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Pipeline Outputs

All SeqNado analysis results are organized within the seqnado_output/ directory (or your custom output directory specified during configuration). This page describes the structure and types of files you can expect from your pipeline runs.

General Output Structure

seqnado_output/{assay}/       # Assay-specific directory
├── seqnado_report.html       # Main interactive QC report (MultiQC)
├── protocol.txt              # Auto-generated data processing protocol
├── aligned/                  # Final BAM alignment files
├── bigwigs/                  # BigWig coverage tracks
├── peaks/                    # Peak calling results (ATAC, ChIP, CUT&Tag)
├── readcounts/               # Quantification files (RNA, CRISPR)
├── qc/                       # Quality control metrics
├── hub/                      # UCSC Genome Browser hub
├── heatmap/                  # DeepTools heatmap and metaplot PDFs
├── motifs/                   # Motif analysis results (if enabled)
├── tag_dirs/                 # HOMER tag directories
├── resources/                # Normalisation factors (spike-in)
├── genome_browser_plots/     # PlotNado visualisations (if configured)
├── geo_submission/           # GEO submission-ready files (if enabled)
├── methylation/              # Methylation calls (METH only)
├── variant/                  # VCF files (SNP only)
└── logs/                     # Process execution logs

Note

Not all directories will be present for every assay. The exact output depends on your assay type and configuration options.

Main Entry Point: SeqNado Report

The seqnado_report.html file is your primary analysis report, generated by MultiQC. It provides:

  • QC Summary: FastQC, alignment statistics, and quality metrics
  • Sample Overview: All samples displayed with key metrics
  • Peak Statistics: Number of peaks called, genomic distribution (for applicable assays)
  • Library Complexity: Duplication rates and unique read counts
  • Multi-Sample Comparisons: Side-by-side QC metrics

A protocol.txt file is also generated, providing an auto-generated data processing protocol describing the steps performed.

Viewing the Report

Open seqnado_report.html in any modern web browser. No server required!

Core Output Files

Alignment Files (aligned/)

The BAM processing pipeline runs through several intermediate stages (sorting, blacklist filtering, duplicate removal, optional Tn5 shifting, quality filtering), but all intermediate files are temporary and deleted after the pipeline completes. Only the final processed BAM files are retained:

aligned/
├── {sample}.bam                  # Final processed BAM
└── {sample}.bam.bai              # BAM index

Processing chain (intermediates are removed automatically):

raw → sorted → blacklist_regions_removed → duplicates_removed → shifted_for_tn5_insertion (ATAC/CUT&Tag only) → filtered → final

File Formats:

  • BAM: Binary alignment format, viewable with samtools/IGV
  • BAI: Index files for rapid random access

Coverage Tracks (bigwigs/)

Genome-wide signal tracks for visualisation, organised by tool and scaling method:

bigwigs/
├── {method}/                         # deeptools, homer, or bamnado
│   ├── unscaled/                     # Default unscaled tracks
│   │   └── {sample}.bigWig
│   ├── csaw/                         # CSAW-normalised (if enabled)
│   │   └── {sample}.bigWig
│   ├── spikein/                      # Spike-in normalised (if applicable)
│   │   └── {spikein_method}/
│   │       └── {sample}.bigWig
│   └── merged/                       # Consensus group merged tracks
│       └── {group}.bigWig

For RNA-seq, stranded bigwigs are produced with _plus and _minus suffixes:

bigwigs/{method}/unscaled/{sample}_plus.bigWig
bigwigs/{method}/unscaled/{sample}_minus.bigWig

Pileup Tools:

  • DeepTools: bamCoverage-based tracks (supports all scaling methods)
  • HOMER: makeBigWig.pl-based tracks (unscaled and merged only)
  • BamNado: Custom pileup tool (unscaled and merged only)

Scaling Methods:

  • unscaled: No normalisation applied
  • csaw: CSAW-based normalisation (DeepTools only)
  • spikein: Spike-in normalisation using external control DNA (DeepTools only)
  • merged: Merged tracks across consensus groups

Peak Calls (peaks/)

Peak calling results are simplified to 3-column BED files (chr, start, end):

peaks/
├── {method}/                  # macs2, macs3, homer, lanceotron, or seacr
│   ├── {sample}.bed           # Individual sample peaks
│   └── merged/                # Consensus peaks across replicates
│       └── {group}.bed

Supported Peak Callers:

  • MACS2: Standard peak calling
  • MACS3: Updated MACS peak calling
  • HOMER: findPeaks-based peak calling
  • LanceOtron: ML-based peak calling
  • SEACR: Sparse Enrichment Analysis for CUT&RUN/CUT&Tag

