Day 3
Day 3 Agenda
Goals
Morning session: Differential expression analysis
- To learn how long-read RNA-seq can be used for differential isoform expression/usage analysis and what are the current challenges
- To learn about specific case of expression analysis: allele-specific expression analysis
Afternoon session: RNA modifications
- Learn how long-read sequencing technologies are utilized for the study of RNA modifications
- Hands-on session for the study of the tRNAome
Timetable
| Time | Activity | Details | Literature |
|---|---|---|---|
| 9:00 - 9:30 | How to build your expression matrix and concept of DE analysis vs DIU | call and join, join and call | |
| 9:30 - 10:30 | Hands on | tappAS, IsoTools | tappAS, IsoTools |
| 10:30 - 11:00 | Coffee Break | ||
| 11:00 - 11:30 | Allele-specific expression | Specificity of long-reads * Introduction. * Biases and limitations. * Main steps. * Minimum input. * Expected output * Existing pipelines: LORALS, FALIR2, IDP-ASE |
Review about allele-specific expression analyis, LORALS |
| 11:30 - 12:30 | Hands on | analyze ASE dataset Hands-on Task 1 Description of the data. QC and filtering of already mapped samples. Hands-on Task 2 Allele-specific expression quantification Hands-on Task 3 Allele-imbalance testing Hands-on Task 4 Interpretation of results and output. Real example application Conclusion and remarks * |
|
| 12:30 - 13:30 | Lunch break | ||
| 13:30 - 14:30 | Introduction to epitranscriptomics | Overview of the field; use of long-read sequencing technologies for mapping RNA modifications; tRNA-specialized protocols and pipelines | |
| 14:30 - 15:00 | Hands-on | Mapping Nano-tRNAseq data | Tools: minimap2 |
| 15:00 - 15:30 | Coffee Break | ||
| 15:30 - 16:30 | Hands-on | Downstream processing: filtering, quality control, batch effect investigation, differential expression, diffenetial modifications | Tools: AMaNITA |
| 16:30 - 17:00 | Closing remarks | Additional pipelines; modification-aware basecallers |
Learning Objectives
Morning session: Differential expression analysis
- understand the difficulties of assigning reads to isoforms
- understand biological mechanisms of allelic imbalance
- understand different sources of bias in allele-specific expression analysis
Afternoon session: RNA modifications
- understand the past and current challenges in the epitranscriptomics field
- understand why and how long-reads sequencing technologies are utilized for RNA modification detection
- familiarize with tools for epitranscriptomics data analysis
Materials
- Differential expression: Slides for Join&Call, Call&Join and differential expression.
- tappAS: Slides for tappAS analysis.
- Haplotype analysis: Slides for allele-specific expression.
- Epitranscriptomics: Slides for epitranscriptomics.
Data
Recommended Bibliography
- Bi, Yalan. et al. IsoTools 2.0: software for comprehensive analysis of long-read transcriptome sequencing data. Journal of Molecular Biology (2025). https://doi.org/10.1016/j.jmb.2025.169049
- Glinos, D.A., Garborcauskas, G., Hoffman, P. et al. Transcriptome variation in human tissues revealed by long-read sequencing. Nature (2022). https://doi.org/10.1038/s41586-022-05035-y
- Cleary, S. and Seoighe, C. Perspectives on Allele-Specific Expression. Annual Reviews (2021). https://doi.org/10.1146/annurev-biodatasci-021621-122219
- De la Fuente, L., Arzalluz-Luque, A., Tardáguila, M., et al. tappAS: a comprehensive computational framework for the analysis of the functional impact of differential splicing. Genome Biology (2020). https://doi.org/10.1186/s13059-020-02028-w
- Lucas, M.C., Pryszcz, L. et al. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nature Biotechnology (2023). https://doi.org/10.1038/s41587-023-01743-6
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