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 8 -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 8 -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