Overview of today’s session:
...
inspect the results from Session 2
run an advanced RNA-seq pipeline to measure the expression of genes
(optional) run statistical analysis to identify differentially expressed genes
Task 1: Evaluation of RNA-seq results using a basic (generic) nextflow pipeline
The nextflow/RNA-seq pipeline automatically generates two output folders:
...
Connect to the work folder via HPC-FS (See session 2). Browse to the fastqc output folder: run1_star_salmon → results → multiqc.
Task 2: Run the nextflow nf-core/rnaseq pipeline by including advanced filtering parameters
Requirements:
index.csv → a file that provides a list of sample IDs and their associated FASTQ files (read 1 and read 2)
launch.pbs → a script to submit the job to the HPC cluster
Example index.csv file for nf-core/rnaseq version 3.3:
| Code Block |
|---|
group,fastq_1,fastq_2,strandedness
control_r1,/work/kenna_team/data/raw_data/SRR1039508_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039508_2.fastq.gz,unstranded
dex_r1,/work/kenna_team/data/raw_data/SRR1039509_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039509_2.fastq.gz,unstranded
control_r2,/work/kenna_team/data/raw_data/SRR1039512_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039512_2.fastq.gz,unstranded
dex_r2,/work/kenna_team/data/raw_data/SRR1039513_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039513_2.fastq.gz,unstranded
control_r3,/work/kenna_team/data/raw_data/SRR1039516_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039516_2.fastq.gz,unstranded
dex_r3,/work/kenna_team/data/raw_data/SRR1039517_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039517_2.fastq.gz,unstranded
control_r4,/work/kenna_team/data/raw_data/SRR1039520_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039520_2.fastq.gz,unstranded
dex_r4,/work/kenna_team/data/raw_data/SRR1039521_1.fastq.gz,/work/kenna_team/data/raw_data/SRR1039521_2.fastq.gz,unstranded
|
Example of a launch.pbs script with advanced parameter options:
| Code Block |
|---|
#!/bin/bash -l
#PBS -N nfrnaseq
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=24:00:00
#Use the current directory to run the workflow
cd $PBS_O_WORKDIR
module load java
NXF_OPTS='-Xms1g -Xmx4g'
#run the nextflow RNA-seq pipeline:
nextflow run nf-core/rnaseq -profile singularity -r 3.3 --aligner star_salmon --input index.csv --genome GRCh38 --clip_r1 10 --clip_r2 10 --three_prime_clip_r1 2 --three_prime_clip_r2 2 --save_trimmed -resume
|
Session 3 exercises:
Run the nf-core/rnaseq pipeline using the Airway smooth muscle public data (PMID: 24926665. GEO: GSE52778) - aligner option set to ‘star_salmon’
Same as above but aligner option set to ‘star_rsem’
Create a new working folder:
| Code Block |
|---|
mkdir session3
cd session3
mkdir run1_star_salmon
cd run1_star_salmon
cd ..
|
Copy index.csv and launch.pbs files to the newly created folder
| Code Block |
|---|
cp /work/kenna_team/scripts/star_salmon/session3/* . |
Check that files were copied into the new working folder
| Code Block |
|---|
ls -a
./ ../ index.csv launch.pbs
#verify the content of index.csv
cat index.csv
#also check the PBS Pro submission script
cat launch.pbs
|
Run the workflow:
| Code Block |
|---|
qsub launch.pbs |
Monitor the progress of the workflow:
| Code Block |
|---|
qjobs |
or
| Code Block |
|---|
qstats -u userID |
Repeat the above process for ‘star_rsem’
The only variation is copying the index.csv and launch.pbs script. As follows:
| Code Block |
|---|
cp /work/kenna_team/scripts/star_rsem/session3/* . |