Hello everyone,
I am trying to use the new Humann4 using the demo.fastq from your tutorial (HUMAnN 4.0.0.alpha.1 Release Notes - Google Docs), but at the end of the process I only get all reads as unmapped and I guess why.
I have humann v4.0.0.alpha.1 and MetaPhlAn version 4.0.6 (1 Mar 2023).
I have produced a sam file using bowtie2 using the following command:
bowtie2 --sam-no-hd --sam-no-sq --no-unal --very-sensitive -S metagenome.sam -x Metaphlan4/mpa_vOct22_CHOCOPhlAnSGB_202403 -U demo.fastq
Then Humann as following:
humann -i metagenome.sam --input-format sam -o metagenome --prescreen-threshold 0.01 --translated-subject-coverage-threshold 40
and this is a copy of my log file (hoping it could help to solve the error):
11/29/2024 10:37:55 AM - humann.humann - INFO: Running humann v4.0.0.alpha.1
11/29/2024 10:37:55 AM - humann.humann - INFO: Output files will be written to: /mnt/nfs/home/minuzzi/Database/metagenome
11/29/2024 10:37:55 AM - humann.humann - INFO: Writing temp files to directory: /mnt/nfs/home/minuzzi/Database/metagenome/metagenome_humann_temp
11/29/2024 10:37:55 AM - humann.config - INFO:
Run config settings:
DATABASE SETTINGS
nucleotide database folder = /mnt/nfs/home/minuzzi/Database/Humann4/chocophlan
protein database folder = /mnt/nfs/home/minuzzi/Database/Humann4/uniref
pathways database file 1 = /mnt/nfs/home/minuzzi/Database/Humann4/utility_mapping/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /mnt/nfs/home/minuzzi/Database/Humann4/utility_mapping/metacyc_pathways_structured_filtered_v24_subreactions
utility mapping database folder = /mnt/nfs/home/minuzzi/Database/Humann4/utility_mapping
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 = 1
Identity thresholds
nucleotide = 0.0
translated = 50.0
ALIGNMENT SETTINGS
bowtie2 options = --very-sensitive --no-hd --no-sq
diamond options = --top 1 --sensitive --outfmt 6
evalue threshold = 1.0
prescreen threshold = 0.01
average read length = 150.0
translated subject coverage threshold = 40.0
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 = sam
output file format = tsv
normalization mode = Adjusted CPMs
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG
11/29/2024 10:37:55 AM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
11/29/2024 10:37:55 AM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
11/29/2024 10:39:04 AM - humann.humann - INFO: Load pathways database part 1: /mnt/nfs/home/minuzzi/Database/Humann4/utility_mapping/metacyc_reactions_level4ec_only.uniref.bz2
11/29/2024 10:39:04 AM - humann.humann - INFO: Load pathways database part 2: /mnt/nfs/home/minuzzi/Database/Humann4/utility_mapping/metacyc_pathways_structured_filtered_v24_subreactions
11/29/2024 10:39:04 AM - humann.humann - INFO: Process the sam mapping results ...
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments where percent identity is not a number: 0
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments where alignment length is not a number: 0
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments where E-value is not a number: 0
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments not included based on large e-value: 0
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments not included based on small percent identity: 0
11/29/2024 10:39:04 AM - humann.utilities - DEBUG: Total alignments not included based on small query coverage: 0
11/29/2024 10:39:04 AM - humann.search.blastx_coverage - INFO: Total alignments without coverage information: 0
11/29/2024 10:39:04 AM - humann.search.blastx_coverage - INFO: Total proteins in blastx output: 1184
11/29/2024 10:39:04 AM - humann.search.blastx_coverage - INFO: Total proteins without lengths: 1184
11/29/2024 10:39:04 AM - humann.search.blastx_coverage - INFO: Proteins with coverage greater than threshold (50.0): 0
11/29/2024 10:39:05 AM - humann.search.nucleotide - DEBUG: Total nucleotide alignments not included based on filtered genes: 16783
11/29/2024 10:39:05 AM - humann.search.nucleotide - DEBUG: Total nucleotide alignments not included based on small percent identities: 0
11/29/2024 10:39:05 AM - humann.search.nucleotide - DEBUG: Total nucleotide alignments not included based on query coverage threshold: 0
11/29/2024 10:39:05 AM - humann.search.nucleotide - DEBUG: Keeping sam file
11/29/2024 10:39:05 AM - humann.humann - INFO: TIMESTAMP: Completed alignment post-processing : 1 seconds
11/29/2024 10:39:05 AM - humann.humann - INFO: Computing gene families ...
11/29/2024 10:39:05 AM - humann.quantify.families - DEBUG: Compute gene families
11/29/2024 10:39:05 AM - humann.store - INFO:
Total gene families : 0
11/29/2024 10:39:05 AM - humann.humann - INFO: TIMESTAMP: Completed computing gene families : 0 seconds
11/29/2024 10:39:05 AM - humann.humann - INFO: Computing reaction and pathway abundance ...
11/29/2024 10:39:05 AM - humann.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath
11/29/2024 10:39:05 AM - humann.quantify.modules - INFO: Compute reaction scores for bug: all
11/29/2024 10:39:07 AM - humann.quantify.modules - DEBUG: Print pathways
11/29/2024 10:39:07 AM - humann.quantify.modules - DEBUG: Print pathways _Abundance
11/29/2024 10:39:07 AM - humann.humann - INFO: TIMESTAMP: Completed computing pathways : 3 seconds
11/29/2024 10:39:07 AM - humann.humann - INFO:
Output files created:
/mnt/nfs/home/minuzzi/Database/metagenome/metagenome_2_genefamilies.tsv
/mnt/nfs/home/minuzzi/Database/metagenome/metagenome_3_reactions.tsv
/mnt/nfs/home/minuzzi/Database/metagenome/metagenome_4_pathabundance.tsv
/mnt/nfs/home/minuzzi/Database/metagenome/metagenome_0.log
11/29/2024 10:39:07 AM - humann.utilities - DEBUG: Remove directory: /mnt/nfs/home/minuzzi/Database/metagenome/metagenome_humann_temp/tmp1j69ch6h
Any suggestions? what am I doing wrong?
thanks