# 2.RNA-seq

* 常规RNA-seq最直接的作用在于对基因表达进行定量。在本章中我们主要来讨论和基因表达定量相关的RNA-seq数据分析。

## 1) Table of Contents

* [2.1 Expression Matrix](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/2.rna-seq/2.1.expression-matrix)
* [2.2 Differential Expression with Cufflinks](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/2.rna-seq/2.2.differential-expression-with-cufflinks)
* [2.2 Differential Expression with DEseq2 and edgeR](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/2.rna-seq/2.3.differential_expression_with_deseq2-edger)
* [2.4 Alternative Splicing](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/6.rna-regulation/alternative-splicing)

## **2) Recommended Tools for RNA-seq**

* Raw Data QC and preprocessing (请参考[mapping](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/1.mapping)一节)
  * [fastqc](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
  * [cutadapt](https://cutadapt.readthedocs.io/en/stable/)
  * [trim\_galore](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)
  * [fastp](https://github.com/OpenGene/fastp)
  * [fastx\_toolkit](http://hannonlab.cshl.edu/fastx_toolkit/)
* Spliced Mapping/Alignment (请参考[mapping](https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/1.mapping)一节)
  * [hisat2](http://daehwankimlab.github.io/hisat2/)
  * [STAR](https://github.com/alexdobin/STAR)
* Differential analysis:
  * [DESeq2](https://bioconductor.org/packages/DESeq2/): 利用负二项分布广义线性模型进行差异分析
  * [edgeR](https://bioconductor.org/packages/edgeR/): 和DESeq2类似，利用负二项分布广义线性模型进行差异分析。
  * [limma](https://bioconductor.org/packages/limma/): 最早针对microarray数据开发的差异分析工具。开发者后来又进行了多次改进使其也适用于RNA-seq的分析。

## 3) Pipelines for RNA-seq

* [RNA-seq analysis pipeline](https://github.com/mgonzalezporta/TeachingMaterial) ([Best practices on the differential expression analysis of multi-species RNA-seq](https://pubmed.ncbi.nlm.nih.gov/33926528/) - *Genome Biology 2021*)
* [lncRNA analysis pipeline](http://webhome.weizmann.ac.il/home/igoru/PLAR/)

## 4) Teaching Videos

* see Videos in the [**Files needed** ](https://courses.ncrnalab.org/files)

## 5) References

* [A survey of best practices for RNA-seq data analysis](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0881-8#Abs1) -- *Genome Biology* 2016
* [Best practices on the differential expression analysis of multi-species RNA-seq](https://pubmed.ncbi.nlm.nih.gov/33926528/) - *Genome Biology 2021*

###


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://book.ncrnalab.org/teaching/part-iii.-ngs-data-analyses/2.rna-seq.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.