RNAseq 1.1

2021-09-07 18:11:43 浏览数 (1)

前言

本教程来自与我保存在github上的RNAseq教程

这是一个RNA-seq分析的教学教程和工作演示流程，包括介绍云计算(不介绍了，直接从第二章开始)、下一代序列文件格式、参考基因组、基因注释、表达分析、差异表达分析、选择性剪接分析、数据可视化和解释。

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

7.Appendix

  1. Saving Your Results
  2. Abbreviations
  3. Lectures
  4. Practical Exercise Solutions
  5. Integrated Assignment
  6. Proposed Improvements
  7. AWS Setup

软件安装（module 1： Installation）

分析所需的软件有:samtools, bamo -readcount, HISAT2, stringtie, gffcompare, htseq-count, flexbar, R, ballgown，fastqc和picard-tools。

设置软件安装位置:

代码语言：javascript复制
mkdir student_tools
cd student_tools
SAMtools
代码语言：javascript复制
wget https://github.com/samtools/samtools/releases/download/1.9/samtools-1.9.tar.bz2
bunzip2 samtools-1.9.tar.bz2
tar -xvf samtools-1.9.tar
cd samtools-1.9
make
./samtools
bam-readcount
代码语言：javascript复制
export SAMTOOLS_ROOT=$RNA_HOME/student_tools/samtools-1.9
git clone https://github.com/genome/bam-readcount.git
cd bam-readcount
cmake -Wno-dev $RNA_HOME/student_tools/bam-readcount
make
./bin/bam-readcount
HISAT2
代码语言：javascript复制
wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.1.0-Linux_x86_64.zip
unzip hisat2-2.1.0-Linux_x86_64.zip
cd hisat2-2.1.0
./hisat2
StringTie
代码语言：javascript复制
wget http://ccb.jhu.edu/software/stringtie/dl/stringtie-1.3.4d.Linux_x86_64.tar.gz
tar -xzvf stringtie-1.3.4d.Linux_x86_64.tar.gz
cd stringtie-1.3.4d.Linux_x86_64
./stringtie
gffcompare
代码语言：javascript复制
wget http://ccb.jhu.edu/software/stringtie/dl/gffcompare-0.10.6.Linux_x86_64.tar.gz
tar -xzvf gffcompare-0.10.6.Linux_x86_64.tar.gz
cd gffcompare-0.10.6.Linux_x86_64
./gffcompare
htseq-count
代码语言：javascript复制
wget https://github.com/simon-anders/htseq/archive/release_0.11.0.tar.gz
tar -zxvf release_0.11.0.tar.gz
cd htseq-release_0.11.0/
python setup.py install --user
chmod  x scripts/htseq-count
./scripts/htseq-count
TopHat
代码语言：javascript复制
wget https://ccb.jhu.edu/software/tophat/downloads/tophat-2.1.1.Linux_x86_64.tar.gz
tar -zxvf tophat-2.1.1.Linux_x86_64.tar.gz
cd tophat-2.1.1.Linux_x86_64/
./gtf_to_fasta
kallisto
代码语言：javascript复制
wget https://github.com/pachterlab/kallisto/releases/download/v0.44.0/kallisto_linux-v0.44.0.tar.gz
tar -zxvf kallisto_linux-v0.44.0.tar.gz
cd kallisto_linux-v0.44.0/
./kallisto
FastQC
代码语言：javascript复制
wget https://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.8.zip --no-check-certificate
unzip fastqc_v0.11.8.zip
cd FastQC/
chmod 755 fastqc
./fastqc --help
MultiQC
代码语言：javascript复制
pip3 install multiqc
multiqc --help
Picard
代码语言：javascript复制
wget https://github.com/broadinstitute/picard/releases/download/2.18.15/picard.jar -O picard.jar
java -jar $RNA_HOME/student_tools/picard.jar
Flexbar
代码语言：javascript复制
wget https://github.com/seqan/flexbar/releases/download/v3.4.0/flexbar-3.4.0-linux.tar.gz
tar -xzvf flexbar-3.4.0-linux.tar.gz
cd flexbar-3.4.0-linux/
export LD_LIBRARY_PATH=$RNA_HOME/student_tools/flexbar-3.4.0-linux:$LD_LIBRARY_PATH
./flexbar
Regtools
代码语言：javascript复制
git clone https://github.com/griffithlab/regtools
cd regtools/
mkdir build
cd build/
cmake ..
make
./regtools
RSeQC
代码语言：javascript复制
pip install RSeQC
read_GC.py
R Libraries
代码语言：javascript复制
#install.packages(c("devtools","dplyr","gplots","ggplot2"),repos="http://cran.us.r-project.org")
#quit(save="no")
Bioconductor
代码语言：javascript复制
#source("http://bioconductor.org/biocLite.R")
#biocLite(c("genefilter","ballgown","edgeR","GenomicRanges","rhdf5","biomaRt"))
#quit(save="no")
Sleuth
代码语言：javascript复制
#install.packages("devtools")
#devtools::install_github("pachterlab/sleuth")
#quit(save="no")

练习

在student_tools下安装bedtools，并编译和测试

express 编程算法

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