Error during mikado serialize

[desmodus1984]

Hi, @gemygk

I commented on here- #450 for an installation problem and somehow, I managed to get it to work but now I got a different error. I am attaching the error file.
MikadoSerial-pick_1187.err.txt
At first it mentions a deprecation error:

/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/subprograms/configure.py:7: UserWarning: pkg_resources is deprecated as an API. See https://setuptools.pypa.io/en/latest/pkg_resources.html. The pkg_resources package is slated for removal as early as 2025-11-30. Refrain from using this package or pin to Setuptools<81.
  from pkg_resources import resource_filename, resource_stream

and then it keeps on reporting error after error after

Process Bed12ParseWrapper-23:
Process Bed12ParseWrapper-30:
Process Bed12ParseWrapper-35:
Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1188, in thick_end
    value = int(value)
ValueError: invalid literal for int() with base 10: ''

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1802, in bed_next
    bed12 = BED12(line,
Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 366, in __init__
    self.__set_values_from_fields()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 536, in __set_values_from_fields
    self.thick_start, self.thick_end, self.rgb, \
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1190, in thick_end
    raise ValueError("Thick end must be an integer, not {}! Value: {}".format(type(value), value))
ValueError: Thick end must be an integer, not <class 'str'>! Value: 
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1188, in thick_end
    value = int(value)

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1866, in run
    row = self.bed_next(line, seq)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1811, in bed_next
    raise InvalidParsingFormat("Invalid line: {}".format(line))
ValueError: invalid literal for int() with base 10: ''
Mikado.exceptions.InvalidParsingFormat: Invalid line: Eviann0_XLOC_003602U_lncRNA-mRNA-1	0	708	ID=Eviann0_XLOC_003602U_lncRNA-mRNA-1.p9;type:complete;len:55;gc:the;standard;code;Eviann0_XLOC_003602U_lncRNA-mRNA-1:218-54(-)	583	-	218		0	1	708	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/multiprocessing/process.py", line 314, in _bootstrap
    self.run()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1880, in run
    raise InvalidParsingFormat(line)
Mikado.exceptions.InvalidParsingFormat: Eviann0_XLOC_003602U_lncRNA-mRNA-1	0	708	ID=Eviann0_XLOC_003602U_lncRNA-mRNA-1.p9;type:complete;len:55;gc:the;standard;code;Eviann0_XLOC_003602U_lncRNA-mRNA-1:218-54(-)	583	-	218		0	1	708	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1802, in bed_next
    bed12 = BED12(line,
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1188, in thick_end
    value = int(value)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 366, in __init__
    self.__set_values_from_fields()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 536, in __set_values_from_fields
    self.thick_start, self.thick_end, self.rgb, \
ValueError: invalid literal for int() with base 10: ''
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1190, in thick_end
    raise ValueError("Thick end must be an integer, not {}! Value: {}".format(type(value), value))
ValueError: Thick end must be an integer, not <class 'str'>! Value: 

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1866, in run
    row = self.bed_next(line, seq)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1811, in bed_next
    raise InvalidParsingFormat("Invalid line: {}".format(line))
Mikado.exceptions.InvalidParsingFormat: Invalid line: Eviann0_LOC_00004922-mRNA-4	0	1059	ID=Eviann0_LOC_00004922-mRNA-4.p12;type:complete;len:59;gc:the;standard;code;Eviann0_LOC_00004922-mRNA-4:248-72(-)	681	-	248		0	1	1059	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/multiprocessing/process.py", line 314, in _bootstrap
    self.run()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1880, in run
    raise InvalidParsingFormat(line)
Mikado.exceptions.InvalidParsingFormat: Eviann0_LOC_00004922-mRNA-4	0	1059	ID=Eviann0_LOC_00004922-mRNA-4.p12;type:complete;len:59;gc:the;standard;code;Eviann0_LOC_00004922-mRNA-4:248-72(-)	681	-	248		0	1	1059	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1802, in bed_next
    bed12 = BED12(line,
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 366, in __init__
    self.__set_values_from_fields()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 536, in __set_values_from_fields
    self.thick_start, self.thick_end, self.rgb, \
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1190, in thick_end
    raise ValueError("Thick end must be an integer, not {}! Value: {}".format(type(value), value))
ValueError: Thick end must be an integer, not <class 'str'>! Value: 

