genome annotation and GFF file formation

Question

[manaswiniparija3]

I have predicted some organism's whole genome annotation and produced gff files by using Meta Euk. but these gff files are not recognizable by feature count.

I am getting error like below

||                                                                            ||
|| Load annotation file dataset_fd73837e-381d-4b9f-bd69-503c68d90c19.dat ...  ||

ERROR: failed to find the gene identifier attribute in the 9th column of the provided GTF file.
The specified gene identifier attribute is 'gene_id ' 
An example of attributes included in your GTF annotation is 'gene_id XP_049336233.1'.

my file looks like below

CM033892.1  gene    MetaEuk 54523435    54530177    656 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";

Can any one help me to figure out the issue in the gff format? (I am using usegalaxy for differential gene expression analysis )

[Pierre Lindenbaum]

my file looks like below

that's not enough, show us the lines associated to this gene (transcript, exons, CDS)

[manaswiniparija3]

CM033892.1  gene    MetaEuk 54523435    54530177    656 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  mRNA    MetaEuk 54523435    54530177    656 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54523435    54523623    136 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54523435    54523623    136 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54525354    54525521    107 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54525357    54525521    107 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54525629    54525694    52  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54525635    54525694    52  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54526582    54526692    75  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54526582    54526692    75  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54527102    54527212    75  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54527117    54527212    75  +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  exon    MetaEuk 54529863    54530177    213 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  CDS MetaEuk 54529869    54530177    213 +   .   gene_id "XP_049336233.1"; transcript_id "XP_049336233.1"; protein_name "XP_049336233.1 uncharacterized protein C14orf93-like [Astyanax mexicanus]";
CM033892.1  gene    MetaEuk 85040217    85046198    1319    +   .   gene_id "XP_049334361.1"; transcript_id "XP_049334361.1"; protein_name "XP_049334361.1 uncharacterized protein LOC125801630 [Astyanax mexicanus]";

[Juke34]

This is not gff but gtf. One rule that this gtf is not following is the presence of transcript_id in gene feature. Removing that might help.

[DGTool]

Would it be possible to post the command you used to run featureCounts (either the command-line command, or the settings in galaxy that you might be using for it - if I understood it correctly). It could be that the extra space i.e. 'gene_id ' could be causing the issue

[manaswiniparija3]

[manaswiniparija3]

[DGTool]

Can you try excluding the GFF gene identifier field (i.e. keep it empty) and rerun it?

[manaswiniparija3]

I have tried it but it is still not working