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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:

  • “assembly_GTF_list.txt” is a text file “manifest” with a list (one per line) of GTF files that you would like to merge together into a single GTF file.
  • ‘-p 8’ tells stringtie to use eight CPUs
  • ‘-o’ tells stringtie to write output to a particular file or directory
  • ‘-G’ tells stringtie where to find reference gene annotations. It will use these annotations to gracefully merge novel isoforms (for de novo runs) and known isoforms and maximize overall assembly quality.

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
De Novo Transcript Assembly | Griffith Lab

RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

De Novo Transcript Assembly


RNA-seq_Flowchart5


Stringtie De Novo

Note, to discover novel transcripts with Stringtie Using the alignments we generated in the previous modules we will now run Stringtie in de novo mode To use de novo mode do NOT specify either of the ‘-G’ OR ‘-e’ options.

Extra options specified below

cd $RNA_HOME/
mkdir -p expression/stringtie/de_novo/
cd expression/stringtie/de_novo/

stringtie -p 8 -l HBR_Rep1 -o HBR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep1.bam
stringtie -p 8 -l HBR_Rep2 -o HBR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep2.bam
stringtie -p 8 -l HBR_Rep3 -o HBR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/HBR_Rep3.bam

stringtie -p 8 -l UHR_Rep1 -o UHR_Rep1/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep1.bam
stringtie -p 8 -l UHR_Rep2 -o UHR_Rep2/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep2.bam
stringtie -p 8 -l UHR_Rep3 -o UHR_Rep3/transcripts.gtf $RNA_ALIGN_DIR/UHR_Rep3.bam