I downloaded the gff3 annotation file for [Zebrafish][1] from NCBI and ran [gff3validator][2] from genometools as such

    gt gff3validator GCF_000002035.5_GRCz10_genomic.gff

and I get the following error:

    gt gff3validator: error: CDS feature on line 621884 in file "GCF_000002035.5_GRCz10_genomic.gff" has the wrong phase 1 (should be 0)

I have not modified the file in any way. What does this error mean? Should I be concerned that it could lead to potential issues? How can this be fixed? Thanks.


  [1]: ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_other/Danio_rerio/latest_assembly_versions/GCF_000002035.5_GRCz10
  [2]: http://www.genometools.org/tools/gt_gff3validator.html
I have become interested in tracking down this and ran what the OP did.  Basically I am trying to determine if this is a GFF file format error or a validation error.

I do get the same error as the OP:

    gt gff3validator: error: CDS feature on line 621884 in file "GCF_000002035.5_GRCz10_genomic.gff" has the wrong phase 1 (should be 0

The obtain the line:

     cat GCF_000002035.5_GRCz10_genomic.gff | awk ' NR==621884 { print $0 } '

This produces the offending line:

    NC_007121.6	BestRefSeq	CDS	21750127	21750129	.	+	1	ID=cds20891;Parent=rna31380;Dbxref=GeneID:553997,Genbank:NP_001019280.1,ZFIN:ZDB-GENE-050609-5;Name=NP_001019280.1;Note=The RefSeq protein has 9 substitutions compared to this genomic sequence;exception=annotated by transcript or proteomic data;gbkey=CDS;gene=pcdh1g9;product=protocadherin 1 gamma 9;protein_id=NP_001019280.1

Looking at the genbank format at https://www.ncbi.nlm.nih.gov/nuccore/NC_007121.6 it shows:

    join(21744267..21746679,21746681,21750127..21750129,
                     21750131..21750164,21855170..21855228,21858956..21859074,
                     21860119..21860155,21860973..21860979)
                     /gene="pcdh1g9"
                     /gene_synonym="DrPcdh1g8"
                     /inference="similar to AA sequence (same
                     species):RefSeq:NP_001019280.1"
                     /exception="annotated by transcript or proteomic data"
                     /note="The RefSeq protein has 9 substitutions compared to
                     this genomic sequence; Derived by automated computational
                     analysis using gene prediction method: BestRefSeq."
                     /codon_start=1
                     /product="protocadherin 1 gamma 9"
                     /protein_id="NP_001019280.1"
                     /db_xref="GI:66773380"
                     /db_xref="GeneID:553997"
                     /db_xref="ZFIN:ZDB-GENE-050609-5"

This shows that it is the third CDS that raises the error. Add up the lengths of the previous  CDS sizes and see how far are we from the multiple of 3. That would be the phase.

    >>> size = 21746679 - 21744267 + 1 + 1
    >>> divmod(size, 3)
    (804, 2)

The remainder is 2, this means that the next codon starts one base in. So phase should be 1.

Basically telling us that the GFF is correct and the validator is incorrect.