Day 3 Agenda

Goals

Morning session: Differential expression and haplotype 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 & haplotype analysis.

Afternoon session: Single-cell transcriptomics with long reads

Learn the differences between bulk experiments and single-cell experiments (library preparation and data).
Understand the different steps involved in going from Long Sequencing Reads to gene and isoform matrices.
Learn the basics of tertiary analysis for single cell data with focus on long-read specific steps.

Time	Activity	Details
09:00 - 10:30	Differential Expression & DIU	Ana: How to build your expression matrix and concept of DE analysis vs DIU. Hands-on with tappAS and IsoTools.
10:30 - 11:00	Coffee Break
11:00 - 12:30	Allele-specific & Haplotype analysis	Nadja: Biases, limitations and pipelines for allele-specific expression. Hands-on ASE quantification and analysis.
12:30 - 13:30	Lunch break
13:30 - 15:00	Single-Cell Long-Read intro & QC	Eamon & Fran: Library preparation overview, Preprocessing and QC for single-cell long-reads.
15:00 - 15:30	Coffee Break
15:30 - 17:00	Tertiary analysis & Hands-on	Eamon & Fran: Filtering, QC, cell typing, gene & isoform level analysis. Pipeline overview and exploring differential isoform usage.

Morning session: Differential expression and haplotype analysis

Understand the difficulties of assigning reads to isoforms.
Understand biological mechanisms of allelic imbalance & different sources of bias in allele-specific expression analysis.

Afternoon session: Single-cell transcriptomics with long reads

Identify and understand differences between bulk and single-cell protocols.
Understand single-cell analysis pipeline steps (preprocessing, QC, tertiary analysis).

Differential expression: Slides for Join&Call, Call&Join and differential expression.
tappAS: Slides for tappAS analysis.
Haplotype analysis: Slides for allele-specific expression.
Single-cell 1: Introductory slides about single-cell long-reads.
Single-cell 2: Slides for single cell analysis pipelines.
Single-cell 3: Slides for tertiary analysis of long-reads single cell.

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
Gupta, P., O’Neill, H., Wolvetang, E.J., Chatterjee, A., Gupta, I., 2024. Advances in single-cell long-read sequencing technologies. NAR Genomics and Bioinformatics 6, lqae047. https://doi.org/10.1093/nargab/lqae047
Heumos, L., Schaar, A.C., Lance, C., Litinetskaya, A., Drost, F., Zappia, L., Lücken, M.D., Strobl, D.C., Henao, J., Curion, F., Schiller, H.B., Theis, F.J., 2023. Best practices for single-cell analysis across modalities. Nat Rev Genet 24, 550–572. https://doi.org/10.1038/s41576-023-00586-w