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

4.4 Differential (Expression) Splicing

Use Ballgown and Stringtie to compare the UHR and HBR conditions against reference guided and de novo transcript assemblies.

参考Stringtie手册获得更详细的解释:https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

Ballgown的github页面也有Ballgown的说明文档:https://github.com/alyssafrazee/ballgown

计算已知/新(参考引导模式)转录本的UHR和HBR表达估计数

使用参考引导的合并GTF和Ballgown的输出表重新运行Stringtie。将结果存储在一个新目录中，这样我们仍然可以检查在没有合并GTF的情况下生成的结果。

mkdir ref_guided_merged
cd ref_guided_merged

stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep3.bam

运行Ballgown使用参考指导，合并转录

mkdir -p ref_guided_merged/
cd ref_guided_merged/
printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep3\"\n" > UHR_vs_HBR.csv

请参阅 Differential Expression 关于运行Ballgown确定DE基因/转录单的详细信息。

计算已知/新的(从头开始模式)转录本的UHR和HBR表达估计:

使用新生合并的GTF重新运行Stringtie，并为Ballgown输出表。将结果存储在一个新目录中，这样我们仍然可以检查在没有合并GTF的情况下生成的结果。

mkdir de_novo_merged
cd de_novo_merged
stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep3.bam

运行Ballgown，使用重新合并的转录本

printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep3\"\n" > UHR_vs_HBR.csv