using gffread for change GFF file to GTF file

Question

[worarado.kan]

Hello everyone,

I try to change GFF file to GTF for analysing in HISAT.

Why when I run following this command;

gffread Blueberry.gff -T -o Blueberry.gtf

Showed this error

GFF Error: overlapping duplicate gene feature (ID=CUFF.71) GFF Error: overlapping duplicate gene feature (ID=CUFF.168) GFF Error: overlapping duplicate gene feature (ID=CUFF.279) GFF Error: overlapping duplicate gene feature (ID=CUFF.330) ....................................

What I am wrong? Please give me the guidance.

Thank you so much

[Kevin Blighe]

Hello, Please run:

grep -e "CUFF.71" Blueberry.gff

grep -e "CUFF.168" Blueberry.gff

grep -e "CUFF.279" Blueberry.gff

grep -e "CUFF.330" Blueberry.gff

Then paste the output here.

[xluo85]

i have the same issue while trying to convert gff3 files to gtf file using gffread. i am new to RNAseq data analysis , i am trying to convert my bam files to raw count so could use DEseq2 to find DEGs, below is the error message after i run the command line,

gffread -E file.gff -T -o file.gtf

"GFF Error: overlapping duplicate mRNA feature (ID=mRNA20293)
GFF Warning: duplicate feature ID gene7319 (1048320-1050627) (discontinuous feature?)
GFF Error: overlapping duplicate mRNA feature (ID=mRNA20294)
GFF Error: overlapping duplicate mRNA feature (ID=mRNA20295)"

"Error parsing value of GFF attribute "Name=", line:
scaffold_6  .   gene    1597619 1612316 .   -   .   ID=gene28515;Name=
Abort trap: 6
"

hope i can get some help.

i did "grep -e "mRNA20294" my file" got run info below:

scaffold_33 .   exon    612943  613025  .   -   .   ID=exon83889;Parent=mRNA20294
scaffold_33 .   exon    613104  613211  .   -   .   ID=exon83890;Parent=mRNA20294
scaffold_33 .   exon    613277  613340  .   -   .   ID=exon83891;Parent=mRNA20294
scaffold_33 .   exon    613404  613561  .   -   .   ID=exon83892;Parent=mRNA20294
scaffold_33 .   exon    613626  613728  .   -   .   ID=exon83893;Parent=mRNA20294
scaffold_33 .   exon    615310  615321  .   -   .   ID=exon83894;Parent=mRNA20294
scaffold_33 .   CDS 612943  613025  .   -   0   ID=CDS83106;Parent=mRNA20294
scaffold_33 .   CDS 613104  613211  .   -   0   ID=CDS83107;Parent=mRNA20294
scaffold_33 .   CDS 613277  613340  .   -   0   ID=CDS83108;Parent=mRNA20294
scaffold_33 .   CDS 613404  613561  .   -   0   ID=CDS83109;Parent=mRNA20294
scaffold_33 .   CDS 613626  613728  .   -   0   ID=CDS83110;Parent=mRNA20294
scaffold_33 .   CDS 615310  615321  .   -   0   ID=CDS83111;Parent=mRNA20294
scaffold_29 .   mRNA    1184259 1185319 .   -   .   ID=mRNA20294;Parent=gene20294;Name=SNAP_00012882
scaffold_29 .   exon    1184259 1184277 .   -   .   ID=exon98427;Parent=mRNA20294
scaffold_29 .   exon    1184412 1184887 .   -   .   ID=exon98428;Parent=mRNA20294
scaffold_29 .   exon    1184942 1185238 .   -   .   ID=exon98429;Parent=mRNA20294
scaffold_29 .   exon    1185305 1185319 .   -   .   ID=exon98430;Parent=mRNA20294
scaffold_29 .   CDS 1184259 1184277 .   -   0   ID=CDS98427;Parent=mRNA20294
scaffold_29 .   CDS 1184412 1184887 .   -   0   ID=CDS98428;Parent=mRNA20294
scaffold_29 .   CDS 1184942 1185238 .   -   0   ID=CDS98429;Parent=mRNA20294
scaffold_29 .   CDS 1185305 1185319 .   -   0   ID=CDS98430;Parent=mRNA20294
scaffold_53 .   mRNA    743863  747177  .   +   .   ID=mRNA20294;Parent=gene18656;Name=fgenesh1_pg.C_scaffold_53000130
scaffold_53 .   exon    743863  743985  .   +   .   ID=exon100811;Parent=mRNA20294
scaffold_53 .   exon    744048  744451  .   +   .   ID=exon100812;Parent=mRNA20294
scaffold_53 .   exon    744540  746113  .   +   .   ID=exon100813;Parent=mRNA20294
scaffold_53 .   exon    746183  747177  .   +   .   ID=exon100814;Parent=mRNA20294
scaffold_53 .   CDS 743863  743985  .   +   0   ID=CDS100063;Parent=mRNA20294
scaffold_53 .   CDS 744048  744451  .   +   0   ID=CDS100064;Parent=mRNA20294
scaffold_53 .   CDS 744540  746113  .   +   0   ID=CDS100065;Parent=mRNA20294
scaffold_53 .   CDS 746183  747177  .   +   0   ID=CDS100066;Parent=mRNA20294