Peak File Format:

  • BED: Simple 3-column genomic coordinates (chr, start, end) extracted from each caller's native output

Read Counts (readcounts/)

Quantification files for RNA-seq and CRISPR assays:

readcounts/
├── feature_counts/
│   └── read_counts.tsv         # Combined featureCounts output
└── salmon/                     # RNA-seq only
    └── salmon_counts.csv       # Combined Salmon quantification

Quality Control (qc/)

Comprehensive QC metrics:

qc/
├── fastqc_raw/                     # FastQC reports on raw reads
│   ├── {sample}_1_fastqc.html      # Read 1 (paired-end)
│   └── {sample}_2_fastqc.html      # Read 2 (paired-end)
├── fastq_screen/                   # Contamination screening (if enabled)
│   ├── {sample}_1_screen.html
│   └── {sample}_2_screen.html
├── qualimap_bamqc/                 # BAM quality metrics (non-RNA assays)
│   └── {sample}/
│       └── qualimapReport.html
├── qualimap_rnaseq/                # RNA-specific BAM QC (RNA assay only)
│   └── {sample}/
│       └── qualimapReport.html
├── alignment_stats.tsv             # Aggregated alignment statistics
├── library_complexity/             # Picard duplicate metrics
│   └── {sample}.metrics
└── frip_enrichment/                # FRiP scores (if enabled, peak-calling assays)
    └── {method}/
        ├── {sample}_frip.pdf
        └── {sample}_frip.txt

Note

For single-end data, FastQC and FastQ Screen files use {sample}_fastqc.html / {sample}_screen.html without the _1/_2 suffix.

QC Metrics Include:

  • Read quality scores per base position
  • GC content distribution
  • Adapter content
  • Duplication rates
  • Mapping statistics
  • Coverage uniformity

UCSC Genome Browser Hub (hub/)

Ready-to-load UCSC track hub generated by TracKNado:

hub/
└── {hub_name}.hub.txt     # Hub description file (default: seqnado_hub.hub.txt)

The hub structure (genomes.txt, trackDb.txt, etc.) is generated by TracKNado and includes links to your bigWig and other track files.

Usage:

  1. Upload the hub/ directory to a web-accessible location
  2. Load in UCSC Genome Browser using the hub URL
  3. Or use locally with IGV/other genome browsers

Heatmaps (heatmap/)

DeepTools-generated heatmaps and metaplots (for assays with peak calling):

heatmap/
├── heatmap.pdf             # Signal heatmap over regions of interest
└── metaplot.pdf            # Average signal profile

Motif Analysis (motifs/)

Motif enrichment analysis for peak-calling assays (if enabled):

motifs/
├── homer/                      # HOMER findMotifsGenome results
│   └── {peak_method}/
│       └── {sample}/
└── meme/                       # MEME-ChIP results (if enabled)
    └── {peak_method}/
        └── {sample}/

Genome Browser Plots (genome_browser_plots/)

Publication-ready visualisations generated with PlotNado (if configured with plotting coordinates):

genome_browser_plots/
├── {region_name}.{format}             # Named regions from BED file
├── {chr}-{start}-{end}.{format}       # Unnamed regions use coordinates
└── template.toml                      # PlotNado configuration template

Output format can be svg, png, or pdf as configured.

GEO Submission (geo_submission/)

Pre-formatted files for GEO/SRA submission (if enabled):

geo_submission/
├── samples_table.txt                 # Sample metadata (TSV format)
├── md5sums.txt                       # Combined checksums
├── raw_data_checksums.txt            # Checksums for raw FASTQ files
├── processed_data_checksums.txt      # Checksums for processed files
├── upload_instructions.txt           # Instructions for GEO upload
├── {sample}_1.fastq.gz               # Symlinks to raw FASTQ files
├── {sample}_2.fastq.gz
├── {sample}_{method}_{scale}.bigWig  # Renamed processed bigWig files
├── {sample}_{method}.bed             # Renamed peak files
└── {assay}/                          # Upload directory

Assay-Specific Outputs

ATAC-seq

ATAC-seq includes Tn5 insertion site correction during BAM processing and supports all peak callers. LanceOtron is the default peak caller.

