Analysis says 100% unknown?

Hi all,

I am trying to run metagenomic profiling on one read (the fwd one) from paired end shot gun sequencing. Everything seems to work- I ran a tutorial with the Supragingival_plaque.fasta and it worked great. When I tried to use my own files I don’t get any error messages but I get this result:

the bowtie output file looks like this:

Here is the code I used for the run:

And if it helps, here’s all the code from downloading MetaPhlAn and its databases:
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % conda config --add channels defaults
Warning: ‘defaults’ already in ‘channels’ list, moving to the top
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % conda config --add channels bioconda
Warning: ‘bioconda’ already in ‘channels’ list, moving to the top
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % conda config --add channels conda-forge
Warning: ‘conda-forge’ already in ‘channels’ list, moving to the top
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % conda create --name mpa -c bioconda python=3.7 metaphlan
Collecting package metadata (current_repodata.json): done
Solving environment: done

==> WARNING: A newer version of conda exists. <==
current version: 4.12.0
latest version: 4.13.0

Please update conda by running

$ conda update -n base conda

Package Plan

environment location: /Users/stephaniegetto/opt/anaconda3/envs/mpa

added / updated specs:
- metaphlan
- python=3.7

The following packages will be downloaded:

package                    |            build
---------------------------|-----------------
biom-format-2.1.12         |   py37h49e79e5_1        10.4 MB  conda-forge
biopython-1.79             |   py37h271585c_1         2.6 MB  conda-forge
bowtie2-2.4.5              |   py37h0f016ca_2         1.4 MB  bioconda
brotlipy-0.7.0             |py37h69ee0a8_1004         366 KB  conda-forge
bx-python-0.8.13           |   py37h2b10027_1         931 KB  bioconda
certifi-2022.5.18.1        |   py37hf985489_0         150 KB  conda-forge
cffi-1.15.0                |   py37h446072c_0         218 KB  conda-forge
click-8.1.3                |   py37hf985489_0         145 KB  conda-forge
cryptography-37.0.2        |   py37h0169fcd_0         1.2 MB  conda-forge
fonttools-4.33.3           |   py37h994c40b_0         1.6 MB  conda-forge
future-0.18.2              |   py37hf985489_5         709 KB  conda-forge
h5py-3.6.0                 |nompi_py37h0ac0de7_100         1.1 MB  conda-forge
importlib-metadata-4.11.4  |   py37hf985489_0          33 KB  conda-forge
kiwisolver-1.4.2           |   py37h18621fa_1          61 KB  conda-forge
matplotlib-base-3.5.2      |   py37h80cb303_0         7.4 MB  conda-forge
metaphlan-3.0.14           |     pyhb7b1952_0         237 KB  bioconda
numpy-1.21.6               |   py37h345d48f_0         6.0 MB  conda-forge
pandas-1.3.4               |   py37h5b83a90_1        11.3 MB  conda-forge
pillow-9.1.1               |   py37h1eb1bbc_1        44.5 MB  conda-forge
pysam-0.19.1               |   py37h64e44a1_0         2.3 MB  bioconda
pysocks-1.7.1              |   py37hf985489_5          28 KB  conda-forge
python-3.7.12              |haf480d7_100_cpython        24.3 MB  conda-forge
python-lzo-1.14            |   py37h1c4811e_1          18 KB  conda-forge
python_abi-3.7             |          2_cp37m           4 KB  conda-forge
scipy-1.7.3                |   py37h4e3cf02_0        19.7 MB  conda-forge
setuptools-59.8.0          |   py37hf985489_1         1.0 MB  conda-forge
statsmodels-0.13.2         |   py37h032687b_0        10.6 MB  conda-forge
typing-extensions-4.2.0    |       hd8ed1ab_1           8 KB  conda-forge
typing_extensions-4.2.0    |     pyha770c72_1          27 KB  conda-forge
unicodedata2-14.0.0        |   py37h69ee0a8_1         497 KB  conda-forge
zipp-3.8.0                 |     pyhd8ed1ab_0          12 KB  conda-forge
------------------------------------------------------------
                                       Total:       148.8 MB

The following NEW packages will be INSTALLED:

