Hello,

I have been following the procedure given in the paper "Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown" with my own examples of course.

1) I have sorted the SAM files to BAM just fine with no errors.


2) Then I assembled transcripts for each of my samples following the example:

    stringtie -p 8 -G chrX_data/genes/chrX.gtf -o ERR188044_chrX.gtf –l ERR188044 ERR188044_chrX.bam

Below is what I used for my samples: 

     stringtie GG1_hisat.sorted.bam -o GG1_hisat.sorted.gtf -m 300 -p5

I did not use the `-G` option because I don't have a reference annotation file yet.

3) Then I merged the transcripts of all my samples:

     stringtie --merge -o stringtie_merged.gtf gtf_files.txt

4) Now, I want to create a table of counts so that I can move on to use Deseq2 but I am unable to do so, and I am not sure why... maybe because I have not provided the `-G` option?

1st try: 

    stringtie –B -p8 -G stringtie_merged.gtf -o ballgown_Ceratodon.gtf

Error: 

>input file –B cannot be found! 

2nd try: 

    stringtie -p8 -G stringtie_merged.gtf -o ballgown_Ceratodon.gtf

Error: 

>no input file provided!

If your goal was to create a new, merged, transcriptome reference and to perform differential expression analysis on the transcripts identified in this, then use *stringtie_merged.gtf* and restart your analysis with *BallGown* and *StringTie*, where you will specify this file with the `-G` option. When using *StringTie*, ensure that you use the `-e` option. To create a counts matrix from *StringTie* that is suitable for DESeq2, you should then use the *prepDE.py* function: <a href="https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq">Using StringTie with DESeq2 and edgeR</a>.

If you need a FASTA file relating to your merge GTF, you can produce that with the `gffread` program that come bundled with StringTie. Yo'ull also need a reference genome in FASTA.

Kevin

