🧬 Getting started with Bulk RNA-Seq Primary, Secondary/Upstream and Tertiary/Downstream analysis?

What are the important concepts, recommendations and best practices for RNA-Seq experimental design? Which pipelines/tools (with and without coding) to go for secondary/upstream analysis?

✨ Concepts, Recommendations and Best Practices: 📚

1. What are the three analysis stages?

1. Primary analysis: The generation of DNA sequencing data from biological samples through the process of converting raw sequencing instrument signals into nucleotides and sequence reads.

2. Secondary analysis: This involves transcriptome profiling through read alignment & gene/transcript quantification.

3. Tertiary analysis: This includes differential gene expression & functional analysis.

   

2. What’s your project goal? Choose the RNA-Seq library prep method that fits it.

1. mRNA only (via Poly-A selection)
2. Whole transcriptome library (all RNA Species except for rRNA via rRNA depletion)
3. https://pubmed.ncbi.nlm.nih.gov/34782601/ Fig. 1

3. What are technical and biological replicates?

1. Technical replicates: Experimental samples isolated from one biological sample (a single mouse)

2. Biological replicates: Extracting RNA from three different mice

3. https://pubmed.ncbi.nlm.nih.gov/26392568/

   

4. How many replicates do you need?

1. https://pubmed.ncbi.nlm.nih.gov/27022035/
2. https://pubmed.ncbi.nlm.nih.gov/26813401/

5. How many reads and read length required?

1. https://knowledge.illumina.com/library-preparation/rna-library-prep/library-preparation-rna-library-prep-reference_material-list/000001243
2. https://genohub.com/recommended-sequencing-coverage-by-application/
3. “Sequencing depth and coverage: key considerations in genomic analyses” https://pubmed.ncbi.nlm.nih.gov/24434847/
4. "A survey of best practices for RNA-seq data analysis" https://pubmed.ncbi.nlm.nih.gov/26813401/

6. Which human reference genome to use?

https://lh3.github.io/2017/11/13/which-human-reference-genome-to-use

🛠️ Pipelines and Tools With and Without Coding:

1. With coding:

1. On HPC cluster: Secondary/Upstream analysis workflow using open-source RNA-Seq pipeline https://nf-co.re/rnaseq/3.21.0/ from nf-core
2. On Seqera platform using nf-core/rnaseq pipeline: https://seqera.io/blog/step-by-step-rna-seq/

2. No coding:

1. Using publicly available Galaxy instances, e.g. UseGalaxy.org, UseGalaxy.org.au, UseGalaxy.fr, UseGalaxy.eu
2. https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/rna-seq-reads-to-counts/tutorial.html