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Pipeline Overview

The SeqNado pipeline is built on Snakemake and handles the end-to-end processing of various sequencing assays. This page details the standard workflow and assay-specific steps.

Usage

Run the pipeline for a given assay (e.g., ATAC-seq):

seqnado pipeline atac --preset le

For all arguments and presets, see the CLI reference: seqnado pipeline.

General Workflow

Regardless of the assay type, all SeqNado runs follow these core stages:

  1. Quality Control: Raw FASTQ validation using FastQC and Fastq Screen.
  2. Adapter Trimming: Automatic detection and removal of adapters via Trim Galore.
  3. Alignment: Mapping reads to the reference genome (Bowtie2 for DNA, STAR for RNA).
  4. Post-processing: Filtering, sorting, and indexing of BAM files.
  5. Signal Generation: BigWig track creation for visualization.
  6. Summarization: Integration of all QC metrics into a final MultiQC report.

Supported Assays

ATAC-seq (ATAC)

The ATAC-seq pipeline focuses on identifying regions of open chromatin.

  • Filtering: Removal of mitochondrial reads and duplicates.
  • Peak Calling: Uses MACS2 or LanceOtron for accessibility peak detection.
  • QC: Calculates TSS enrichment scores and fragment size distributions.

ChIP-seq (ChIP)

Designed for Protein-DNA interaction mapping.

  • Background Correction: Support for Input/IgG controls.
  • Peak Calling: MACS2 (narrow or broad) and SEACR options.
  • Normalization: Supports spike-in normalization if specified in the config.

RNA-seq (RNA)

Standard transcriptomic profiling.

  • Alignment: Splice-aware mapping using STAR.
  • Quantification: Gene-level counts via featureCounts.
  • Analysis: Optional automated DESeq2 differential expression results.

CUT&Tag (CAT)

Low-input chromatin profiling targeting protein-DNA interactions.

  • Specialized Filtering: Optimized for small fragment sizes.
  • Callers: SEACR integration for sparse signal.

SNP Analysis (SNP)

Variant detection workflows.

  • Alignment/Processing: Standard mapping and BAM post-processing.
  • Variant Calling: Uses established variant calling utilities; outputs VCF/BCF.

Methylation (METH)

Bisulfite sequencing and methylation calling.

  • Processing: Bisulfite-aware read handling.
  • Methylation Calls: Generates cytosine methylation metrics and summaries.

MCC (MCC)

Multiplex chromatin conformation capture.

  • Processing: Contact map-oriented post-processing.
  • Analysis: MCC-specific peak/enrichment outputs and QC metrics.

CRISPR Screens (CRISPR)

Guide-level quantification and screen analysis.

  • Quantification: Count guides/targets across samples.
  • Analysis: Screen statistics and summary tables.

Multiomics (MULTIOMICS)

Run multiple assay types together in one project for integrated outputs. Configure per-assay sections and enable multiomics mode in the config.

Technical Details

Resource Management

SeqNado automatically calculates required cores and memory for each step based on your provided configuration and the available system resources.

Parallelization

The pipeline leverages Snakemake's ability to run samples in parallel, scaling from local machines to large high-performance computing (HPC) clusters.

Next Steps

Once your pipeline is running, you can monitor the progress in the terminal. After completion, visit the Outputs page to understand the result structure.

For command-line options (presets, queues, scaling), see seqnado pipeline.