error when run “perl METABOLIC-C.pl -test true”

[green34567]

When I was running the test script METABOLIC_C.pl, the following error occurred. I checked that the dependencies such as bowtie2, samtools, and diamond were all installed. However, the MW score in the output file is blank.
I noticed that others have also encountered this problem, but it seems that it has not been resolved.

ls: 无法访问METABOLIC_out/intermediate_files/MEROPS_Files/*.MEROPSout.m8: 没有那个文件或目录
[2025-06-03 12:57:42] MEROPS peptidase searching is done
[2025-06-03 12:57:44] METABOLIC table has been generated
[2025-06-03 12:57:44] Drawing element cycling diagrams...
No output file specified!
Bowtie 2 version 2.4.5 by Ben Langmead (langmea@cs.jhu.edu, www.cs.jhu.edu/~langmea)
Usage: bowtie2-build [options]* <reference_in> <bt2_index_base>
    reference_in            comma-separated list of files with ref sequences
    bt2_index_base          write bt2 data to files with this dir/basename
*** Bowtie 2 indexes will work with Bowtie v1.2.3 and later. ***
Options:
    -f                      reference files are Fasta (default)
    -c                      reference sequences given on cmd line (as
                            <reference_in>)
    --large-index           force generated index to be 'large', even if ref
                            has fewer than 4 billion nucleotides
    --debug                 use the debug binary; slower, assertions enabled
    --sanitized             use sanitized binary; slower, uses ASan and/or UBSan
    --verbose               log the issued command
    -a/--noauto             disable automatic -p/--bmax/--dcv memory-fitting
    -p/--packed             use packed strings internally; slower, less memory
    --bmax <int>            max bucket sz for blockwise suffix-array builder
    --bmaxdivn <int>        max bucket sz as divisor of ref len (default: 4)
    --dcv <int>             diff-cover period for blockwise (default: 1024)
    --nodc                  disable diff-cover (algorithm becomes quadratic)
    -r/--noref              don't build .3/.4 index files
    -3/--justref            just build .3/.4 index files
    -o/--offrate <int>      SA is sampled every 2^<int> BWT chars (default: 5)
    -t/--ftabchars <int>    # of chars consumed in initial lookup (default: 10)
    --threads <int>         # of threads
    --seed <int>            seed for random number generator
    -q/--quiet              verbose output (for debugging)
    --h/--help              print this message and quit
    --version               print version information and quit
(ERR): "METABOLIC_out/All_gene_collections.gene.scaffold" does not exist or is not a Bowtie 2 index
Exiting now ...
rm: 无法删除"METABOLIC_out/All_gene_collections_mapped.1.sam": 没有那个文件或目录
rm: 无法删除"METABOLIC_out/*.bt2": 没有那个文件或目录
rm: 无法删除"METABOLIC_out/*.bam": 没有那个文件或目录
rm: 无法删除"METABOLIC_out/*.bai": 没有那个文件或目录
[2025-06-03 12:57:50] Drawing element cycling diagrams finished
[2025-06-03 12:57:50] Drawing metabolic handoff diagrams...
[2025-06-03 12:57:54] Drawing metabolic handoff diagrams finished
[2025-06-03 12:57:54] Drawing energy flow chart...
mv: 无法获取"METABOLIC_out/Output_energy_flow/Energy_plot/network.plot.pdf" 的文件状态(stat): 没有那个文件或目录
[2025-06-03 12:57:59] Drawing energy flow chart finished
[2025-06-03 12:57:59] Calculating MW-score ...
[2025-06-03 12:57:59] Calculating MW-score is done
METABOLIC-C was done, the total running time: 00:18:48 (hh:mm:ss)

[daerbiubiu]

Issue Summary: When running METABOLIC-C.pl (v4.0) with the -r option for community analysis, the pipeline failed during the coverage calculation step due to a bowtie2-build error ("No output file specified"). This caused downstream failures in generating metabolic handoff diagrams, energy flow charts, and MW-score calculations, with additional "uninitialized value $cat" errors related to taxonomy (-tax phylum).
Root Cause: The bowtie2-build command in the script used the --quiet flag, which suppressed error messages and caused the command to fail silently, likely due to environment-specific issues (e.g., Conda wrapper or file permissions). Additionally, missing or incomplete taxonomy data led to $cat errors.
Resolution:

Removed the --quiet flag from the bowtie2-build command in METABOLIC-C.pl, replacing it with --verbose or omitting it to ensure proper execution.
Verified that All_gene_collections.gene was valid and non-empty.
Confirmed read alignment (Bowtie2) and coverage calculation (CoverM) produced valid outputs.
Addressed taxonomy issues by either providing valid taxonomy data (e.g., GTDB-Tk output) or omitting the -tax option.

Suggestion: Consider removing or making the --quiet flag optional in bowtie2-build commands to improve error reporting. Additionally, add checks for taxonomy data availability to prevent $cat errors when using -tax.