STAR RNA-seq Alignment

Hard rules

1. No fabricated citations. Every cited work must resolve to a verifiable
2. No claim without provenance. Every quantitative or factual claim
3. No silent failure. Every script invocation, API call, or tool use must declare its exit status and what to do on non-zero. A skill that silently swallows errors is a violation.

When to use

• Bulk RNA-seq with novel junction discovery (most cases).
• Single-cell RNA-seq (STARsolo).
• Chimeric / fusion detection (with --chimOutType).
• Long-read cDNA / ONT direct RNA (use --alignEndsType Extend...).

When NOT to use

• Genome with no annotation and small memory budget → use HISAT2 (this category) or minimap2 -ax splice.
• DNA-seq → use bwa-mem or bwa-mem2.
• Quantification-only → use pseudo-alignment (salmon / kallisto).

Prerequisites

• STAR ≥ 2.7.11
• samtools ≥ 1.19
• Reference FASTA + GTF
• ~40 GB RAM and 50 GB disk for human index

Core workflow

1. Generate the genome index with STAR --runMode genomeGenerate.
2. Align reads with STAR --runMode alignReads.
3. 2-pass mapping for sensitive novel junction discovery: first pass produces a SJ.out.tab, second pass uses it.
4. Sort and index the BAM (STAR can output sorted BAM directly with --outSAMtype BAM SortedByCoordinate).
5. Index the BAM with samtools index.

Code patterns

Generate the genome index (one-time, ~40 GB RAM for human)

STAR --runMode genomeGenerate \
    --runThreadN 16 \
    --genomeDir star_index/ \
    --genomeFastaFiles reference/genome.fa \
    --sjdbGTFfile reference/genes.gtf \
    --sjdbOverhang 149 \
    --genomeSAindexNbases 14

--sjdbOverhang 149 for 150 bp reads; for shorter reads, set to read_length - 1.

Paired-end alignment (most common)

STAR --runMode alignReads \
    --runThreadN 16 \
    --genomeDir star_index/ \
    --readFilesIn reads_R1.fq.gz reads_R2.fq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample1/ \
    --outSAMtype BAM SortedByCoordinate \
    --outSAMattrRGline ID:sample1 SM:sample1 PL:ILLUMINA LB:lib1 \
    --quantMode GeneCounts

Output:

• sample1/Aligned.sortedByCoord.out.bam — coordinate-sorted BAM
• sample1/ReadsPerGene.out.tab — gene-level counts (use this for DESeq2/edgeR)
• sample1/SJ.out.tab — splice junctions
• sample1/Log.final.out — alignment stats

2-pass mapping for novel junction discovery

# Pass 1: align and produce splice junctions
STAR --runMode alignReads ... --outFileNamePrefix pass1/
mv pass1/SJ.out.tab pass1_SJ.tab

# Pass 2: re-align with discovered junctions
STAR --runMode alignReads \
    --sjdbFileChrStartEnd pass1_SJ.tab \
    --genomeDir star_index_with_pass1_SJ/ \
    ... --outFileNamePrefix pass2/

Or use the simpler workflow: regenerate the index with the new SJ file and re-align.

Single-cell RNA-seq (STARsolo)

STAR --runMode alignReads \
    --genomeDir star_index/ \
    --readFilesIn sc_R1.fastq.gz sc_R2.fastq.gz \
    --soloType CB_UMI_Simple \
    --soloCBstart 1 --soloCBlen 16 \
    --soloUMIstart 17 --soloUMIlen 10 \
    --soloBarcodeReadLength 0 \
    --soloCellFilter EmptyDrops_CR \
    --outFileNamePrefix sc_outs/

Long-read cDNA / ONT

STAR --runMode alignReads \
    --genomeDir star_index/ \
    --readFilesIn long_reads.fq.gz \
    --outFilterMismatchNmax 5 \
    --outFilterMatchNmin 10 \
    --alignEndsType ExtendSoftClip

Output sorted BAM and skip the post-alignment sort

STAR ... --outSAMtype BAM SortedByCoordinate ...

This writes the BAM sorted and lets you skip samtools sort. You still need samtools index.

Multi-sample parallel alignment (shell loop)

for r1 in reads/*_R1.fq.gz; do"
  base=$(basename "$r1" _R1.fq.gz)
  r2="reads/${base}_R2.fq.gz"
  mkdir -p "star_out/${base}"
  STAR --runMode alignReads \
      --runThreadN 8 \
      --genomeDir star_index/ \
      --readFilesIn "$r1" "$r2" \
      --readFilesCommand zcat \
      --outFileNamePrefix "star_out/${base}/" \
      --outSAMtype BAM SortedByCoordinate \
      --outSAMattrRGline ID:${base} SM:${base} PL:ILLUMINA \
      --quantMode GeneCounts
done

Index the output BAM

samtools index sample1/Aligned.sortedByCoord.out.bam

Extract splice junctions for visualization

# Convert STAR SJ.out.tab to BED12 for IGV
awk 'BEGIN{OFS="\t"} $1 !~ /#/ {print $1, $2-1, $3, ".", $7, $4}' SJ.out.tab > sj.bed

Important flags

Common pitfalls

• Not enough RAM for human index. STAR --runMode genomeGenerate needs ~40 GB. STAR 2.7.11+ has --limitGenomeGenerateRAM to bound it, but at the cost of slower index generation.
• Forgetting --readFilesCommand zcat for .fq.gz inputs. STAR will try to read them as text and fail.
• Using a default --sjdbOverhang for non-150 bp reads. Set to read_length - 1 for best sensitivity.
• Ignoring 2-pass mapping. The first pass discovers novel junctions, the second pass is more sensitive. For novel transcript discovery, 2-pass is essential.
• STARsolo barcode config. CBstart + CBlen and UMIstart + UMIlen must match your library prep. Misconfiguration gives 0% barcodes-in-cells.

Validation

• samtools flagstat sample1/Aligned.sortedByCoord.out.bam — high mapping rate (≥85% for bulk RNA-seq).
• head sample1/Log.final.out — look for Uniquely mapped reads % ≥ 75%.
• awk '$4=="Uniquely mapped reads %"' Log.final.out should be ≥ 70% for most samples.
• ReadsPerGene.out.tab should have non-zero counts for known reference genes.

Open alternatives

References

• STAR paper: Dobin et al. 2013, Bioinformatics — 10.1093/bioinformatics/bts635
• STAR 2.7.11+ release notes: https://github.com/alexdobin/STAR/releases
• Companion: ors-bioinformatics-sequence-hisat2-alignment, ors-bioinformatics-omics-rna-seq-count-matrix-qc.

Changelog

• 1.0.0 (2026-06-10): Initial adaptation by Pradyumna Jayaram from bio-star-alignment (bioSkills-main/read-alignment/star-alignment).

Cross-references

Other skills in this category:

• batch-processing
• bowtie2-alignment
• bwa-alignment
• bwa-mem2-alignment
• codon-usage
• compressed-sequence-files
• fastq-quality-scores
• filter-sequences
• format-conversion
• hisat2-alignment
• motif-search
• paired-end-fastq
• pysam-genomics
• read-write-sequences
• reverse-complement
• sam-bam-basics
• samtools-bam-processing
• seq-objects
• sequence-properties
• sequence-slicing
• sequence-statistics
• transcription-translation