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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)

Copy the metadata “samplesheet.csv” we just created into the working folder and paste the code below to the terminal:

cp $HOME/workshop/2024/rnaseq/data/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/rnaseq/scripts/launch_nf-core_RNAseq_pipeline.pbs $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 "

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
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