# 目录
1.Module 1 - Introduction to RNA sequencing
2.Module 2 - RNA-seq Alignment and Visualization
3.Module 3 - Expression and Differential Expression
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
6.Module 6 - Functional Annotation of Transcripts
# 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。
r 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 |
