How to create GFF file for HT seq

Question: How to create GFF file for HT seq

2.3 years ago by

Hello there, I am dealing with some RNA seq data and was trying to run the raw data in bam file format thorugh HTseq. But it demands for GFF file format. So I wanted to know: What is GFF file and why do I need to create it? How am I suppose to create it? How to know if I have correctly created and ran that file?

It will be really great if you could let me know about it.

Thanks, Shradha

rna-seq bam • 2.2k views

ADD COMMENT • link •

modified 10 months ago by Joey Zou • 0 • written 2.3 years ago by shraddha.adamane • 10

The appropriate GTF file to use depends on the genome used to create the BAM file. What species did you align against and what version of the genome?

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

2.2 years ago by

fate.gh • 10

fate.gh • 10 wrote:

I'm not very professional but as I know from HTSeq 0.6.1p2 documentation , In GTF files generated by the Table Browser function of the UCSC Genome Browser, the gene_id attribute incorrectly contains the same value as the transcript_id attribute and hence a different value for each transcript of the same gene. Hence, if a read maps to an exon shared by several transcripts of the same gene, this will appear to htseq-count as and overlap with several genes. Therefore, these GTF files cannot be used as is. Either correct the incorrect gene_id attributes with a suitable script, or use a GTF file from a different source.

For the reason above, maybe it's not appropriate to use GTF file from UCSC.

Maybe using GFT files from Gencode works better.

ADD COMMENT • link modified 2.2 years ago • written 2.2 years ago by fate.gh • 10

2.3 years ago by

shraddha.adamane • 10

shraddha.adamane • 10 wrote:

Hi Devon Ryan, I guess it has been aligned to hg19 (Human Genome version 19).

ADD COMMENT • link written 2.3 years ago by shraddha.adamane • 10

If UCSC chromosome names were used (e.g., "chr1" and "chr2"), then get your GTF file from UCSC via the table browser (unless this is available under "shared data" in Galaxy, in which case just use it). If you instead used Ensembl chromosome names (e.g., 1 and 2), you can download the a GTF file from Ensembl.

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

2.3 years ago by

shraddha.adamane • 10

shraddha.adamane • 10 wrote:

Hello Devon Ryan. I have got the GFF file. But I am not getting how to upload it from my computer into the RNAanalysis--> HTseq--> GFF file folder. It will be great if you could let me know this. Thanks

ADD COMMENT • link written 2.3 years ago by shraddha.adamane • 10

Upload it to Galaxy so it's in the same history. Then it'll be selectable.

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

hi Devon Ryan. I was finally able to upload the GTF file. But I am getting error, which is as follow:

"Fatal error: Unknown error occured 0 GFF lines processed. Warning: No features of type 'exon' found. Error occured when processing SAM input (record #583 in file /galaxy-repl/main/files/016/925/dataset_16925703.dat): tid -1 out of range 0<=tid<25 [Ex"

It will be great if you could help explain me the error and solution for the same..

ADD REPLY • link written 2.3 years ago by shraddha.adamane • 10

Can you post a link to where you got the GFF/GTF file?

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

http://genome.ucsc.edu/cgi-bin/hgTables

ADD REPLY • link written 2.3 years ago by shraddha.adamane • 10

Right, could you post the first 10-20 lines then?

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene chr1 hg19_knownGene CDS 12190 chr1 hg19_knownGene exon chr1 hg19_knownGene CDS 12595 chr1 hg19_knownGene exon chr1 hg19_knownGene CDS 13403 chr1 hg19_knownGene chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon chr1 hg19_knownGene exon 11874 12227 0.000000 + . gene_id "uc001aaa.3"; transcript_id "uc001aaa.3"; 12613 12721 0.000000 + . gene_id "uc001aaa.3"; transcript_id "uc001aaa.3"; 13221 14409 0.000000 + . gene_id "uc001aaa.3"; transcript_id "uc001aaa.3"; 11874 12227 0.000000 + . gene_id "uc010nxr.1"; transcript_id "uc010nxr.1"; 12646 12697 0.000000 + . gene_id "uc010nxr.1"; transcript_id "uc010nxr.1"; 13221 14409 0.000000 + . gene_id "uc010nxr.1"; transcript_id "uc010nxr.1"; start_codon 12190 12192 0.000000 + . gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 12227 0.000000 + 0 gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 11874 12227 0.000000 + . gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 12721 0.000000 + 1 gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 12595 12721 0.000000 + . gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 13636 0.000000 + 0 gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; stop_codon 13637 13639 0.000000 + . gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 13403 14409 0.000000 + . gene_id "uc010nxq.1"; transcript_id "uc010nxq.1"; 14362 14829 0.000000 - . gene_id "uc009vis.3"; transcript_id "uc009vis.3"; 14970 15038 0.000000 - . gene_id "uc009vis.3"; transcript_id "uc009vis.3"; 15796 15942 0.000000 - . gene_id "uc009vis.3"; transcript_id "uc009vis.3"; 16607 16765 0.000000 - . gene_id "uc009vis.3"; transcript_id "uc009vis.3"; 16858 17055 0.000000 - . gene_id "uc009vjc.1"; transcript_id "uc009vjc.1"; 17233 17751 0.000000 - . gene_id "uc009vjc.1"; transcript_id "uc009vjc.1"; 15796 15947 0.000000 - . gene_id "uc009vjd.2"; transcript_id "uc009vjd.2"; 16607 16765 0.000000 - . gene_id "uc009vjd.2"; transcript_id "uc009vjd.2"; 16858 17055 0.000000 - . gene_id "uc009vjd.2"; transcript_id "uc009vjd.2"; 17233 17368 0.000000 - . gene_id "uc009vjd.2"; transcript_id "uc009vjd.2"; 17606 18061 0.000000 - . gene_id "uc009vjd.2"; transcript_id "uc009vjd.2"; 14362 14829 0.000000 - . gene_id "uc009vit.3"; transcript_id "uc009vit.3"; 14970 15038 0.000000 - . gene_id "uc009vit.3"; transcript_id "uc009vit.3";

ADD REPLY • link written 2.3 years ago by shraddha.adamane • 10

Odd, your file obviously has exon entries. You might submit a bug report so the admin of the server can directly have a look. This will take him/her all of a couple minutes to debug, since the files are immediately at hand.

