STAR RNA-seq Alignment

Generate Genome Index

# Basic index generation
STAR --runMode genomeGenerate \
    --runThreadN 8 \
    --genomeDir star_index/ \
    --genomeFastaFiles reference.fa \
    --sjdbGTFfile annotation.gtf \
    --sjdbOverhang 100    # Read length - 1

Index with Specific Read Length

# For 150bp reads, use sjdbOverhang=149
STAR --runMode genomeGenerate \
    --runThreadN 8 \
    --genomeDir star_index_150/ \
    --genomeFastaFiles reference.fa \
    --sjdbGTFfile annotation.gtf \
    --sjdbOverhang 149

Basic Alignment

# Paired-end alignment
STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn reads_1.fq.gz reads_2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate

Single-End Alignment

STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn reads.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate

Two-Pass Mode

# Two-pass mode for better novel junction detection
STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn r1.fq.gz r2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate \
    --twopassMode Basic

Quantification Mode

# Output gene counts (like featureCounts)
STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn r1.fq.gz r2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate \
    --quantMode GeneCounts

Output: sample_ReadsPerGene.out.tab with columns:

1. Gene ID
2. Unstranded counts
3. Forward strand counts
4. Reverse strand counts

ENCODE Options

# ENCODE recommended settings
STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn r1.fq.gz r2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate \
    --outSAMunmapped Within \
    --outSAMattributes NH HI AS NM MD \
    --outFilterType BySJout \
    --outFilterMultimapNmax 20 \
    --outFilterMismatchNmax 999 \
    --outFilterMismatchNoverReadLmax 0.04 \
    --alignIntronMin 20 \
    --alignIntronMax 1000000 \
    --alignMatesGapMax 1000000 \
    --alignSJoverhangMin 8 \
    --alignSJDBoverhangMin 1

Fusion Detection

# For chimeric/fusion detection
STAR --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn r1.fq.gz r2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate \
    --chimSegmentMin 12 \
    --chimJunctionOverhangMin 8 \
    --chimOutType Junctions WithinBAM SoftClip \
    --chimMainSegmentMultNmax 1

Output Files

Memory Requirements

# Reduce memory for limited systems
STAR --genomeLoad NoSharedMemory \
    --limitBAMsortRAM 10000000000 \  # 10GB for sorting
    ...

# For very large genomes, limit during index generation
STAR --runMode genomeGenerate \
    --limitGenomeGenerateRAM 31000000000 \  # 31GB
    ...

Shared Memory Mode

# Load genome into shared memory (for multiple samples)
STAR --genomeLoad LoadAndExit --genomeDir star_index/

# Run alignments (faster startup)
STAR --genomeLoad LoadAndKeep --genomeDir star_index/ ...

# Remove from memory when done
STAR --genomeLoad Remove --genomeDir star_index/

Key Parameters

Related Skills

• rna-quantification/featurecounts-counting – Alternative counting
• rna-quantification/alignment-free-quant – Salmon/kallisto alternative
• differential-expression/deseq2-basics – Downstream DE analysis
• read-qc/fastp-workflow – Preprocess reads