Bio workflows rnaseq to de

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RNA-seq to Differential Expression Workflow

Complete pipeline from raw FASTQ files to differential expression results.

Workflow Overview

FASTQ files
    |
    v
[1. QC & Trimming] -----> fastp
    |
    v
[2. Quantification] ----> Salmon (recommended) or STAR + featureCounts
    |
    v
[3. Import to R] -------> tximport (for Salmon) or direct counts
    |
    v
[4. DE Analysis] -------> DESeq2
    |
    v
[5. Visualization] -----> Volcano, MA, heatmaps
    |
    v
Significant gene list

Primary Path: Salmon + DESeq2

Step 1: Quality Control with fastp

# Single sample
fastp -i sample_R1.fastq.gz -I sample_R2.fastq.gz \
    -o sample_R1.trimmed.fq.gz -O sample_R2.trimmed.fq.gz \
    --detect_adapter_for_pe \
    --qualified_quality_phred 20 \
    --length_required 35 \
    --html sample_fastp.html

# Batch processing
for sample in sample1 sample2 sample3; do
    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
        -o trimmed/${sample}_R1.fq.gz -O trimmed/${sample}_R2.fq.gz \
        --detect_adapter_for_pe \
        --html qc/${sample}_fastp.html
done

QC Checkpoint 1: Check fastp reports

• Q30 bases >80%
• Adapter content <5%
• Duplication rate reasonable for library type

Step 2: Salmon Quantification

# Build index (once per transcriptome)
salmon index -t transcriptome.fa -i salmon_index -k 31

# Quantify each sample
for sample in sample1 sample2 sample3; do
    salmon quant -i salmon_index \
        -l A \
        -1 trimmed/${sample}_R1.fq.gz \
        -2 trimmed/${sample}_R2.fq.gz \
        -o quants/${sample} \
        --validateMappings \
        --gcBias \
        --seqBias \
        -p 8
done

QC Checkpoint 2: Check Salmon logs

• Mapping rate >70%

• 10 million reads mapped

Step 3: Import with tximport

library(tximport)
library(DESeq2)

# Create tx2gene mapping (Ensembl example)
tx2gene <- read.csv('tx2gene.csv')  # columns: TXNAME, GENEID

# List quantification files
samples <- c('sample1', 'sample2', 'sample3', 'sample4', 'sample5', 'sample6')
files <- file.path('quants', samples, 'quant.sf')
names(files) <- samples

# Import transcript-level estimates
txi <- tximport(files, type = 'salmon', tx2gene = tx2gene)

# Create sample metadata
coldata <- data.frame(
    condition = factor(c('control', 'control', 'control', 'treated', 'treated', 'treated')),
    row.names = samples
)

Step 4: DESeq2 Analysis

# Create DESeqDataSet from tximport
dds <- DESeqDataSetFromTximport(txi, colData = coldata, design = ~ condition)

# Pre-filter low count genes
keep <- rowSums(counts(dds)) >= 10
dds <- dds[keep,]

# Set reference level
dds$condition <- relevel(dds$condition, ref = 'control')

# Run DESeq2
dds <- DESeq(dds)

# Get results with shrinkage
res <- lfcShrink(dds, coef = 'condition_treated_vs_control', type = 'apeglm')

# Summary
summary(res)

QC Checkpoint 3: Check DESeq2 diagnostics

• Dispersion plot shows expected trend
• PCA separates conditions
• No severe outliers in sample distances

Step 5: Visualization and Export

library(ggplot2)
library(pheatmap)
library(ggrepel)

# Volcano plot
res_df <- as.data.frame(res)
res_df$gene <- rownames(res_df)
res_df$significant <- res_df$padj < 0.05 & abs(res_df$log2FoldChange) > 1

ggplot(res_df, aes(x = log2FoldChange, y = -log10(pvalue), color = significant)) +
    geom_point(alpha = 0.5) +
    scale_color_manual(values = c('grey', 'red')) +
    theme_minimal() +
    labs(title = 'Volcano Plot', x = 'Log2 Fold Change', y = '-Log10 P-value')

# Heatmap of top genes
vsd <- vst(dds, blind = FALSE)
top_genes <- head(order(res$padj), 50)
pheatmap(assay(vsd)[top_genes,], scale = 'row', show_rownames = FALSE)

# Export significant genes
sig_genes <- subset(res, padj < 0.05 & abs(log2FoldChange) > 1)
write.csv(as.data.frame(sig_genes), 'significant_genes.csv')

Alternative Path: STAR + featureCounts + DESeq2

Step 2 Alternative: STAR Alignment

# Build STAR index (once)
STAR --runMode genomeGenerate \
    --genomeDir star_index \
    --genomeFastaFiles genome.fa \
    --sjdbGTFfile genes.gtf \
    --sjdbOverhang 100 \
    --runThreadN 8

