RNA-Seq

RNA-Seq is an high throughput sequencing method commonly used to assess gene expression.

Keywords

Alignment, Annotation, BAM, De-novo-transcriptome-assembly, Differential-expression, Exploratory-analysis, Expression-estimation, FASTA, FASTQ, Feature-summarisation, Pre-processing, QC, Statistical-model

Global learning objectives

• Performing RNA-Seq analysis
• Being able to choose an appropriate strategy {Advanced}
• Recognizing the challenges and pitfalls
• Recognizing issues with the data
• Troubleshooting issues with the data {Advanced}
• Recognizing the importance of experimental designs

Modules

Every module has had an Essential/Optional state assigned by the consortium.
Essential conveys the opinion that these modules should always be adressed in
any course/workshop about RNA-Seq, i.e. they are the minimal set of modules
that a trainee has to learn about. The optional modules on the other hand may
be addressed depending on the workshop/course program/duration.

Preprocessing (Essential)

• Applying QC software and interpreting the output
• Deciding/performing necessary preprocessing steps

Alignment (Essential)

• Producing alignment
• Differentiate genome and trancriptome alignment
• Selecting the appropriate tool
• Recognizing the challenges and pitfalls
• Interpreting the aligners output

Alignment QC (Essential)

• Applying QC software and interpreting the output
• Deciding/performing necessary filtering steps

Feature summarization (Essential)

• Producing a table of counts
• Identifying proper strategy for the biological question
• Interpreting output

Feature summarization QC (Essential)

• Applying QC software and interpreting the output
• Deciding/performing necessary QC steps

Differential expression (Optional)

• Choosing an appropriate strategy
• Performing DE analysis
• Interpreting the output
• Recognizing the need for normalization

Exploratory analysis (Optional)

• Being able to visualise alignments
• Identifying confounding effects and taking necessary action

De-novo transcriptome assembly (Optional)

• Performing the analysis
• Recognizing the challenges and pitfalls
• Interpreting the assemblers output