I am trying to run MetaPhlAn on the demo dataset, but it keeps giving me the error that --bowtie2out is an unrecognized argument. When I ran MetaPhlAn the first time and it downloaded the MetaPhlAn database, it also gave me the warning that “[Warning] Downloaded indexes are not compatible with the installed version of Bowtie2”. I have HUMAnN downloaded in its own conda environment, so there shouldn’t be anything interfering with it. I installed it from bioconda.
I am using MetaPhlAn version 4.2.2 (4 Jun 2025) and Bowtie2 version 2.2.3.
The only arguments I specified when running MetaPhlAn were the input type and the demo fastq file.
metaphlan --input_type fastq /scratch/trytten.e/demo_humann/examples/demo.fastq
I tried updating Bowtie2 but 2.2.3 was the newest version that I could download. Is there a different version of Bowtie2 that I should be using to fix this?
Here is the full error message it gave me in the log file:
06/13/2025 04:00:47 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
06/13/2025 04:00:47 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
06/13/2025 04:01:06 PM - humann.humann - INFO: Load pathways database part 1: /home/trytten.e/.local/lib/python3.12/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
06/13/2025 04:01:06 PM - humann.humann - INFO: Load pathways database part 2: /home/trytten.e/.local/lib/python3.12/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered_v24_subreactions
06/13/2025 04:01:06 PM - humann.search.prescreen - INFO: Running metaphlan ........
06/13/2025 04:01:06 PM - humann.utilities - DEBUG: Using software: /projects/vollmer/software/.conda/envs/humann/bin/metaphlan
06/13/2025 04:01:06 PM - humann.utilities - INFO: Execute command: /projects/vollmer/software/.conda/envs/humann/bin/metaphlan /scratch/trytten.e/demo_humann/examples/demo.fastq -t rel_ab -o /scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bowtie2.txt
06/13/2025 04:01:08 PM - humann.utilities - CRITICAL: Error executing: /projects/vollmer/software/.conda/envs/humann/bin/metaphlan /scratch/trytten.e/demo_humann/examples/demo.fastq -t rel_ab -o /scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bowtie2.txt
Error message returned from metaphlan :
usage: metaphlan --input_type {fastq,fasta,mapout,sam} [--force]
[--db_dir METAPHLAN_DB] [-x INDEX] [--mapout FILE_NAME]
[--min_mapq_val MIN_MAPQ_VAL] [--no_map] [--tmp_dir]
[--bt2_ps BowTie2 presets] [--bowtie2_exe BOWTIE2_EXE]
[--bowtie2_build BOWTIE2_BUILD] [--tax_lev TAXONOMIC_LEVEL]
[--min_alignment_len] [--ignore_eukaryotes]
[--ignore_bacteria] [--ignore_archaea] [--ignore_ksgbs]
[--ignore_usgbs] [--stat_q] [--perc_nonzero]
[--ignore_markers IGNORE_MARKERS] [--avoid_disqm] [--stat]
[-t ANALYSIS TYPE] [--nreads NUMBER_OF_READS]
[--pres_th PRESENCE_THRESHOLD] [-o output file]
[--sample_id_key name] [--use_group_representative]
[--sample_id value] [-s sam_output_file]
[--CAMI_format_output] [--skip_unclassified_estimation]
[--biom_format_output] [--biom_mdelim mdelim] [--profile_vsc]
[--vsc_out VSC_OUT] [--vsc_breadth VSC_BREADTH]
[--long_reads] [--max_gcsd MAX_GCSD]
[--minimap2_exe MINIMAP2_EXE]
[--minimap2_ps minimap2 presets] [--nbases NUMBER_OF_BASES]
[--split_reads] [--split_readlen SPLIT_READLEN] [--nproc N]
[--subsampling SUBSAMPLING] [--mapping_subsampling]
[--subsampling_seed SUBSAMPLING_SEED]
[--subsampling_output SUBSAMPLING_OUTPUT]
[--subsampling_paired SUBSAMPLING_PAIRED] [-1 FORWARD_READS]
[-2 REVERSE_READS] [--install] [--offline] [--force_download]
[--read_min_len READ_MIN_LEN] [--verbose] [-v] [-h]
[INPUT_FILE]
metaphlan: error: unrecognized arguments: --bowtie2out /scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bowtie2.txt
06/13/2025 04:01:08 PM - humann.utilities - CRITICAL: TRACEBACK:
Traceback (most recent call last):
File "/home/trytten.e/.local/lib/python3.12/site-packages/humann/utilities.py", line 761, in execute_command
p_out = subprocess.check_output(cmd, stderr=subprocess.STDOUT)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/projects/vollmer/software/.conda/envs/humann/lib/python3.12/subprocess.py", line 466, in check_output
return run(*popenargs, stdout=PIPE, timeout=timeout, check=True,
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/projects/vollmer/software/.conda/envs/humann/lib/python3.12/subprocess.py", line 571, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['/projects/vollmer/software/.conda/envs/humann/bin/metaphlan', '/scratch/trytten.e/demo_humann/examples/demo.fastq', '-t', 'rel_ab', '-o', '/scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bugs_list.tsv', '--input_type', 'fastq', '--bowtie2out', '/scratch/trytten.e/demo_humann/demo_humann_temp/demo_metaphlan_bowtie2.txt']' returned non-zero exit status 2.