Subject of RNA-Seq
High throughput sequencing tells us which genes are activated, and how much they are transcribed.
3 Main Steps for RNA-Seq
- Prepare a sequencing library
- Sequence
Sequence
Raw data
Each sequencing “Read” consists of four lines of data.
- 1st line started with “@” A unique ID for the sequence that follows
- 2nd line Bases called for the sequenced fragments.
- 3rd line A “+” character.
Filter out garbage reads
garbage reads
- Reads with low quality base calls.
Count the gene reads
Data analysis
Step 1. Plot the data
PCA (Principal Component Analysis) reduces the number of axes you need to display the important aspects of the data.
Step 2. Identify differentially expressed genes (DEGs)
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