Overview
Use modified launch script to run the full pipeline, including trimming parameters based on the QC output.
Inspect precomputed results
Run full nf-core/rnaseq pipeline
STEP1: copy metadata (sample sheet.csv) into the working folder (run2_RNAseq)
cp $HOME/workshop/2024-2/session4_RNAseq/data/mouse/samplesheet.csv $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq cd $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq
Line 1: Copy the samplesheet.csv file to the working directory
Line 2: move to the working directory
Copy the PBS Pro script to run the nf-core/rnaseq pipeline:
cp $HOME/workshop/2024-2/session4_RNAseq/scripts/launch_nf-core_RNAseq_pipeline.pbs $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq
NOTE: if you had issues with the above lines. Alternatively, run the following code to copy the sample sheet.csv and launch files:
cp /work/training/2024/rnaseq/data/samplesheet.csv $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq cp /work/training/2024/rnaseq/scripts/launch_nf-core_RNAseq_pipeline.pbs cd $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq
Adjusting the Trim Galore (read trimming) options
Print the content of the launch_RNAseq.pbs script:
cat launch_nf-core_RNAseq_pipeline.pbs
#!/bin/bash -l #PBS -N nfRNAseq #PBS -l select=1:ncpus=2:mem=4gb #PBS -l walltime=48:00:00 #work on current directory cd $PBS_O_WORKDIR
#load java and set up memory settings to run nextflow module load java export NXF_OPTS='-Xms1g -Xmx4g'
nextflow run nf-core/rnaseq --input samplesheet.csv \ --outdir results \ -r 3.14.0 \ --genome GRCm38-local \ -profile singularity \ --aligner star_salmon \ --extra_trimgalore_args "--quality 30 --clip_r1 10 --clip_r2 10 --three_prime_clip_r1 1 --three_prime_clip_r2 1 " |
|---|
where:
Submitting the job
qsub launch_nf-core_RNAseq_pipeline.pbs
Monitoring the Run
qjobs
Outputs
The pipeline will produce two folders, one called “work,” where all the processing is done, and another called “results,” where we can find the pipeline's outputs. The content of the results folder is as follows:
/work/training/2024/rnaseq/runs/run3_RNAseq/results/
├── fastqc
│ ├── SRR20622172_fastqc.html
│ ├── SRR20622172_fastqc.zip
│ ├── SRR20622173_fastqc.html
│ ├── SRR20622173_fastqc.zip
│ ├── SRR20622174_fastqc.html
│ ├── SRR20622174_fastqc.zip
│ ├── SRR20622175_fastqc.html
│ ├── SRR20622175_fastqc.zip
│ ├── SRR20622176_fastqc.html
│ ├── SRR20622176_fastqc.zip
│ ├── SRR20622177_fastqc.html
│ ├── SRR20622177_fastqc.zip
│ ├── SRR20622178_fastqc.html
│ ├── SRR20622178_fastqc.zip
│ ├── SRR20622179_fastqc.html
│ ├── SRR20622179_fastqc.zip
│ ├── SRR20622180_fastqc.html
│ └── SRR20622180_fastqc.zip
├── multiqc
│ └── star_salmon
├── pipeline_info
│ ├── execution_report_2024-08-08_12-45-46.html
│ ├── execution_timeline_2024-08-08_12-45-46.html
│ ├── execution_trace_2024-08-08_12-45-46.txt
│ ├── params_2024-08-08_14-01-19.json
│ ├── pipeline_dag_2024-08-08_12-45-46.html
│ └── software_versions.yml
├── star_salmon
│ ├── bigwig
│ ├── deseq2_qc
│ ├── dupradar
│ ├── featurecounts
│ ├── log
│ ├── metadata.xlsx
│ ├── picard_metrics
│ ├── qualimap
│ ├── rseqc
│ ├── salmon.merged.gene_counts_length_scaled.rds
│ ├── salmon.merged.gene_counts_length_scaled.tsv
│ ├── salmon.merged.gene_counts.rds
│ ├── salmon.merged.gene_counts_scaled.rds
│ ├── salmon.merged.gene_counts_scaled.tsv
│ ├── salmon.merged.gene_counts.tsv
│ ├── salmon.merged.gene_lengths.tsv
│ ├── salmon.merged.gene_tpm.tsv
│ ├── salmon.merged.transcript_counts.rds
│ ├── salmon.merged.transcript_counts.tsv
│ ├── salmon.merged.transcript_lengths.tsv
│ ├── salmon.merged.transcript_tpm.tsv
│ ├── samtools_stats
│ ├── SRR20622172
│ ├── SRR20622172.markdup.sorted.bam
│ ├── SRR20622172.markdup.sorted.bam.bai
│ ├── SRR20622173
│ ├── SRR20622173.markdup.sorted.bam
│ ├── SRR20622173.markdup.sorted.bam.bai
│ ├── SRR20622174
│ ├── SRR20622174.markdup.sorted.bam
│ ├── SRR20622174.markdup.sorted.bam.bai
│ ├── SRR20622175
│ ├── SRR20622175.markdup.sorted.bam
│ ├── SRR20622175.markdup.sorted.bam.bai
│ ├── SRR20622176
│ ├── SRR20622176.markdup.sorted.bam
│ ├── SRR20622176.markdup.sorted.bam.bai
│ ├── SRR20622177
│ ├── SRR20622177.markdup.sorted.bam
│ ├── SRR20622177.markdup.sorted.bam.bai
│ ├── SRR20622178
│ ├── SRR20622178.markdup.sorted.bam
│ ├── SRR20622178.markdup.sorted.bam.bai
│ ├── SRR20622179
│ ├── SRR20622179.markdup.sorted.bam
│ ├── SRR20622179.markdup.sorted.bam.bai
│ ├── SRR20622180
│ ├── SRR20622180.markdup.sorted.bam
│ ├── SRR20622180.markdup.sorted.bam.bai
│ ├── stringtie
│ └── tx2gene.tsv
└── trimgalore
├── fastqc
├── SRR20622172.fastq.gz_trimming_report.txt
├── SRR20622173.fastq.gz_trimming_report.txt
├── SRR20622174.fastq.gz_trimming_report.txt
├── SRR20622175.fastq.gz_trimming_report.txt
├── SRR20622176.fastq.gz_trimming_report.txt
├── SRR20622177.fastq.gz_trimming_report.txt
├── SRR20622178.fastq.gz_trimming_report.txt
├── SRR20622179.fastq.gz_trimming_report.txt
└── SRR20622180.fastq.gz_trimming_report.txt
The quantification of the gene and transcript expressions can be found in the ‘star_salmon’ directory.
cd results/star_salmon
The following feature count tables are generated:
#gene level expression salmon.merged.gene_counts_length_scaled.rds salmon.merged.gene_counts_length_scaled.tsv salmon.merged.gene_counts.rds salmon.merged.gene_counts_scaled.rds salmon.merged.gene_counts_scaled.tsv salmon.merged.gene_counts.tsv <--- This file will be used for differential expression analysis using DESeq2 salmon.merged.gene_tpm.tsv #transcript level expression salmon.merged.transcript_counts.rds salmon.merged.transcript_counts.tsv salmon.merged.transcript_tpm.tsv