The output of `damid-seq` can be found in the `results/` directory. When run with the test data provided in the GitHub repository (.test/reads), the directory structure will be as follows: .. code-block:: console results/ ├── bam │ ├── exp1 │ │ ├── Dam.sorted.bam │ │ ├── Dam.sorted.bam.bai │ │ ├── Piwi.sorted.bam │ │ └── Piwi.sorted.bam.bai │ ├── exp2 │ │ ├── Dam.sorted.bam │ │ ├── Dam.sorted.bam.bai │ │ ├── Piwi.sorted.bam │ │ └── Piwi.sorted.bam.bai │ └── exp3 │ ├── Dam.sorted.bam │ ├── Dam.sorted.bam.bai │ ├── Piwi.sorted.bam │ └── Piwi.sorted.bam.bai ├── bedgraph │ ├── exp1 │ │ ├── Piwi-vs-Dam-norm.gatc.bedgraph │ │ ├── Piwi-vs-Dam.quantile-norm.gatc.bedgraph │ │ └── Piwi-vs-Dam.rev_log2.bedgraph │ ├── exp2 │ │ ├── Piwi-vs-Dam-norm.gatc.bedgraph │ │ ├── Piwi-vs-Dam.quantile-norm.gatc.bedgraph │ │ └── Piwi-vs-Dam.rev_log2.bedgraph │ └── exp3 │ ├── Piwi-vs-Dam-norm.gatc.bedgraph │ ├── Piwi-vs-Dam.quantile-norm.gatc.bedgraph │ └── Piwi-vs-Dam.rev_log2.bedgraph ├── bigwig │ ├── average_bw │ │ └── Piwi.bw │ ├── bam2bigwig │ │ ├── exp1 │ │ │ ├── Dam.bw │ │ │ └── Piwi.bw │ │ ├── exp2 │ │ │ ├── Dam.bw │ │ │ └── Piwi.bw │ │ └── exp3 │ │ ├── Dam.bw │ │ └── Piwi.bw │ ├── exp1 │ │ └── Piwi.bw │ ├── exp2 │ │ └── Piwi.bw │ └── exp3 │ └── Piwi.bw ├── bigwig_rev_log2 │ ├── average_bw │ │ └── Piwi.bw │ ├── exp1 │ │ └── Piwi.bw │ ├── exp2 │ │ └── Piwi.bw │ └── exp3 │ └── Piwi.bw ├── deeptools │ ├── average_bw_matrix.gz │ ├── correlation_bam.tab │ ├── correlation.tab │ ├── heatmap_matrix.gz │ ├── PCA_bam.tab │ ├── PCA.tab │ ├── scores_per_bin_bam.npz │ └── scores_per_bin.npz ├── peaks │ └── fdr0.01 │ ├── consensus_peaks │ │ ├── enrichment_analysis │ │ │ └── Piwi.xlsx │ │ ├── Piwi.annotated.txt │ │ ├── Piwi.filtered.bed │ │ ├── Piwi.geneIDs.txt │ │ └── Piwi.overlap.bed │ ├── exp1 │ │ ├── Piwi.bed │ │ ├── Piwi.data │ │ ├── Piwi.peaks.gff │ │ └── Piwi.sorted.bed │ ├── exp2 │ │ ├── Piwi.bed │ │ ├── Piwi.data │ │ ├── Piwi.peaks.gff │ │ └── Piwi.sorted.bed │ ├── exp3 │ │ ├── Piwi.bed │ │ ├── Piwi.data │ │ ├── Piwi.peaks.gff │ │ └── Piwi.sorted.bed │ ├── frip.csv │ └── read_counts │ ├── exp1 │ │ ├── Piwi.peak.count │ │ └── Piwi.total.count │ ├── exp2 │ │ ├── Piwi.peak.count │ │ └── Piwi.total.count │ └── exp3 │ ├── Piwi.peak.count │ └── Piwi.total.count ├── plots │ ├── heatmap.pdf │ ├── mapping_rates.pdf │ ├── PCA_bam.pdf │ ├── PCA.pdf │ ├── peaks │ │ └── fdr0.01 │ │ ├── distance_to_tss.pdf │ │ ├── enrichment_analysis │ │ │ └── Piwi │ │ │ ├── GO_Biological_Process_2018.pdf │ │ │ ├── GO_Molecular_Function_2018.pdf │ │ │ └── KEGG_2019.pdf │ │ ├── feature_distributions.pdf │ │ └── frip.pdf │ ├── profile_plot.pdf │ ├── sample_correlation_bam.pdf │ ├── sample_correlation.pdf │ ├── scree_bam.pdf │ └── scree.pdf ├── qc │ ├── fastqc │ │ ├── exp1 │ │ │ ├── Dam_fastqc.zip │ │ │ ├── Dam.html │ │ │ ├── Piwi_fastqc.zip │ │ │ └── Piwi.html │ │ ├── exp2 │ │ │ ├── Dam_fastqc.zip │ │ │ ├── Dam.html │ │ │ ├── Piwi_fastqc.zip │ │ │ └── Piwi.html │ │ └── exp3 │ │ ├── Dam_fastqc.zip │ │ ├── Dam.html │ │ ├── Piwi_fastqc.zip │ │ └── Piwi.html │ └── multiqc │ ├── multiqc_data │ │ ├── multiqc_citations.txt │ │ ├── multiqc_data.json │ │ ├── multiqc_fastqc.txt │ │ ├── multiqc_general_stats.txt │ │ ├── multiqc.log │ │ ├── multiqc_software_versions.txt │ │ └── multiqc_sources.txt │ └── multiqc.html └── trimmed ├── exp1 │ ├── Dam.fastq.gz_trimming_report.txt │ ├── Dam.flag │ ├── Piwi.fastq.gz_trimming_report.txt │ └── Piwi.flag ├── exp2 │ ├── Dam.fastq.gz_trimming_report.txt │ ├── Dam.flag │ ├── Piwi.fastq.gz_trimming_report.txt │ └── Piwi.flag └── exp3 ├── Dam.fastq.gz_trimming_report.txt ├── Dam.flag ├── Piwi.fastq.gz_trimming_report.txt └── Piwi.flag 50 directories, 118 files Quality control --------------- FastQC/MultiQC ============== FastQC is used to do some control check on the trimmed reads. The output of FastQC is summarized in a MultiQC report (results/qc/multiqc/multiqc.html). .. figure:: images/multiqc.png :align: center :width: 1000 Alignment rates =============== The Bowtie2 alignment rates are summarised in results/plots/mapping_rates.pdf. .. figure:: images/mapping_rates.png :align: center :width: 1000 PCA plot of BAM files ===================== A PCA plot of the BAM files is generated to check the consistency of biological replicates. The plot is saved in results/plots/PCA_bam.pdf. A scree plot is also generated to show the variance explained by each principal component (results/plots/scree_bam.pdf). .. figure:: images/PCA_bam.png :align: center :width: 1000 .. figure:: images/scree_bam.png :align: center :width: 1000 Sample correlation heatmap ========================== A correlation (Spearman) heatmap of the BAM files is generated to also check the consistency of biological replicates. The plot is saved in results/plots/sample_correlation_bam.pdf. .. figure:: images/sample_correlation_bam.png :align: center :width: 1000 Fraction of reads in peaks (FRiP) ================================= The FRiP is calculated for each sample and plotted in results/plots/peaks/frip.pdf, when using the Perl peak calling script, or results/plots/macs2\_[broad or narrow]/fdr[value]/frip.pdf. .. figure:: images/frip.png :align: center :width: 1000 Visualization of damid-seq data ------------------------------- Profile plot ============ Using deepTools, a profile plot is generated to show the average coverage of the reads around defined features of the genome (TSS or gene body). The plot is saved in results/plots/profile_plot.pdf. .. figure:: images/profile_plot.png :align: center :width: 1000 Heatmap A heatmap of the coverage of the reads around defined features of the genome (TSS or gene body) is also generated. The plot is saved in results/plots/heatmap.pdf. .. figure:: images/heatmap.png :align: center :width: 1000 Peak-related plots ------------------ Various plot are created relating to peak data. Distance to TSS =============== A plot showing the distance of the peaks to the nearest transcription start site (TSS) is generated. The plot is saved in results/plots/[peaks, macs2_broad, macs2_narrow]/fdr[value]/distance_to_tss.pdf. .. figure:: images/distance_to_tss.png :align: center :width: 1000 Binding site distributions ========================== A plot showing the distribution of the peaks across different genomic features is generated. The plot is saved in results/plots/[peaks, macs2_broad, macs2_narrow]/fdr[value]/feature_distributions.pdf. .. figure:: images/feature_distributions.png :align: center :width: 1000