4.Normalization

Background

Sparsity of data and technical noise ("batch effects") --> will mask the signal of interest

Causes:

RNA content (total amount and species) varies

for mRNAs:

for sRNAs:

Caveat:

Typically only half of the spike-in were detected.

for Single cell RNA-seq (and exRNA-seq)

scran:
1. pools multiple cells (samples) in order to estimate cell-specific size factors in the presence of zero inflation and unbalanced differential expression of genes across groups of cells;
2. precluster (using e.g. rank-based clustering) the cells into smaller, more homogeneous sets
SCnorm
Census