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marcelauliano / MitoHiFi

Find, circularise and annotate mitogenome from PacBio assemblies
MIT License
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mitohifi.py: No such file or directory: 'gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa' #26

Closed junaruga closed 2 years ago

junaruga commented 2 years ago

Perhaps, the issue might be essentially the same with the https://github.com/marcelauliano/MitoHiFi/issues/10 .

I am using MitoHiFi's latest docker container image.

First, I created the reference .fasta and .db files by findMitoReference.py.

$ mkdir data

$ docker run --rm -v $(pwd)/data/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  findMitoReference.py --species "human" --email jun.aruga@gmail.com --outfolder /data/ --min_length 16000
Looking for mitochondrion for human
output is written to /data/ON980565.1.[gb,fast]

$ ls -hl data/
total 56K
-rw-r--r--. 1 root root 17K Oct 11 18:15 ON980565.1.fasta
-rw-r--r--. 1 root root 35K Oct 11 18:15 ON980565.1.gb

Then then I copied a fast file that was originally created by hifiasm, and that I modified. The generated_asm.bp.r_utg.fa file is here.

$ cp -p ~/git/GargGroup/BioDivGenomics/test/data/hifiasm/generated_asm.bp.r_utg.fa data/

Here is the MitoHifi's version.

$ docker run --rm -t docker.io/biocontainers/mitohifi:2.2_cv1 mitohifi.py --version
MitoHiFi 2.14.2

Then I executed the mitohifi.py with the fast file above and reference .fasta and .db files as input files. I got the following error. Do you know what's wrong? You can reproduce it in your environment, as I shared my data. Thank you.

$ docker run --rm -w /data/ -v $(pwd)/data/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  mitohifi.py -r /data/generated_asm.bp.r_utg.fa -f /data/ON980565.1.fasta -g /data/ON980565.1.gb -t 4 -o 2
2022-10-11 16:31:54 [INFO] Welcome to MitoHifi v2. Starting pipeline...
2022-10-11 16:31:54 [INFO] Length of related mitogenome is: 16574 bp
2022-10-11 16:31:54 [INFO] Number of genes on related mitogenome: 37
2022-10-11 16:31:54 [INFO] Running MitoHifi pipeline in reads mode...
2022-10-11 16:31:54 [INFO] 1. First we map your Pacbio HiFi reads to the close-related mitogenome
2022-10-11 16:31:54 [INFO] minimap2 -t 4 --secondary=no -ax map-pb /data/ON980565.1.fasta /data/generated_asm.bp.r_utg.fa | samtools view -@ 4 -S -b -F4 -F 0x800 > reads.HiFiMapped.bam
2022-10-11 16:31:54 [INFO] 2. Now we filter out any mapped reads that are larger than the reference mitogenome to avoid NUMTS
2022-10-11 16:31:54 [INFO] 2.1 First we convert the mapped reads from BAM to FASTA format:
2022-10-11 16:31:54 [INFO] samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta
2022-10-11 16:31:54 [INFO] Total number of mapped reads: 0
2022-10-11 16:31:54 [INFO] 2.2 Then we filter reads that are larger than 16574 bp
2022-10-11 16:31:54 [INFO] Number of filtered reads: 0
2022-10-11 16:31:54 [INFO] 3. Now let's run hifiasm to assemble the mapped and filtered reads!
2022-10-11 16:31:54 [INFO] hifiasm --primary -t 4 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta
Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 139, in main
    f1 = open("gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa")
FileNotFoundError: [Errno 2] No such file or directory: 'gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa'

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 143, in main
    An error may have occurred when assembling reads with HiFiasm.""")
SystemExit: No gbk.HiFiMapped.bam.filtered.assembled.[a/p]_ctg.gfa file(s).
            An error may have occurred when assembling reads with HiFiasm.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 377, in <module>
    main()
  File "/bin/MitoHiFi/mitohifi.py", line 145, in main
    f1.close()
UnboundLocalError: local variable 'f1' referenced before assignment

Here is the working directory's status after executing the command mitohifi.py above.

