single-Cell Workflow (scRNA-seq / scATAC-seq)

• Other single-cell protocol → see Single-Cell Analysis

Step 3: Bulk or single-cell ATAC-seq?

• Bulk ATAC-seq → Bulk Workflow (RNA-seq / ATAC-seq) (use BED peak file as --region)

• 10x scATAC-seq → Single-Cell Workflow (scRNA-seq / scATAC-seq)

Do I Need to Run the WASP Remapping Step?

The remapping step (wasp2-map) corrects reference mapping bias — reads carrying the alternative allele are harder to map than reference-allele reads, causing false-positive imbalance signals.

You need remapping if:

• Your BAM was aligned with a standard aligner (BWA-MEM, STAR, HISAT2, bowtie2)

• You want the most rigorous allele-specific analysis

• You are studying regions near known variants (high variant density)

You can skip remapping if:

• Your BAM was already produced by an unbiased pipeline

• You are doing a quick exploratory analysis

• You are using simulated or controlled data

See Mapping Module for the full remapping workflow.

What VCF Do I Need?

WASP2 requires a phased VCF with heterozygous variants for the sample(s) you are analyzing. Supported formats:

• VCF/BCF (bgzip + tabix indexed)

• PLINK2 PGEN files (with .pvar + .psam)

See Counting Module for VCF format requirements and examples using bcftools to subset and phase.

Nextflow Pipelines

If you prefer a managed workflow with automatic parallelization, containerization, and output publishing, use the bundled Nextflow pipelines instead of the CLI:

• nf-rnaseq — bulk RNA-seq allele-specific expression

• nf-atacseq — bulk ATAC-seq allele-specific chromatin accessibility

• nf-scatac — single-cell ATAC-seq allelic imbalance

• nf-outrider — outlier expression detection with allele-aware correction

See the pipeline-specific documentation for samplesheet format and parameter reference.