nf-core/rnaseq: Gene Expression Analysis

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

Further details on the workflow can be found at:

Install Nextflow

The Sarek 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

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