FASTA to GFA

[juanjo255]

Hello developers!

Thanks for this great work.

Due to how Strainy works, I find attractive to try to use it for haplotype assembly of human mitochondrial genome, do you think it might work?

Anyways, when I tried to use it I am facing problems when converting from fasta to gfa it always fail with this:

[2024-10-17 19:18:46] [Root] INFO:  Checking which sequences need to be phased
[2024-10-17 19:18:46] [Root] INFO:  0/0 unitigs will NOT be phased.
Using processor(s):  Intel(R) Xeon(R) CPU E5-2683 v4 @ 2.10GHz
[2024-10-17 19:18:46] [Root] INFO:  Starting phasing
[2024-10-17 19:18:46] [Root] INFO:  CMD: --unitig-split-length 0 -t 32 --min-unitig-coverage 20 --min-unitig-length 0.05 -m nano --gfa /home/jjpiconc/COL-HUMAN-PROJECT/chrMT.gfa --stage phase --fastq /home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/reads_MT_20240815.fastq --bam /home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/aln_human_CHM13_20240815.onlyMT.sorted.bam --snp /home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/VariantCall/gatk_mutect2/mitochondria_20240815.filtered.vcf.gz -o /home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasin --debug
[2024-10-17 19:18:46] [Root] INFO:  Total number of key hits and misses for consensus computation:
[2024-10-17 19:18:46] [Root] INFO:   H:0, M:0
[2024-10-17 19:18:46] [Root] INFO:  Position hit/miss
[2024-10-17 19:18:46] [Root] INFO:   H:0, M:0
[2024-10-17 19:18:46] [Root] INFO:  Alignment cache hit/miss
[2024-10-17 19:18:46] [Root] INFO:   H:0, M:0
[2024-10-17 19:18:46] [Root] INFO:  Creating phased bam
[E::hts_open_format] Failed to open file "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasin/intermediate/bam/coloredSAM.sam" : No such file or directory

I tried to add a fasta and I tried converting using minigraph chrMT.fna MT/MT_20240815/reads_MT_20240815.fastq > chrMT.gfa, but when I check for the preprocessing_data folder, it always shows an empty gfa_converted.fasta. Maybe this is the cause of the program failing? or it just cannot work in this context?

I am using my own VCF file and BAM file.

Here the command:

strainy.py         --unitig-split-length 0 -t 32 --min-unitig-coverage 20 --min-unitig-length 0.05 -m nano         --gfa ~/COL-HUMAN-PROJECT/chrMT.gfa --stage phase         --fastq ~/COL-HUMAN-PROJECT/MT/MT_20240815/reads_MT_20240815.fastq         --bam ~/COL-HUMAN-PROJECT/MT/MT_20240815/aln_human_CHM13_20240815.onlyMT.sorted.bam         --snp ~/COL-HUMAN-PROJECT/MT/MT_20240815/VariantCall/gatk_mutect2/mitochondria_20240815.filtered.vcf.gz        -o ~/COL-HUMAN-PROJECT/MT/MT_20240815/phasin --debug

Thank you very much for the help,

Juan

[katerinakazantseva]

Hi Juan!

Thanks for your interest in Strainy! It looks like the problem is with the gfa file, but we just added support for input fasta, please try doing a pull and running Strainy with the --fasta_ref parameter (gfa is not needed).

Katya

[juanjo255]

Hello @katerinakazantseva,

Thanks for the reply!

It worked! unfortunately, I'm facing another problem, I think that maybe it has to do with this issue #75. I tried downgrading to 3.10, but it failed using 10 threads.

The error:

ERROR:  Worker thread exception! Cannot save file into a non-existent directory: '/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy_out/intermediate/adj_M'
Traceback (most recent call last):
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy/phase.py", line 30, in _thread_fun
    cluster(i, shared_flye_consensus)
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy/clustering/cluster.py", line 106, in cluster
    m.to_csv("%s/adj_M/adj_M_%s_%s_%s.csv" % (StRainyArgs().output_intermediate, edge, I, StRainyArgs().AF))
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/util/_decorators.py", line 333, in wrapper
    return func(*args, **kwargs)
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/core/generic.py", line 3967, in to_csv
    return DataFrameRenderer(formatter).to_csv(
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/io/formats/format.py", line 1014, in to_csv
    csv_formatter.save()
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/io/formats/csvs.py", line 251, in save
    with get_handle(
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/io/common.py", line 749, in get_handle
    check_parent_directory(str(handle))
  File "/home/jjpiconc/.conda/envs/strainy/lib/python3.10/site-packages/pandas/io/common.py", line 616, in check_parent_directory
    raise OSError(rf"Cannot save file into a non-existent directory: '{parent}'")
OSError: Cannot save file into a non-existent directory: '/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy_out/intermediate/adj_M'

Traceback (most recent call last):
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy.py", line 32, in <module>
    main()
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy.py", line 26, in main
    sys.exit(strainy.main.main())
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy/main.py", line 127, in main
    sys.exit(phase_main(args))
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy/phase.py", line 134, in phase_main
    consensus_dict = phase(StRainyArgs().edges_to_phase, args)
  File "/home/jjpiconc/COL-HUMAN-PROJECT/MT/MT_20240815/phasing/strainy/strainy/phase.py", line 57, in phase
    raise Exception("Error in worker thread, exiting")
Exception: Error in worker thread, exiting

Juan

[juanjo255]

Update: Actually, maybe it is not a threads problem, when I used -t 1 It failed too. It was that it was not been able to create the folder intermediate/adj_M, so I did it manually and it worked, same happened latter with intermediate/graphs/linear_phase_NC_012920.1.png. Probably a permission issue (?)

[katerinakazantseva]

Hi Juan, can you please send command you use to run Strainy and log file ({output_dir}/log_phase/phase_root.log)?

Thank you, Katya