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qbic-pipelines/rnadeseq: Output

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report (if provided), which summarises results at the end of the pipeline.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

  • DE analysis - differential expression analysis with DESeq2
  • Pathway analysis - pathway analysis with gProfileR
  • Report - QBiC Report describing results of the whole pipeline

DE analysis

Differential expression analysis is performed with the DESeq2 R package.

Output directory: results/DESeq2

This directory contains the zipped results. When unzipping them, the following subfolders can be found.

  • metadata/
    • metadata.tsv: metadata sheet used by the pipeline.
    • contrasts.tsv: contrasts used for DE analysis (if provided).
    • linear_model.txt: linear model used for DE analysis.
    • gene_list.txt: provided list of interesting genes (if provided).
    • star_[rsem/salmon]_files.txt: list of counts files that were provided as input (only if --input_type is rsem or salmon).
    • smrnaseq_files.txt: list of counts files that were provided as input (only if --input_type is smrnaseq).
  • gene_counts_tables/
    • raw_gene_counts.txt: raw gene counts table from the nf-core/rnaseq pipeline and used for the differential gene expression analysis (only if --input_type is featurecounts or smrnaseq; in the second case, the pipeline will internally build a counts table from the input files, taking the third column of each .sorted.idxstats file (second column if counting rownames) and combining all values from both hairpin and non-hairpin mature files into one table).
    • rlog_transformed_gene_counts.tsv: normalized gene counts with the "regularized logarithm" approach. This normalization is used prior to PCA analysis and heatmap plotting of the gene counts (only when rlog transformation is used).
    • vst_transformed_gene_counts.tsv: normalized gene counts with the "variance stabilizing" transformation (only when vst transformation is used).
    • sizeFactor_libraries.tsv: size factors for each sample.
  • DE_genes_tables/: folder containing one tab-separated table for each of the contrasts in the analysis. Each table contains a list of all differentially expressed genes in the contrast, specifying the mean gene expression across all samples (baseMean), and the log2 fold change value and p-adjusted values (padj) for this contrast.
  • final_gene_table/final_gene_list_DESeq2.tsv: table containing a list of all genes considered in the analysis. Here a summary of the log2 Fold Change and p-adjusted values for all contrasts is displayed. Additionally, the column filter shows if this gene was differentially expressed (DE) in any of the contrasts, or not (not_DE). The column contrast_vector contains for each contrast considered in the analysis a 1 if the gene was differentially expressed for this contrast or a 0 if it was not.
  • plots/:
    • Heatmaps_of_distances.pdf/.svg: heatmap of the pairwise euclidean distances among samples, when the rlog normalized gene counts are considered.
    • PCA_plot.pdf: PCA of the rlog normalized gene counts.
    • boxplots_example_genes/: boxplots of the normalized gene counts for each of the sample groups for example genes.
    • boxplots_requested_genes/: boxplots of the normalized gene counts for each of the sample groups for the list of requested genes.
    • further_diagnostics_plots/: plots for diagnostics of the differential gene analysis procedure.

Pathway analysis

Pathway analysis with gProfileR R package.

Output directory: results/pathway_analysis/

If pathway analysis was run, this directory contains the zipped pathway analysis results (gProfileR.zip). When unzipping them, a subfolder for each contrast used for the differential gene expression analysis is found. Inside each contrast folder, there is the following output:

  • *_gost_pathway_venn_diagram.pdf/png
    • Venn diagrams showing the numbers of enriched pathways when using a background gene list vs when not using a bg list.
  • enrichment_plots/*_pathway_enrichment_plot.{pdf/png/svg}
    • Barplots showing the proportion of differentially expressed genes in the pathway for a certain pathway database.
  • gost_pathway_gostplot.{pdf/png/svg}
    • Manhattan plots displaying all enriched pathways.
  • KEGG_pathways/
    • Contains the KEGG pathways graphs with the log fold change of the differentially expressed genes.
  • pathway_heatmaps
    • Contains heatmaps of the normalized gene counts for each of the differentially expressed pathways.

Report

QBiC report for RNAseq analysis.

Output directory: results/report/

In this directory the zipped report is contained. This file is ready for upload to the project in the QBiC portal. Once unzipped, this directory contains:

  • Report.html
    • QBiC report describing the RNAseq results.
  • DESeq2/
    • DESeq2 results to be attached to the report (see DESeq2 output description).
  • QC/
    • Quality control results. Contains:
    • fastqc.zip: zipped fastqc results for each sample.
    • multiqc_data: If multiQC input was provided: data from the multiQC process of the RNAseq pipeline which is incorporated into the report.
    • multiqc_plots: If multiQC input was provided: plots from the multiQC process of the RNAseq pipeline which are incorporated into the report.
    • qc_summary.tsv: If multiQC input was provided: summary of the multiQC process in a table format.