# 6.RNA Regulation - I

## 1) Features from <mark style="color:red;">conventional RNA-seq</mark>

### 1a) Introduction

在本节中，我们将介绍一系列从二代测序数据出发分析RNA调控的生物信息学方法。基因表达的调控指的是细胞通过改变不同蛋白的表达水平来响应外界的变化。这里的RNA调控指的是在RNA水平上的基因调控。利用二代测序来对RNA调控进行分析，有些利用常规的RNA-seq数据就可以完成，例如:

* 在不同组织中或不同条件下，真核生物的转录本普遍存在可变剪接(alternative splicing)，这就可能导致蛋白产物也相应的发生变化。除此之外，还存在一些跨转录本的剪接事件，导致不同基因的转录本被嵌合到一条RNA中，产生嵌合RNA(chimeric RNA)；以及反向剪接(back splicing)事件，使得一部分转录本首尾相连形成环状(circular RNA)等。这些剪接产物有些会被表达出来，直接导致蛋白的改变；也有一些会发挥调控作用。常规RNA-seq数据就可以用来做以上这些剪接事件的分析。
* 真核生物mRNA的polyA加尾的位置也会有可变性，这一般被称为alternative poly adenylation(APA)。这样产生的转录本3' UTR就会存在序列差异，从而可能导致mRNA翻译效率，稳定性，定位等发生变化，最终调控蛋白的表达。常规RNA-seq数据可以用来做APA的分析，也有专门针对APA分析设计的测序方法，如PAT-seq等。

### 1b) Pipelines

Multiple **features** can be derived from **conventional RNA-seq** data:

1. [Abundance](/teaching/part-iii.-ngs-data-analyses/2.rna-seq/2.1.expression-matrix.md) (Expression Level)
2. **Alternative Splicing (AS)**
3. **Alternative Polyadenylation (APA)**
4. **Chimeric RNA**
5. **RNA editing**
6. **Mutation (SNV/INDEL of DNA)**
7. Alternative Promoter
8. Allele-specific expression (ASE)
9. TE-RNA (**T**ransposable **E**lements, i.e., Repeats)
10. mbRNA (**m**icro**b**ial RNA)
11. ...

> * Basic methods of RNA feature calculation for *features* *2-6*  will be introduced in *6.1-6.5* of this book.&#x20;
> * Advanced analysis pipelines for more RNA features are introduced in *Github* by Lulab members:
>   * [**RNA analyses**](https://github.com/uaauaguga/NGS-Analysis-Notes/blob/master/RNA-analysis.md) by *YF Jin*
>   * [**RNA/Multi-Omics analyses** ](https://github.com/tyh-19/Pipeline-for-multiomics/blob/main/README.md)by *YH Tao*

### 1c) Literatures

* [RNA Regulation - Lu Lab Docs](https://docs.ncrnalab.org/docs/literature-reading/ai/rna/postar)
* [cfRNA-seq Analysis - Lu Lab Docs](https://docs.ncrnalab.org/docs/literature-reading/med/2.-data-mining/cfrna)

## 2) Files Needed <a href="#files" id="files"></a>

### Method 1: Use Docker

docker images的下载链接如[附表](/teaching/appendix/appendix-iv.-teaching.md#teaching-docker)所示：

(1) **6.1 Alternative Splicing**: bioinfo\_PartI-PartII-PartIII1-3.tar.gz

```bash
#与PART I + II类似
docker load -i ~/Desktop/bioinfo_PartI-PartII-PartIII1-3.tar.gz  #load the image
docker run --name=bioinfo_tsinghua -dt  -h bioinfo_docker --restart unless-stopped -v ~/Desktop/bioinfo_tsinghua_share:/home/test/share xfliu1995/bioinfo_tsinghua:2

docker exec -it rnaeditor bash

cd /home/test/alter-spl/
```

(2) **6.2 APA, 6.8 Ribo-seq, 6.9 Structure-seq**：bioinfo\_tsinghua\_6.2\_apa\_6.3\_ribo\_6.4\_structure.tar.gz

```bash
docker load -i ~/Desktop/bioinfo_tsinghua_6.2_apa_6.3_ribo_6.4_structure.tar.gz

#注意：下面的文件挂载目录为~/Desktop/bioinfo_tsinghua_share, 自己运行时记得改为自己新建的一个目录名称。
docker run --name=rnaregulation -dt -h bioinfo_docker --restart unless-stopped -v ~/Desktop/bioinfo_tsinghua_share:/home/test/share gangxu/bioinfo_tsinghua_6.2_apa_6.3_ribo_6.4_structure:latest

docker exec -u root -it rnaregulation bash


# APA
cd /home/test/rna_regulation/apa

# Ribo-seq
cd /home/test/rna_regulation/ribo-wave

# Structure-seq
cd /home/test/rna_regulation/structure_seq
```

(3) **6.3.Chimeric RNA Detection**: bioinfo\_chimeric.tar.gz

需要下载原始数据和基因组文件[ctat\_genome\_lib\_build\_X\_docker.zip,ref\_genome.fa.star.idx.zip](https://lulab2.gitbook.io/teaching/appendix/appendix-iv.-teaching#teaching-files)，请把 ref\_genome.fa.star.idx 移到 ctat\_genome\_lib\_build\_X\_docker文件中。下载解压后需要挂载。

```bash
docker load -i ~/Desktop/bioinfo_chimeric.tar.gz
mv ~/Downloads/ref_genome.fa.star.idx ~/Downloads/ctat_genome_lib_build_X_docker
docker run -dt -v ~/Downloads/ctat_genome_lib_build_X_docker:/data --name=bioinfo_starfusion gangxu/starfusion:latest
```

(4) **6.4 RNA Editing**: bioinfo\_rnaeditor.1.8.tar.gz

```bash
docker load -i ~/Desktop/bioinfo_rnaeditor.tar.gz

#注意：下面的文件挂载目录为/Users/xugang/Downloads/data2, 自己运行时记得改为自己新建的一个目录名称。
docker run --name=rnaeditor -dt -h bioinfo_docker --restart unless-stopped -v /Users/xugang/Downloads/data2:/data2 gangxu/rnaeditor:1.8

docker exec -it rnaeditor bash

cd /home/test
```

(5) **6.6.SNV/INDEL Detection**: bioinfo\_snv.tar.gz

```bash
docker load -i ~/Downloads/bioinfo_snv.tar.gz
docker run -dt --name=snv -v ~/Downloads/data:/data gangxu/snv:2.0
docker exec -it snv bash
```

### Method 2: Directly Download

* 如果不使用docker，也可以直接从 [**Files needed** ](https://courses.ncrnalab.org/files)中的**Files/** 路径下的相应文件夹中下载所需要的文件。


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