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Old 08-16-2018, 12:34 PM   #1
Volklor
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Location: Pacific Northwest

Join Date: Sep 2010
Posts: 13
Unhappy stringtie error: GFF not recognized

Hello,
I'm trying to run stringtie with a reference genome, and I'm getting the following error:

WARNING: no reference transcripts were found for the genomic sequences where reads were mapped!
Please make sure the -G annotation file uses the same naming convention for the genome sequences.

Similar posts indicate that there is some inconsistency between the reference genome fasta file that was used in the hisat2 step and the gff3 or gtf file that is used for stringtie. I can't see any inconsistency in my case. I'm stumped. I really, really hope someone can help!

my stringtie command:
Code:
stringtie 585_10CR_1.bam -p 8 -B -G /nfs0/ROOTS/Vining_Lab/GENOMES/Mlong585/Mlong_CMEN585_v3.01/Mlong585_v3.01.gff -o 585_10CR_1.gtf
The reference genome fasta headers look like this:
Code:
>scaffold_1_length_46699537
>scaffold_2_length_45526029
>scaffold_3_length_45460755
>scaffold_4_length_44284954
>scaffold_5_length_43231337

The first few lines of the gff look like this:
Code:
##gff-version 3
scaffold1_length_46699537       .       contig  1       46699537        .       .       .       ID=scaffold1_length_46699537;Name=scaffold1_length_46699537
scaffold1_length_46699537       maker   gene    1701    4304    .       -       .       ID=Mlong585_00001;Name=Mlong585_00001;Alias=augustus-scaffold1_length_46699537-processed-gene-0.17;
scaffold1_length_46699537       maker   mRNA    1701    4304    .       -       .       ID=Mlong585_00001-RA;Parent=Mlong585_00001;Name=Mlong585_00001-RA;Alias=augustus-scaffold1_length_46699537-processed-gene-0.17-mRNA-1;_AED=1.00
scaffold1_length_46699537       maker   exon    1701    3837    .       -       .       ID=Mlong585_00001-RA:exon:78;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   CDS     1701    3837    .       -       1       ID=Mlong585_00001-RA:cds;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   exon    4012    4097    .       -       .       ID=Mlong585_00001-RA:exon:79;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   CDS     4012    4097    .       -       0       ID=Mlong585_00001-RA:cds;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   CDS     4173    4304    .       -       0       ID=Mlong585_00001-RA:cds;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   exon    4173    4304    .       -       .       ID=Mlong585_00001-RA:exon:80;Parent=Mlong585_00001-RA;
scaffold1_length_46699537       maker   gene    4819    8990    .       +       .       ID=Mlong585_00002;Name=Mlong585_00002;Alias=snap-scaffold1_length_46699537-processed-gene-0.25;
scaffold1_length_46699537       maker   mRNA    4819    8990    .       +       .       ID=Mlong585_00002-RA;Parent=Mlong585_00002;Name=Mlong585_00002-RA;Alias=snap-scaffold1_length_46699537-processed-gene-0.25-mRNA-1;_AED=1.00;_QI
scaffold1_length_46699537       maker   exon    4819    6558    .       +       .       ID=Mlong585_00002-RA:exon:48;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     4819    6558    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     7278    7432    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    7278    7432    .       +       .       ID=Mlong585_00002-RA:exon:49;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     7462    7531    .       +       1       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    7462    7531    .       +       .       ID=Mlong585_00002-RA:exon:50;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     7567    7629    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    7567    7629    .       +       .       ID=Mlong585_00002-RA:exon:51;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     7639    7974    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    7639    7974    .       +       .       ID=Mlong585_00002-RA:exon:52;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     8000    8056    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    8000    8056    .       +       .       ID=Mlong585_00002-RA:exon:53;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     8800    8856    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    8800    8856    .       +       .       ID=Mlong585_00002-RA:exon:54;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   CDS     8958    8990    .       +       0       ID=Mlong585_00002-RA:cds;Parent=Mlong585_00002-RA;
scaffold1_length_46699537       maker   exon    8958    8990    .       +       .       ID=Mlong585_00002-RA:exon:55;Parent=Mlong585_00002-RA
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Old 10-05-2018, 08:50 AM   #2
rajarapupriya
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Location: OH, USA

Join Date: Oct 2013
Posts: 16
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Hi, I ran into the same error recently. Fortunately was able to fix. My gtf file looks the same, however the genome fasta had extra information in the name. For example, your fasta file should also have the SAME name as the gtf/gff file "scaffold1_length_46699537". Anything before or after will give you that error. I changes the fasta name to match the gff file and it worked!
Good luck!
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