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1866, in run
    row = self.bed_next(line, seq)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1811, in bed_next
    raise InvalidParsingFormat("Invalid line: {}".format(line))
Mikado.exceptions.InvalidParsingFormat: Invalid line: Tiberius_g8972.t1	0	786	ID=Tiberius_g8972.t1.p2;type:internal;len:262;gc:the;standard;code;Tiberius_g8972.t1:786-1(-)	999	-	786		0	1	786	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/multiprocessing/process.py", line 314, in _bootstrap
    self.run()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1880, in run
    raise InvalidParsingFormat(line)
Mikado.exceptions.InvalidParsingFormat: Tiberius_g8972.t1	0	786	ID=Tiberius_g8972.t1.p2;type:internal;len:262;gc:the;standard;code;Tiberius_g8972.t1:786-1(-)	999	-	786		0	1	786	0

Process Bed12ParseWrapper-5:
Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1188, in thick_end
    value = int(value)
ValueError: invalid literal for int() with base 10: ''

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1802, in bed_next
    bed12 = BED12(line,
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 366, in __init__
    self.__set_values_from_fields()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 536, in __set_values_from_fields
    self.thick_start, self.thick_end, self.rgb, \
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1190, in thick_end
    raise ValueError("Thick end must be an integer, not {}! Value: {}".format(type(value), value))
ValueError: Thick end must be an integer, not <class 'str'>! Value: 

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1866, in run
    row = self.bed_next(line, seq)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1811, in bed_next
    raise InvalidParsingFormat("Invalid line: {}".format(line))
Mikado.exceptions.InvalidParsingFormat: Invalid line: Eviann0_LOC_00011369-mRNA-2	0	774	ID=Eviann0_LOC_00011369-mRNA-2.p6;type:complete;len:65;gc:the;standard;code;Eviann0_LOC_00011369-mRNA-2:290-96(-)	523	-	290		0	1	774	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/multiprocessing/process.py", line 314, in _bootstrap
    self.run()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1880, in run
    raise InvalidParsingFormat(line)
Mikado.exceptions.InvalidParsingFormat: Eviann0_LOC_00011369-mRNA-2	0	774	ID=Eviann0_LOC_00011369-mRNA-2.p6;type:complete;len:65;gc:the;standard;code;Eviann0_LOC_00011369-mRNA-2:290-96(-)	523	-	290		0	1	774	0

after error

Process Bed12ParseWrapper-10:
Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1188, in thick_end
    value = int(value)
ValueError: invalid literal for int() with base 10: ''

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1802, in bed_next
    bed12 = BED12(line,
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 366, in __init__
    self.__set_values_from_fields()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 536, in __set_values_from_fields
    self.thick_start, self.thick_end, self.rgb, \
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1190, in thick_end
    raise ValueError("Thick end must be an integer, not {}! Value: {}".format(type(value), value))
ValueError: Thick end must be an integer, not <class 'str'>! Value: 

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1866, in run
    row = self.bed_next(line, seq)
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1811, in bed_next
    raise InvalidParsingFormat("Invalid line: {}".format(line))
Mikado.exceptions.InvalidParsingFormat: Invalid line: Eviann0_LOC_00002944-mRNA-1	0	279	ID=Eviann0_LOC_00002944-mRNA-1.p2;type:complete;len:66;gc:the;standard;code;Eviann0_LOC_00002944-mRNA-1:213-16(-)	871	-	213		0	1	279	0


During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/multiprocessing/process.py", line 314, in _bootstrap
    self.run()
  File "/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10/site-packages/Mikado/parsers/bed12.py", line 1880, in run
    raise InvalidParsingFormat(line)
Mikado.exceptions.InvalidParsingFormat: Eviann0_LOC_00002944-mRNA-1	0	279	ID=Eviann0_LOC_00002944-mRNA-1.p2;type:complete;len:66;gc:the;standard;code;Eviann0_LOC_00002944-mRNA-1:213-16(-)	871	-	213		0	1	279	0

Any reason for these errors?
They are the direct output files from EviAnn and Tiberius, which should be fine.
I am using SLURM job management and I assigned 60 cores and 5 GB of ram per core. It gave it 4 days and it didn't finish. I ran Tiberius in a similar fashion, and it finished everything in 27 hours. Any reason why the annotation files are failing? Any way to "validate" them before?
I did proteome QC - agat - to retrieve the longest isoform and then I used gffread to get the transcripts and the protein. I don't know how else to validate them.

