GPTomics/bio-read-alignment-star-alignment

Version Compatibility

Reference examples tested with: STAR 2.7.11+, Subread 2.0+, fastp 0.23+, kallisto 0.50+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

STAR RNA-seq Alignment

"Align RNA-seq reads with STAR" -> Map RNA-seq reads to a reference genome with fast, sensitive splice-aware alignment. Preferred for large datasets and downstream fusion/chimeric read detection.

• CLI: STAR --runMode alignReads --genomeDir index/ --readFilesIn R1.fq R2.fq --outSAMtype BAM SortedByCoordinate

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

| File | Description | |------|-------------| | *Aligned.sortedByCoord.out.bam | Sorted BAM file | | *Log.final.out | Alignment summary statistics | | *Log.out | Detailed log | | *SJ.out.tab | Splice junctions | | *ReadsPerGene.out.tab | Gene counts (if --quantMode) | | *Chimeric.out.junction | Fusion candidates (if chimeric) |

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

| Parameter | Default | Description | |-----------|---------|-------------| | --runThreadN | 1 | Number of threads | | --sjdbOverhang | 100 | Read length - 1 | | --outFilterMultimapNmax | 10 | Max multi-mapping | | --alignIntronMax | 0 | Max intron size | | --outFilterMismatchNmax | 10 | Max mismatches | | --outSAMtype | SAM | Output format | | --quantMode | - | GeneCounts for counting | | --twopassMode | None | Basic for two-pass |

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