QC + Mapping + Counting (single+paired) - Ref Based RNA Seq - Transcriptomics - GTN

transcriptomics-ref-based/qc-mapping-counting-paired-and-single

Author(s)
Bérénice Batut, Mallory Freeberg, Mo Heydarian, Anika Erxleben, Pavankumar Videm, Clemens Blank, Maria Doyle, Nicola Soranzo, Peter van Heusden, Lucille Delisle
Version
6
Last updated
Feb 11, 2025
License
MIT
Tags
transcriptomics

Features
Tutorial
Reference-based RNA-Seq data analysis
Other workflows associated with this material
Workflow Testing
Tests: ✅
Results: Not yet automated
FAIRness PURL
https://gxy.io/GTN:W00245
RO-Crate logo with flask Download Workflow RO-Crate Workflowhub cloud with gears logo View on (Dev) WorkflowHub
Launch in Tutorial Mode
Download 
output
output
output
output
output
output
out1
output
output
report
report
output
out_pairs
mapped_reads
output
output
output
output_log
output_log
reads_per_gene
reads_per_gene
mapped_reads
output
mapped_reads
mapped_reads
signal_unique_str1
signal_unique_str1
signal_unique_str2
signal_unique_str2
text_file
output
output
output
output_short
output_short
output_summary
output_summary
output
bed_file
output
bed_file
output
bed_file
output
output
output
output
output
output
outfile
output
output
output
outputsam
bed_file
output
metrics_file
out_file1
outputtxt
ℹ️ Input Collection
single fastqs
ℹ️ Input Collection
paired fastqs
ℹ️ Input Dataset
Drosophila_melanogaster.BDGP6.32.109_UCSC.gtf.gz
Cutadapt: remove bad quality bp
Flatten paired collection for Falco
Cutadapt
Get gene length
convert gtf to bed12
STAR: map single reads
Merge fastqs for Falco
Merge Cutadapt reports
STAR: map paired reads
count reads per gene for SR
Falco
Combine cutadapt results
Merge STAR logs
Merge STAR counts
count fragments per gene for PE
Merge STAR BAM
merge coverage unique strand 1
merge coverage unique strand 2
Combine FastQC results
Combine STAR Results
Remove statistics from STAR counts
Determine library strandness with STAR
merge counts from featureCounts
merge featureCounts summary
Determine library strandness with Infer Experiment
Read Distribution
Compute read distribution statistics
sample BAM
Get reads number per chromosome
Remove duplicates
Determine library strandness with STAR coverage
Select unstranded counts
Sort counts to get gene with highest count on feature Counts
Combine read asignments statistics
Combine read distribution on known features
Get gene body coverage
Combine results on reads per chromosome
Combine results of duplicate reads
Sort counts to get gene with highest count on STAR
Combine gene body coverage
Output
Gene length
Output
multiqc_cutadapt_html
Output
featureCounts_gene_length
Output
STAR_BAM
Output
multiqc_falco_html
Output
multiqc_star_html
Output
multiqc_star_counts_html
Output
featureCounts
Output
inferexperiment
Output
pgt
Output
counts_from_star
Output
featureCounts_sorted
Output
multiqc_featureCounts_html
Output
multiqc_read_distrib
Output
multiqc_reads_per_chrom
Output
multiqc_dup
Output
counts_from_star_sorted
Output
multiqc_gene_body_cov

Inputs

Input Label
Input dataset collection single fastqs
Input dataset collection paired fastqs
Input dataset Drosophila_melanogaster.BDGP6.32.109_UCSC.gtf.gz

Outputs

From Output Label
toolshed.g2.bx.psu.edu/repos/iuc/length_and_gc_content/length_and_gc_content/0.1.2 Gene length and GC content Get gene length
toolshed.g2.bx.psu.edu/repos/iuc/gtftobed12/gtftobed12/357 Convert GTF to BED12 convert gtf to bed12
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine cutadapt results
toolshed.g2.bx.psu.edu/repos/iuc/featurecounts/featurecounts/2.0.3+galaxy2 featureCounts count fragments per gene for PE
__MERGE_COLLECTION__ Merge collections Merge STAR BAM
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine FastQC results
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine STAR Results
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Determine library strandness with STAR
__MERGE_COLLECTION__ Merge collections merge counts from featureCounts
toolshed.g2.bx.psu.edu/repos/nilesh/rseqc/rseqc_infer_experiment/5.0.3+galaxy0 Infer Experiment Determine library strandness with Infer Experiment
toolshed.g2.bx.psu.edu/repos/nilesh/rseqc/rseqc_read_distribution/5.0.3+galaxy0 Read Distribution
toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_MarkDuplicates/3.1.1.0 MarkDuplicates Remove duplicates
toolshed.g2.bx.psu.edu/repos/iuc/pygenometracks/pygenomeTracks/3.8+galaxy2 pyGenomeTracks Determine library strandness with STAR coverage
Cut1 Cut Select unstranded counts
toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_sort_header_tool/9.3+galaxy1 Sort Sort counts to get gene with highest count on feature Counts
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine read asignments statistics
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine read distribution on known features
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine results on reads per chromosome
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine results of duplicate reads
toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_sort_header_tool/9.3+galaxy1 Sort Sort counts to get gene with highest count on STAR
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 MultiQC Combine gene body coverage