Also I want to mention that TransDecoder is no longer actively supported or maintained by the developer. There is a new version: https://github.com/Markusjsommer/TD2/wiki - TransDecoder2 is now released. Hope Mikado is compatible with TD2.

[gemygk]

[edit: update git markdown: version -> __version__]

Hi @desmodus1984,

The error appears to be related to the BED12 thickEnd field (8th column), which is missing/empty in your file, specifically between 213 and 0, as highlighted below:

Eviann0_LOC_00002944-mRNA-1 0 279 ID=Eviann0_LOC_00002944-mRNA-1.p2;type:complete;len:66;gc:the;standard;code;Eviann0_LOC_00002944-mRNA-1:213-16(-) 871 - 213 0 1 279 0

If you are able to share a small sample of the input data along with the commands you ran, I would be happy to reproduce the issue with BED12 and investigate it further on my end as well.

Regarding the deprecation warning: we should not be seeing this, as the dependency is pinned to setuptools<80.9. Could you please run the following command and share the installed setuptools version?

python -c "import setuptools; print(setuptools.__version__)"

Thanks also for flagging TD2. We are aware of it and do make use of it (not in the Mikado context yet). At present, we use Prodigal, which is considerably faster for ORF identification. That said, it does not perform as well as TransDecoder when it comes to accurately identifying the correct ORFs (as Prodigal was originally developed for bacterial genomes).

[desmodus1984]

Hi,
I ran the command and the output was this:
"python -c "import setuptools; print(setuptools.version)"
<module 'setuptools.version' from '/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado2/lib/python3.9/site-packages/setuptools/version.py'>
"
I did conda list, and I got found this:
setuptools 80.8.0 pyhff2d567_0 conda-forge

The code that I ran was this:

mikado serialise \
 -p 60 --start-method spawn \
 --transcripts mikado_prepared.fasta \
 --json-conf conf.yaml \
 --genome_fai Lepwed.softmasked.fasta.fai \
 --junctions /data/common/juanpablo.aguilar/analysis/RNA/portcullis_out/3-filt/portcullis_filtered.pass.junctions.bed \
 --tsv LW3_blast_results.tsv \
 --blast-targets uniprot_sprot.fasta \
 --max-target-seqs 5 \
 --orfs mikado_prepared.fasta.TD2.bed \
 --log mikado_serialise.log

Let me know which file do you need, and if possible, a code to extract the minimum information to reproduce the issue. Badly, I couldn't find a code to subsample a gff file.

Thanks.

[gemygk]

Hi @desmodus1984

Apologies, Git Markdown changed my command a bit, and I didn't notice. I have updated my comment earlier, please see here or below

python -c "import setuptools; print(setuptools.__version__)"

If you can paste the first few lines of the mikado_prepared.fasta.TD2.bed file here, that would be helpful

$ head mikado_prepared.fasta.TD2.bed

According to your logs, I can see that Mikado is installed at two locations. You might need to ensure you're sourcing the correct Mikado environment.

From comment here

/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado/lib/python3.10

From comment here

/data/common/juanpablo.aguilar/apps/Miniforge3/envs/mikado2/lib/python3.9

[desmodus1984]

Hi,

The new code output:
python -c "import setuptools; print(setuptools.version)"
80.8.0

• was the same as checking the conda app list.

