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Differential Splicing
#4-iv. Differential Splicing
Use Cuffdiff to compare the UHR and HBR conditions.
Refer to the Cufflinks manual for a more detailed explanation: http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html
Cuffdiff basic usage:
cuffdiff [options] <transcripts.gtf> <sample1_hits.sam> <sample2_hits.sam> [... sampleN_hits.sam]
- Supply replicate SAMs as comma separated lists for each condition: sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.sam
- '-p 8' tells cuffdiff to use eight CPUs
- '-L' tells cuffdiff the labels to use for samples
Perform UHR vs. HBR comparison, for known/novel (reference guided mode) transcripts:
cd $RNA_HOME/
mkdir -p de/cufflinks/ref_guided/
cd $RNA_ALIGN_DIR
cuffdiff -p 8 -L UHR,HBR -o $RNA_HOME/de/cufflinks/ref_guided/ --no-update-check $RNA_HOME/expression/cufflinks/ref_guided/merged/merged.gtf UHR_Rep1.bam,UHR_Rep2.bam,UHR_Rep3.bam HBR_Rep1.bam,HBR_Rep2.bam,HBR_Rep3.bam
Perform UHR vs. HBR comparison, for known/novel (de novo mode) transcripts:
cd $RNA_HOME/
mkdir -p de/cufflinks/de_novo/
cd $RNA_ALIGN_DIR
cuffdiff -p 8 -L UHR,HBR -o $RNA_HOME/de/cufflinks/de_novo/ --no-update-check $RNA_HOME/expression/cufflinks/de_novo/merged/merged.gtf UHR_Rep1.bam,UHR_Rep2.bam,UHR_Rep3.bam HBR_Rep1.bam,HBR_Rep2.bam,HBR_Rep3.bam
| Previous Section | This Section | Next Section | |:----------------------------------------------:|:-----------------------------------------------:|:-----------------------------------------------------------------:| | Merging | Differential Splicing | Splicing Visualization |
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