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Differential Splicing | Griffith Lab

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 4 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 4 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 4 -G ../ref_guided/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

stringtie -p 4 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 4 -G ../ref_guided/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 4 -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 4 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 4 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 4 -G ../de_novo/stringtie_merged.gtf -e -B -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

stringtie -p 4 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 4 -G ../de_novo/stringtie_merged.gtf -e -B -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 4 -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.

Transcript Assembly Merge | Griffith Lab

RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Transcript Assembly Merge


RNA-seq_Flowchart5


Stringtie Merge

Use Stringtie to merge predicted transcripts from all libraries into a unified transcriptome. Refer to the Stringtie manual for a more detailed explanation:

https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

Options specified below:

Merge all 6 Stringtie results so that they will have the same set of transcripts for comparison purposes:

For reference guided mode:

cd $RNA_HOME/expression/stringtie/ref_guided/
ls -1 *Rep*/transcripts.gtf > assembly_GTF_list.txt
cat assembly_GTF_list.txt
stringtie --merge -p 4 -o stringtie_merged.gtf -G $RNA_REF_GTF assembly_GTF_list.txt

What do the resulting transcripts look like?

awk '{if($3=="transcript") print}' stringtie_merged.gtf | cut -f 1,4,9 | less

Press q to exit the less viewer

Compare reference guided transcripts to the known annotations. This allows us to assess the quality of transcript predictions made from assembling the RNA-seq data. For more details, refer to the Stringtie GFF Utilities and Cuffcompare manuals.

gffcompare -r $RNA_REF_GTF -o gffcompare stringtie_merged.gtf
cat gffcompare.stats

What does the merged annotation look like after comparing it to known annotation? How are the GTF lines different?

awk '{if($3=="transcript") print}' gffcompare.annotated.gtf | cut -f 1,4,9 | less

Press ‘q’ to exit the less viewer

For de novo mode (again, we do not provide an Ensembl GTF):

cd $RNA_HOME/expression/stringtie/de_novo/
ls -1 *Rep*/transcripts.gtf > assembly_GTF_list.txt
cat assembly_GTF_list.txt
stringtie --merge -p 4 -o stringtie_merged.gtf assembly_GTF_list.txt

Compare the de novo merged transcripts to the known annotations:

gffcompare -r $RNA_REF_GTF -o gffcompare stringtie_merged.gtf
cat gffcompare.stats