RNAseq 教程 (4.3)

# 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

# 4.3 Stringle Merge

使用 Stringtie 将所有库中的预测转录本合并成一个统一的转录组。参考 Stringtie 手册获得更详细的解释：https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

Options specified below:

• "assembly_GTF_list.txt" is a text file "manifest" with a list (one per line) of GTF files that you would like to merge together into a single GTF file.

• '-p 8' tells stringtie to use eight CPUs

• '-o' tells stringtie to write output to a particular file or directory

• '-G' tells stringtie where to find reference gene annotations. It will use these annotations to gracefully merge novel isoforms (for de novo runs) and known isoforms and maximize overall assembly quality.

合并所有 6 个 Stringtie 结果：

ls -1 *Rep*/transcripts.gtf > assembly_GTF_list.txt

cat assembly_GTF_list.txt

stringtie --merge -p 8 -o stringtie_merged.gtf -G $RNA_REF_GTF assembly_GTF_list.txt

stringtie --merge -p 8 -o stringtie_merged.gtf -G ../chr22_with_ERCC92.gtf assembly_GTF_list.txt

结果的转录本是什么样子的？

awk '{if($3=="transcript") print}' stringtie_merged.gtf | cut -f 1,4,9 | less

将参考引导的转录本与已知的注释进行比较。这使我们能够评估组装 RNA-seq 数据的转录预测的质量。更多细节，请参考 Stringtie GFF 和 Cuffcompare 手册。

gffcompare -r ../chr22_with_ERCC92.gtf -o gffcompare stringtie_merged.gtf

cat gffcompare.stats

将合并的注释与已知的注释进行比较后，它是什么样子的？与一般的 gtf 有什么不同？

awk '{if($3=="transcript") print}' gffcompare.annotated.gtf | cut -f 1,4,9 | less

对于 de novo 模式:

ls -1 *Rep*/transcripts.gtf > assembly_GTF_list.txt

cat assembly_GTF_list.txt

stringtie --merge -p 8 -o stringtie_merged.gtf assembly_GTF_list.txt

将重新合并的转录本与已知的注释进行比较:

gffcompare -r ../chr22_with_ERCC92.gtf -o gffcompare stringtie_merged.gtf

cat gffcompare.stats