I followed the specific workflow which is fastq–>tophats–>HTseq. I ran tophat and it worked well. But now I am confused in the HTseq. The tool asks for BAM file which is from the tophats while it also asks for GTF which is I think the genome reference GTF file…? Does that mean I have to load the human reference GTF file which I used for tophats (used in build index)?
You need to use a GFF/GTF file (which is a feature list) that corresponds to the reference sequence you used as the reference (a sequence) you used in Tophat. So if you used the human h19 reference genome you should use a GFF/GTF file corresponding to the h19 reference. You can probably find the file you need here: http://useast.ensembl.org/info/data/ftp/index.html