Error when running HUMAnN

Microbial community profiling HUMAnN
1

Hi, I am keep getting an error when running humann v3.7

humann v3.7 was installed with conda conda install humann -c biobakery --solver=libmamba
Before installing the environment was created and channels were configured as per instructions

I downloaded the databases as follow

humann_databases --download chocophlan full /home/swijegun/humann/databases/ --update-config yes

humann_databases --download uniref uniref90_diamond /home/swijegun/humann/databases/ --update-config yes

Humann was run as

#!/bin/bash

for i in *_unmapped2HU_cat.fq.gz
    do
        humann --input $i --output /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/ --threads 48 --memory-use maximum 
        done

I have added a log file for one samples

10/02/2023 02:28:39 AM - humann.humann - INFO: Running humann v3.7
10/02/2023 02:28:39 AM - humann.humann - INFO: Output files will be written to: /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out
10/02/2023 02:28:39 AM - humann.humann - INFO: Writing temp files to directory: /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp
10/02/2023 02:28:39 AM - humann.utilities - INFO: File ( /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/10_S237_unmapped2HU_cat.fq.gz ) is of format:  fastq.gz
10/02/2023 02:28:39 AM - humann.utilities - INFO: Decompressing gzipped file ...
10/02/2023 02:28:39 AM - humann.utilities - DEBUG: Check software, metaphlan, for required version, 3.0
10/02/2023 02:28:57 AM - humann.utilities - INFO: Using metaphlan version 4.0
10/02/2023 02:28:57 AM - humann.utilities - DEBUG: Check software, bowtie2, for required version, 2.2
10/02/2023 02:28:58 AM - humann.utilities - INFO: Using bowtie2 version 2.5
10/02/2023 02:28:58 AM - humann.humann - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected
10/02/2023 02:28:58 AM - humann.utilities - DEBUG: Check software, diamond, for required version, 2.0.15
10/02/2023 02:28:58 AM - humann.utilities - INFO: Using diamond version 2.1.8
10/02/2023 02:28:58 AM - humann.config - INFO: 
Run config settings: 

DATABASE SETTINGS
nucleotide database folder = /home/swijegun/humann/databases/chocophlan
protein database folder = /home/swijegun/humann/databases/uniref
pathways database file 1 = /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered_v24
utility mapping database folder = /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/misc

RUN MODES
resume = False
verbose = False
bypass prescreen = False
bypass nucleotide index = False
bypass nucleotide search = False
bypass translated search = False
translated search = diamond
threads = 48

SEARCH MODE
search mode = uniref90
nucleotide identity threshold = 0.0
translated identity threshold = 80.0

ALIGNMENT SETTINGS
bowtie2 options = --very-sensitive
diamond options = --top 1 --outfmt 6
evalue threshold = 1.0
prescreen threshold = 0.01
translated subject coverage threshold = 50.0
translated query coverage threshold = 90.0
nucleotide subject coverage threshold = 50.0
nucleotide query coverage threshold = 90.0

PATHWAYS SETTINGS
minpath = on
xipe = off
gap fill = on

INPUT AND OUTPUT FORMATS
input file format = fastq.gz
output file format = tsv
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG

10/02/2023 02:28:58 AM - humann.store - DEBUG: Initialize Alignments class instance to maximize memory use
10/02/2023 02:28:58 AM - humann.store - DEBUG: Initialize Reads class instance to maximize memory use
10/02/2023 02:29:18 AM - humann.humann - INFO: Load pathways database part 1: /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
10/02/2023 02:29:18 AM - humann.humann - INFO: Load pathways database part 2: /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered_v24
10/02/2023 02:29:18 AM - humann.search.prescreen - INFO: Running metaphlan ........
10/02/2023 02:29:18 AM - humann.utilities - DEBUG: Using software: /home/swijegun/miniconda3/envs/biobakery3/bin/metaphlan
10/02/2023 02:29:18 AM - humann.utilities - INFO: Execute command: /home/swijegun/miniconda3/envs/biobakery3/bin/metaphlan /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/tmpve747l53/tmp3rwe6rot -t rel_ab -o /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bowtie2.txt --nproc 48
10/02/2023 02:29:40 AM - humann.utilities - CRITICAL: Error executing: /home/swijegun/miniconda3/envs/biobakery3/bin/metaphlan /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/tmpve747l53/tmp3rwe6rot -t rel_ab -o /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bowtie2.txt --nproc 48

