Unexpected output (format)

Hello,

I’m using the latest version of MetaPhlAn3 (version 3.0.1 -> 25 Jun 2020) and I’m getting an output that I am not sure that the format is correct or how to interpret interpret it

cmd:
zcat sample.pair.1.fq.gz sample.pair.2.fq.gz | metaphlan --input_type fastq -t rel_ab --add_viruses --nproc 8 --tax_lev a --sample_id_key ‘#’ --no_map -o met301_output

It runs successfully but with the following warnings:

Use of uninitialized value $bt2_args[2] in join or string at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 423.
Use of uninitialized value $bt2_args[3] in join or string at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 423.
Use of uninitialized value $_[2] in string eq at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 360.
Use of uninitialized value $_[3] in string eq at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 360.
Use of uninitialized value in exists at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 81.
Use of uninitialized value in exists at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 81.
Use of uninitialized value $bt2_args[2] in join or string at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 459.
Use of uninitialized value $bt2_args[3] in join or string at /g/scb2/bork/mocat/software/metaphlan/3.0.1/bowtie2 line 459.
WARNING: The metagenome profile contains clades that represent multiple species merged into a single representant.
An additional column listing the merged species is added to the MetaPhlAn output.

output:

#mpa_v30_CHOCOPhlAn_201901
#/g/scb2/bork/mocat/software/metaphlan/3.0.1/metaphlan --input_type fastq -t rel_ab --add_viruses --nproc 8 --tax_lev a --sample_id_key # --no_map -o met301_output
##	Metaphlan_Analysis
#clade_name	NCBI_tax_id	relative_abundance	additional_species
k__Bacteria	2	98.5612
k__Archaea	2157	1.4388
k__Bacteria|p__Bacteroidetes	2|976	86.06614
k__Bacteria|p__Firmicutes	2|1239	7.13839
k__Bacteria|p__Actinobacteria	2|201174	2.8684
k__Bacteria|p__Proteobacteria	2|1224	2.48827
k__Archaea|p__Euryarchaeota	2157|28890	1.4388
k__Bacteria|p__Bacteroidetes|c__Bacteroidia	2|976|200643	86.06614
k__Bacteria|p__Firmicutes|c__Bacilli	2|1239|91061	3.79753
k__Bacteria|p__Firmicutes|c__Clostridia	2|1239|186801	2.84848
k__Bacteria|p__Actinobacteria|c__Actinobacteria	2|201174|1760	2.55458
.
.
.
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Odoribacteraceae|g__Butyricimonas	2|976|200643|171549|1853231|574697	0.05371
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_uniformis	2|976|200643|171549|815|816|820	30.08586	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_D20,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_AR29,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_uniformis_CAG_3,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_4_1_36
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_caccae	2|976|200643|171549|815|816|47678	21.5374	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_43_46
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_dorei	2|976|200643|171549|815|816|357276	7.57107	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_3_1_33FAA,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_9_1_42FAA
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_distasonis	2|976|200643|171549|2005525|375288|823	7.28879	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_20_3,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_2_1_7,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Porphyromonadaceae|g__Porphyromonas|s__Porphyromonas_sp_31_2,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_AT13,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_2_1_33B,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_D25,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_AM17_47,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_D13,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_CT06,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_D26,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_AM25_14,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__Bacteroides|s__Bacteroides_sp_3_1_19,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Tannerellaceae|g__Parabacteroides|s__Parabacteroides_sp_CAG_2
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Rikenellaceae|g__Alistipes|s__Alistipes_putredinis	2|976|200643|171549|171550|239759|28117	6.93637	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Rikenellaceae|g__Alistipes|s__Alistipes_sp,k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Rikenellaceae|g__Alistipes|s__Alistipes_putredinis_CAG_67
k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_faecium	2|1239|91061|186826|81852|1350|1352	3.79753	k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC034B11,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC056C08,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_10F3_DIV0382,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC060D07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC060E05,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC069A01,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_GMD5E,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_GMD1E,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC077E07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC34G12,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC072F02,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_3G1_DIV0629,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC063C12,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC067C01,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC076D08,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC063D12,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC077E04,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC065H03,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC070F12,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC063H10,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC073E07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC074F07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HPCN18,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC035B04,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC058D07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC072F07,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC035C10,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC061C05,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC055G03,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC076E04,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC073E08,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC072D11,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC072G01,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC060D09,k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Enterococcaceae|g__Enterococcus|s__Enterococcus_sp_HMSC065H12
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Odoribacteraceae|g__Odoribacter|s__Odoribacter_splanchnicus	2|976|200643|171549|1853231|283168|28118	2.79578	k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Odoribacteraceae|g__Odoribacter|s__Odoribacter_splanchnicus_CAG_14
k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_bromii	2|1239|186801|186802|541000|1263|40518	2.71565	k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AM28_29LB,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AF43_11,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AM31_32,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AF37_3AC,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AM36_18,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AF42_10,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AF17_11,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_CAG_108,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AM34_10LB,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_TF12_19AC,k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Ruminococcaceae|g__Ruminococcus|s__Ruminococcus_sp_AF16_40

Can you please clarify that the output is what is expected and if yes, how is it to be interpreted

The command used and the output obtained are OK, this is the new format introduced in version 3.

These warnings are not affecting the MetaPhlAn run, they come from bowtie2, the latest version available on bioconda has some issues.

These are from MetaPhlAn, they just inform you that some species found can have “alternative” taxonomies (the list of species in the additional_species column). All the species listed under additional_species are not represented by any markers but they were found to be <5% ANI distant from the “reference” species (clade_name).

Thank you for the quick reply.

Can you please advice on how to interpret the new format in comparison to the old metaphlan2 format

Best
W

The first two columns are linked to the taxonomy of the clade, the first one is the seven-level taxonomy with the clade name, the second one is the same seven-level taxonomy but instead the clade name, each level is the associated NCBI taxonomy ID.
Relative abundance of the clade is present in the third column.
The latest column lists the alternative" species name as I described you in the previous post.

You can see that are present more header lines: first one is the name and version of the database used for the profiling. We introduced also the command line used to perform the analysis.

That helps greatly.

Thank you again for the time