$ ls -lh data
total 3.7M
-rw-r--r--. 1 root   root      0 Oct 11 18:31 gbk.HiFiMapped.bam.fasta
-rw-r--r--. 1 root   root   7.9K Oct 11 18:31 gbk.HiFiMapped.bam.filtered.assembled.ec.bin
-rw-r--r--. 1 root   root      8 Oct 11 18:31 gbk.HiFiMapped.bam.filtered.assembled.ovlp.reverse.bin
-rw-r--r--. 1 root   root      8 Oct 11 18:31 gbk.HiFiMapped.bam.filtered.assembled.ovlp.source.bin
-rw-r--r--. 1 root   root      0 Oct 11 18:31 gbk.HiFiMapped.bam.filtered.fasta
-rw-r--r--. 1 jaruga jaruga 3.7M Oct 10 14:02 generated_asm.bp.r_utg.fa
-rw-r--r--. 1 root   root    217 Oct 11 18:31 hifiasm.log
-rw-r--r--. 1 root   root    17K Oct 11 18:15 ON980565.1.fasta
-rw-r--r--. 1 root   root    35K Oct 11 18:15 ON980565.1.gb
-rw-r--r--. 1 root   root    212 Oct 11 18:31 reads.HiFiMapped.bam
junaruga commented 2 years ago

Note that the generated_asm.bp.r_utg.fa (this file) was created from the asm.bp.r_utg.gfa (output of the hifiasm) by the following steps.

Here is the used hifiasm version. Maybe it is the latest version.

$ docker run --rm -t quay.io/biocontainers/hifiasm:0.16.1--h5b5514e_1 hifiasm --version
0.16.1-r375
$ awk '/^S/{print ">"$2; print $3}' tmp/hifiasm/asm.bp.r_utg.gfa > tmp/hifiasm/generated_asm.bp.r_utg.fa
ksenia-krasheninnikova commented 2 years ago

Hi @junaruga You might try typing the full path to your current folder instead of $(pwd). Let me know if it works for you.

junaruga commented 2 years ago

You might try typing the full path to your current folder instead of $(pwd). Let me know if it works for you.

I tried the full path instead of the $(pwd). But the result is the same. Here is the log.

$ pwd
/home/jaruga/tmp/mitohifi

$ ls -hl data
total 3.7M
-rw-r--r--. 1 jaruga jaruga 3.7M Oct 10 14:02 generated_asm.bp.r_utg.fa
-rw-rw-rw-. 1 jaruga jaruga  17K Oct 11 18:15 ON980565.1.fasta
-rw-rw-rw-. 1 jaruga jaruga  35K Oct 11 18:15 ON980565.1.gb

$ docker run --rm -w /data/ -v /home/jaruga/tmp/mitohifi/data/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  mitohifi.py -r /data/generated_asm.bp.r_utg.fa -f /data/ON980565.1.fasta -g /data/ON980565.1.gb -t 4 -o 2
2022-10-17 13:28:17 [INFO] Welcome to MitoHifi v2. Starting pipeline...
2022-10-17 13:28:17 [INFO] Length of related mitogenome is: 16574 bp
2022-10-17 13:28:17 [INFO] Number of genes on related mitogenome: 37
2022-10-17 13:28:17 [INFO] Running MitoHifi pipeline in reads mode... 
2022-10-17 13:28:17 [INFO] 1. First we map your Pacbio HiFi reads to the close-related mitogenome 
2022-10-17 13:28:17 [INFO] minimap2 -t 4 --secondary=no -ax map-pb /data/ON980565.1.fasta /data/generated_asm.bp.r_utg.fa | samtools view -@ 4 -S -b -F4 -F 0x800 > reads.HiFiMapped.bam
2022-10-17 13:28:17 [INFO] 2. Now we filter out any mapped reads that are larger than the reference mitogenome to avoid NUMTS
2022-10-17 13:28:17 [INFO] 2.1 First we convert the mapped reads from BAM to FASTA format:
2022-10-17 13:28:17 [INFO] samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta
2022-10-17 13:28:17 [INFO] Total number of mapped reads: 0
2022-10-17 13:28:17 [INFO] 2.2 Then we filter reads that are larger than 16574 bp
2022-10-17 13:28:17 [INFO] Number of filtered reads: 0
2022-10-17 13:28:17 [INFO] 3. Now let's run hifiasm to assemble the mapped and filtered reads!
2022-10-17 13:28:17 [INFO] hifiasm --primary -t 4 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta
Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 139, in main
    f1 = open("gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa")
FileNotFoundError: [Errno 2] No such file or directory: 'gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa'

