RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Differential Splicing


RNA-seq_Flowchart5


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

Re-run Stringtie using the reference guided merged GTF, and output tables for Ballgown. Store the results in a new directory so that we can still examine the results generated without the merged GTF.

    cd $RNA_HOME/expression/stringtie/
    mkdir ref_guided_merged
    cd ref_guided_merged

    stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
    stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
    stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

    stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
    stringtie -p 8 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
    stringtie -p 8 -G ../ref_guided/stringtie_merged.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_merged/
    cd $RNA_HOME/de/ballgown/ref_guided_merged/

    printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/ref_guided_merged/HBR_Rep3\"\n" > UHR_vs_HBR.csv

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:

Re-run Stringtie using the de novo merged GTF, and output tables for Ballgown. Store the results in a new directory so that we can still examine the results generated without the merged GTF.

    cd $RNA_HOME/expression/stringtie/de_novo
    cd $RNA_HOME/expression/stringtie/
    mkdir de_novo_merged
    cd de_novo_merged

    stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
    stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
    stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

    stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
    stringtie -p 8 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
    stringtie -p 8 -G ../de_novo/stringtie_merged.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_merged/
    cd $RNA_HOME/de/ballgown/de_novo_merged/

    printf "\"ids\",\"type\",\"path\"\n\"UHR_Rep1\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep1\"\n\"UHR_Rep2\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep2\"\n\"UHR_Rep3\",\"UHR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/UHR_Rep3\"\n\"HBR_Rep1\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep1\"\n\"HBR_Rep2\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep2\"\n\"HBR_Rep3\",\"HBR\",\"$RNA_HOME/expression/stringtie/de_novo_merged/HBR_Rep3\"\n" > UHR_vs_HBR.csv

Please see Differential Expression for details on running ballgown to determine a DE gene/transcript list.