Key outputs:

  • aligned/{sample}.bam -- Tn5-shifted, filtered alignments
  • peaks/lanceotron/{sample}.bed -- ML-based peak calls (default)
  • bigwigs/{method}/unscaled/{sample}.bigWig -- Coverage tracks

Key Metrics:

  • Fragment size distribution (nucleosome periodicity visible in QC report)
  • FRiP (Fraction of Reads in Peaks) score (if enabled)

ChIP-seq

Standard ChIP-seq with support for input controls:

Key outputs:

  • aligned/{sample}.bam -- Final alignments
  • peaks/{method}/{sample}.bed -- Peak calls (MACS2, HOMER, LanceOtron)
  • peaks/{method}/merged/{group}.bed -- Consensus peaks
  • tag_dirs/{sample}/ -- HOMER tag directories
  • motifs/homer/{method}/{sample}/ -- Motif analysis (if enabled)

Spike-in Normalisation (if applicable):

  • resources/{method}/normalisation_factors.tsv -- Scaling factors
  • bigwigs/deeptools/spikein/{spikein_method}/{sample}.bigWig -- Normalised tracks

CUT&Tag

CUT&Tag is a separate assay from ChIP-seq, with SEACR as the default peak caller and optional Tn5 shift correction:

Key outputs:

  • aligned/{sample}.bam -- Final alignments (optionally Tn5-shifted)
  • peaks/seacr/{sample}.bed -- SEACR peak calls (default)
  • bigwigs/{method}/unscaled/{sample}.bigWig -- Coverage tracks

RNA-seq

RNA-seq alignment uses STAR, with quantification by featureCounts and/or Salmon:

Key outputs:

  • aligned/{sample}.bam -- STAR-aligned, processed BAM
  • readcounts/feature_counts/read_counts.tsv -- Combined gene-level counts
  • readcounts/salmon/salmon_counts.csv -- Salmon quantification (if enabled)
  • bigwigs/{method}/unscaled/{sample}_plus.bigWig -- Stranded coverage (plus strand)
  • bigwigs/{method}/unscaled/{sample}_minus.bigWig -- Stranded coverage (minus strand)
  • qc/qualimap_rnaseq/{sample}/qualimapReport.html -- RNA-specific QC

Methylation (METH)

Methylation calling uses MethylDackel, with support for both bisulfite and TAPS methods:

methylation/
├── methyldackel/
│   ├── {sample}_{genome}_CpG.bedGraph             # CpG methylation calls
│   ├── {sample}_{genome}_CpG_inverted.bedGraph    # TAPS-inverted calls (TAPS method only)
│   └── bias/
│       └── {sample}_{genome}.txt                  # M-bias data
├── methylation_conversion.tsv                     # Conversion rate statistics
└── methylation_conversion.png                     # Conversion rate plot

Samples are split by genome (reference vs spike-in) with split BAMs at aligned/spikein/{sample}_{genome}.bam.

SNP / Variant Calling

Variant calling using bcftools:

variant/
├── {sample}.vcf.gz             # Called variants
└── {sample}.anno.vcf.gz        # Annotated variants (if annotation enabled)

QC stats are produced at qc/variant/{sample}.stats.txt.

Micro Capture-C (MCC)

Chromatin contact analysis:

mcc/
└── contacts/
    └── {group}/
        └── {group}.mcool      # Multi-resolution contact matrix

CRISPR Screens

Guide RNA quantification with optional MAGeCK analysis:

readcounts/
├── feature_counts/
│   └── read_counts.tsv                     # featureCounts guide counts (always produced)
└── mageck/                                 # MAGeCK analysis (if enabled)
    ├── mageck_count.count.txt              # Raw guide counts
    ├── mageck_count.count_normalized.txt   # Normalised counts
    ├── mageck_count.countsummary.txt       # Count summary
    ├── mageck_mle.gene_summary.txt         # MAGeCK MLE gene-level results
    ├── mageck_mle.sgrna_summary.txt        # MAGeCK MLE sgRNA-level results
    └── design_matrix.txt                   # Design matrix used

Accessing Your Results

Command Line

# Navigate to output directory
cd seqnado_output/

# View main report
firefox {assay}/seqnado_report.html &

# List peaks
ls -lh {assay}/peaks/macs2/

# Load BAM in IGV
igv {assay}/aligned/{sample}.bam

Opening Reports

The HTML reports can be opened directly in your browser:

# On local machine
open seqnado_output/chip/seqnado_report.html

# Via X11 forwarding on HPC
firefox seqnado_output/chip/seqnado_report.html &

# Transfer to local machine
scp -r user@hpc:path/to/seqnado_output/ ./

Finding Specific Outputs

Peak calling results

find seqnado_output/ -name "*.bed"

Coverage tracks for visualisation

find seqnado_output/ -name "*.bigWig"

QC HTML reports

find seqnado_output/ -name "*.html"

See Also: