Session IV starts with a discussion about different methods for differential expression analysis of RNA-seq data. It then segues into a discussion on the considerations for isoform level differential expression (lecture), followed by an interactive lesson on the quantification of isoforms using pseudo-alignment tools and analysis of isoform level differential expression of RNA-seq data. Transcriptome expression abundances for the MOV10 dataset are estimated using Salmon. The output of Salmon is used as input to Sleuth to obtain lists of differentially expressed transcripts/genes. The first half of the session ends with a lecture talking about advanced concepts related to using bash, and more specifically using the Orchestra cluster.
The second half of Session IV begins with combining the different components of RNA-seq analysis to create an automated workflow/pipeline using shell scripting. Following this, we introduce HBC's bcbio-nextgen pipeline for RNA-seq analysis with a hands-on demonstration using the Mov10 dataset. Finally, we explore other NGS technologies related to RNA-seq, with a focus on small RNA-seq and single cell RNA-seq (lectures).
These materials were developed for a trainer-led workshop, but are also amenable to self-guided learning.
| Lessons | Estimated Duration |
|---|---|
| RNA-seq analysis methods | 15 min |
| Considerations for isoform-level differential expression analysis | 75 min |
| Alignment-free expression estimation using Salmon | 45 min |
| Isoform-level differential expression using Sleuth | 100 min |
| Revisiting Orchestra | 50 min |
| Automating the RNA-seq workflow | 60 min |
| bcbio-nextgen RNA-seq | 80 min |
| Accessing genomic data - genome builds and FTP sites | 35 min |
| Other applications of RNA sequencing | 20 min |
| Small RNA-seq analysis | 75 min |
| Single-cell RNA-seq analysis (lecture) and Seurat RMarkdown demo | 120 min |