Here are some lines from the bed file:
head mikado_prepared.fasta.TD2.bed -n 20
track name='mikado_prepared.fasta.TD2.gff3'
Eviann0_LOC_00016619-mRNA-4 0 3562 ID=Eviann0_LOC_00016619-mRNA-4.p6;type:complete;len:641;gc:the;standard;code;Eviann0_LOC_00016619-mRNA-4:326-2248(+) 986 + 326 2248 0 1 3562 0
Eviann0_LOC_00000667-mRNA-5 0 815 ID=Eviann0_LOC_00000667-mRNA-5.p2;type:complete;len:271;gc:the;standard;code;Eviann0_LOC_00000667-mRNA-5:2-814(+) 950 + 2 814 0 1 815 0
Tiberius_g16701.t1 0 735 ID=Tiberius_g16701.t1.p1;type:complete;len:245;gc:the;standard;code;Tiberius_g16701.t1:1-735(+) 992 + 1 735 01 735 0
Eviann0_LOC_00021090-mRNA-9 0 6207 ID=Eviann0_LOC_00021090-mRNA-9.p1;type:complete;len:2069;gc:the;standard;code;Eviann0_LOC_00021090-mRNA-9:1-6207(+) 996 + 1 6207 0 1 6207 0
Eviann0_LOC_00007585-mRNA-7 0 1797 ID=Eviann0_LOC_00007585-mRNA-7.p2;type:complete;len:593;gc:the;standard;code;Eviann0_LOC_00007585-mRNA-7:19-1797(+) 871 + 19 1797 0 1 1797 0
Eviann0_LOC_00011475-mRNA-11 0 4528 ID=Eviann0_LOC_00011475-mRNA-11.p1;type:complete;len:268;gc:the;standard;code;Eviann0_LOC_00011475-mRNA-11:1-804(+) 992 + 1 804 0 1 4528 0
Eviann0_XLOC_010655U_lncRNA-mRNA-1 0 406 ID=Eviann0_XLOC_010655U_lncRNA-mRNA-1.p2;type:3prime_partial;len:94;gc:the;standard;code;Eviann0_XLOC_010655U_lncRNA-mRNA-1:123-404(+) 899 + 123 404 0 1 406 0
Eviann0_LOC_00011741-mRNA-9 0 8978 ID=Eviann0_LOC_00011741-mRNA-9.p2;type:complete;len:1878;gc:the;standard;code;Eviann0_LOC_00011741-mRNA-9:2116-7749(+)919 + 2116 7749 0 1 8978 0
Eviann0_LOC_00005169-mRNA-2 0 2038 ID=Eviann0_LOC_00005169-mRNA-2.p2;type:complete;len:466;gc:the;standard;code;Eviann0_LOC_00005169-mRNA-2:241-1638(+) 992 + 241 1638 0 1 2038 0
Eviann0_LOC_00018175-mRNA-11 0 2025 ID=Eviann0_LOC_00018175-mRNA-11.p1;type:complete;len:675;gc:the;standard;code;Eviann0_LOC_00018175-mRNA-11:1-2025(+) 996 + 1 2025 0 1 2025 0
Eviann0_LOC_00015629-mRNA-6 0 2224 ID=Eviann0_LOC_00015629-mRNA-6.p5;type:complete;len:448;gc:the;standard;code;Eviann0_LOC_00015629-mRNA-6:359-1702(+) 995 + 359 1702 0 1 2224 0
Eviann1_LOC_00016232-mRNA-6 0 7596 ID=Eviann1_LOC_00016232-mRNA-6.p1;type:complete;len:2532;gc:the;standard;code;Eviann1_LOC_00016232-mRNA-6:1-7596(+) 990 + 1 7596 0 1 7596 0
Eviann0_LOC_00004110-mRNA-1 0 70336 ID=Eviann0_LOC_00004110-mRNA-1.p75;type:complete;len:203;gc:the;standard;code;Eviann0_LOC_00004110-mRNA-1:542-1150(+) 989 + 542 1150 0 1 70336 0
Eviann0_LOC_00014445-mRNA-31 0 9723 ID=Eviann0_LOC_00014445-mRNA-31.p12;type:complete;len:1135;gc:the;standard;code;Eviann0_LOC_00014445-mRNA-31:377-3781(+) 997 + 377 3781 0 1 9723 0
Eviann0_LOC_00015601-mRNA-6 0 4042 ID=Eviann0_LOC_00015601-mRNA-6.p4;type:complete;len:855;gc:the;standard;code;Eviann0_LOC_00015601-mRNA-6:161-2725(+) 995 + 161 2725 0 1 4042 0
Eviann1_LOC_00009685-mRNA-3 0 921 ID=Eviann1_LOC_00009685-mRNA-3.p1;type:complete;len:307;gc:the;standard;code;Eviann1_LOC_00009685-mRNA-3:1-921(+) 962 + 1 921 0 1 921 0
Eviann0_LOC_00008831-mRNA-1 0 588 ID=Eviann0_LOC_00008831-mRNA-1.p1;type:complete;len:196;gc:the;standard;code;Eviann0_LOC_00008831-mRNA-1:1-588(+) 986 + 1 588 0 1 588 0
Eviann0_LOC_00008911-mRNA-1 0 4174 ID=Eviann0_LOC_00008911-mRNA-1.p6;type:complete;len:419;gc:the;standard;code;Eviann0_LOC_00008911-mRNA-1:117-1373(+) 953 + 117 1373 0 1 4174 0
Eviann0_LOC_00016199-mRNA-1 0 2214 ID=Eviann0_LOC_00016199-mRNA-1.p1;type:complete;len:738;gc:the;standard;code;Eviann0_LOC_00016199-mRNA-1:1-2214(+) 911 + 1 2214 0 1 2214 0

