Errors when running test MrHAMER2 installation

[bioinfo123]

Hi,
I got the error after I installed MrHAMER2 and ran the test.
Any suggestions? Thanks.

To test if the installation was successful run:
conda activate MrHAMER2
snakemake -j 4 --configfile config.yml

Errors:
[Mon Sep 30 17:45:01 2024]
Finished job 7.
6 of 17 steps (35%) done
Select jobs to execute...

[Mon Sep 30 17:45:01 2024]
rule polish_clusters:
input: MrHAMER2_test/clustering/Env/clusters_fa, MrHAMER2_test/clustering/Env/smolecule_clusters.fa
output: MrHAMER2_test/fasta/Env_consensus_tmp, MrHAMER2_test/fasta/Env_consensus.bam, MrHAMER2_test/fasta/Env_consensus.fasta
jobid: 6
reason: Missing output files: MrHAMER2_test/fasta/Env_consensus.fasta; Input files updated by another job: MrHAMER2_test/clustering/Env/smolecule_clusters.fa, MrHAMER2_test/clustering/Env/clusters_fa
wildcards: name=MrHAMER2_test, target=Env
threads: 4
resources: tmpdir=/tmp

[Mon Sep 30 17:47:28 2024]
Error in rule polish_clusters:
jobid: 6
input: MrHAMER2_test/clustering/Env/clusters_fa, MrHAMER2_test/clustering/Env/smolecule_clusters.fa
output: MrHAMER2_test/fasta/Env_consensus_tmp, MrHAMER2_test/fasta/Env_consensus.bam, MrHAMER2_test/fasta/Env_consensus.fasta
shell:

    rm -rf MrHAMER2_test/fasta/Env_consensus_tmp
    medaka smolecule --threads 4 --length 50 --min_depth 2 --model ./test_files/r1041_e82_400bps_sup_v4.2.0_model.tar.gz --method racon --qualities MrHAMER2_test/fasta/Env_consensus_tmp MrHAMER2_test/clustering/Env/smolecule_clusters.fa 2> MrHAMER2_test/fasta/Env_consensus.bam_smolecule.log
    cp MrHAMER2_test/fasta/Env_consensus_tmp/consensus.fastq MrHAMER2_test/fasta/Env_consensus.fasta
    cp MrHAMER2_test/fasta/Env_consensus_tmp/subreads_to_spoa.bam MrHAMER2_test/fasta/Env_consensus.bam && cp MrHAMER2_test/fasta/Env_consensus_tmp/subreads_to_spoa.bam.bai MrHAMER2_test/fasta/Env_consensus.bam.bai
    
    (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

Removing output files of failed job polish_clusters since they might be corrupted:
MrHAMER2_test/fasta/Env_consensus_tmp
Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Complete log: .snakemake/log/2024-09-30T174454.718555.snakemake.log

===============
I looked at the log file "../MrHAMER2/MrHAMER2_test/fasta/Env_consensus.bam_smolecule.log" shows the following error.

[17:47:27 - POAManager] Created 0 consensus with 0 alignments.
[17:47:27 - Smolecule] Writing medaka input bam for 0 reads.
[17:47:27 - Smolecule] Running medaka consensus.
[17:47:27 - Predict] Reducing threads to 2, anymore is a waste.
[17:47:27 - Predict] It looks like you are running medaka without a GPU and attempted to set a high number of threads. We have scaled this down to an optimal number. If you wish to improve performance please see https://nanoporetech.github.io/medaka/installation.html#improving-parallelism.
[17:47:27 - Predict] Setting tensorflow inter/intra-op threads to 2/1.
concurrent.futures.process._RemoteTraceback:
"""
Traceback (most recent call last):
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/concurrent/futures/process.py", line 239, in _process_worker
r = call_item.fn(*call_item.args, **call_item.kwargs)
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/site-packages/medaka/prediction.py", line 119, in predict
bam_regions = medaka.common.get_bam_regions(
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/site-packages/medaka/common.py", line 706, in get_bam_regions
with pysam.AlignmentFile(bam) as bam_fh:
File "pysam/libcalignmentfile.pyx", line 747, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 996, in pysam.libcalignmentfile.AlignmentFile._open
ValueError: file has no sequences defined (mode='r') - is it SAM/BAM format? Consider opening with check_sq=False
"""

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "/home/ppham/.conda/envs/MrHAMER2/bin/medaka", line 8, in
sys.exit(main())
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/site-packages/medaka/medaka.py", line 724, in main
args.func(args)
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/site-packages/medaka/smolecule.py", line 472, in main
_ = fut.result()
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/concurrent/futures/_base.py", line 444, in result
return self.__get_result()
File "/home/ppham/.conda/envs/MrHAMER2/lib/python3.8/concurrent/futures/_base.py", line 389, in __get_result
raise self._exception
ValueError: file has no sequences defined (mode='r') - is it SAM/BAM format? Consider opening with check_sq=False

[gallardo-seq]

Thanks for reaching out, and apologies for the delay. We've been finalizing the manuscript for the pipeline. Thanks for providing the information on the log file. That is not an error I have ever observed before. I'm getting the sense that the file being fed into the smolecule module is empty, which means that there's something wrong happening upstream to the medaka step. Just to be sure, would you mind running "medaka smolecule --help" and pasting here the resulting screen. I want to make sure that the issue is not related to a mismatched medaka version. Thanks!