# Align each sample
for sample in sample1 sample2 sample3; do
    STAR --genomeDir star_index \
        --readFilesIn trimmed/${sample}_R1.fq.gz trimmed/${sample}_R2.fq.gz \
        --readFilesCommand zcat \
        --outFileNamePrefix aligned/${sample}_ \
        --outSAMtype BAM SortedByCoordinate \
        --quantMode GeneCounts \
        --runThreadN 8
done

Step 3 Alternative: featureCounts

# Count reads per gene
featureCounts -T 8 -p --countReadPairs \
    -a genes.gtf \
    -o counts.txt \
    aligned/*_Aligned.sortedByCoord.out.bam

Step 4 Alternative: Load Counts Directly

# Load featureCounts output
counts <- read.table('counts.txt', header = TRUE, row.names = 1, skip = 1)
counts <- counts[, 6:ncol(counts)]  # Remove annotation columns
colnames(counts) <- gsub('_Aligned.sortedByCoord.out.bam', '', colnames(counts))

# Create DESeqDataSet directly
dds <- DESeqDataSetFromMatrix(countData = counts, colData = coldata, design = ~ condition)

Parameter Recommendations

Step	Parameter	Recommendation
fastp	--qualified_quality_phred	20 (standard)
fastp	--length_required	35 for 2x100, 50 for 2x150
Salmon	-l	A (auto-detect library type)
Salmon	--gcBias	Enable for better accuracy
STAR	--sjdbOverhang	read_length - 1
featureCounts	-s	0=unstranded, 1=stranded, 2=reversely stranded
DESeq2	lfcShrink type	apeglm (recommended)
DESeq2	alpha	0.05 (standard significance)

Troubleshooting

Issue	Likely Cause	Solution
Low mapping rate (<50%)	Wrong reference, contamination	Check species, run FastQ Screen
High duplication	Low complexity library, over-sequencing	Check library prep, may be normal for low-input
No DE genes	Low power, batch effects	Add replicates, include batch in design
All genes DE	Normalization issue, sample swap	Check sample metadata, rerun normalization
Outlier samples	Technical failure, sample swap	Remove or investigate, check PCA

Complete Bash Pipeline Script

#!/bin/bash
set -e

THREADS=8
SAMPLES="sample1 sample2 sample3 sample4 sample5 sample6"
SALMON_INDEX="salmon_index"
OUTDIR="results"

mkdir -p ${OUTDIR}/{trimmed,quants,qc}

# Step 1: QC and trim
for sample in $SAMPLES; do
    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
        -o ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
        -O ${OUTDIR}/trimmed/${sample}_R2.fq.gz \
        --detect_adapter_for_pe \
        --html ${OUTDIR}/qc/${sample}_fastp.html \
        -w ${THREADS}
done

# Step 2: Quantify
for sample in $SAMPLES; do
    salmon quant -i ${SALMON_INDEX} -l A \
        -1 ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
        -2 ${OUTDIR}/trimmed/${sample}_R2.fq.gz \
        -o ${OUTDIR}/quants/${sample} \
        --validateMappings --gcBias -p ${THREADS}
done

echo "Quantification complete. Run R script for DE analysis."

Complete R Analysis Script

library(tximport)
library(DESeq2)
library(apeglm)
library(ggplot2)
library(pheatmap)

# Configuration
samples <- c('sample1', 'sample2', 'sample3', 'sample4', 'sample5', 'sample6')
conditions <- c('control', 'control', 'control', 'treated', 'treated', 'treated')
quant_dir <- 'results/quants'

# Import
tx2gene <- read.csv('tx2gene.csv')
files <- file.path(quant_dir, samples, 'quant.sf')
names(files) <- samples
txi <- tximport(files, type = 'salmon', tx2gene = tx2gene)

# DESeq2
coldata <- data.frame(condition = factor(conditions), row.names = samples)
dds <- DESeqDataSetFromTximport(txi, colData = coldata, design = ~ condition)
dds <- dds[rowSums(counts(dds)) >= 10,]
dds$condition <- relevel(dds$condition, ref = 'control')
dds <- DESeq(dds)

# Results
res <- lfcShrink(dds, coef = 'condition_treated_vs_control', type = 'apeglm')
sig <- subset(res, padj < 0.05 & abs(log2FoldChange) > 1)

cat('Significant genes:', nrow(sig), '\n')
write.csv(as.data.frame(sig), 'significant_genes.csv')

Related Skills

• read-qc/fastp-workflow - Detailed QC options and parameters
• rna-quantification/alignment-free-quant - Salmon and kallisto details
• rna-quantification/tximport-workflow - tximport options and tx2gene creation
• differential-expression/deseq2-basics - Complete DESeq2 reference
• differential-expression/de-visualization - Advanced visualization options
• pathway-analysis/go-enrichment - Next step: functional enrichment

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