> For the complete documentation index, see [llms.txt](https://lulab1.gitbook.io/training/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://lulab1.gitbook.io/training/archive/archive-2019/part-iii.-case-studies/case-study-1.exrna-seq/1.4.normalization-issues.md).

# 1.4 Normalization Issues

## Background

### Problems and issues

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

**Causes:**

![](/files/-LApP5BTGlhGxP0yvsit)

### Spike-in for Normalization

**RNA content (total amount and species) varies**

![](/files/-LApP5Qqho3lpI6lMTJP)

**for mRNAs:**

* 92 ERCC molecules
* 8 mRNAs
* whole transcriptome HeLa RNAs

**for sRNAs:**

* 52(?) sRNA sequences

**Caveat:**

Typically only half of the spike-in were detected.

### Computational Normalization Tools

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

1. **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
2. SCnorm
3. Census

If considering spike-ins:

1. SAMstrt
2. GRM

## **References**

* [Normalizing single-cell RNA sequencing data: challenges and opportunities, *Nature Methods*, 2017](https://www.ncbi.nlm.nih.gov/pubmed/28504683)

## **More Reading & Practice**

See more about **normalization**, **imputation** and **confounder** (e.g. batch effect) in

* [Additional Tutorial ](/training/archive/archive-2019/getting-startted.md#learning-materials): 4.QC and Normalization; 5. Imputation and Confounders

## Video

### a) Normalization 1

[@Youtube](https://youtu.be/QCxNIGrTWh0)

[@Bilibili](https://player.bilibili.com/player.html?aid=30592584\&cid=53396844\&page=1)

### b) Normalization 2

[@Youtube](https://youtu.be/QDy2grY78DE)

[@Bilibili](https://player.bilibili.com/player.html?aid=30592643\&cid=53396972\&page=1)