# Metaphlan3 output issues

**URL:** <https://forum.biobakery.org/t/metaphlan3-output-issues/444>\
**Category:** MetaPhlAn\
**Created:** [May 8, 2020, 2:05pm UTC](https://forum.biobakery.org/t/metaphlan3-output-issues/444 "2020-05-08T14:05:47Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![brucesteel](https://avatars.discourse-cdn.com/v4/letter/b/3bc359/32.png) [@brucesteel](https://forum.biobakery.org/u/brucesteel)\
**Post date:** [May 8, 2020, 2:05pm UTC](https://forum.biobakery.org/t/metaphlan3-output-issues/444/1 "2020-05-08T14:05:47Z")

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I have installed metaphlan3 according to the instructions on the website ([https://github.com/biobakery/MetaPhlAn/wiki/MetaPhlAn-3.0](https://github.com/biobakery/MetaPhlAn/wiki/MetaPhlAn-3.0)) using Miniconda2 in the bioconda channel, and have created a separate environment for metaphlan3. I am using MetaPhlAn on trimmed paired and unpaired reads created by trimmomatic and Metaphlan seems to run with no problems with the following command:

metaphlan MG2\_trimmomatic\_R1\_paired.fastq.gz,MG2\_trimmomatic\_R1\_unpaired.fastq.gz,MG2\_trimmomatic\_R2\_paired.fastq.gz,MG2\_trimmomatic\_R2\_unpaired.fastq.gz --bowtie2out MG2.bowtie2.bz2 --input\_type fastq \> MG2\_profiled\_metagenome.txt

However, when I view the MG2\_profiled\_metagenome.txt file generated by MetaPhlAn, only two species of microorganism have been identified. Furthermore, when I assemble these reads using Megahit and look at the MetaQUAST assembly report, it appears that there are more than 2 species of microorganism within the metagenome, therefore suggesting that the MetaPhlAn profiled metagenome file is incorrect.

I was wondering if there are any problems that can cause this in MetaPhlAn3?

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**Author:** ![brucesteel](https://avatars.discourse-cdn.com/v4/letter/b/3bc359/32.png) [@brucesteel](https://forum.biobakery.org/u/brucesteel)\
**Post date:** [May 8, 2020, 2:19pm UTC](https://forum.biobakery.org/t/metaphlan3-output-issues/444/2 "2020-05-08T14:19:59Z")

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Sorry, I should have shown the output .txt file:

#mpa\_v30\_CHOCOPhlAn\_201901  
#/home/brucesteel/miniconda2/envs/mpa/bin/metaphlan MB2\_trimmomatic\_R1\_paired.fastq.gz,MB2\_trimmomatic\_R1\_unpaired.fastq.gz,MB2\_trimmomatic\_R2\_paired.fastq.gz,MB2\_trimmomatic\_R2\_unpaired.fastq.gz --bowtie2out MB2.bowtie2.bz2 --input\_type fastq  
#SampleID Metaphlan\_Analysis  
#clade\_name NCBI\_tax\_id relative\_abundance additional\_species  
k\_\_Bacteria 2 100.0   
k\_\_Bacteria|p\_\_Firmicutes 2|1239 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria 2|1224 13.42752   
k\_\_Bacteria|p\_\_Firmicutes|c\_\_Bacilli 2|1239|91061 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria|c\_\_Deltaproteobacteria 2|1224|28221 13.42752   
k\_\_Bacteria|p\_\_Firmicutes|c\_\_Bacilli|o\_\_Lactobacillales 2|1239|91061|186826 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria|c\_\_Deltaproteobacteria|o\_\_Desulfobacterales 2|1224|28221|213118 13.42752   
k\_\_Bacteria|p\_\_Firmicutes|c\_\_Bacilli|o\_\_Lactobacillales|f\_\_Streptococcaceae 2|1239|91061|186826|1300 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria|c\_\_Deltaproteobacteria|o\_\_Desulfobacterales|f\_\_Desulfobacteraceae 2|1224|28221|213118|213119 13.42752   
k\_\_Bacteria|p\_\_Firmicutes|c\_\_Bacilli|o\_\_Lactobacillales|f\_\_Streptococcaceae|g\_\_Lactococcus 2|1239|91061|186826|1300|1357 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria|c\_\_Deltaproteobacteria|o\_\_Desulfobacterales|f\_\_Desulfobacteraceae|g\_\_Desulfobacter 2|1224|28221|213118|213119|2289 13.42752   
k\_\_Bacteria|p\_\_Firmicutes|c\_\_Bacilli|o\_\_Lactobacillales|f\_\_Streptococcaceae|g\_\_Lactococcus|s\_\_Lactococcus\_chungangensis 2|1239|91061|186826|1300|1357|451457 86.57248   
k\_\_Bacteria|p\_\_Proteobacteria|c\_\_Deltaproteobacteria|o\_\_Desulfobacterales|f\_\_Desulfobacteraceae|g\_\_Desulfobacter|s\_\_Desulfobacter\_hydrogenophilus 2|1224|28221|213118|213119|2289|2291 13.42752

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<div class="post-metadata">

**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\
**Post date:** [May 25, 2020, 11:12am UTC](https://forum.biobakery.org/t/metaphlan3-output-issues/444/3 "2020-05-25T11:12:05Z")

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It is possible that the third species is present in the metagenome in low abundance and/or there are not present markers for identify the species. You could try lowering the `stat_q` value for letting MetaPhlAn considering more markers