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creating gtf file for bam file generated by Hisat2 using Stringtie

hello, I am involved in doing DE calculations of miRNA present in control and stress condition, i completed the step till hisat2 and also i get output for hisat2 but stringtie is not working if i enter the command empty .gtf document only created by stringtie i am having shortreads data without reference gtf file

assembly r next-gen rna-seq gene
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written by selkash2016 • selkash2016

Hey, please show all commands that you used.

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written by Kevin Blighe Kevin Blighe

hisat2 command

hisat2-build Nseqref.fa NseqRef

hisat2 -p 8 -q -x NseqRef -U control_all.fq -S ControlNseq.sam

stringtie command

stringtie -o sahaControlNseq.gtf ControlNseq.sorted.bam

I used this command to print output but what i get was an empty output file with the command i used to print output

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written by selkash2016 • selkash2016

Just to humour me, can you try this:

stringtie ControlNseq.sorted.bam -o sahaControlNseq.gtf

Also, you are evidently not showing all of your commands, including those from intermediate steps

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written by Kevin Blighe Kevin Blighe

I already tried that sir but same output was generated an empty .gtf file with the command

sir another information to your knowledge my refernce genome is MIRNA data predicted using MIRDEEP2 software and I want to do DEG for those so that only i following this pipeline

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written by selkash2016 • selkash2016

I see, so, Nseqref.fa contains FASTA mRNA sequences (?)

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written by Kevin Blighe Kevin Blighe

No, sir it contains miRNA sequences sir. mature, star, and precursor sequences of miRNA.

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written by selkash2016 • selkash2016

Okay, can you show some of these sequences here (paste the data)?

Also confirm that there is output in ControlNseq.sam (show some alignment statistics)?

Also show the log(s) from your stringtie command?

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written by Kevin Blighe Kevin Blighe

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