Stringtie issue. "Error: no valid ID found for GFF record"

Question

[Pegasus]

Hi all,

Using Linux, I successfully aligned my rna-seq data using Hisat2, converted sam to bam files, sorted them using samtools, and include these bam files with gtf.file in stringtie command line.

However, I got this "Error: no valid ID found for GFF record"

I consulted a previous post; stringtie Error: input file cannot be found

But I am still facing the same problem.

Here is the head -10 result;

![Thank you]

[barslmn]

Could you post the output instead of a screenshot? There seems to be an empty line there. Could you try to read the file with gffread? http://ccb.jhu.edu/software/stringtie/gff.shtml

[Pegasus]

Thank you barslmn,

I checked both gff.file and gtf.file for the same RG using gffread as below;

gff

*JAFJXZ010000001.1  Genbank gene    1   237 .   +   .   ID=gene-JYU28_00005;geneID=gene-JYU28_00005
JAFJXZ010000001.1   Genbank CDS 1   237 .   +   0   Parent=gene-JYU28_00005
JAFJXZ010000001.1   Genbank pseudogene  412 934 .   +   .   ID=gene-JYU28_00010;geneID=gene-JYU28_00010
JAFJXZ010000001.1   Genbank CDS 412 934 .   +   0   Parent=gene-JYU28_00010
JAFJXZ010000010.1   cmsearch    rRNA    1   224 .   -   .   ID=rna-JYU28_03015;geneID=gene-JYU28_03015
JAFJXZ010000010.1   cmsearch    exon    1   224 .   -   .   Parent=rna-JYU28_03015
JAFJXZ010000010.1   cmsearch    rRNA    480 577 .   -   .   ID=rna-JYU28_03020;geneID=gene-JYU28_03020;gene_name=rrf
JAFJXZ010000010.1   cmsearch    exon    480 577 .   -   .   Parent=rna-JYU28_03020
JAFJXZ010000010.1   cmsearch    rRNA    1764    4363    .   -   .   ID=rna-JYU28_03025;geneID=gene-JYU28_03025
JAFJXZ010000010.1   cmsearch    exon    1764    4363    .   -   .   Parent=rna-JYU28_03025
JAFJXZ010000011.1   cmsearch    rRNA    1   453 .   +   .   ID=rna-JYU28_03030;geneID=gene-JYU28_03030
JAFJXZ010000011.1   cmsearch    exon    1   453 .   +   .   Parent=rna-JYU28_03030
JAFJXZ010000012.1   cmsearch    rRNA    1   228 .   +   .   ID=rna-JYU28_03035;geneID=gene-JYU28_03035
JAFJXZ010000012.1   cmsearch    exon    1   228 .   +   .   Parent=rna-JYU28_03035
JAFJXZ010000012.1   cmsearch    rRNA    381 497 .   +   .   ID=rna-JYU28_03040;geneID=gene-JYU28_03040;gene_name=rrf
JAFJXZ010000012.1   cmsearch    exon    381 497 .   +   .   Parent=rna-JYU28_03040
JAFJXZ010000012.1   Genbank gene    712 1704    .   +   .   ID=gene-JYU28_03045;geneID=gene-JYU28_03045
JAFJXZ010000012.1   Genbank CDS 712 1704    .   +   0   Parent=gene-JYU28_03045
JAFJXZ010000012.1   Genbank gene    1834    3969    .   +   .   ID=gene-JYU28_03050;geneID=gene-JYU28_03050
JAFJXZ010000012.1   Genbank CDS 1834    3969    .   +   0   Parent=gene-JYU28_03050
JAFJXZ010000012.1   Genbank gene    4079    4216    .   +   .   ID=gene-JYU28_03055;geneID=gene-JYU28_03055
JAFJXZ010000012.1   Genbank CDS 4079    4216    .   +   0   Parent=gene-JYU28_03055
JAFJXZ010000012.1   Genbank gene    4399    5565    .   -   .   ID=gene-JYU28_03060;geneID=gene-JYU28_03060
JAFJXZ010000012.1   Genbank CDS 4399    5565    .   -   0   Parent=gene-JYU28_03060
JAFJXZ010000012.1   Genbank gene    5914    6042    .   -   .   ID=gene-JYU28_03065;geneID=gene-JYU28*

gtf using gffread

no valid ID found for GFF record

gtf using head -10

!gtf-version 2.2
!genome-build ASM1758969v1
!genome-build-accession NCBI_Assembly:GCA_017589695.1
!annotation-date 02/26/2021 15:56:28
!annotation-source NCBI 
JAFJXZ010000001.1   Genbank gene    1   237 .   +   .   gene_id "JYU28_00005"; transcript_id ""; gbkey "Gene"; gene_biotype "protein_coding"; locus_tag "JYU28_00005"; partial "true"; 
JAFJXZ010000001.1   GeneMarkS-2+    CDS 1   234 .   +   0   gene_id "JYU28_00005"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; inference "COORDINATES: ab initio prediction:GeneMarkS-2+"; locus_tag "JYU28_00005"; partial "true"; product "IS5/IS1182 family transposase"; protein_id "MBO3282641.1"; transl_table "11"; 
JAFJXZ010000001.1   GeneMarkS-2+    start_codon 1   3   .   +   0   gene_id "JYU28_00005"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; inference "COORDINATES: ab initio prediction:GeneMarkS-2+"; locus_tag "JYU28_00005"; partial "true"; product "IS5/IS1182 family transposase"; protein_id "MBO3282641.1"; transl_table "11"; 
JAFJXZ010000001.1   GeneMarkS-2+    stop_codon  235 237 .   +   0   gene_id "JYU28_00005"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; inference "COORDINATES: ab initio prediction:GeneMarkS-2+"; locus_tag "JYU28_00005"; partial "true"; product "IS5/IS1182 family transposase"; protein_id "MBO3282641.1"; transl_table "11"; 
JAFJXZ010000001.1   Genbank gene    412 934 .   +   .   gene_id "JYU28_00010"; transcript_id ""; gbkey "Gene"; gene_biotype "pseudogene"; locus_tag "JYU28_00010"; pseudo "true";*

Both files did not show a similar pattern to the one posted in the GFF utilities;

http://ccb.jhu.edu/software/stringtie/gff.shtml

Following the thread,

StringTIe Error: no valid ID found for GFF record

I believe Stringtie could not read my gtf.file (because gffread couldn't read it), maybe there is a problem in the trancript_ID, and gene_ID, so I still need recommendations ( which is the right command line to fix it)

Thank you,

[barslmn]

Could you try to create your annotation file from the table browser? https://genome.ucsc.edu/cgi-bin/hgTables

[Pegasus]

Hi barslmn,

It is a bacterial genome, which I believe is not supported by this website. I just updated the previous thread (gffread could not read my gtf.file)