Error message returned from metaphlan :

Downloading MetaPhlAn database
Please note due to the size this might take a few minutes

\Downloading and uncompressing indexes

File /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/metaphlan/metaphlan_databases/mpa_vOct22_CHOCOPhlAnSGB_202212_bt2.tar already present!

File /home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/metaphlan/metaphlan_databases/mpa_vOct22_CHOCOPhlAnSGB_202212_bt2.md5 already present!
MD5 checksums do not correspond! If this happens again, you should remove the database files and rerun MetaPhlAn so they are re-downloaded

10/02/2023 02:29:40 AM - humann.utilities - CRITICAL: TRACEBACK: 
Traceback (most recent call last):
  File "/home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/site-packages/humann/utilities.py", line 761, in execute_command
    p_out = subprocess.check_output(cmd, stderr=subprocess.STDOUT)
  File "/home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/subprocess.py", line 411, in check_output
    **kwargs).stdout
  File "/home/swijegun/miniconda3/envs/biobakery3/lib/python3.7/subprocess.py", line 512, in run
    output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['/home/swijegun/miniconda3/envs/biobakery3/bin/metaphlan', '/home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/tmpve747l53/tmp3rwe6rot', '-t', 'rel_ab', '-o', '/home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bugs_list.tsv', '--input_type', 'fastq', '--bowtie2out', '/home/swijegun/test/clean-reads-fastp/analysis/unmapped2HU_fastq/cat_all_unmapped/humann_out/10_S237_unmapped2HU_cat_humann_temp/10_S237_unmapped2HU_cat_metaphlan_bowtie2.txt', '--nproc', '48']' returned non-zero exit status 1.
2

It looks like something may have gone wrong when downloading / indexing your MetaPhlAn databases. Per the instructions in the log, I would recommend removing the MetaPhlAn databases and then doing a MetaPhlAn demo run to download and build its index outside of this workflow. Then you could try executing the workflow again and see if you can get past this error.

3

Dear users,

I had the same error [MD5 checksums do not correspond!] but I think I made it work.
First, I took (transferred) humann databases from another machine and relinked them to my installation with humann_config.

humann_config --update database_folders utility_mapping databases/utility_mapping/                                  
humann_config --update database_folders nucleotide databases/chocophlan
humann_config --update database_folders protein databases/uniref

I manually downloaded the MPA databases and I passed metaphlan options and strictly pointed to a that folder.

humann --input demo.fastq --output OUT --threads 6 --metaphlan-options "--bowtie2db /home/andrespara/HUMANN/nowmetaphlan"

BTW, I have humann v3.8 + MetaPhlAn version 4.0.6 (1 Mar 2023), both installed through mamba.

Cheers

Edited to add: I removed the database from this folder ~/mambaforge/envs/humano/lib/python3.10/site-packages/metaphlan/metaphlan_databases/ as @sachiwije mentioned below

A

4

Thanks All,

I removed the databases at

/home/user/miniconda3/envs/biobakery3/lib/python3.7/site-packages/metaphlan/metaphlan_databases/

And added the OCT22 database downloaded from the googledrive and it worked
https://drive.google.com/drive/u/1/folders/1EeM0evDiXDImwgWYsoiZzBQltK7ggk6C

1 Like
5

Hi Andres,
Could you please specify what versions of humann and metaphlan databases are you using? I keep getting different errors and I just want to try one setting that works.
Thanks,
Erick

6

Hi Erick,

Welcome! Here are the version I have:

Chocoplan == v201901_v31, uniref == uniref90_201901b_full.dmnd AND MetaPhlAn (in a different folder) == vOct22_CHOCOPhlAnSGB_202212

Cheers

Andrés