How to create EGFP GTF file?

Question

[Payal]

Hi,

I am analyzing RNA Seq data with EGFP. I need to get the count of EGFPs for samples. So I am thinking to align the fastq files to the EGFP sequence concatenated to the reference genome and then get the counts using HTseq counts. I have the EGFP.fa file, but how to create the EGFP GTF file as thats required for Htseq counts and for annotation!!

EGFP Sequence:(Is this ok?)

>EGFP
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA
GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGC
TGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG
CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA
CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG
ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC
GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC
CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG
AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

GTF: (Is this ok?)

EGFP    EGFP    exon    1   720 0.000000    +   .   gene_id "EGFP"; transcript_id "EGFP";

Thanks, Payal

[GenoMax]

If your EGFP.fa file looks like this

>EGFP
AGCTCAGATCGATAGCTACG

then make your GTF file to have a line at the end with something like this (chromosome name EGFP needs to match in both, add right length of sequence in place of 1000)

EGFP    unknown exon    1   1000   .       +       .       gene_id "EGFP"; gene_name "EGFP"

[deeptimanchatterjee]

This helped me a lot.

Just another update on this: You'll need to add "transcript_id" to the GTF file to get it to work.