bcbio-gff bioconda/noarch::bcbio-gff-0.6.9-pyh5e36f6f_0
biom-format conda-forge/osx-64::biom-format-2.1.12-py37h49e79e5_1
biopython conda-forge/osx-64::biopython-1.79-py37h271585c_1
blast bioconda/osx-64::blast-2.6.0-boost1.64_2
boost-cpp conda-forge/osx-64::boost-cpp-1.74.0-h8b082ac_8
bowtie2 bioconda/osx-64::bowtie2-2.4.5-py37h0f016ca_2
brotli conda-forge/osx-64::brotli-1.0.9-h5eb16cf_7
brotli-bin conda-forge/osx-64::brotli-bin-1.0.9-h5eb16cf_7
brotlipy conda-forge/osx-64::brotlipy-0.7.0-py37h69ee0a8_1004
bx-python bioconda/osx-64::bx-python-0.8.13-py37h2b10027_1
bzip2 conda-forge/osx-64::bzip2-1.0.8-h0d85af4_4
c-ares conda-forge/osx-64::c-ares-1.18.1-h0d85af4_0
ca-certificates conda-forge/osx-64::ca-certificates-2022.5.18.1-h033912b_0
cached-property conda-forge/noarch::cached-property-1.5.2-hd8ed1ab_1
cached_property conda-forge/noarch::cached_property-1.5.2-pyha770c72_1
capnproto conda-forge/osx-64::capnproto-0.9.1-h45c0eea_5
certifi conda-forge/osx-64::certifi-2022.5.18.1-py37hf985489_0
cffi conda-forge/osx-64::cffi-1.15.0-py37h446072c_0
charset-normalizer conda-forge/noarch::charset-normalizer-2.0.12-pyhd8ed1ab_0
click conda-forge/osx-64::click-8.1.3-py37hf985489_0
cmseq bioconda/noarch::cmseq-1.0.4-pyhb7b1952_0
cryptography conda-forge/osx-64::cryptography-37.0.2-py37h0169fcd_0
cycler conda-forge/noarch::cycler-0.11.0-pyhd8ed1ab_0
dendropy bioconda/noarch::dendropy-4.5.2-pyh3252c3a_0
diamond bioconda/osx-64::diamond-2.0.15-h9d1909e_0
fasttree bioconda/osx-64::fasttree-2.1.11-hdcdfbac_1
fonttools conda-forge/osx-64::fonttools-4.33.3-py37h994c40b_0
freetype conda-forge/osx-64::freetype-2.10.4-h4cff582_1
future conda-forge/osx-64::future-0.18.2-py37hf985489_5
giflib conda-forge/osx-64::giflib-5.2.1-hbcb3906_2
gsl conda-forge/osx-64::gsl-2.7-h93259b0_0
h5py conda-forge/osx-64::h5py-3.6.0-nompi_py37h0ac0de7_100
hdf5 conda-forge/osx-64::hdf5-1.12.1-nompi_ha60fbc9_104
htslib bioconda/osx-64::htslib-1.15.1-hc057d7f_0
icu conda-forge/osx-64::icu-70.1-h96cf925_0
idna conda-forge/noarch::idna-3.3-pyhd8ed1ab_0
importlib-metadata conda-forge/osx-64::importlib-metadata-4.11.4-py37hf985489_0
iqtree bioconda/osx-64::iqtree-2.2.0.3-h135ad0d_0
jpeg conda-forge/osx-64::jpeg-9e-h5eb16cf_1
kiwisolver conda-forge/osx-64::kiwisolver-1.4.2-py37h18621fa_1
krb5 conda-forge/osx-64::krb5-1.19.3-hb49756b_0
lcms2 conda-forge/osx-64::lcms2-2.12-h577c468_0
lerc conda-forge/osx-64::lerc-3.0-he49afe7_0
libblas conda-forge/osx-64::libblas-3.9.0-14_osx64_openblas
libbrotlicommon conda-forge/osx-64::libbrotlicommon-1.0.9-h5eb16cf_7
libbrotlidec conda-forge/osx-64::libbrotlidec-1.0.9-h5eb16cf_7
libbrotlienc conda-forge/osx-64::libbrotlienc-1.0.9-h5eb16cf_7
libcblas conda-forge/osx-64::libcblas-3.9.0-14_osx64_openblas
libcurl conda-forge/osx-64::libcurl-7.83.1-h372c54d_0
libcxx conda-forge/osx-64::libcxx-14.0.4-hc203e6f_0
libdeflate conda-forge/osx-64::libdeflate-1.10-h0d85af4_0
libedit conda-forge/osx-64::libedit-3.1.20191231-h0678c8f_2
libev conda-forge/osx-64::libev-4.33-haf1e3a3_1
libffi conda-forge/osx-64::libffi-3.4.2-h0d85af4_5
libgfortran conda-forge/osx-64::libgfortran-5.0.0-9_3_0_h6c81a4c_23
libgfortran5 conda-forge/osx-64::libgfortran5-9.3.0-h6c81a4c_23
liblapack conda-forge/osx-64::liblapack-3.9.0-14_osx64_openblas
libnghttp2 conda-forge/osx-64::libnghttp2-1.47.0-h942079c_0
libopenblas conda-forge/osx-64::libopenblas-0.3.20-openmp_hb3cd9ec_0
libpng conda-forge/osx-64::libpng-1.6.37-h7cec526_2
