title: How to create a .GTF file?
body: <p>I am new to bioinformatics and programming. I would greatly appreciate some help with step-by-step instructions on how to create a <code>.GTF</code> file. I have two cancer cell lines with different green fluorescent protein (GFP) variants knocked-in to the genome of each cell line. The idea is that the expression of GFP can be used to distinguish cancer cells from non-cancer cells. I would like to count GFP reads in all cancer cells in a single cell RNA-seq experiment. The single cell experiment was performed on the 10X Chromium platform, on organoids composed of a mix of these cancer cells and non-cancer cells. Next generation sequencing was then performed and the reference genome is the human genome sequence, GRCh38. To 'map' and count GFP reads I was told to create a .GTF file which holds the location information, and this file will be used retrospectively to add GFP to the human genome sequence. I have the FASTA sequences for both GFP variants, which I can upload if requested. Where do I start with creation of a .GTF file? Do I create this file in Excel, or with, for example BASH script in a Terminal? I have a link to a Wellcome Trust genome website (<a href="https://www.ensembl.org/info/website/upload/gff.html?redirect=no" rel="nofollow noreferrer">https://www.ensembl.org/info/website/upload/gff.html?redirect=no</a>) but it is not clear what practical/programming steps are needed. From my reading it seems a GFF (GFF3?) file is needed as an intermediate step. Step-by-step instructions would be very welcome to create the <code>.GTF</code> file. Thanks in advance.</p>