I have Illumina metagenomic shotgun sequence data in fastq format (quality controlled paired-end R1 and R2 files were converted to a single file running cat). Metagenomic DNA was isolated from a lepidopteran insect’s gut.
Now, when I ran $ humann --input myinput.fastq --output humann3_output, it showed No species were selected from the prescreen.
Because of this the custom ChocoPhlAn database is empty.
This will result in zero species-specific gene families and pathways.
Below is the log.
Is there any problem with my input data? Pls guide me.
08/14/2021 06:53:09 PM - humann.humann - INFO: Running humann v3.0.0
08/14/2021 06:53:09 PM - humann.humann - INFO: Output files will be written to: /home/researchscholar/Analysis_new/Shotgun/humann3/humann3_output
08/14/2021 06:53:09 PM - humann.humann - INFO: Writing temp files to directory: /home/researchscholar/Analysis_new/Shotgun/humann3/humann3_output/trimmed_SLG_humann_temp
08/14/2021 06:53:09 PM - humann.utilities - INFO: File ( /home/researchscholar/Analysis_new/Shotgun/humann3/trimmed_SLG.fastq ) is of format: fastq
08/14/2021 06:53:09 PM - humann.humann - INFO: Removing spaces from identifiers in input file
08/14/2021 06:55:47 PM - humann.utilities - DEBUG: Check software, metaphlan, for required version, 3.0
08/14/2021 06:55:56 PM - humann.utilities - INFO: Using metaphlan version 3.0
08/14/2021 06:55:56 PM - humann.utilities - DEBUG: Check software, bowtie2, for required version, 2.2
08/14/2021 06:55:56 PM - humann.utilities - INFO: Using bowtie2 version 2.4
08/14/2021 06:55:56 PM - humann.humann - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected
08/14/2021 06:55:56 PM - humann.utilities - DEBUG: Check software, diamond, for required version, 0.9.36
08/14/2021 06:55:56 PM - humann.utilities - INFO: Using diamond version 2.0.8
08/14/2021 06:55:56 PM - humann.config - INFO:
Run config settings:
DATABASE SETTINGS
nucleotide database folder = /home/researchscholar/Analysis_new/Shotgun/humann3/chocophlan/
protein database folder = /home/researchscholar/Analysis_new/Shotgun/humann3/uniref/
pathways database file 1 = /home/researchscholar/anaconda3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /home/researchscholar/anaconda3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered
utility mapping database folder = /home/researchscholar/Analysis_new/Shotgun/humann3/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
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
output file format = tsv
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG
08/14/2021 06:55:56 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
08/14/2021 06:55:56 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
08/14/2021 06:56:12 PM - humann.humann - INFO: Load pathways database part 1: /home/researchscholar/anaconda3/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
08/14/2021 06:56:13 PM - humann.humann - INFO: Load pathways database part 2: /home/researchscholar/anaconda3/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered
08/14/2021 06:56:13 PM - humann.search.prescreen - INFO: Running metaphlan …
08/14/2021 06:56:13 PM - humann.utilities - DEBUG: Using software: /home/researchscholar/anaconda3/bin/metaphlan
08/14/2021 06:56:13 PM - humann.utilities - INFO: Execute command: /home/researchscholar/anaconda3/bin/metaphlan /home/researchscholar/Analysis_new/Shotgun/humann3/humann3_output/trimmed_SLG_humann_temp/tmp12mq9uc3/tmp1wb29re0 -t rel_ab -o /home/researchscholar/Analysis_new/Shotgun/humann3/humann3_output/trimmed_SLG_humann_temp/trimmed_SLG_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /home/researchscholar/Analysis_new/Shotgun/humann3/humann3_output/trimmed_SLG_humann_temp/trimmed_SLG_metaphlan_bowtie2.txt
08/14/2021 07:07:46 PM - humann.utilities - DEBUG: b’’
08/14/2021 07:07:46 PM - humann.humann - INFO: TIMESTAMP: Completed prescreen : 693 seconds
08/14/2021 07:07:46 PM - humann.search.prescreen - INFO: Total species selected from prescreen: 0
08/14/2021 07:07:46 PM - humann.search.prescreen - DEBUG:
No species were selected from the prescreen.
Because of this the custom ChocoPhlAn database is empty.
This will result in zero species-specific gene families and pathways.
08/14/2021 07:07:46 PM - humann.humann - INFO: TIMESTAMP: Completed custom database creation : 0 seconds
08/14/2021 07:07:46 PM - humann.humann - DEBUG: Custom database is empty
08/14/2021 07:07:46 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
Any help is much appreciated.
Thank you.