gffread error

Question

[rhksgudbc]

hello

I am currently trying to do RNA-seq using public data in brassica juncea.

To use htseq-count for making count table, I have to convert gff file which downloaded in brassica database to gtf file.

So I used gffread for converting gff file with below command

gffread Bju.genome.gff -T -o Bju.genome.gtf

But when I type above command, I got a lot of error message like below

Error: discarding overlapping duplicate mRNA feature (24650-31472) with ID=BjuO004617
Error: discarding overlapping duplicate mRNA feature (39979-40535) with ID=BjuO004618
Error: discarding overlapping duplicate mRNA feature (30824-34680) with ID=BjuO001651
Error: discarding overlapping duplicate mRNA feature (56651-57957) with ID=BjuO001652
Error: discarding overlapping duplicate mRNA feature (78160-80198) with ID=BjuO001654
Error: discarding overlapping duplicate mRNA feature (74204-77329) with ID=BjuO001653
Error: discarding overlapping duplicate mRNA feature (4453-4737) with ID=BjuO006559
Error: discarding overlapping duplicate mRNA feature (10094-10618) with ID=BjuO009958
Error: discarding overlapping duplicate mRNA feature (15520-17470) with ID=BjuO010812
Error: discarding overlapping duplicate mRNA feature (22118-22816) with ID=BjuO010813
Error: discarding overlapping duplicate mRNA feature (5722-6432) with ID=BjuO010811
Error: discarding overlapping duplicate mRNA feature (3944-4429) with ID=BjuO007439
Error: discarding overlapping duplicate mRNA feature (790-2307) with ID=BjuO007438
Error: discarding overlapping duplicate mRNA feature (6457-6978) with ID=BjuO005411

To check if the file was created properly, I checked file size. And it looks like ok.

40M Bju.genome.gff 56M Bju.genome.gtf

and I also used 'head' command, and there seems to be no problem

A01     GeneWise        gene    3352    5985    1195.88 +       .       ID=BjuA000594;
A01     GeneWise        mRNA    3352    5985    1195.88 +       .       ID=BjuA000594;Parent=BjuA000594;
A01     GeneWise        CDS     3352    3675    1195.88 +       0       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        CDS     4225    4447    1195.88 +       0       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        CDS     4579    4949    1195.88 +       2       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        CDS     5091    5326    1195.88 +       0       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        CDS     5421    5691    1195.88 +       1       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        CDS     5785    5985    1195.88 +       0       ID=BjuA000594.cds;Parent=BjuA000594;
A01     GeneWise        gene    8012    14242   1731.69 +       .       ID=BjuA002557;
A01     GeneWise        mRNA    8012    14242   1731.69 +       .       ID=BjuA002557;Parent=BjuA002557;

So, I wonder why the error message appeared and whether it is okay to use the created file. I would be really grateful if someone reply.

[dariober]

I just wanted to note that the input GFF is not a valid GFF3 file since the ID field should be unique (see http://gmod.org/wiki/GFF3) and this is not the case.

[Juke34]

Right between mRNA and gene. That might be the reason of the errors thrown by gffread.

Fortunately AGAT deals well with that type of problem.

Answer 1

[Juke34]

Might be full overlap duplicated mRNA, so should be fine if removed... but not 100% sure. Did you look or ask on the gffread github repo?

Some ideas what you could do:
Check features you have before and after the converting, using awk or agat_sq_stat_basic.pl from AGAT.
You can check the potential problem from you input file using agat_convert_sp_gxf2gxf.pl from AGAT.
You might try and prefer converting your gtf file with agat_convert_sp_gff2gtf.pl

[rhksgudbc]

Thank you for the reply!

I downloaded AGAT with

conda install -c bioconda agat 

And I followed the method below link

conda create -n agat

and I got this error message

NotWritableError: The current user does not have write permissions to a required path.
  path: /anaconda3/envs/.conda_envs_dir_test
  uid: 1000
  gid: 1000

If you feel that permissions on this path are set incorrectly, you can manually
change them by executing

  $ sudo chown 1000:1000 /anaconda3/envs/.conda_envs_dir_test

In general, it's not advisable to use 'sudo conda'.

So I tried

sudo chown 1000:1000 /anaconda3/envs/.conda_envs_dir_test

but it show me below error message

chown: cannot access '/anaconda3/envs/.conda_envs_dir_test': No such file or directory

Because I don't know well about the conda, I can't solve this problem Can you help me?

[dariober]

/anaconda3/ is an unusual installation path for conda/anaconda/miniconda. Did you install it yourself? If not and you are on shared server or cluster, I would install conda and miniconda by yourself and put it in your home directory (miniconda's default) or where you have your working space. One of the nice things of the conda system is that you don't need admin rights to install and manage it.

[rhksgudbc]

Thank you! I reinstalled miniconda and finally I can excute AGAT