ASPEN Outputs¶

Workdir¶

The workdir which is supplied as -w while running aspen init, dryrun and run commands will contain the following files:

WORKDIR
├── cluster.json
├── config.yaml
├── contrasts.tsv
├── dryrun_git_commit.txt
├── dryrun.log
├── fastqs
├── logs
├── results
├── runinfo.yaml
├── sampleinfo.txt
├── samples.tsv
├── scripts
├── slurm-48768339.out
├── snakemake.log
├── snakemake.stats
├── stats
├── submit_script.sbatch
└── tools.yaml

Here are more details about these files:

Resultsdir¶

The results directory contains the actual output files. Below are the folders that you may find within it.

WORKDIR
├── results
    ├── dedupBam
    ├── peaks
    ├── QC
    ├── qsortedBam
    ├── tagAlign
    └── tmp

Content details:

The QC folder contains the multiqc_report.html file which provides a comprehensive summary of the quality control metrics across all samples, including read quality, duplication rates, and other relevant statistics. This report aggregates results from various QC tools such as FastQC, FastqScreen, FLD, TSS enrichment, Peak Annotations, and others, presenting them in an easy-to-read format with interactive plots and tables. It helps in quickly identifying any issues with the sequencing data and ensures that the data quality is sufficient for downstream analysis.

Most of the above folders are self-explanatory. The peaks folder has this hierarchy:

WORKDIR
├── results
    ├── peaks
        ├── genrich
        │   ├── <replicateName>.genrich.narrowPeak
        │   ├── <replicateName>.genrich.narrowPeak.annotated
        │   ├── <replicateName>.genrich.narrowPeak.genelist
        │   ├── <replicateName>.genrich.narrowPeak.annotation_summary
        │   ├── <replicateName>.genrich.narrowPeak.annotation_distribution
        │   ├── <sampleName>.genrich.pooled.narrowPeak
        │   ├── <sampleName>.genrich.consensus.bed
        │   ├── ROI.counts.tsv
        │   ├── bigwig
        │   ├── <replicateName>.genrich.narrowPeak_motif_enrichment
        │   │   └── knownResults
        │   ├── DiffATAC
        │   ├── <replicateName>.genrich.consensus.bed_motif_enrichment
        │   └── tn5nicks
        └── macs2
        │   ├── <replicateName>.macs2.narrowPeak
        │   ├── <replicateName>.macs2.narrowPeak.annotated
        │   ├── <replicateName>.macs2.narrowPeak.genelist
        │   ├── <replicateName>.macs2.narrowPeak.annotation_summary
        │   ├── <replicateName>.macs2.narrowPeak.annotation_distribution
        │   ├── <sampleName>.macs2.pooled.narrowPeak
        │   ├── <sampleName>.macs2.consensus.bed
        │   ├── ROI.counts.tsv
            ├── bigwig
            ├── <replicateName>.macs2.narrowPeak_motif_enrichment
            │   └── knownResults
            ├── DiffATAC
        │   ├── <replicateName>.macs2.consensus.bed_motif_enrichment
            ├── fixed_width
            └── tn5nicks

Some of the important folders and files are highlighted below:

Folders¶

• bigwig:

For easy visualization, they are converted to bigWig format and saved in respective bigwig folders. The bigWig files can be directly loaded into UCSC Browser or IGV.

• tn5nicks:

This folder host the per-replicate BAM files containing the Tn5 nicking sites in Genrich or MACS2 "peakcalling" reads, respectively.

• DiffATAC:

Contains the DESeq2 differential accessiblity results, both per-contrast and aggregated accross all contrasts in contrasts.tsv. These results are solely based on tn5 nick counts.

• fixed_width:

This folder contains fixed-width consensus peaks across replicates and samples, represented in the "Regions-Of-Interest" files. The ROI.bed file lists genomic regions where chromatin accessibility is analyzed using DESeq2, with results stored in the DiffATAC folder.

• <replicateName>.macs2.narrowPeak_motif_enrichment;<replicateName>.genrich.narrowPeak_motif_enrichment;<replicateName>.macs2.consensus.bed_motif_enrichment;<replicateName>.genrich.consensus.bed_motif_enrichment:

Contains the motif enrichments calculated using HOMER and AME for peaks called for each replicate, sample consensus peaks using both MACS2 and Genrich. Specifically, two types of motif enrichments are performed:

• Enrichment of known HOCOMOCO (version 11) motifs for HUMAN or MOUSE or BOTH using HOMER. See file knownResults.html.

• de novo motif enrichment using AME from MEME suite. See file ame_results.txt. Custom parallelization is used to optimize AME based enrichment analysis.

Files¶

• *.narrowPeak:

Called peaks from Genrich or MACS2

• Annotated peak files:

Peaks are annotated with ChIPSeeker and results are saved in the following files:

• .annotated

  Tab-delimited txt file with the following columns:

• .genelist

This is a tab-delimited file with names (Ensembl ID, gene symbol) of genes which have ATAC-seq peaks in their promotor regions. This file can be used downstream for gene enrichment analysis (ORA or over-representation analysis).

• .annotation_summary; .annotation_distribution

Tab-delimited files that provide statistics on peak annotations, quantifying the number of peaks found in Promoters, Exonic regions, Distal Intergenic regions, etc. The .annotation_distribution is use to create visualization of these annotation-distributions in the MultiQC report.

• ROI.counts.tsv

This file contains the read counts for each Region-Of-Interest (ROI) across all replicates of all samples. It is a tab-delimited file with the following columns:

Each row represents a specific ROI, and the columns contain the read counts for each sample, allowing for differential accessibility analysis.