Hands-on Labs
Practical exercises for the RNA-seq Data Analysis Course
Lab Overview
Genomic File Formats
Explore common RNA-seq file formats: FASTQ, FASTA, GTF/GFF, BAM/SAM, BED, VCF. Understand header structures, quality encoding, and how each format is used in the analysis pipeline.
Open LabPublic Data Retrieval
Retrieve the GRCh38 reference genome and GTF annotation using NCBI datasets. Download GSE136366 FASTQ files via sra-toolkit and subsample to 1M reads using seqkit.
QC, Trimming & MultiQC
Assess raw read quality with FastQC, trim adapters and low-quality bases with fastp, and aggregate reports across all GSE136366 samples using MultiQC.
Open LabRead Alignment with STAR
Build a STAR genome index (GRCh38 + Ensembl GTF), align subsampled GSE136366 reads, index BAM with samtools, mark duplicates with Picard, and visualize alignments in IGV.
Open LabSalmon Quantification & Exploratory Analysis
Build a Salmon transcript index, quantify all GSE136366 samples with GC-bias correction, import counts into R with tximport, and perform PCA and sample-distance heatmap.
Open Labnf-core/rnaseq Workflow
Write a samplesheet for GSE136366, launch nf-core/rnaseq (STAR + Salmon) on Ibex with Singularity and SLURM, monitor progress, and explore the MultiQC report and merged count matrix.
Differential Expression with DESeq2
Load Salmon counts via tximport, build a DESeqDataSet, run DESeq2 for KO vs control, apply apeglm LFC shrinkage, and produce volcano plots, MA plots, and a heatmap of top 50 DE genes.
Open LabFunctional Enrichment & GSEA
Run GO (BP) and KEGG enrichment with clusterProfiler on DE genes from GSE136366. Perform GSEA using fgsea with MSigDB Hallmarks. Produce dotplots and enrichment plots.
Open Labnf-core/differentialabundance
Prepare samplesheet and contrasts CSV for GSE136366, run nf-core/differentialabundance on Ibex, and interpret the automated DESeq2 results, MultiQC dashboard, and interactive Shiny app.
Real-world Analysis Capstone
End-to-end independent analysis: download data, run QC, align, quantify, perform DEA with DESeq2, run functional enrichment, and optionally execute both nf-core workflows on GSE136366 or your own dataset.
Open Lab