All unclassified with Metaphlan4

I’m using MetaPhlAn version 4.0.6 (1 Mar 2023), installed according to the manual using conda. I have installed the database using:

metaphlan --install --bowtie2db ${db}

And repeating the command confirms it is installed. I then try to profile a trimmed, human filtered Illumina dataset (a mock sample containing human and bacterial/viral DNA) using this command:

metaphlan input.fastq \
--bowtie2db ${db} \
--bowtie2out input.bowtie2.bz2 \
--input_type fastq  \
--output_file output.txt 

However, the resulting output.txt file says everything is unclassified:

#236482 reads processed
#SampleID       Metaphlan_Analysis
#clade_name     NCBI_tax_id     relative_abundance      additional_species
UNCLASSIFIED    -1      100.0 

The only error message was:

WARNING: MetaPhlAn did not detect any microbial taxa in the sample.

I have analysed the same input file using other classifiers, e.g. Kraken2, and I see the organisms I am expecting. Could you let me know what I might be doing wrong here? Thanks!

Hi @sarahb
It seems there is not enough depth of coverage of your bacterial DNA to be able to be detected by metaphlan. Kraken2 is a more sensitive (but less precise) method and this might be the reason you are able to profile your sample with that tool

OK, thanks for your answer!