PROKKA.gff file is not compatible with featureCounts

Hi all,

I am trying to count the number of reads that map to each gene using FeatureCounts. (RNA-Seq PE, linux)

my input;

  • GFF. file generated using Prokka
  • GTF.file generated by NCBI annotation
  • Sorted.bam files generated by bowtie2 and samtools.

When I used gtf.file generated by NCBI, featurecounts run without any issue, however, I am interested in PROKKA.gff because it showed more comprehensive features and a higher mapping rate (compared to NCBI.gtf). So, when I used prokke.gff file, I received this error;

ERROR: no features were loaded in format GTF. The annotation format can be specified by the ‘-F’ option, and the required feature type can be specified by the ‘-t’ option..

Part of the content of prokka.gff file as below;

##sequence-region JAFJXZ010000052.1 1 250
##sequence-region JAFJXZ010000053.1 1 48755
##sequence-region JAFJXZ010000054.1 1 255
##sequence-region JAFJXZ010000055.1 1 465
##sequence-region JAFJXZ010000056.1 1 355
##sequence-region JAFJXZ010000008.1 1 618
##sequence-region JAFJXZ010000057.1 1 255
##sequence-region JAFJXZ010000058.1 1 271
##sequence-region JAFJXZ010000059.1 1 223
##sequence-region JAFJXZ010000009.1 1 354
JAFJXZ010000010.1 Prodigal:002006 CDS 3477 3851 . - 0 ID=GOHBADNI_00001;inference=ab initio prediction:Prodigal:002006;locus_tag=GOHBADNI_00001;product=hypothetical protein
JAFJXZ010000012.1 Prodigal:002006 CDS 712 1704 . + 0 ID=GOHBADNI_00002;eC_number=2.3.1.180;Name=fabHB;db_xref=COG:COG0332;gene=fabHB;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:007600;locustag=GOHBADNI_00002;product=3-oxoacylqacyl-carrier-protein] synthase 3 protein 2
JAFJXZ010000012.1 Prodigal:002006 CDS 1834 3969 . + 0 ID=GOHBADNI_00003;eC_number=5.6.2.1;Name=topB 1;db_xref=COG:COG0550;gene=topB 1;inference=ab initio prediction:Prodigal:002006,similar to AA 

I checked several threads discussing this issue and couldn’t find an appropriate answer so far.

I tried these steps to solve the error;

  • used stringtie to generate merge gtf.file (prokka.gff as input). However, I counted the same error when I used the merged.gtf file in featureCounts instead of ncbi.gtf or PROKKA.gff.

  • converted gff to gtf or gff3 > gffread resulted 0 bytes files, so I couldn’t try them

Kindly provide guidance on resolving this error. Appreciate your help greatly.

Read more here: Source link