I deleted the first environment - where mikado worked - and I asked chatgpt to fix it. Then, I tried following the instructions and I got the gcc error.

[gemygk]

[Edit: Added all the models that have been reported]

Hi @desmodus1984

Thank you for sharing a few lines from mikado_prepared.fasta.TD2.bed

I ran an internal Mikado test using the TD2-generated BED12 file, and the test completed successfully on my end.

Based on this, it appears that the issue is related to the models listed below, which seem to cause TD2 to generate an incomplete BED12 entry (missing BED12 thickEnd field (8th column))

Eviann0_XLOC_003602U_lncRNA-mRNA-1
Eviann0_LOC_00004922-mRNA-4
Tiberius_g8972.t1
Eviann0_LOC_00011369-mRNA-2
Eviann0_LOC_00002944-mRNA-1

Could you please paste the FASTA sequences for the models below so that I can run TD2 locally and compare the outputs?

Eviann0_LOC_00016619-mRNA-4   -- TD2 BED12 is correct
Tiberius_g16701.t1            -- TD2 BED12 is correct
Eviann0_LOC_00002944-mRNA-1   -- TD2 BED12 is **NOT** correct
Tiberius_g8972.t1             -- TD2 BED12 is **NOT** correct

In parallel, you could try the following two steps.

1. Test Mikado serialise with a small BED12 subset

Create a test BED12 file using the same lines you shared with me:

head -n 20 mikado_prepared.fasta.TD2.bed > mikado_prepared.fasta.TD2.test.bed

Then run mikado serialise using this test file:

mikado serialise \
 -p 60 --start-method spawn \
 --transcripts mikado_prepared.fasta \
 --json-conf conf.yaml \
 --genome_fai Lepwed.softmasked.fasta.fai \
 --junctions /data/common/juanpablo.aguilar/analysis/RNA/portcullis_out/3-filt/portcullis_filtered.pass.junctions.bed \
 --tsv LW3_blast_results.tsv \
 --blast-targets uniprot_sprot.fasta \
 --max-target-seqs 5 \
 --orfs mikado_prepared.fasta.TD2.test.bed \
 --log mikado_serialise.log

If this runs successfully, it further confirms that the issue originates with the models I listed earlier.

