Testing F2

[edwhisnant]

A test of Funannotate2

I have run funannotate2 as suggested previously. I have run 4 different tests: the A. nidulans genome 1) without and 2) with GeneMark AND the same programs on a Cladonia grayi genome I downloaded from JGI, 3) without GeneMark and 4) with GeneMark.

Aspergillus nidulans

Script:

source $(conda info --base)/etc/profile.d/conda.sh
conda activate funannotate2

cd /hpc/group/bio1/ewhisnant/armaleo-data/funannotate2

mkdir -p test-genome

cd test-genome

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/011/425/GCF_000011425.1_ASM1142v1/GCF_000011425.1_ASM1142v1_genomic.fna.gz

cd ..

funannotate2 train -f test-genome/GCF_000011425.1_ASM1142v1_genomic.fna.gz --cpus 12  -s "Aspergillus nidulans" --strain FGSCA4 -o anid_f2

funannotate2 predict -i anid_f2/ --cpus 12

funannotate2 annotate -i anid_f2/ --cpus 12

conda deactivate

1. A. nidulans without GeneMark:

• See output: link

2) A. nidulans with GeneMark

• See output: link

Cladonia grayi

This genome sequence had been cleaned, sorted, and masked previously. The input for the training is the masked genome after running through RepeatMasker.

Script:

source $(conda info --base)/etc/profile.d/conda.sh
conda activate funannotate2

funannotate2 train \
    -f /hpc/group/bio1/ewhisnant/armaleo-data/Clagr3/funannotations/masked-assemblies/repeat-masker/Clagr3_AssemblyScaffolds_cleaned_RMtest.masked.fasta \
    --cpus 12  \
    -s "Cladonia grayi" \
    -o clagr3_f2

funannotate2 predict -i clagr3_f2/ --cpus 12

funannotate2 annotate -i clagr3_f2/ --cpus 12

conda deactivate

3. C. grayi without GeneMark

See the output: link

4. C grayi w/ GeneMark

See the final output: link

My notes:

1. BUSCO databases

Currently, F2 is using BUSCO _odb10 gene models. I can see in your scripts that _odb10 is specified directly. However, BUSCO recently released the _odb12 gene models. Would it be possible for buscolite to call the updated models? These were released very recently, 4/11/25 if I remember correctly. As an example, ascomycota now include 2826 models vs ~1700.

2. Issue with GeneMark

During my first couple of runs including GeneMark, both A. nidulans and 'C grayi` returned this error after attempting to execute GeneMark.

�[92m[Apr 21 02:37 PM] �[0m�[38;20mTraining GeneMark-ES using self-training�[0m
�[92m[Apr 21 02:37 PM] �[0m�[31;20mCMD ERROR: gmes_petap.pl --ES --cores 12 --work_dir genemark --training --fungus --sequence genome.fasta�[0m
�[92m[Apr 21 02:37 PM] �[0m�[31;20mCan't locate Hash/Merge.pm in @INC (you may need to install the Hash::Merge module) (@INC contains: /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/5.32/site_perl /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/site_perl /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/5.32/vendor_perl /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/vendor_perl /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/5.32/core_perl /hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/perl5/core_perl .) at /hpc/group/bio1/ewhisnant/software/gene-mark-software/gmes_linux_64/gmes_petap.pl line 88.
BEGIN failed--compilation aborted at /hpc/group/bio1/ewhisnant/software/gene-mark-software/gmes_linux_64/gmes_petap.pl line 88.
�[0m
WARNING: unable to find params file in clagr3_f2/train_results
['Clagr3_AssemblyScaffolds_cleaned_RMtest.masked.fasta']Traceback (most recent call last):
  File "/hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/bin/funannotate2", line 8, in <module>
    sys.exit(main())
             ^^^^^^
  File "/hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/python3.12/site-packages/funannotate2/__main__.py", line 26, in main
    predict(args)
  File "/hpc/group/bio1/ewhisnant/miniconda3/envs/funannotate2/lib/python3.12/site-packages/funannotate2/predict.py", line 81, in predict
    args.species = params.get("species")
                   ^^^^^^^^^^
AttributeError: 'NoneType' object has no attribute 'get'

This error was solved by installing the missing perl modules, which allowed the programs to run GeneMark without error.

conda install -c bioconda perl-hash-merge
conda install -c bioconda perl-mce

[nextgenusfs]

Great thanks. Of course they just updated BUSCO😩.... shouldn't be too hard to change/update that.

[nextgenusfs]

I don't intend to add the Perl modules to the conda recipe for f2 as part of this whole rewrite was to reduce dependencies and make install easier. But I will include that info in the documentation. Thanks.

So glad that it ran okay. Another an initio predictor that seems to work pretty well is Helixer, I wrote and repacked a version of that called helixerlite and you can install with pip. I will also not include that as a dependency for reasons above.

There are also some functional annotation tools that are useful and f1 had parsers for (interproscan, eggnog, etc) and f2 will not natively support those, instead I have a separate tool that will try to support this stuff, effectively allowing f2 to incorporate any annotations but I don't have the time/resources to continuously update the parsers for changing formats. Hopefully these design changes make f2 more stable and flexible.

[nextgenusfs]

Looks like odb12 release changed a file, so will need to update underlying buscolite package. running some tests now.

[nextgenusfs]

Okay, should be resolved now with buscolite v25.4.24 and the latest on f2 repo.

[edwhisnant]

Sweet. I will update this on my end and get helixerlite installed. Thanks for the updates!