---
name: read-alignment-star-alignment
description: Align RNA-seq reads with STAR (Spliced Transcripts Alignment to a Reference). Supports two-pass mode for novel splice junction discovery. Use when aligning RNA-seq data requiring splice-aware alignment.
tool_type: cli
primary_tool: STAR
---
## 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
```bash
# 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
```bash
# 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
```bash
# 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
```bash
STAR --runThreadN 8 \
--genomeDir star_index/ \
--readFilesIn reads.fq.gz \
--readFilesCommand zcat \
--outFileNamePrefix sample_ \
--outSAMtype BAM SortedByCoordinate
```
## Two-Pass Mode
```bash
# 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
```bash
# 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
```bash
# 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
```bash
# 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
```bash
# 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
```bash
# 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