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Transcript Assembly Merge
#4-iii. Stringtie Merge Use Stringtie to merge predicted transcripts from all libraries into a unified transcriptome. Refer to the Stringtie manual for a more detailed explanation: 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.
Merge all 6 Stringtie results so that they will have the same set of transcripts for comparison purposes:
For reference guided mode:
cd $RNA_HOME/expression/stringtie/ref_guided/
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
What do the resulting transcripts look like?
awk '{if($3=="transcript") print}' stringtie_merged.gtf | cut -f 1,4,9 | less
Press 'q' to exit the less viewer
Compare reference guided transcripts to known annotation:
gffcompare -r $RNA_REF_GTF -o merged stringtie_merged.gtf
cat merged.stats
What does the merged annotation look like after comparing it to known annotation? How are the GTF lines different?
awk '{if($3=="transcript") print}' merged.annotated.gtf | cut -f 1,4,9 | less
Press 'q' to exit the less viewer
For de novo mode:
cd $RNA_HOME/expression/stringtie/de_novo/
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
Compare de novo to known annotation:
gffcompare -r $RNA_REF_GTF -o merged stringtie_merged.gtf
cat merged.stats
| Previous Section | This Section | Next Section | |:------------------------------------------------:|:------------------------------------------:|:----------------------------------------------------:| | De novo | Merging | Differential Splicing |
NOTICE: This resource has been moved to rnabio.org. The version here will be maintained for legacy use only. All future development and maintenance will occur only at rnabio.org. Please proceed to rnabio.org for the current version of this course.
Table of Contents
Module 0: Authors | Citation | Syntax | Intro to AWS | Log into AWS | Unix | Environment | Resources
Module 1: Installation | Reference Genomes | Annotations | Indexing | Data | Data QC
Module 2: Adapter Trim | Alignment | IGV | Alignment Visualization | Alignment QC
Module 3: Expression | Differential Expression | DE Visualization
Module 4: Alignment Free - Kallisto
Module 5: Ref Guided | De novo | Merging | Differential Splicing | Splicing Visualization
Module 6: Trinity
Module 7: Trinotate
Appendix: Saving Results | Abbreviations | Lectures | Practical Exercise Solutions | Integrated Assignment | Proposed Improvements | AWS Setup