STAR–Salmon–StringTie–DEXSeq RNA-seq Analysis Pipeline

This entry documents a fully automated Bulk RNA-seq Auto Pipeline (BRAP) that performs read alignment, transcript quantification, splicing analysis, transcriptome assembly, and statistical analysis. The pipeline dynamically adapts to input data quality and structure, ensuring robust and reproducible results.

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Short reads mapping

📋 Workflow Overview

This pipeline performs:

• ✅ FASTQ quality control and trimming
• ✅ STAR-based genome alignment
• ✅ Salmon transcript quantification (2 rounds)
• ✅ Transcriptome assembly via StringTie
• ✅ Transcriptome refinement via SQANTI3
• ✅ circRNA detection (CIRCexplorer2)
• ✅ Alternative splicing (LeafCutter)
• ✅ Exon-level quantification (DEXSeq)
• ✅ Final analysis and visualization in R

🗺️ Workflow Diagram

graph TD

  

subgraph Preprocessing

A1[Start Script and Load Config] --> A2[Download genome.fa and genome.gtf]

A2 --> A3[Filter GTF - cellranger mkgtf]

A3 --> A4[Extract transcriptome - genomeTx.fa]

A4 --> A5[Build Salmon index 1 - Ensembl GTF]

end

  

subgraph FASTQ Processing

B1[Scan FASTQ files] --> B2[Run FastQC]

B2 --> B3["Detect read quality and adapter content"]

B3 --> B4{Is QC pass?}

B4 -- Yes --> B5[Use raw FASTQ]

B4 -- No or auto --> B6[Run trim_galore and re-FastQC]

B1 --> B7["Detect Phred encoding (score)"]

B7 --> B6

B6 --> B8["Use trimmed FASTQ"]

end

  

subgraph Alignment and Quantification Round 1

B5 --> C1[STAR mapping]

B8 --> C1

C1 --> C2[Aligned.sortedByCoord.out.bam]

C1 --> C3[Chimeric.out.junction]

C2 --> C4[samtools index]

C2 --> C5[regtools junctions extract → .junc]

C4 --> C6[featureCounts]

A5 --> D1[Salmon quant 1 - Ensembl GTF]

B5 --> D1

B8 --> D1

D1 --> D2["Detect library type (strandness) from Salmon log"]

D2 --> C6

end

  

subgraph Alternative Splicing Analysis

C5 --> L1[LeafCutter: junction clustering]

end

  

subgraph circRNA Detection

C3 --> M1[CIRCexplorer2 parse]

M1 --> M2[CIRCexplorer2 annotate]

end

  

subgraph Transcriptome Assembly and Refinement

C2 --> E1[StringTie per-sample using STAR BAM]

E1 --> E2[StringTie merge - stdout.gtf]

E2 --> E3[SQANTI3 QC → stdout_final.gtf]

E3 --> E4[Extract transcriptome - genomeTx_stdout.fa]

E3 --> E5[Convert GTF to GFF for DEXSeq]

end

  

subgraph Alignment and Quantification Round 2

E4 --> F1[Build Salmon index 2 - Merged GTF]

F1 --> F2["Salmon quant 2 - Merged GTF (overwrites round 1)"]

B5 --> F2

B8 --> F2

end

  

subgraph DEXSeq Exon-Level Counting

E5 --> Z1[Prepare exon bins - genome_stdoutFinal.gff]

C2 --> Z2[Use STAR BAM for exon counting]

D2 --> Z3[Use strand info from Salmon log]

Z1 --> Z4[Run dexseq_count.py]

Z2 --> Z4

Z3 --> Z4

Z4 --> Z5[Generate exon count matrix]

end

  

subgraph Downstream Analysis

F2 --> Z6[MultiQC input ← final Salmon output]

Z5 --> Z6

Z6 --> Z7[MultiQC and Final Report]

Z7 --> Z8[Run 000.Analysis.R for all downstream analysis]

end

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🔁 Step-by-Step Description

1. Environment Setup

• Activates SQANTI3.env Conda environment
• Parses settings from ExConfiguration_bashScript.txt

2. Genome Preparation

• Downloads genome FASTA and GTF from Ensembl
• Optionally adds exogenous sequences (e.g., GFP)
• Filters GTF using cellranger mkgtf
• Builds:
  • STAR index
  • Salmon transcriptome index (with decoy)
  • Pfam HMM database

