Why Normalize?

Normalization smooths out technical variations among the samples we are comparing so that we can more confidently attribute variations we see to biological reasons. 

We usually normalize for:

1. Sequencing depth: Say we are comparing gene counts in sample A against sample B.  If you start out with 10 million reads in sample A  vs 1 million reads in sample B, a 10 fold increase in expression in sample A is going to be purely due to its sequencing depth.

2. Gene length: A gene that is twice as long is likely to have twice as many reads sampling it.

3. GC content

Some Normalization Methods

RPKM: 

Median scaling (DESeq method):

TMM:

Quantile: