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