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Discipline 05 of 18 32 Tools Stage 5: Transcriptome Abundance & Pathway Analysis

📈 5. Bulk RNA-Seq: Quantification, DE, Splicing & Networks

Transcript-level abundance quantification, gene count matrix generation, differential expression modeling, alternate splicing resolution, and pathway network enrichment.

Essential Software: featureCounts HTSeq DESeq2 edgeR limma-voom clusterProfiler
Comparative Analysis Production Benchmarks

Algorithm Architecture & Decision Matrices

Detailed comparison cards highlighting algorithmic trade-offs, strengths, weaknesses, and recommended production setups.

Quantification & Differential Expression
featureCounts HTSeq RSEM StringTie Scallop Subread DESeq2 edgeR limma-voom sleuth NOISeq tximport
Negative binomial GLM fitting, dispersion shrinkage, non-parametric modeling, transcript-to-gene abundance aggregation.
Functional Pathways & Enrichment
clusterProfiler GSEA / fgsea g:Profiler Enrichr DAVID Metascape ReactomePA STRING IPA (Ingenuity)
Ranked GSEA, Gene Ontology over-representation, KEGG/Reactome cascades, and protein-protein network enrichment.
Splicing, Fusions & Co-Expression
rMATS LeafCutter MAJIQ DEXSeq / DRIMSeq SUPPA2 STAR-Fusion Arriba FusionCatcher WGCNA CEMiTool Cytoscape
Differential exon/transcript usage, alternative splicing, gene fusion discovery, co-expression modules and network layout.
Hardware & Execution Profiles

Primary Software & Algorithms in Bulk RNA-Seq Transcriptomics

Direct command-line recipes, Conda environment specifications, memory footprints, and license metadata.

Differential Expression
💻 < 8GB RAM R • Bioconductor • GLM

DESeq2

Docs ↗ By Love, Huber & Anders (EMBL / UNC)
(License: LGPL-3.0)

Gold-standard package for differential gene expression analysis of RNA-seq read counts using negative binomial generalized linear models and empirical Bayes dispersion shrinkage.

Formats: Input: Raw Count Matrix • Output: Log2FC, p-adj, Volcano Plots
💻 4-8 CPU Cores • 8-16 GB RAM
R -e 'BiocManager::install("DESeq2")'
Digital Gene Expression
💻 < 8GB RAM R • Bioconductor • Quasi-Likelihood

edgeR

Docs ↗ By Robinson, McCarthy & Smyth (WEHI)
(License: GPL-3.0)

Differential expression analysis for RNA-seq, ChIP-seq, and CITE-seq count data using overdispersed Poisson and negative binomial generalized linear models with quasi-likelihood F-tests.

Formats: Input: Raw Count Table • Output: DEG Tables, MDS Plots
💻 4 CPU Cores • 8 GB RAM
R -e 'BiocManager::install("edgeR")'
Variance Modeling RNA-Seq
💻 < 8GB RAM R • Bioconductor • Empirical Bayes

limma-voom

Docs ↗ By Charity Law & Gordon Smyth (WEHI)
(License: GPL-3.0)

Transforms RNA-seq count data into log2-CPM with precision weights based on the observed mean-variance relationship, enabling standard linear modeling and complex multi-factor designs.

Formats: Input: Count Matrix • Output: Moderated t-statistics, DE list
💻 4 CPU Cores • 8 GB RAM
R -e 'BiocManager::install("limma")'
Transcript Assembly & Quant
💻 < 8GB RAM C++ • Network Flow Optimization

StringTie

Docs ↗ By Mihaela Pertea et al. (JHU)
(License: MIT)

Highly efficient transcript assembler and abundance estimator for RNA-seq. Uses maximum flow network algorithms to assemble complex spliced isoforms simultaneously.

Formats: Input: Spliced BAM (STAR/HISAT2) • Output: GTF, FPKM, TPM
💻 4-8 CPU Cores • 8-16 GB RAM
conda install -c bioconda stringtie
Alternative Splicing Analysis
💻 16-32GB RAM Python / C • Hierarchical Model

rMATS

Docs ↗ By Shen Lab (UCLA / Penn)
(License: GPL-3.0)

Statistical detection of differential alternative splicing events: skipped exons (SE), alternative 5'/3' splice sites (A5SS/A3SS), mutually exclusive exons (MXE), and retained introns (RI).

Formats: Input: Spliced BAM, GTF • Output: Splicing percentage (PSI), p-values
💻 8-16 CPU Cores • 16-32 GB RAM
conda install -c bioconda rmats
Transcript-Level Differential Analysis
💻 < 8GB RAM R • Measurement Error Models

Sleuth

Docs ↗ By Harold Pimentel & Lior Pachter (Caltech)
(License: GPL-3.0)

Statistical model for differential analysis of RNA-Seq that directly incorporates inferential variance (bootstrap uncertainty) calculated during kallisto pseudoalignment.

Formats: Input: kallisto output directories • Output: Wald test, Likelihood Ratio tests
💻 4-8 CPU Cores • 8-16 GB RAM
R -e 'install.packages("sleuth")'
Read Summarization & Counting
💻 < 8GB RAM C • Ultra-fast Hash Table

featureCounts (Subread)

Docs ↗ By Yang Liao, Gordon Smyth & Wei Shi (WEHI)
(License: GPL-3.0)

Blazing-fast read summarization program for counting mapped RNA-seq or genomic DNA reads to genomic features (exons, genes, promoters). Substantially faster than HTSeq-count.

Formats: Input: SAM, BAM, SAF/GTF • Output: Count Matrix, Summary stats
💻 4-8 CPU Cores • 4-8 GB RAM
conda install -c bioconda subread
Recommended Workflow Progression

Proceed to Stage 6: Single-Cell & Spatial Transcriptomics

Sequencing pipelines proceed sequentially. Move forward to the next analytical domain to explore downstream algorithms and execution profiles.

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