# Running Metaphlan....Total species selected from prescreen: 0 **URL:** \ **Category:** HUMAnN\ **Created:** [August 14, 2021, 2:59pm UTC](https://forum.biobakery.org/t/running-metaphlan-total-species-selected-from-prescreen-0/2485 "2021-08-14T14:59:46Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![Ivy](https://avatars.discourse-cdn.com/v4/letter/i/bc8723/32.png) [@Ivy](https://forum.biobakery.org/u/Ivy)\ **Post date:** [August 14, 2021, 2:59pm UTC](https://forum.biobakery.org/t/running-metaphlan-total-species-selected-from-prescreen-0/2485/1 "2021-08-14T14:59:47Z")
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. ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [August 16, 2021, 8:29pm UTC](https://forum.biobakery.org/t/running-metaphlan-total-species-selected-from-prescreen-0/2485/2 "2021-08-16T20:29:59Z")
It might be that (unfortunately) we just don’t have marker genes for any species in that sample (because they are not well characterized). If true, I would recommend running HUMAnN in UniRef50 mode rather than UniRef90. Although HUMAnN will do all it’s work during translated search (since no known species were identified), you can use the `infer_taxonomy` script to attach approximate taxonomy to the resulting gene families.