Differential Splicing
#4-iv. Differential (Expression) Splicing
Use Ballgown and Stringtie to compare the UHR and HBR conditions against reference guided and de novo transcript assemblies.
Refer to the Stringtie manual for a more detailed explanation: https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual
The Ballgown github page also has documentation for getting started with ballgown: https://github.com/alyssafrazee/ballgown
Calculate UHR and HBR expression estimates, for known/novel (reference guided mode) transcripts
Run Stringtie using the reference guided, merged GTF and output tables for Ballgown
cd $RNA_HOME/expression/stringtie/ref_guided
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep3.bam
Run Ballgown using the reference guided, merged transcripts
mkdir -p $RNA_HOME/de/ballgown/ref_guided/
cd $RNA_HOME/de/ballgown/ref_guided/
printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided/HBR_Rep3\"\n" > UHR_vs_HBR.csv
R
Please see Differential Expression for details on running ballgown to determine a DE gene/transcript list.
Calculate UHR and HBR expression estimates, for known/novel (de novo mode) transcripts:
Run Stringtie using the de novo, merged GTF and output tables for Ballgown
cd $RNA_HOME/expression/stringtie/de_novo
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 8 -G merged.annotated.gtf -e -B -o UHR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep3.bam
Run Ballgown using the de novo, merged transcripts
mkdir -p $RNA_HOME/de/ballgown/de_novo/
cd $RNA_HOME/de/ballgown/de_novo/
printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo/HBR_Rep3\"\n" > UHR_vs_HBR.csv
R
Please see Differential Expression for details on running ballgown to determine a DE gene/transcript list.
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