Day 4
Day 4 Agenda
Goals
Morning 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.
Afternoon session: Metatranscriptomics
- Provide participants with a comprehensive understanding of the metatranscriptomics workflow
- Introduce practical skills for analyzing long-read metatranscriptomic data
- Highlight the challenges and considerations unique to environmental metatranscriptomics
Timetable
| Time | Activity | Details | |
|---|---|---|---|
| 09:00 - 09:15 | Introduction & Objectives | Eamon: Introduction | |
| 09:15 - 09:30 | Long-Read Single-cell Library Preparation | Fran: Differences between bulk library preparation and single-cell library preparation. | |
| 09:30 - 10:00 | Preprocessing and QC | Fran: Commercial analysis pipelines available, overview of the analysis steps and additional tools available. | |
| 10:00 - 10:30 | Coffee Break | Break | |
| 10:30 - 11:00 | Tertiary analysis | Eamon: Filtering, QC, cell typing, gene level analysis, isoform level analysis. | |
| 11:00 - 12:30 | Hands-on | Eamon and Fran: 1) Iso-Seq pipeline overview: commands, intermediate files and report. 2) Tertiary analysis: exploring differential isoform usage across different cell types. | |
| 12:30 - 13:30 | Lunch Break | Lunch and informal networking | |
| 13:30 - 13:40 | Introduction & Objectives | Carmen: Welcome participants, overview of the workshop goals and structure. Learning outcomes and expectations. | |
| 13:40 - 14:00 | Introduction to Metatranscriptomics | Carmen: What is metatranscriptomics? Difference with classical transcriptomics, applications, short vs long reads, challenges (rRNA contamination, read orientation issues, incomplete annotations) | |
| 14:00 - 15:00 | Metatranscriptomics workflow | Carmen: Understand all steps of the metatranscriptomic workflow: Experimental design, RNA extraction and purification, library preparation (prokaryotic vs eukaryotic), RNASeq, Filtering and QC, Downstream analysis (mapping and quantification, taxonomic classification, functional annotation). | |
| 15:00 - 15:30 | Coffee Break & Group Discussion | Break and open discussion | |
| 15:30 - 15:45 | Practical Setup & Dataset Overview | Carmen: Description of environmental samples, sequencing platform used, introduction to data files, preview of analysis tasks. | |
| 15:45 - 16:15 | Task 1: | Carmen: Pre-processing metatranscriptomic reads. | |
| 16:15 - 16:30 | Q&A Break | Open discussion, troubleshooting, clarification of Task1. | |
| 16:30 - 17:00 | Task 2 & 3: Mapping, Taxonomic assignation and Functional analysis | Carmen: Mapping metatranscriptomic reads, handling alignment files, perform taxonomic assignation and functional analysis. |
Learning Outcomes
Morning session: Single-cell and spatial transcriptomics with long reads
- Identify and understand differences between bulk and single-cell protocols
- Understand single-cell analysis pipeline steps
Afternoon session: Metatranscriptomics
- Explore applications of metatranscriptomics in environmental studies
- Understand what is metatranscriptomics workflow from RNA extraction to data interpretation
- Recognize challenges and biases regarding metatranscriptomics
- Perform quality control of raw metatranscriptomic reads
- Explore taxonomic diversity in different communities and characterise active functions
Materials
- 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.
- Metatranscriptomics: Slides for metatranscriptomics.
Data
Recommended Bibliography
- 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
- Peimbert, M. and Alcaraz, L. D. A hitchhiker’s guide to metatranscriptomics. Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing (2016). https://doi.org/10.1007/978-3-319-31350-4_13
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