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Differential Splicing

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malachig edited this page Nov 16, 2014 · 27 revisions

RNA-seq Flowchart - Module 5

#DIFFERENTIAL SPLICING Use Cuffdiff to compare the tumor and normal conditions. Refer to the Cufflinks manual for a more detailed explanation: http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff

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

For reference guided mode: cd $RNA_HOME/ mkdir -p de/tophat_cufflinks/ref_guided/ cd $RNA_HOME/alignments/tophat/

Perform Tumor vs. Normal comparison, using just the capture libraries, for known/novel (reference guided mode) transcripts cuffdiff -p 8 -L Normal,Tumor -o $RNA_HOME/de/tophat_cufflinks/ref_guided/ --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/expression/tophat_cufflinks/ref_guided/merged/merged.gtf Normal_cDNA1_lib2/accepted_hits.bam,Normal_cDNA2_lib2/accepted_hits.bam Tumor_cDNA1_lib2/accepted_hits.bam,Tumor_cDNA2_lib2/accepted_hits.bam

For de novo mode: cd $RNA_HOME/ mkdir de/tophat_cufflinks/de_novo/ cd $RNA_HOME/alignments/tophat/

Perform Tumor vs. Normal comparison, using just the capture libraries, for known/novel (de novo mode) transcripts cuffdiff -p 8 -L Normal,Tumor -o $RNA_HOME/de/tophat_cufflinks/de_novo/ --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/expression/tophat_cufflinks/de_novo/merged/merged.gtf Normal_cDNA1_lib2/accepted_hits.bam,Normal_cDNA2_lib2/accepted_hits.bam Tumor_cDNA1_lib2/accepted_hits.bam,Tumor_cDNA2_lib2/accepted_hits.bam

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

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