Differential Splicing

#5-iv. Differential Splicing

Use Cuffdiff to compare the UHR and HBR 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 UHR vs. HBR comparison, for known/novel (reference guided mode) transcripts:

cuffdiff -p 8 -L UHR,HBR -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 UHR_Rep1_ERCC-Mix1/accepted_hits.bam,UHR_Rep2_ERCC-Mix1/accepted_hits.bam,UHR_Rep3_ERCC-Mix1/accepted_hits.bam HBR_Rep1_ERCC-Mix2/accepted_hits.bam,HBR_Rep2_ERCC-Mix2/accepted_hits.bam,HBR_Rep3_ERCC-Mix2/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 UHR,HBR -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 UHR_Rep1_ERCC-Mix1/accepted_hits.bam,UHR_Rep2_ERCC-Mix1/accepted_hits.bam,UHR_Rep3_ERCC-Mix1/accepted_hits.bam HBR_Rep1_ERCC-Mix2/accepted_hits.bam,HBR_Rep2_ERCC-Mix2/accepted_hits.bam,HBR_Rep3_ERCC-Mix2/accepted_hits.bam

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