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

HI Devon Ryan, If I am getting right, when you asked me to upload 10-20 lines, you meant those from GTF file and not from my study file right? Just thought of checking it with you.

ADD REPLY • link written 2.3 years ago by shraddha.adamane14 • 0

Right, you uploaded exactly what I wanted to see. That's actually the weird thing about it, since what you have should work perfectly. My only guess is that there's something like having spaces instead of tabs or some other weird sort of thing that's mucking everything up. The server admin might need to play around with the file a bit to see what the issue is.

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

2.3 years ago by

shraddha.adamane • 10

shraddha.adamane • 10 wrote:

HI Devon Ryan, I guess I tried uploading both Ensembl and UCSC GTF files. And The errors I got are : Error with Ensembl GTF file: error An error occurred with this dataset: Fatal error: Unknown error occured 0 GFF lines processed. Warning: No features of type 'exon' found. Error occured when processing SAM input (record #583 in file /galaxy-repl/main/files/016/925/dataset_16925703.dat): tid -1 out of range 0<=tid<25 [Ex

The error with UCSC GTF files is: error An error occurred with this dataset: Fatal error: Unknown error occured 100000 GFF lines processed. 200000 GFF lines processed. 300000 GFF lines processed. 400000 GFF lines processed. 500000 GFF lines processed. 600000 GFF lines processed. 700000 GFF lines processed. 800000 GFF lines proces

I have reported the admin about the error in UCSC GTF files. Would you suggest anything else or I should await for the reply from Admin?

Thanks

ADD COMMENT • link written 2.3 years ago by shraddha.adamane • 10

You'll have to wait for the admin to respond.

ADD REPLY • link written 2.3 years ago by Devon Ryan • 1.9k

Okk.. I will wait.. Thanks ..

ADD REPLY • link written 2.3 years ago by shraddha.adamane • 10

10 months ago by

Joey Zou • 0

Joey Zou • 0 wrote:

Hello，Devon Ryan When I ran htseq with my data. I am getting the error:

Warning: No features of type 'exon' found.
Warning: Read E00488:175:H3CYCCCXY:3:1101:1610:63173 claims to have an aligned mate which could not be found in an adjacent line.
Warning: 2235041 reads with missing mate encountered.

Could you please help me to solve this problem. I don't konw if the gff file was broken or something problem, and I should need to convert gff file to gtf file? I really appreciate it, if you can do me a favor. My gff files looks like the following below:

scaffold1002    GLEAN   mRNA    20257   25173   0.970574    +   .   ID=DUH000001.2;source_id=RHOqdgD_GLEAN_10019967;
scaffold1002    GLEAN   CDS 20257   20352   .   +   0   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 20458   20579   .   +   0   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 20917   21050   .   +   1   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 21578   21728   .   +   2   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 22844   23115   .   +   1   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 24360   24475   .   +   2   Parent=DUH000001.2;
scaffold1002    GLEAN   CDS 24871   25173   .   +   0   Parent=DUH000001.2;
scaffold1002    Cuff    mRNA    29262   31510   1000    +   .   ID=DUH000002.1;source_id=CUFF1.6.1;
scaffold1002    Cuff    UTR_5   29262   29749   1000    +   .   Parent=DUH000002.1;support_id=CUFF1.6.1;
scaffold1002    Cuff    CDS 29750   31108   1000    +   0   Parent=DUH000002.1;
scaffold1002    Cuff    UTR_3   31109   31510   1000    +   .   Parent=DUH000002.1;support_id=CUFF1.6.1;
scaffold1002    Cuff    mRNA    78137   83175   1000    +   .   ID=DUH000003.1;source_id=CUFF1.7.1;
scaffold1002    Cuff    UTR_5   81341   81400   1000    +   .   Parent=DUH000003.1;support_id=CUFF1.7.1;
scaffold1002    Cuff    CDS 81401   81403   1000    +   0   Parent=DUH000003.1;
scaffold1002    Cuff    CDS 82430   82660   1000    +   0   Parent=DUH000003.1;
scaffold1002    Cuff    UTR_3   82661   83175   1000    +   .   Parent=DUH000003.1;support_id=CUFF1.7.1;

ADD COMMENT • link written 10 months ago by Joey Zou • 0

Please post new questions as new posts in the future and not as answers to old posts.

As the first warning indicates, you don't have any exons in your annotation. I have no idea how that happened, but you'll need to figure it out.

ADD REPLY • link written 10 months ago by Devon Ryan • 1.9k

10 months ago by

Joey Zou • 0

Joey Zou • 0 wrote:

Hi,Devon Ryanhanks. Thanks for your help. Please excuse me. I'll follow the rule of biostar community. Thanks a lot.

ADD COMMENT • link written 10 months ago by Joey Zou • 0

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