You can then remove the test Mikado serialise database

rm mikado.db serialise.log

2. Run Mikado serialise, excluding the problematic models

Remove the models from the TD2 BED12 file

cat mikado_prepared.fasta.TD2.bed | egrep -v "Eviann0_XLOC_003602U_lncRNA-mRNA-1|Eviann0_LOC_00004922-mRNA-4|Tiberius_g8972.t1|Eviann0_LOC_00011369-mRNA-2|Eviann0_LOC_00002944-mRNA-1" > mikado_prepared.fasta.TD2.filtered.bed

Then run mikado serialise using the filtered BED12 file:

mikado serialise \
 -p 60 --start-method spawn \
 --transcripts mikado_prepared.fasta \
 --json-conf conf.yaml \
 --genome_fai Lepwed.softmasked.fasta.fai \
 --junctions /data/common/juanpablo.aguilar/analysis/RNA/portcullis_out/3-filt/portcullis_filtered.pass.junctions.bed \
 --tsv LW3_blast_results.tsv \
 --blast-targets uniprot_sprot.fasta \
 --max-target-seqs 5 \
 --orfs mikado_prepared.fasta.TD2.filtered.bed \
 --log mikado_serialise.log

If this completes successfully, it again confirms that the issue arises from the models I listed earlier.
If you still encounter an error with another model, please check whether it is related to the missing BED12 thickEnd field (8th column). In that case, you can repeat Step 2 I mentioned earlier.

Finally, with the current version of setuptools (80.8.0) in your conda environment, you should not be seeing a deprecation warning. Could you please confirm this?

[desmodus1984]

Hey @gemygk
I tried to follow the installation instructions on site and now mikado can't be installed.
I made a file with the gene models that you requested.
I am in contact with a researcher who made a singularity image of mikado to try it.

models.zip

[desmodus1984]

I was able to install mikado with conda on a workstation.
I ran mikado prepare and I got this error:

mikado prepare \
 --json-conf conf.yaml
 --start-method spawn \
 -p 12

Mikado prepare has finished correctly with seed 0. The output ./mikado_prepared.fasta FASTA file can now be used for BLASTX and/or ORF calling before the next step in the pipeline, `mikado serialise`.
--start-method: command not found

I ran help and it is there:

mikado prepare -h
usage: Mikado prepare [-h] [--fasta REFERENCE] [--verbose | --quiet | -lv {DEBUG,INFO,WARN,ERROR}] [--start-method {fork,spawn,forkserver}]
                      [-s | -sa STRAND_SPECIFIC_ASSEMBLIES] [--list LIST] [-l LOG] [--lenient] [-m MINIMUM_CDNA_LENGTH] [-MI MAX_INTRON_LENGTH]
                      [-p PROCS] [-scds] [--labels LABELS] [--codon-table CODON_TABLE] [--single] [-od OUTPUT_DIR] [-o OUT] [-of OUT_FASTA]
                      [--configuration CONFIGURATION] [-er] [--strip-faulty-cds] [--seed SEED | --random-seed]
                      [gff ...]

positional arguments:
  gff                   Input GFF/GTF file(s).

optional arguments:
  -h, --help            show this help message and exit
  --fasta REFERENCE, --reference REFERENCE, --genome REFERENCE
                        Genome FASTA file. Required.
  --verbose
  --quiet
  -lv {DEBUG,INFO,WARN,ERROR}, --log-level {DEBUG,INFO,WARN,ERROR}
                        Log level. Default: derived from the configuration; if absent, INFO
  **--start-method {fork,spawn,forkserver}
                        Multiprocessing start method.**

[cschu]

mikado prepare \
 --json-conf conf.yaml <--
 --start-method spawn \
 -p 12

@desmodus1984, you're missing a backslash after --json-conf conf.yaml.

[desmodus1984]

Hi @gemygk @cschu
For some reason, it still ran and finished. Nonetheless, I ran it again with the fixes.
Then, I ran TransDecoder.LongOrfs and it finished,