thanks,

[Kevin Blighe]

Hey, well, just looking at your output, I can see that mRNA20294 is indeed duplicated:

scaffold_29 .   mRNA    1184259 1185319 .   -   .   ID=mRNA20294;Parent=gene20294;Name=SNAP_00012882
...
scaffold_53 .   mRNA    743863  747177  .   +   .   ID=mRNA20294;Parent=gene18656;Name=fgenesh1_pg.C_scaffold_53000130

How did you produce this data?

[xluo85]

thanks for the quick reply, i downloaded the reference file from JGI website for Daphnia pulex. The only option is either a .gff file or . gff3 file. The data used downloaded .gff3 file with command line gffread -E my file.gff3 -T -o my file. gtf, which i got nothing in my output. this is the gff3 file looks like :

gff-version 3

scaffold_1 . gene 62188 73952 . + . ID=gene1;Name=gw1.1.25.1 scaffold_1 . mRNA 62188 73952 . + . ID=mRNA1;Parent=gene1;Name=gw1.1.25.1 scaffold_1 . exon 62188 62346 . + . ID=exon1;Parent=mRNA1 scaffold_1 . exon 62647 62736 . + . ID=exon2;Parent=mRNA1 scaffold_1 . exon 67111 67169 . + . ID=exon3;Parent=mRNA1 scaffold_1 . exon 67365 67525 . + . ID=exon4;Parent=mRNA1

[xluo85]

but if i use .gff file , scaffold_1 JGI exon 264824 264890 . + . name "estExt_fgenesh1_kg.C_10002"; transcriptId 230065 scaffold_1 JGI CDS 264882 264890 . + 0 name "estExt_fgenesh1_kg.C_10002"; proteinId 230065; exonNumber 1 scaffold_1 JGI start_codon 264882 264884 . + 0 name "estExt_fgenesh1_kg.C_10002" scaffold_1 JGI exon 265102 265158 . + . name

then i got no error for the same command line with gffread, but i still got nothing on my output file. i don't know what should i suppose to do. I used the fasta and gff3 to generate the genome index in STAR and mapped my reads to generate the BAM file, i tried to use HTseq to convert bam file to raw counts so i could do the DEG analysis , i know HTseq except the gtf file, but no such files available, so i am trying to use gffread to generate a gtf file form gff or gff3 file. thanks

[xluo85]

i tried gff file with gffread-0.9.12.OSX_x86_64, i got lots of warning on the same command:

Warning: invalid GTF record, transcript_id not found: scaffold_999 JGI CDS 40436 40726 . - 0 name "SNAP_00037760"; proteinId 269702; exonNumber 2 Warning: invalid GTF record, transcript_id not found: scaffold_999 JGI stop_codon 40436 40438 . - 0 name "SNAP_00037760" Warning: invalid GTF record, transcript_id not found: scaffold_999 JGI exon 41056 41220 . - . name "SNAP_00037760"; transcriptId 269702 Warning: invalid GTF record, transcript_id not found: scaffold_999 JGI CDS 41056 41220 . - 0 name "SNAP_00037760"; proteinId 269702; exonNumber 1 Warning: invalid GTF record, transcript_id not found: scaffold_999 JGI start_codon 41218 41220 . - 0 name "SNAP_00037760"