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This page provides a guide to QUT users to run the nf-core/sarek workflow on the QUT HPC.

 Further Further details on the workflow can be found at:

...

• Preprocessing

  • FastQC - Raw read QC

  • UMI-tools extract - UMI barcode extraction

  • TrimGalore - Adapter and quality trimming

  • SortMeRNA - Removal of ribosomal RNA (optional)

• Alignment and quantification

  • STAR and Salmon - Fast spliced aware genome alignment and transcriptome quantification

  • STAR via RSEM  - Alignment and quantification of expression levels

  • HISAT2  - Memory efficient splice aware alignment to a reference

• Alignment post-processing

  • SAMtools - Sort and index alignments

  • UMI-tools dedup - UMI-based deduplication

  • picard MarkDuplicates - Duplicate read marking

• Other steps

  • StringTie - Transcript assembly and quantification

  • BEDTools and bedGraphToBigWig - Create bigWig coverage files

• Quality control

  • RSeQC - Various RNA-seq QC metrics

  • Qualimap - Various RNA-seq QC metrics

  • dupRadar - Assessment of technical / biological read duplication

  • Preseq - Estimation of library complexity

  • featureCounts - Read counting relative to gene biotype

  • DESeq2 - PCA plot and sample pairwise distance heatmap and dendrogram

  • MultiQC - Present QC for raw reads, alignment, read counting and sample similiarity

• Pseudo-alignment and quantification

  • Salmon - Wicked fast gene and isoform quantification relative to the transcriptome

• Workflow reporting and genomes

  • Reference genome files - Saving reference genome indices/files

  • Pipeline information - Report metrics generated during the workflow execution

Getting Started

Download and run the workflow using a minimal data provided by nf-core/rnaseq. We recommend using singularity as the profile for QUT’s HPC. Other profile option can be ‘conda’. Note: the profile option ‘docker’ is not available on the HPC.

nextflow run nf-core/rnaseq -profile test,singularity

Running the pipeline using custom data

Example of a typical command to run a RNA-seq analysis for mouse samples:

  nextflow run nf-core/rnaseq \
      --input samplesheet.csv \
      --genome GRCm38 \
      -profile conda

Note, if the running was interrupted or did not complete a particular step or you want to modify a parameter for a particular step, instead of re-running all process nextflow enables to “-resume” the workflow.

  nextflow run nf-core/rnaseq \
      --input samplesheet.csv \
      --genome GRCm38 \
      -profile conda
      -resume

Preparing a ‘samplesheet.csv’ file

A samplesheet.csv file tells the workflow the location of the read 1 (R1), read 2 (R2) and other information about the samples including ‘group’ (i.e., control or infected), replicate number and the orientation of the reads (i.e., forward, reverse, unstranded).

Example samplesheet.csv:

group,replicate,fastq_1,fastq_2,strandedness
control,1,/path/to/fastq/control-1_R1.fastq.gz,/path/to/fastq/control-1_R2.fastq.gz,unstranded
control,2,/path/to/fastq/control-2_R1.fastq.gz,/path/to/fastq/control-2_R2.fastq.gz,unstranded
control,3,/path/to/fastq/control-3_R1.fastq.gz,/path/to/fastq/control-3_R2.fastq.gz,unstranded
infected,1,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-1_R2.fastq.gz,unstranded
infected,2,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-2_R2.fastq.gz,unstranded
infected,3,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-3_R2.fastq.gz,unstranded

Preparing to run on the HPC

To run this on the HPC a PBS submission script needs to be created.

In the folder you have created for this run create launch.pbs using a text editor (i.e., vim, nano)

#!/bin/bash -l
#PBS -N nfrna2
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=24:00:00
cd $PBS_O_WORKDIR
module load java
NXF_OPTS='-Xms1g -Xmx4g'
nextflow run nf-core/rnaseq -profile conda --input samplesheet.csv --genome GRCm38 --aligned star_rsem --min_mapped_reads 5 

Submitting the job

Once you have created the folder for the run, the input.tsv file, nextflow.config and launch.pbs you are ready to submit.

Submit the run with this command (On Lyra)

qsub launch.pbs

Monitoring the Run

You can use the command

qstat -u $USER

To check on the jobs you are running. Nextflow will launch additional jobs during the run.

You can also check the .nextflow.log file for details on what is going on.

Finally, if you have configured the connection to the NFTower you can logon and check your run.