Tools

Tool Links
Cut1
__FLATTEN__
__MERGE_COLLECTION__
toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_sort_header_tool/9.3+galaxy1 View in ToolShed
toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_tail_tool/9.3+galaxy1 View in ToolShed
toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_MarkDuplicates/3.1.1.0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/devteam/samtools_idxstats/samtools_idxstats/2.0.5 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/falco/falco/1.2.4+galaxy0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/featurecounts/featurecounts/2.0.3+galaxy2 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/gtftobed12/gtftobed12/357 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/length_and_gc_content/length_and_gc_content/0.1.2 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.24.1+galaxy0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/pygenometracks/pygenomeTracks/3.8+galaxy2 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/rgrnastar/rna_star/2.7.11a+galaxy0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/iuc/samtools_view/samtools_view/1.15.1+galaxy2 View in ToolShed
toolshed.g2.bx.psu.edu/repos/lparsons/cutadapt/cutadapt/4.9+galaxy1 View in ToolShed
toolshed.g2.bx.psu.edu/repos/nilesh/rseqc/rseqc_geneBody_coverage/5.0.3+galaxy0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/nilesh/rseqc/rseqc_infer_experiment/5.0.3+galaxy0 View in ToolShed
toolshed.g2.bx.psu.edu/repos/nilesh/rseqc/rseqc_read_distribution/5.0.3+galaxy0 View in ToolShed

To use these workflows in Galaxy you can either click the links to download the workflows, or you can right-click and copy the link to the workflow which can be used in the Galaxy form to import workflows.

Importing into Galaxy

Below are the instructions for importing these workflows directly into your Galaxy server of choice to start using them!
Hands On: Importing a workflow
  1. Click on Workflows in the Galaxy activity bar (on the left side of the screen, or in the top menu bar of older Galaxy instances). You will see a list of all your workflows
  2. Click on Import at the top-right of the screen
  3. Provide your workflow
    • Option 1: Paste the URL of the workflow into the box labelled “Archived Workflow URL”
    • Option 2: Upload the workflow file in the box labelled “Archived Workflow File”
  4. Click the Import workflow button

Below is a short video demonstrating how to import a workflow from GitHub using this procedure:

Video: Importing a workflow from URL

Version History

Version Commit Time Comments
11 f81845b85 2025-01-21 10:07:17 Use Falco instead of FastQC in ref-based tutorial
10 9a19075e2 2024-10-18 13:22:04 Update ref-based workflows
9 a1251f286 2024-07-05 09:38:54 Removed 'comments' tags
8 d804d52ac 2024-07-05 09:22:56 Updated tools in 'QC + Mapping + Counting (single+paired)' workflow
7 41dead43e 2023-05-02 10:31:07 add mo orcid to workflows
6 36eb5cf82 2023-04-28 17:26:00 update workflows and tests
5 8fc9c9026 2023-04-25 07:46:15 add creators and licence to workflows
4 dc21d9ddb 2023-04-22 08:29:08 update images and results, rearrange workflow for part1
3 9921a8623 2023-04-21 12:37:10 Update first part of the tutorial
2 4d2f611a6 2022-04-28 15:20:51 subset BAM before gene body coverage
1 8bf6877e4 2022-04-15 11:16:13 add workflow for PE and SE in parallel

For Admins

Installing the workflow tools

wget https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/workflows/qc-mapping-counting-paired-and-single.ga -O workflow.ga
workflow-to-tools -w workflow.ga -o tools.yaml
shed-tools install -g GALAXY -a API_KEY -t tools.yaml
workflow-install -g GALAXY -a API_KEY -w workflow.ga --publish-workflows