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 143, in main
    An error may have occurred when assembling reads with HiFiasm.""")
SystemExit: No gbk.HiFiMapped.bam.filtered.assembled.[a/p]_ctg.gfa file(s).
            An error may have occurred when assembling reads with HiFiasm.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 377, in <module>
    main()
  File "/bin/MitoHiFi/mitohifi.py", line 145, in main
    f1.close()
UnboundLocalError: local variable 'f1' referenced before assignment

Debugging

I debugged the code a bit. In the container, the mitohifi.py has 377 lines. I expected the code is on the v2.2 git tag. However, on the tag v2.2, the code has 377 lines. Anyway, I use the mitohifi.py on the v2.2 to explain for convenience.

$ docker run --rm -it docker.io/biocontainers/mitohifi:2.2_cv1 bash

root@393dbe4cedb7:/# wc -l /bin/MitoHiFi//mitohifi.py
377 /bin/MitoHiFi//mitohifi.py

The error happens on the

https://github.com/marcelauliano/MitoHiFi/blob/852cd1d27c92534c90993d387354f5fb868b0f0e/mitohifi.py#L138

The file gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa is excepted to be created as an output by the hifiasm command below. But it is actually not created.

hifiasm --primary -t 4 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta

The gbk.HiFiMapped.bam.filtered.fasta file used as an input of the hifiasm command exists as zero byte.

$ ls -hl gbk.HiFiMapped.bam.filtered.fasta
-rw-r--r--. 1 root root 0 Oct 11 18:31 gbk.HiFiMapped.bam.filtered.fasta

The gbk.HiFiMapped.bam.filtered.fasta file is the output from the command below.

https://github.com/marcelauliano/MitoHiFi/blob/852cd1d27c92534c90993d387354f5fb868b0f0e/mitohifi.py#L113-L114

And the gbk.HiFiMapped.bam.fasta as an input of the command exists as zero byte.

$ ls -hl gbk.HiFiMapped.bam.fasta
-rw-r--r--. 1 root root 0 Oct 11 18:31 gbk.HiFiMapped.bam.fasta
$ ls -hl reads.HiFiMapped.bam
-rw-r--r--. 1 root root 212 Oct 11 18:31 reads.HiFiMapped.bam

So, the log shows that the samtools fasta created from the non-zero byte reads.HiFiMapped.bam to zero byte gbk.HiFiMapped.bam.fasta.

samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta

And this is the mitohifi.py command line option -r.

https://github.com/marcelauliano/MitoHiFi/blob/852cd1d27c92534c90993d387354f5fb868b0f0e/mitohifi.py#L83

mutually_exclusive_group.add_argument("-r", help= "-r: Pacbio Hifi Reads from your species", metavar='<reads>.fasta')

My concern is the specified generated_asm.bp.r_utg.fa above is nuclear DNA, not mitocondria DNA. Can this cause the error above?

Running with the exampleFiles data

I tested the following example command mentioned in this repository's README.md. It succeeded. However, the command doesn't use the mitohifi.py's -r option. So, it doesn't be a clue for this issue.

