---
title: Epigenomics inputs and parameters
description: >-
  BAM file requirements, peak calling parameters, genome assemblies, statistical
  thresholds, and methylation options for all epigenomics tools.
tags:
  - epigenomics
  - inputs
---

You provide an aligned BAM file per sample. The peak- and methylation-calling tools (`atacseq_peaks`, `chipseq_peaks`, `methylation_call`) take a BAM directly; the differential and DMR tools (`atacseq_differential`, `chipseq_differential`, `methylation_dmr`) instead take the per-sample peak or methylation files those calling tools produced, plus a sample metadata file and a contrast string — run the calling step on each replicate first. BAM quality (sorted, indexed, deduplicated) and running the calling step before the differential step are what most affect whether the results are trustworthy.

## Epigenomics inputs and parameters

## BAM file requirements

All epigenomics tools accept aligned BAM files. For best results, BAM files should meet these criteria:

| Requirement | Details |
|-------------|---------|
| Format | BAM (Binary Alignment Map) |
| Sorted | Coordinate-sorted |
| Indexed | BAI index file present |
| Duplicates | Marked (Picard or samtools markdup) |
| Alignment | Aligned to a supported reference genome |
| Quality | MAPQ ≥ 30 recommended (filtered by tools) |

### Recommended preprocessing

| Step | Tool | Command Example |
|------|------|----------------|
| Alignment | BWA-MEM2 or Bowtie2 | `bwa-mem2 mem ref.fa reads.fq` |
| Sort | samtools | `samtools sort -o sorted.bam` |
| Mark duplicates | Picard | `picard MarkDuplicates` |
| Index | samtools | `samtools index sorted.bam` |

## Supported reference genomes

The calling tools (`atacseq_peaks`, `chipseq_peaks`, `methylation_call`) accept a `genome` parameter restricted to two values; the differential and DMR tools work from files already tied to a genome and don't take a separate genome parameter.

| Genome | Organism | Assembly |
|--------|----------|----------|
| `hg38` | Human | GRCh38 (default) |
| `mm10` | Mouse | GRCm38 |

## ATAC-seq Peak Calling parameters

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `bam_file_uri` | string | Yes | -- | URI of the aligned ATAC-seq BAM file |
| `genome` | string | No | `"hg38"` | Reference genome: `"hg38"` or `"mm10"` |
| `peak_caller` | string | No | `"macs2"` | Peak calling algorithm: `"macs2"`, `"hmmratac"`, `"genrich"` |
| `broad_peaks` | boolean | No | `false` | Call broad peaks instead of narrow |

### Peak mode selection

| `broad_peaks` | Best For | Typical Width |
|----------------|----------|---------------|
| `false` (narrow) | Promoters, TF footprints, NFRs | 100-500 bp |
| `true` (broad) | Large regulatory domains, super-enhancers | 1,000-50,000 bp |

## ATAC-seq Differential parameters

`atacseq_differential` takes the peak files `atacseq_peaks` produced — it does not take raw BAMs.

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `peak_files` | string[] | Yes | -- | URIs of peak files from `atacseq_peaks`, one per sample |
| `metadata_file_uri` | string | Yes | -- | URI of a sample metadata CSV (`sample`, `condition` columns) |
| `contrast` | string | Yes | -- | Contrast to test, e.g. `"treated_vs_control"` |
| `method` | string | No | `"diffbind"` | Differential method: `"diffbind"` or `"deseq2"` |

## ChIP-seq Peak Calling parameters

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `treatment_bam_uri` | string | Yes | -- | URI of the ChIP treatment BAM file |
| `control_bam_uri` | string | No | -- | URI of the input/control BAM file (strongly recommended) |
| `genome` | string | No | `"hg38"` | Reference genome: `"hg38"` or `"mm10"` |
| `histone_mark` | string | No | -- | Histone mark name (e.g. `H3K4me3`, `H3K27ac`) — informs peak calling |
| `peak_caller` | string | No | `"macs3"` | `"macs3"` (default, successor to MACS2), `"macs2"`, or `"sicer2"` |

### Peak caller by mark

| Histone Mark / Factor | Recommended `peak_caller` | Signal Shape |
|-----------------------|---------------------------|--------------|
| H3K4me3 | `macs3` (default) | Sharp, promoter-associated |
| H3K27ac | `macs3` (default) | Sharp, enhancer/promoter |
| H3K4me1 | `macs3` (default) | Moderate, enhancer |
| H3K27me3 | `sicer2` | Diffuse, repressive domains |
| H3K36me3 | `sicer2` | Diffuse, gene body |
| H3K9me3 | `sicer2` | Diffuse, heterochromatin |
| CTCF | `macs3` (default) | Sharp, insulator binding |

## ChIP-seq Differential parameters

`chipseq_differential` takes the peak files `chipseq_peaks` produced — it does not take raw BAMs.

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `peak_files` | string[] | Yes | -- | URIs of peak files from `chipseq_peaks`, one per sample |
| `metadata_file_uri` | string | Yes | -- | URI of a sample metadata CSV (`sample`, `condition` columns) |
| `contrast` | string | Yes | -- | Contrast to test, e.g. `"treated_vs_control"` |

## Methylation Calling parameters

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `bam_file_uri` | string | Yes | -- | URI of the bisulfite-aligned BAM file |
| `genome` | string | No | `"hg38"` | Reference genome: `"hg38"` or `"mm10"` |
| `method` | string | No | `"bismark"` | Methylation calling method: `"bismark"` or `"bsmap"` |
| `min_coverage` | integer | No | `5` | Minimum read coverage to report a CpG |

The tool reports CpG methylation; it does not take a context parameter to switch to CHG/CHH (plant-only contexts).

## Methylation DMR Detection parameters

`methylation_dmr` takes the methylation files `methylation_call` produced — it does not take raw BAMs.

| Parameter | Type | Required | Default | Description |
|-----------|------|----------|---------|-------------|
| `methylation_files` | string[] | Yes | -- | URIs of methylation files from `methylation_call`, one per sample |
| `metadata_file_uri` | string | Yes | -- | URI of a sample metadata CSV (`sample`, `condition` columns) |
| `contrast` | string | Yes | -- | Contrast to test, e.g. `"tumor_vs_normal"` |
| `method` | string | No | `"dmrseq"` | DMR detection method: `"dmrseq"`, `"bsseq"`, `"methylkit"` |

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