# 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。
# 目录
1.Module 1 - Introduction to RNA sequencing
- Installation
- Reference Genomes
- Annotations
- Indexing
- RNA-seq Data
- Pre-Alignment QC
2.Module 2 - RNA-seq Alignment and Visualization
- Adapter Trim
- Alignment
- IGV
- Alignment Visualization
- Alignment QC
3.Module 3 - Expression and Differential Expression
- Expression
- Differential Expression
- DE Visualization
- Kallisto for Reference-Free Abundance Estimation
4.Module 4 - Isoform Discovery and Alternative Expression
- Reference Guided Transcript Assembly
- de novo Transcript Assembly
- Transcript Assembly Merge
- Differential Splicing
- Splicing Visualization
5.Module 5 - De novo transcript reconstruction
- De novo RNA-Seq Assembly and Analysis Using Trinity
6.Module 6 - Functional Annotation of Transcripts
- Functional Annotation of Assembled Transcripts Using Trinotate
# 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:
| mkdir student_tools |
| cd student_tools |
| 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
| 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
| 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
| 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
| 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
| 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
| 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
| 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
| 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
| pip3 install multiqc |
| multiqc --help |
# Picard
| wget https://github.com/broadinstitute/picard/releases/download/2.18.15/picard.jar -O picard.jar |
| java -jar $RNA_HOME/student_tools/picard.jar |
# Flexbar
| wget https://github.com/seqan/flexbar/releases/download/v3.4.0/flexbar-3.4.0-linux.tar.gz |
| tar -xzvf flexbar-3.4.0-linux.tar.gz |
| cd flexbar-3.4.0-linux/ |
| export LD_LIBRARY_PATH=$RNA_HOME/student_tools/flexbar-3.4.0-linux:$LD_LIBRARY_PATH |
| ./flexbar |
| git clone https://github.com/griffithlab/regtools |
| cd regtools/ |
| mkdir build |
| cd build/ |
| cmake .. |
| make |
| ./regtools |
# RSeQC
| pip install RSeQC |
| read_GC.py |
# R Libraries
| #install.packages(c("devtools","dplyr","gplots","ggplot2"),repos="http://cran.us.r-project.org") |
| #quit(save="no") |
# Bioconductor
| #source("http://bioconductor.org/biocLite.R") |
| #biocLite(c("genefilter","ballgown","edgeR","GenomicRanges","rhdf5","biomaRt")) |
| #quit(save="no") |
# Sleuth
| #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