$ docker run --rm -w / -v /home/jaruga/tmp/mitohifi/exampleFiles/:/exampleFiles/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  mitohifi.py -c exampleFiles/test.fa -f exampleFiles/NC_016067.1.fasta -g exampleFiles/NC_016067.1.gb -t 1 -o 5

And when using the `-r option with the example data, I saw the same error with the case that I executed with my data. Could you show me the 

$ docker run --rm -w / -v /home/jaruga/tmp/mitohifi/exampleFiles/:/exampleFiles/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  mitohifi.py -r exampleFiles/test.fa -f exampleFiles/NC_016067.1.fasta -g exampleFiles/NC_016067.1.gb -t 1 -o 5
2022-10-17 14:21:57 [INFO] Welcome to MitoHifi v2. Starting pipeline...
2022-10-17 14:21:57 [INFO] Length of related mitogenome is: 15659 bp
2022-10-17 14:21:57 [INFO] Number of genes on related mitogenome: 37
2022-10-17 14:21:57 [INFO] Running MitoHifi pipeline in reads mode...
2022-10-17 14:21:57 [INFO] 1. First we map your Pacbio HiFi reads to the close-related mitogenome
2022-10-17 14:21:57 [INFO] minimap2 -t 1 --secondary=no -ax map-pb exampleFiles/NC_016067.1.fasta exampleFiles/test.fa | samtools view -@ 1 -S -b -F4 -F 0x800 > reads.HiFiMapped.bam
2022-10-17 14:22:05 [INFO] 2. Now we filter out any mapped reads that are larger than the reference mitogenome to avoid NUMTS
2022-10-17 14:22:05 [INFO] 2.1 First we convert the mapped reads from BAM to FASTA format:
2022-10-17 14:22:05 [INFO] samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta
2022-10-17 14:22:06 [INFO] Total number of mapped reads: 3
2022-10-17 14:22:06 [INFO] 2.2 Then we filter reads that are larger than 15659 bp
2022-10-17 14:22:06 [INFO] Number of filtered reads: 0
2022-10-17 14:22:06 [INFO] 3. Now let's run hifiasm to assemble the mapped and filtered reads!
2022-10-17 14:22:06 [INFO] hifiasm --primary -t 1 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta
Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 139, in main
    f1 = open("gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa")
FileNotFoundError: [Errno 2] No such file or directory: 'gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa'

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 143, in main
    An error may have occurred when assembling reads with HiFiasm.""")
SystemExit: No gbk.HiFiMapped.bam.filtered.assembled.[a/p]_ctg.gfa file(s).
            An error may have occurred when assembling reads with HiFiasm.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 377, in <module>
    main()
  File "/bin/MitoHiFi/mitohifi.py", line 145, in main
    f1.close()
UnboundLocalError: local variable 'f1' referenced before assignment
$ ls -hl exampleFiles
total 39M
-rw-r--r--. 1 jaruga jaruga   1 Oct 11 17:17 .gitkeep
-rw-r--r--. 1 jaruga jaruga 15M Oct 11 17:17 ilDeiPorc1.reads.fa
-rw-r--r--. 1 jaruga jaruga 16K Oct 11 17:17 MW539688.1.fasta
-rw-r--r--. 1 jaruga jaruga 33K Oct 11 17:17 MW539688.1.gb
-rw-r--r--. 1 jaruga jaruga 16K Oct 11 17:17 NC_016067.1.fasta
-rw-r--r--. 1 jaruga jaruga 38K Oct 11 17:17 NC_016067.1.gb
-rw-r--r--. 1 jaruga jaruga 24M Oct 11 17:17 test.fa
junaruga commented 2 years ago

I checked the bam file above with the samtools on my local environment.

