STAR RNA-seq Alignment - Usage Guide

STAR RNA-seq Alignment - Usage Guide

Overview

STAR (Spliced Transcripts Alignment to a Reference) is the most widely used RNA-seq aligner. It's extremely fast, supports splice-aware alignment, and can detect novel junctions with two-pass mode.

Prerequisites

conda install -c bioconda star samtools

Quick Start

Tell your AI agent what you want to do:

• "Align my RNA-seq reads using STAR"
• "Generate a STAR index for my reference genome and GTF annotation"
• "Run two-pass STAR alignment with gene counting"

Example Prompts

Index Generation

"Create a STAR index for genome.fa with genes.gtf annotation for 150bp reads"

"Generate STAR genome index with appropriate sjdbOverhang for my 100bp reads"

Basic Alignment

"Align my paired-end RNA-seq reads to the STAR index"

"Run STAR alignment on sample_R1.fq.gz and sample_R2.fq.gz"

Two-Pass Mode

"Align RNA-seq reads using STAR two-pass mode for novel junction discovery"

"Run STAR with twopassMode Basic and generate gene counts"

Multi-Sample Studies

"Collect splice junctions from all samples and realign with combined junctions"

"Run two-pass alignment across multiple samples for my population study"

What the Agent Will Do

1. Generate STAR index if not present (with correct sjdbOverhang)
2. Align reads with appropriate parameters
3. Run two-pass mode if requested for novel junction discovery
4. Generate gene counts if quantMode is enabled
5. Sort and index output BAM file

Tips

• Set sjdbOverhang to (read length - 1) for optimal junction detection
• Use --twopassMode Basic for most RNA-seq analyses
• STAR requires ~30GB RAM for human genome; use HISAT2 for low-memory systems
• Use --quantMode GeneCounts for built-in gene counting
• Check Log.final.out for alignment quality metrics

Index Parameters

Complete RNA-seq Pipeline

GENOME=genome.fa
GTF=genes.gtf
INDEX_DIR=star_index
R1=sample_R1.fq.gz
R2=sample_R2.fq.gz
PREFIX=sample_
THREADS=16

if [ ! -d "$INDEX_DIR" ]; then
    mkdir -p $INDEX_DIR
    STAR --runMode genomeGenerate \
        --runThreadN $THREADS \
        --genomeDir $INDEX_DIR \
        --genomeFastaFiles $GENOME \
        --sjdbGTFfile $GTF \
        --sjdbOverhang 149
fi

STAR --runThreadN $THREADS \
    --genomeDir $INDEX_DIR \
    --readFilesIn $R1 $R2 \
    --readFilesCommand zcat \
    --outFileNamePrefix $PREFIX \
    --outSAMtype BAM SortedByCoordinate \
    --twopassMode Basic \
    --quantMode GeneCounts

samtools index ${PREFIX}Aligned.sortedByCoord.out.bam

Stranded Libraries

Troubleshooting

Low Unique Mapping

• Check read quality
• Verify correct reference genome
• Check for contamination

Memory Errors

--limitBAMsortRAM 10000000000
--outSAMtype BAM Unsorted

STAR vs HISAT2