3. FASTQ Processing

• Detects FASTQ files and normalizes extensions
• Runs FastQC on raw reads
• Detects:
  • Phred encoding from first 5k reads
  • Read quality and adapter contamination
• Triggers trim_galore if needed
• Reruns FastQC on trimmed reads

4. Alignment & Quantification (Round 1)

• Aligns reads using STAR
• Outputs:
  • *.bam (sorted and indexed)
  • *.Chimeric.out.junction (for circRNA)
  • *.junc (from regtools for LeafCutter)
• Quantifies transcripts using Salmon (based on Ensembl GTF)
• Detects library strandness from salmon_quant.log
• Applies strandness to featureCounts

5. Splicing and circRNA

• LeafCutter analyzes alternative splicing from .junc files
• CIRCexplorer2 annotates circRNAs using STAR chimeric junctions

6. Transcriptome Assembly & Refinement

• StringTie assembles transcripts per sample using STAR BAMs
• Merges GTFs into a master transcriptome
• SQANTI3 filters and corrects transcript models
• Generates:
  • stdout_final.gtf
  • genomeTx_stdout.fa
  • genome_stdoutFinal.gff for DEXSeq

7. Quantification (Round 2)

• Deletes first Salmon output
• Builds new Salmon index using stdout_final.gtf
• Re-runs Salmon quantification (round 2) — overwrites round 1
• These outputs are used for MultiQC and downstream analysis

8. DEXSeq Exon Counting

• Uses:
  • Refined GFF (genome_stdoutFinal.gff)
  • STAR BAMs
  • Strandness from Salmon
• Runs dexseq_count.py to generate *.exon.txt files

9. MultiQC Report

• Runs multiqc . to collect:
  • FastQC, Salmon, STAR, featureCounts, trim_galore, etc.
  • Includes only final Salmon outputs

10. Final R Analysis

• Executes: Rscript 000.Analysis.R
• Performs:
  • Normalization
  • PCA, RLE, clustering
  • DEG or splicing analysis
  • HTML and table outputs

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🔍 Dynamic Features

Property	Detected From	Applied In
Phred encoding	First 5k reads (ASCII)	trim_galore
Read quality	FastQC	Triggers trimming
Adapter presence	FastQC	Triggers trimming
Library strandness	salmon_quant.log	featureCounts, DEXSeq

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📦 Output Summary

Output	Description
*.bam, *.bai	STAR-aligned and indexed BAM files
Salmon_quants/	Final transcript quantification (TPM, etc)
*.counts	Gene-level quantification from featureCounts
*.exon.txt	DEXSeq exon bin counts
LeafCutter.Output/	Splicing analysis results
*_CIRCexplorer2.ce	circRNA annotations
stdout_final.gtf	Refined transcriptome annotation
multiqc_report.html	Aggregated QC and alignment metrics
00.Analysis_Report.html	Custom final R analysis report

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🚀 Getting Started

1. Prepare the input directory with:

   • ExConfiguration_bashScript.txt
   • Raw or trimmed FASTQ files

2. Run:

   bash 00.STAR.sh <path_to_your_project>

3. Inspect outputs:

   • BAMs, counts, and QC
   • HTML reports
   • Final analysis results from 000.Analysis.R

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📚 Dependencies

• STAR, Salmon, StringTie, samtools, featureCounts
• trim_galore, FastQC, regtools, CIRCexplorer2
• SQANTI3, LeafCutter, DEXSeq, MultiQC
• R with custom script: 000.Analysis.R

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🧠 Notes

• The pipeline is idempotent: already-completed steps are skipped unless missing.
• Highly suitable for both canonical and custom transcriptome exploration.
• Ideal for both bulk RNA-seq and pseudo-bulk analyses.

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Comprehensive analysis in R

Example results from this workflow

1. https://d3dcaz4rv8jgb4.cloudfront.net/ from Zheng Y, Wang Z, Weng Y, Sitosari H, He Y, Zhang X, Shiotsu N, Fukuhara Y, Ikegame M, Okamura H. Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis. Scientific reports. 2025 Mar 26;15(1):10462.
2. https://dndy5us1uro9a.cloudfront.net/BulkRNAseq/00.Analysis_Report.html from Weng Y, Wang Z, Sitosari H, Ono M, Okamura H, Oohashi T. O‐GlcNAcylation regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum. BioFactors. 2025 Jan;51(1):e2131.