$ ls -hl reads.HiFiMapped.bam 
-rw-r--r--. 1 jaruga jaruga 212 Oct 11 18:31 reads.HiFiMapped.bam

$ samtools --version | head -1
samtools 1.13

$ samtools tview reads.HiFiMapped.bam 
[E::idx_find_and_load] Could not retrieve index file for 'reads.HiFiMapped.bam'
samtools tview: cannot read index for "reads.HiFiMapped.bam"

$ samtools idxstats reads.HiFiMapped.bam
[E::idx_find_and_load] Could not retrieve index file for 'reads.HiFiMapped.bam'
samtools idxstats: fail to load index for "reads.HiFiMapped.bam", reverting to slow method
ON980565.1  16574   0   0
*   0   0   0
junaruga commented 2 years ago

I prepared the reproducing script for you to check this issue on your local environment. If you have docker or other container tools, you can check it in your environment. The instruction is on the README.md.

https://github.com/junaruga/report-mitohifi-no-such-file

@marcelauliano Do you have any comments? I appreciate it.

ksenia-krasheninnikova commented 2 years ago

Hi @junaruga

Thanks for checking.

MitoHiFi was designed to look for organelles, in particular for mitochodrion.

-r is the flag used to perform assembly from the raw reads, while -c is used to look for a mitogenome among the input contigs. The name generated_asm.bp.r_utg.fa suggests that it's not reads, but the contigs. Did you try to run with -c flag?

With the example files the command line should be as following see here :

assemble from contigs: docker run --rm -w /data/ -v /local/path/to/exampleFiles/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 mitohifi.py -c /data/test.fa -f /data/NC_016067.1.fasta -g /data/NC_016067.1.gb -t 4 -o 2

assemble from raw reads: docker run --rm -w /data/ -v /local/path/to/exampleFiles/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 mitohifi.py -r /data/ilDeiPorc1.reads.fa -f /data/MW539688.1.fasta -g /data/MW539688.1.gb -t 4 -o 2

Hope this helps.

junaruga commented 2 years ago

@ksenia-krasheninnikova Thanks for your info and help.

I was not clear to run what kind of FASTA file, reads, or contigs file. I want to run with the reads file, but I was using the configs file. So, using this repository's example files, I was able to run the mitohifi.py -r <reads file> .... The result is below. The full log is here.

$ docker run --rm -w /data/ -v /home/jaruga/tmp/mitohifi/exampleFiles/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 mitohifi.py -r /data/ilDeiPorc1.reads.fa -f /data/MW539688.1.fasta -g /data/MW539688.1.gb -t 4 -o 2
...
2022-10-18 12:35:21 [INFO] Pipeline finished!
2022-10-18 12:35:21 [INFO] Run time: 4500.39 seconds

$ echo $?
0

$ ls -lh exampleFiles
total 39M
drwxr-xr-x. 1 root   root     72 Oct 18 14:35 contigs_circularization/
drwxr-xr-x. 1 root   root    130 Oct 18 14:35 contigs_filtering/
-rw-r--r--. 1 root   root    17K Oct 18 14:35 contigs_stats.tsv
drwxr-xr-x. 1 root   root    352 Oct 18 14:35 final_mitogenome.annotation/
-rw-r--r--. 1 root   root   1.4K Oct 18 14:35 final_mitogenome.annotation_MitoFinder.log
drwxr-xr-x. 1 root   root    168 Oct 18 14:35 final_mitogenome_choice/
-rw-r--r--. 1 root   root    15K Oct 18 14:35 final_mitogenome.fasta
-rw-r--r--. 1 root   root    30K Oct 18 14:35 final_mitogenome.gb
-rw-r--r--. 1 jaruga jaruga    1 Oct 11 17:17 .gitkeep
-rw-r--r--. 1 jaruga jaruga  15M Oct 11 17:17 ilDeiPorc1.reads.fa
-rw-r--r--. 1 jaruga jaruga  16K Oct 11 17:17 MW539688.1.fasta
-rw-r--r--. 1 jaruga jaruga  33K Oct 11 17:17 MW539688.1.gb
-rw-r--r--. 1 jaruga jaruga  16K Oct 11 17:17 NC_016067.1.fasta
-rw-r--r--. 1 jaruga jaruga  38K Oct 11 17:17 NC_016067.1.gb
drwxr-xr-x. 1 root   root   1.5K Oct 18 14:35 potential_contigs/
drwxr-xr-x. 1 root   root   1.9K Oct 18 14:35 reads_mapping_and_assembly/
-rw-r--r--. 1 jaruga jaruga  24M Oct 11 17:17 test.fa

