RNAseq教程（2.3）

目录

1.Module 1 - Introduction to RNA sequencing

1. Installation
2. Reference Genomes
3. Annotations
4. Indexing
5. RNA-seq Data
6. Pre-Alignment QC

2.Module 2 - RNA-seq Alignment and Visualization

1. Adapter Trim
2. Alignment
3. IGV
4. Alignment Visualization
5. Alignment QC

3.Module 3 - Expression and Differential Expression

1. Expression
2. Differential Expression
3. DE Visualization
4. Kallisto for Reference-Free Abundance Estimation

4.Module 4 - Isoform Discovery and Alternative Expression

1. Reference Guided Transcript Assembly
2. de novo Transcript Assembly
3. Transcript Assembly Merge
4. Differential Splicing
5. Splicing Visualization

5.Module 5 - De novo transcript reconstruction

1. De novo RNA-Seq Assembly and Analysis Using Trinity

6.Module 6 - Functional Annotation of Transcripts

1. Functional Annotation of Assembled Transcripts Using Trinotate

2.3 IGV

1.introduction

Description of the lab

高通量测序最受欢迎的工具-IGV(Integrative Genomics Viewer)

伴随本教程的文件

• IGV Lecture - Brief
• IGV Lecture - Long, from Broad Institute

完成本次教程可实现以下工作

• 可视化各种基因组数据

• 快速导航基因组

• 可视化reads比对情况

• 肉眼验证SNP/SNV

Requirements

• Integrative Genomics Viewer

• Ability to run Java

• Note that while most tutorials in this course are performed on the cloud, IGV will always be run on your local machine

Compatibility

本教程是为IGV v2.3准备的，可以在IGV下载页面上找到。强烈建议使用这个版本。

Data Set for IGV

使用公开的来自HCC1143细胞系的Illumina序列数据。HCC1143细胞系是从一名患有乳腺癌的52岁白人妇女体内产生的。这个细胞系的附加信息可以在这里找到:HCC1143(tumor, TNM stage IIA, grade 3, primary ductal carcinoma)以及HCC1143/BL(matched normal EBV transformed lymphoblast cell line).

• 从细胞系HCC1143产生的reads比对到这个区域

• Chromosome 21: 19,000,000-20,000,000

• HCC1143.normal.21.19M-20M.bam

• HCC1143.normal.21.19M-20M.bam.bai

2. Getting familiar with IGV

Get familiar with the interface

载入一个基因组:

默认情况下，IGV加载Human hg19。如果你研究的是另一个版本的人类基因组，或者另一种物种，你可以通过点击左上角的下拉菜单来改变基因组。在这个教程中，我们将使用人类hg19。

也可以采用以下方式(File -> Load from Server...):

• Ensembl genes (or your favourite source of gene annotations)
• GC Percentage
• dbSNP 1.3.1 or 1.3.7

Navigation：

在这个参考基因组中可以看到染色体列表，选择1号染色体。

location字段(在界面的左上角)中输入，导航到chr1:10 000- 11000，然后单击Go。这显示了1号染色体的窗口宽1000个碱基对，从10000号位置开始。

IGV以颜色序列的形式显示基因组中的碱基序列(例如A=绿色，C =蓝色，等等)。这使得重复序列，比如在这个区域开始处发现的那些序列，很容易识别。放大一点使用+按钮看到参考基因组序列的单个碱基。

你可以在基因组坐标所在的框中输入你感兴趣的基因，然后按Enter/Return键。试试你最喜欢的基因，或者BRCA1。

基因用线和框表示。线代表内含子区域，框代表外显子区域。箭头表示该基因的转录方向/链。当一个外显子框变窄，这表示一个UTR。

Region Lists

有时，保存当前位置或加载感兴趣的区域真的很有用。为此，IGV中有一个区域导航器。要访问它，单击Regions > Region Navigator。在浏览基因组时，可以随时按Add按钮保存一些书签。

Loading Read Alignments

我们将使用乳腺癌细胞系HCC1143来可视化比对结果。在速度方面，只有一小部分chr21将装载(19M:20M)。

HCC1143 Alignments to hg19:

• HCC1143.normal.21.19M-20M.bam
• HCC1143.normal.21.19M-20M.bam.bai

复制文件到你的本地，并在IGV中选择File > Load from File...，选择bam文件，并单击OK。注意，为了让IGV正确地加载它们，bam文件和索引文件必须在同一个目录中。

Visualizing read alignments

选择染色体位点：chr21:19,480,041-19,480,386

To start our exploration, right click on the track-name, and select the following options:

• Sort alignments by start location
• Group alignments by pair orientation

通过右键点击比对界面和切换选项来试验各种设置。想想哪一种方法最适合特定的任务(例如，质量控制、SNP调用、CNV查找)。

3.Inspecting SNPs, SNVs, and SVs

Two neighbouring SNPs

• Navigate to region chr21:19,479,237-19,479,814
• Note two heterozygous variants, one corresponds to a known dbSNP (G/T on the right) the other does not (C/T on the left)
• Zoom in and center on the C/T SNV on the left, sort by base (window chr21:19,479,321 is the SNV position)
• Sort alignments by base
• Color alignments by read strand

Homopolymer region with indel

Navigate to position chr21:19,518,412-19,518,497

Coverage by GC

Navigate to position chr21:19,611,925-19,631,555. Note that the range contains areas where coverage drops to zero in a few places.

**Example **

• Use Collapsed view
• Use Color alignments by -> insert size and pair orientation
• Load GC track
• See concordance of coverage with GC content

Heterozygous SNPs on different alleles

Navigate to region chr21:19,666,833-19,667,007

**Example **

• Sort by base (at position chr21:19,666,901)

对于这两个snp，等位基因之间没有联系，因为两个snp的reads都只包含一个或另一个

4.Automating Tasks in IGV

我们可以使用Tools菜单调用运行批处理脚本。IGV网站描述了批处理脚本:

• Batch file requirements: https://www.broadinstitute.org/igv/batch

• Commands recognized in a batch script: https://www.broadinstitute.org/software/igv/PortCommands

• We also need to provide sample attribute file as described here: http://www.broadinstitute.org/software/igv/?q=SampleInformation

下载数据集的批处理脚本和属性文件:

• Batch script: Run_batch_IGV_snapshots.txt
• Attribute file: Igv_HCC1143_attributes.txt