Mapping with STAR

Owned by Dhivya Arasappan
Last updated: Jun 13, 2024
2 min read

Objectives

In this lab, you will explore a faster splice aware mapper called STAR. Simulated RNA-seq data will be provided to you; the data contains 75 bp paired-end reads that have been generated in silico to replicate real gene count data from Drosophila. The data simulates two biological groups with three biological replicates per group (6 samples total).  The objectives of this lab is mainly to:


  1. Learn how STAR works and how to use it.

Introduction

STAR '‘Spliced Transcripts Alignment to a Reference" is a faster alternative  for splice-aware read alignment. STAR can align the non-contiguous sequences directly to the genome. The STAR algorithm consists of two major steps: seed searching step and clustering/stitching/scoring step. STAR is more memory intensive (30 gb of RAM required for human genome as compared to ~5 gb required by hisat2), but it is fast.




Get your data

Six raw data files have been provided for all our further RNA-seq analysis:

  • c1_r1, c1_r2, c1_r3 from the first biological condition
  • c2_r1, c2_r2, and c2_r3 from the second biological condition


Get set up for the exercises
cds
cd my_rnaseq_course/day_2
cd star_exercise

Run STAR

See if STAR is a module that is available on lonestar6.

module spider star


#if not already loaded, module load biocontainers
module load star
#get the full path to STAR
type STAR

#the below command would need to be RUN IN AN IDEV SESSION since you cannot run biocontainer executables on login nodes.
STAR --help

Part 1. Create a index of your reference

NO NEED TO RUN THIS NOW- YOUR INDEX HAS ALREADY BEEN BUILT!

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b--h43eeafb_1.sif STAR --runMode genomeGenerate --genomeDir STAR_genome/ --genomeFastaFiles genome.fa --sjdbGTFfile genes.formatted.gtf --sjdbOverhang 74 --genomeChrBinNbits 14


Part 2. Align the samples to reference 

Submit to the TACC queue or run in an idev shell

Create a commands file and use launcher_creator.py followed by sbatch.

 Click here to expand...

nano commands.star


Put this in your commands file:

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b--h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C1_R1 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794483_C1_R1_1.fq ../data/GSM794483_C1_R1_2.fq

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b--h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C1_R2 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794484_C1_R2_1.fq ../data/GSM794484_C1_R2_2.fq

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b--h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C1_R3 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794485_C1_R3_1.fq ../data/GSM794485_C1_R3_2.fq

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b--h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C2_R1 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794486_C2_R1_1.fq ../data/GSM794486_C2_R1_2.fq

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b–h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C2_R2 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794487_C2_R2_1.fq ../data/GSM794487_C2_R2_2.fq

apptainer exec ${BIOCONTAINER_DIR}/biocontainers/star/star-2.7.11b–h43eeafb_1.sif STAR --runThreadN 1 --outFileNamePrefix C2_R3 --outSAMstrandField intronMotif --genomeDir ../reference/STAR_genome --outSAMtype BAM Unsorted --readFilesIn ../data/GSM794488_C2_R3_1.fq ../data/GSM794488_C2_R3_2.fq

 Use this Launcher_creator command

launcher_creator.py -j commands.star -n starmap -q normal -t 08:00:00 -a OTH21164 -l star_launcher.slurm -m "module load biocontainers; module load star" -N 6 -w 1

 Submit using sbatch

sbatch --reservation=RNAseq star_launcher.slurm

This brings up the concept of wayness when running things on lonestar6!  Let's go back to Submitting Jobs to lonestar6 to discuss that.

Back to Course Outline


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