$ ls -lh exampleFiles/final_mitogenome.fasta
-rw-r--r--. 1 root root 15K Oct 18 14:35 exampleFiles/final_mitogenome.fasta

$ ls -lh exampleFiles/final_mitogenome.gb
-rw-r--r--. 1 root root 30K Oct 18 14:35 exampleFiles/final_mitogenome.gb

$ ls -lh exampleFiles/contigs_stats.tsv
-rw-r--r--. 1 root root 17K Oct 18 14:35 exampleFiles/contigs_stats.tsv

Right now I only have the FASTA file created from the nuclear DNA. So, when using the reads file, I still go an error above with the message "Total number of mapped reads: 0". So, I will find a mitocondria DNA FASTA file. So, I would close this issue ticket. Thanks for your help.

$ ls -lh data
total 36M
-rw-r--r--. 1 jaruga jaruga 17K Oct 11 18:15 ON980565.1.fasta
-rw-r--r--. 1 jaruga jaruga 35K Oct 11 18:15 ON980565.1.gb
-rw-r--r--. 1 jaruga jaruga 36M Oct 18 12:22 test.hifi.fasta

$ docker run --rm -w /data -v /home/jaruga/tmp/mitohifi/data/:/data/ -t docker.io/biocontainers/mitohifi:2.2_cv1 \
  mitohifi.py -r /data/test.hifi.fasta -f /data/ON980565.1.fasta -g /data/ON980565.1.gb -t 4 -o 2
2022-10-18 11:11:14 [INFO] Welcome to MitoHifi v2. Starting pipeline...
2022-10-18 11:11:14 [INFO] Length of related mitogenome is: 16574 bp
2022-10-18 11:11:14 [INFO] Number of genes on related mitogenome: 37
2022-10-18 11:11:14 [INFO] Running MitoHifi pipeline in reads mode...
2022-10-18 11:11:14 [INFO] 1. First we map your Pacbio HiFi reads to the close-related mitogenome
2022-10-18 11:11:14 [INFO] minimap2 -t 4 --secondary=no -ax map-pb /data/ON980565.1.fasta /data/test.hifi.fasta | samtools view -@ 4 -S -b -F4 -F 0x800 > reads.HiFiMapped.bam
2022-10-18 11:11:14 [INFO] 2. Now we filter out any mapped reads that are larger than the reference mitogenome to avoid NUMTS
2022-10-18 11:11:14 [INFO] 2.1 First we convert the mapped reads from BAM to FASTA format:
2022-10-18 11:11:14 [INFO] samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta
2022-10-18 11:11:14 [INFO] Total number of mapped reads: 0
2022-10-18 11:11:14 [INFO] 2.2 Then we filter reads that are larger than 16574 bp
2022-10-18 11:11:14 [INFO] Number of filtered reads: 0
2022-10-18 11:11:14 [INFO] 3. Now let's run hifiasm to assemble the mapped and filtered reads!
2022-10-18 11:11:14 [INFO] hifiasm --primary -t 4 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta
Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 139, in main
    f1 = open("gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa")
FileNotFoundError: [Errno 2] No such file or directory: 'gbk.HiFiMapped.bam.filtered.assembled.p_ctg.gfa'

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 143, in main
    An error may have occurred when assembling reads with HiFiasm.""")
SystemExit: No gbk.HiFiMapped.bam.filtered.assembled.[a/p]_ctg.gfa file(s).
            An error may have occurred when assembling reads with HiFiasm.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/bin/MitoHiFi/mitohifi.py", line 377, in <module>
    main()
  File "/bin/MitoHiFi/mitohifi.py", line 145, in main
    f1.close()
UnboundLocalError: local variable 'f1' referenced before assignment