TransDecoder.LongOrfs  -t mikado_prepared.fasta  -m 30

I ran TransDecoder.Predict , and it failed

TransDecoder.Predict  -t mikado_prepared.fasta  --retain_long_orfs_length 30

* [Sun Mar  1 20:07:57 2026] Running CMD: /home/jpac1984/miniconda3/envs/annotation/bin/util/gff3_file_to_proteins.pl --gff3 /home/jpac1984/Lwed/mikado/mikado_prepared.fasta.transdecoder.gff3 --fasta mikado_prepared.fasta  --genetic_code Universal > /home/jpac1984/Lwed/mikado/mikado_prepared.fasta.transdecoder.pep
Making pep file: /home/jpac1984/Lwed/mikado/mikado_prepared.fasta.transdecoder.pep
Killed
Error, cmd: /home/jpac1984/miniconda3/envs/annotation/bin/util/gff3_file_to_proteins.pl --gff3 /home/jpac1984/Lwed/mikado/mikado_prepared.fasta.transdecoder.gff3 --fasta mikado_prepared.fasta  --genetic_code Universal > /home/jpac1984/Lwed/mikado/mikado_prepared.fasta.transdecoder.pep died with ret 35072 No such file or directory at /home/jpac1984/miniconda3/envs/annotation/bin/PerlLib/Pipeliner.pm line 187.
	Pipeliner::run(Pipeliner=HASH(0x5e078b76f340)) called at /home/jpac1984/miniconda3/envs/annotation/bin/TransDecoder.Predict line 369

There are a bed and a pep, any way to check if those files are okay and proper to run Mikado serialize?

Thanks

[desmodus1984]

Hey @gemygk @cschu

I blasted the mikado fasta against the UniProt protein file,
wget ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz

I created the database:

makeblastdb \
 -in uniprot_sprot.fasta \
 -dbtype prot \
 -out uniprot_sprot

and then I blasted the mikado prepared fasta to it, using this code:

blastx \
 -max_target_seqs 5 \
 -num_threads 100 \
 -query mikado_prepared.fasta \
 -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore ppos btop" \
 -db uniprot_sprot \
 -evalue 0.000001 \
 -out LW.3_blast_results.tsv

Since Blast takes a long time I ran blast on the server since it has access to more cores (64/100) than my laptop (16).

I used this code to run serialize - in my workstation:

mikado serialise  -p 15 --start-method spawn  --transcripts mikado_prepared.fasta  --json-conf conf.yaml  --genome_fai Lepwed.softmasked.fasta.fai  --junctions portcullis_filtered.pass.junctions.bed  --tsv LW.3_blast_results.tsv  --blast-targets uniprot_sprot.fasta  --max-target-seqs 5  --orfs mikado_prepared.fasta.transdecoder.bed  --log mikado_serialise_3.log

and I got this error:

             sid  slength
target_name              
Q6GZX4         1      256
Q6GZX3         2      320
Q197F8         3      458
Q197F7         4      156
Q6GZX2         5      438

0    O43688
1    Q2HJ61
2    Q8K593
3    Q9DAX2
4    P60588
Name: sseqid, dtype: object

Mikado crashed, cause:

'The tabular file passed in and the targets in the database differ. Please rerun BLAST and Mikado serialise with the correct target file.'
Traceback (most recent call last):
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/__main__.py", line 68, in main
    args.func(args)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/subprograms/serialise.py", line 384, in serialise
    load_blast(mikado_configuration, logger)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/subprograms/serialise.py", line 159, in load_blast
    part_launcher(filenames)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/subprograms/serialise.py", line 87, in xml_launcher
    xml_serializer()
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/blast_serialiser.py", line 376, in __call__
    self.serialize()
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/blast_serialiser.py", line 350, in serialize
    self.__serialise_tabular()
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/blast_serialiser.py", line 354, in __serialise_tabular
    _serialise_tabular(self)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/tab_serialiser.py", line 43, in _serialise_tabular
    parser(bname=fname)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/tabular_utils.py", line 472, in parse_tab_blast
    data = sanitize_blast_data(data, queries, targets, qmult=qmult, tmult=tmult)
  File "/home/jpac1984/miniconda3/envs/mikado2/lib/python3.9/site-packages/Mikado/serializers/blast_serializer/tabular_utils.py", line 287, in sanitize_blast_data
    raise KeyError("The tabular file passed in and the targets in the database differ. Please rerun BLAST and "
KeyError: 'The tabular file passed in and the targets in the database differ. Please rerun BLAST and Mikado serialise with the correct target file.'

I didn't get any error for the blastx. Don't understand why mikado is failing now.
I am running it on a workstation where I can update linux libraries since the damn HPC where I run things uses old linux libraries.
I check the "target" files, and they are the same