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This page provides a guide to QUT users on how to install and run the nextflow nf-core/rnaseq workflow on the HPC.

Further details on the workflow can be found at:

https://nf-co.re/rnaseq/3.0

https://nf-co.re/rnaseq/3.0/usage

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Pre-requisites

• Installed conda3 or miniconda3 ( https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html )

• Basic unix command line knowledge (example: https://researchcomputing.princeton.edu/education/external-online-resources/linux ; https://swcarpentry.github.io/shell-novice/ )

• Familiarity with one unix text editors (example Vi/Vim or Nano):

  • VIM ( https://bioinformatics.uconn.edu/vim-guide/ ; https://missing.csail.mit.edu/2020/editors/)

  • Nano (https://engineering.purdue.edu/ECN/Support/KB/Docs/BasictutorialforNanou ; https://www.howtogeek.com/howto/42980/the-beginners-guide-to-nano-the-linux-command-line-text-editor/ )

• Have an HPC account on QUT’s lyra

Install Nextflow

The nf-core/rnaseq workflow requires Nextflow to be installed in your account on the HPC. Find details on how to install and test Nextflow here prepare a cextflow.config file and run a PBS pro submission script for Nextflow pipelines.

Additional information available here: https://nf-co.re/usage/installation

Additional details on the workflow can be found at:

https://nf-co.re/rnaseq/3.0

https://nf-co.re/rnaseq/3.0/usage

https://nf-co.re/rnaseq/3.0/output

Pipeline summary

• 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

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