STAR aligns RNA-Seq data to reference genomes. It is designed to be fast and accurate for known and novel splice junctions. In addition, it has no limit on the read size and can align reads with multiple splice junctions. This is becoming more important as read lengths increase. As a result of the alignment algorithm, poor quality tails and poly-A tails are clipped in the alignment.

Starting with version 2.4.1a, annotations can be included at the mapping stage instead/in addition to the indexing stage.

STAR-Fusion uses the STAR aligner to identify candidate fusion transcripts.

There are now separate modules for STAR and STAR-Fusion.

References:

• Alexander Dobin et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013, 29:15-21. Pubmed |  PMC |  Journal

• STAR Home page
• STAR Manual
• STAR Google group
• STAR-Fusion wiki

• Module Name: STAR (see the modules page for more information)
• STAR is a multithreaded application. Please match the number of allocated CPUs with the number of threads
• Example files in $STAR_TEST_DATA
• When creating sorted BAM output (--outSAMtype BAM SortedByCoordinate) please use lscratch for temporary file storage. Allocate about 6 x compressed input fastq size of lscratch.

STAR mades backwards incompatible changes to it's index structure in some versions.

For that reason star indices for each version of STAR available on biowulf are stored in

• /fdb/STAR_indices/[STAR VERSION]
• /fdb/STAR_current → /fdb/STAR_indices/[current default STAR version]

Various CTAT libraries are available at /fdb/CTAT. Some of the libraries are directly at the top level. Starting from STAR-Fusion 1.7, CTAT libraries in the correct format are available in version specific subdirectories such as

/fdb/CTAT/__genome_libs_StarFv1.7
/fdb/CTAT/__genome_libs_StarFv1.9
/fdb/CTAT/__genome_libs_StarFv1.10

Note that 1.11.0 and 1.12.0 are still compatible with __genome_libs_StarFv1.10

The following table shows the minimum version of STAR for recent versions of STAR-Fusion:

STAR is used to create genome indices as well as to align and map short reads to the indexed genome. A GTF format annotation of transcripts can be provided during indexing or, since version 2.4.1a, on the fly during mapping.

A simple example of indexing a small genome with annotation. If annotation is provided, a overhang depending on the readlength to be used has to be provided as well. In this example we use a small genome, so 30g is more than enough memory. For example, for 100nt reads:

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --cpus-per-task=12 --mem=30g --gres=lscratch:20
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144 ~]$ module load STAR
[user@cn3144 ~]$ mkdir -p indices/star100-EF4
[user@cn3144 ~]$ GENOME=/fdb/igenomes/Saccharomyces_cerevisiae/Ensembl/EF4
[user@cn3144 ~]$ STAR \
    --runThreadN 2 \
    --runMode genomeGenerate \
    --genomeDir indices/star100-EF4 \
    --genomeFastaFiles $GENOME/Sequence/WholeGenomeFasta/genome.fa \
    --sjdbGTFfile $GENOME/Annotation/Genes/genes.gtf \
    --sjdbOverhang 99 \
    --genomeSAindexNbases 10

However, a default value of 100 will work well in many cases. Note that for the output of sorted bam files, the temp directory should be placed in lscratch.

Align single end 50nt RNA-Seq data to the mouse genome:

[user@cn3144 ~]$ mkdir -p bam/rnaseq_STAR
[user@cn3144 ~]$ GENOME=/fdb/STAR_current/UCSC/mm10/genes-50
[user@cn3144 ~]$ STAR \
    --runThreadN 12 \
    --genomeDir $GENOME \
    --sjdbOverhang 50 \
    --readFilesIn $STAR_TEST_DATA/ENCFF138LJO_1M.fastq.gz \
    --readFilesCommand zcat \
    --outSAMtype BAM SortedByCoordinate \
    --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp \
    --outFileNamePrefix bam/rnaseq_STAR/test

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

To use STAR-Fusion, please load the separately available STAR-Fusion module

[user@3144 ~]$ module load STAR-Fusion

For more details on STAR-Fusion see the STAR-Fusion wiki.

Batch scripts should use the $SLURM_CPUS_PER_TASK environment variable to determine the number of threads to use. This variable is set by slurm according to the number of requested cores. An example script for aligning single end RNA-Seq data with STAR might look like the following:

#! /bin/bash
# this file is STAR.sh
set -o pipefail
set -e

function fail {
    echo "$@" >&2
    exit 1
}

module load STAR/2.7.10b || fail "could not load STAR module"
cd /data/$USER/test_data || fail "no such directory"
mkdir -p bam/rnaseq_STAR
GENOME=/fdb/STAR_indices/2.7.10b/UCSC/mm10/genes-50
STAR \
    --runThreadN $SLURM_CPUS_PER_TASK \
    --genomeDir $GENOME \
    --sjdbOverhang 50 \
    --readFilesIn fastq/rnaseq_500k.fastq.gz \
    --readFilesCommand zcat \
    --outSAMtype BAM SortedByCoordinate \
    --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp \
    --outFileNamePrefix bam/rnaseq_STAR/test

Submit this job using the Slurm sbatch command. STAR requires 30-45g for mammalian genomes. Human genomes generally require about 45GB.

sbatch --cpus-per-task=12 --mem=38g --gres=lscratch:20 STAR.sh

Create a swarmfile (e.g. STAR.swarm). For example:

cd /data/$USER/test_data \
  && mkdir -p bam/rnaseq_STAR \
  && STAR \
    --runThreadN $SLURM_CPUS_PER_TASK \
    --genomeDir /fdb/STAR_indices/2.7.10b/UCSC/mm10/genes-50 \
    --sjdbOverhang 50 \
    --readFilesIn fastq/rnaseq_500k.fastq.gz \
    --readFilesCommand zcat \
    --outSAMtype BAM SortedByCoordinate \
    --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp \
    --outFileNamePrefix bam/rnaseq_STAR/test
cd /data/$USER/test_data \
  && mkdir -p bam/rnaseq_STAR2 \
  && STAR \
    --runThreadN $SLURM_CPUS_PER_TASK \
    --genomeDir /fdb/STAR_indices/2.7.10b/UCSC/mm10/genes-50 \
    --sjdbOverhang 50 \
    --readFilesIn fastq/rnaseq_250k.fastq.gz \
    --readFilesCommand zcat \
    --outSAMtype BAM SortedByCoordinate \
    --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp \
    --outFileNamePrefix bam/rnaseq_STAR2/test

Note the use of line continuation. Submit this job using the swarm command.

swarm -f STAR.swarm -t 12 -g 38 --module STAR/2.7.10b --gres=lscratch:100