Can't identify the error in Funannotate

usegalaxy.eu support
1

Hi all.
I am trying to run Funannotate predict annotation in galaxy.eu.
My inputs are assembled genome (using Spades and cleaned with funannotate assembly clean), merged BAM files (from 12 RNA seq data sets), BUSCO on Sordariomycetes (orthoDB10) and magnaporthe_grisea (on 2023-9 database).
It comes with this error:
[Mar 28 01:45 PM]: OS: Debian GNU/Linux 10, 120 cores, ~ 438 GB RAM. Python: 3.8.15
[Mar 28 01:45 PM]: Running funannotate v1.8.15
[Mar 28 01:48 PM]: Parsed training data, run ab-initio gene predictors as follows:
e[4mProgram Training-Methode[0m
augustus busco
codingquarry rna-bam
glimmerhmm busco
snap busco
[Mar 28 01:58 PM]: Loading genome assembly and parsing soft-masked repetitive sequences
Traceback (most recent call last):
File “/usr/local/lib/python3.8/multiprocessing/process.py”, line 315, in _bootstrap
self.run()
File “/usr/local/lib/python3.8/multiprocessing/process.py”, line 108, in run
self._target(*self._args, **self._kwargs)
File “/usr/local/lib/python3.8/multiprocessing/managers.py”, line 616, in _run_server
server.serve_forever()
File “/usr/local/lib/python3.8/multiprocessing/managers.py”, line 182, in serve_forever
sys.exit(0)
SystemExit: 0

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 300, in _run_finalizers
finalizer()
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 224, in call
res = self._callback(*self._args, **self._kwargs)
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 133, in _remove_temp_dir
rmtree(tempdir)
File “/usr/local/lib/python3.8/shutil.py”, line 718, in rmtree
_rmtree_safe_fd(fd, path, onerror)
File “/usr/local/lib/python3.8/shutil.py”, line 675, in _rmtree_safe_fd
onerror(os.unlink, fullname, sys.exc_info())
File “/usr/local/lib/python3.8/shutil.py”, line 673, in _rmtree_safe_fd
os.unlink(entry.name, dir_fd=topfd)
OSError: [Errno 16] Device or resource busy: ‘.nfs00000000083674ac0007f7a8’
Traceback (most recent call last):
File “/usr/local/lib/python3.8/multiprocessing/process.py”, line 315, in _bootstrap
self.run()
File “/usr/local/lib/python3.8/multiprocessing/process.py”, line 108, in run
self._target(*self._args, **self._kwargs)
File “/usr/local/lib/python3.8/multiprocessing/managers.py”, line 616, in _run_server
server.serve_forever()
File “/usr/local/lib/python3.8/multiprocessing/managers.py”, line 182, in serve_forever
sys.exit(0)
SystemExit: 0

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 300, in _run_finalizers
finalizer()
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 224, in call
res = self._callback(*self._args, **self._kwargs)
File “/usr/local/lib/python3.8/multiprocessing/util.py”, line 133, in _remove_temp_dir
rmtree(tempdir)
File “/usr/local/lib/python3.8/shutil.py”, line 718, in rmtree
_rmtree_safe_fd(fd, path, onerror)
File “/usr/local/lib/python3.8/shutil.py”, line 675, in _rmtree_safe_fd
onerror(os.unlink, fullname, sys.exc_info())
File “/usr/local/lib/python3.8/shutil.py”, line 673, in _rmtree_safe_fd
os.unlink(entry.name, dir_fd=topfd)
OSError: [Errno 16] Device or resource busy: ‘.nfs00000000083679980007f7a9’
[Mar 28 02:01 PM]: Genome loaded: 282 scaffolds; 51,267,046 bp; 2.90% repeats masked
[Mar 28 02:01 PM]: Extracting hints from RNA-seq BAM file using bam2hints
[Mar 28 02:20 PM]: Mapping 553,202 proteins to genome using diamond and exonerate
Traceback (most recent call last):
File “/usr/local/lib/python3.8/site-packages/funannotate/aux_scripts/funannotate-p2g.py”, line 252, in
os.makedirs(tmpdir)
File “/usr/local/lib/python3.8/os.py”, line 223, in makedirs
mkdir(name, mode)
OSError: [Errno 30] Read-only file system: ‘/tmp/p2g_533df5e6-5076-4cfa-9670-6037d4f24411’
Traceback (most recent call last):
File “/usr/local/bin/funannotate”, line 10, in
sys.exit(main())
File “/usr/local/lib/python3.8/site-packages/funannotate/funannotate.py”, line 716, in main
mod.main(arguments)
File “/usr/local/lib/python3.8/site-packages/funannotate/predict.py”, line 1558, in main
lib.exonerate2hints(Exonerate, hintsP)
File “/usr/local/lib/python3.8/site-packages/funannotate/library.py”, line 4600, in exonerate2hints
with open(file, “r”) as input:
FileNotFoundError: [Errno 2] No such file or directory: ‘/data/jwd02f/main/068/340/68340633/working/output/predict_misc/protein_alignments.gff3’

I’ve tried working around the BUSCO models and database (2022 database → sordariomycetes, or other species magnaporther_grisea → fusarium_graminearum) but it seems to be not the problem.
As I have successfully run this tool months ago using different assembled genome (2022 database, BUSCO leotiomycetes (orthodb 10) and Botrytis_cinerea), is it possible that the problem is in my assembled genome?

I would really appreciate any tips and advices.

2

Welcome, @Jewish

This part is intersting:

Other than that, your job seems pretty large. I am wondering if the tool is running out of resources during execution. The EU server can chomp through even the largest of data, so you are working at a good public server choice.

So, yes, the assembly itself could be a factor.

The only advice I have to make sure the assembly is super-clean. Maybe try more stringent parameters with the Funannotate assembly clean tool, then relax the parameters in reruns, once you have a baseline that processes.

Others can comment more if they have alternative advice :slight_smile:

3

Thank you @jennaj !

I also thought of it being heavy so I tried removing the BAM files but it came back with the same error. Also this OSError was also present in the logs of my previous successful funannotate using different assembled genome so I guess it is not the problem.

I was wondering if the genome was the problem though since I said yes to the option for checking the quality of genome… I will try more stringent parameters with the Funannotate assembly clean tool like what you have advised.

Thanks again!