libssh2 conda-forge/osx-64::libssh2-1.10.0-h52ee1ee_2
libtiff conda-forge/osx-64::libtiff-4.4.0-hfca7e8f_0
libwebp conda-forge/osx-64::libwebp-1.2.2-h28dabe5_0
libwebp-base conda-forge/osx-64::libwebp-base-1.2.2-h0d85af4_1
libxcb conda-forge/osx-64::libxcb-1.13-h0d85af4_1004
libzlib conda-forge/osx-64::libzlib-1.2.12-h6c3fc93_0
llvm-openmp conda-forge/osx-64::llvm-openmp-14.0.4-ha654fa7_0
lz4-c conda-forge/osx-64::lz4-c-1.9.3-he49afe7_1
lzo conda-forge/osx-64::lzo-2.10-haf1e3a3_1000
mafft bioconda/osx-64::mafft-7.505-ha5712d3_0
mash bioconda/osx-64::mash-2.3-hf785b45_2
matplotlib-base conda-forge/osx-64::matplotlib-base-3.5.2-py37h80cb303_0
metaphlan bioconda/noarch::metaphlan-3.0.14-pyhb7b1952_0
munkres bioconda/noarch::munkres-1.0.7-py_1
muscle bioconda/osx-64::muscle-5.1-hb339e23_1
ncurses conda-forge/osx-64::ncurses-6.3-h96cf925_1
numpy conda-forge/osx-64::numpy-1.21.6-py37h345d48f_0
openjpeg conda-forge/osx-64::openjpeg-2.4.0-h6e7aa92_1
openssl conda-forge/osx-64::openssl-1.1.1o-hfe4f2af_0
packaging conda-forge/noarch::packaging-21.3-pyhd8ed1ab_0
pandas conda-forge/osx-64::pandas-1.3.4-py37h5b83a90_1
patsy conda-forge/noarch::patsy-0.5.2-pyhd8ed1ab_0
perl conda-forge/osx-64::perl-5.32.1-2_h0d85af4_perl5
phylophlan bioconda/noarch::phylophlan-3.0.2-py_0
pillow conda-forge/osx-64::pillow-9.1.1-py37h1eb1bbc_1
pip conda-forge/noarch::pip-22.1.2-pyhd8ed1ab_0
pthread-stubs conda-forge/osx-64::pthread-stubs-0.4-hc929b4f_1001
pycparser conda-forge/noarch::pycparser-2.21-pyhd8ed1ab_0
pyopenssl conda-forge/noarch::pyopenssl-22.0.0-pyhd8ed1ab_0
pyparsing conda-forge/noarch::pyparsing-3.0.9-pyhd8ed1ab_0
pysam bioconda/osx-64::pysam-0.19.1-py37h64e44a1_0
pysocks conda-forge/osx-64::pysocks-1.7.1-py37hf985489_5
python conda-forge/osx-64::python-3.7.12-haf480d7_100_cpython
python-dateutil conda-forge/noarch::python-dateutil-2.8.2-pyhd8ed1ab_0
python-lzo conda-forge/osx-64::python-lzo-1.14-py37h1c4811e_1
python_abi conda-forge/osx-64::python_abi-3.7-2_cp37m
pytz conda-forge/noarch::pytz-2022.1-pyhd8ed1ab_0
raxml bioconda/osx-64::raxml-8.2.12-ha5712d3_4
readline conda-forge/osx-64::readline-8.1-h05e3726_0
requests conda-forge/noarch::requests-2.27.1-pyhd8ed1ab_0
samtools bioconda/osx-64::samtools-1.15.1-h9f30945_0
scipy conda-forge/osx-64::scipy-1.7.3-py37h4e3cf02_0
seaborn conda-forge/noarch::seaborn-0.11.2-hd8ed1ab_0
seaborn-base conda-forge/noarch::seaborn-base-0.11.2-pyhd8ed1ab_0
setuptools conda-forge/osx-64::setuptools-59.8.0-py37hf985489_1
six conda-forge/noarch::six-1.16.0-pyh6c4a22f_0
sqlite conda-forge/osx-64::sqlite-3.38.5-hd9f0692_0
statsmodels conda-forge/osx-64::statsmodels-0.13.2-py37h032687b_0
tbb conda-forge/osx-64::tbb-2020.2-h940c156_4
tk conda-forge/osx-64::tk-8.6.12-h5dbffcc_0
trimal bioconda/osx-64::trimal-1.4.1-hcd10b59_6
typing-extensions conda-forge/noarch::typing-extensions-4.2.0-hd8ed1ab_1
typing_extensions conda-forge/noarch::typing_extensions-4.2.0-pyha770c72_1
unicodedata2 conda-forge/osx-64::unicodedata2-14.0.0-py37h69ee0a8_1
urllib3 conda-forge/noarch::urllib3-1.26.9-pyhd8ed1ab_0
wheel conda-forge/noarch::wheel-0.37.1-pyhd8ed1ab_0
xorg-libxau conda-forge/osx-64::xorg-libxau-1.0.9-h35c211d_0
xorg-libxdmcp conda-forge/osx-64::xorg-libxdmcp-1.1.3-h35c211d_0
xz conda-forge/osx-64::xz-5.2.5-haf1e3a3_1
zipp conda-forge/noarch::zipp-3.8.0-pyhd8ed1ab_0
zlib conda-forge/osx-64::zlib-1.2.12-h6c3fc93_0
zstd conda-forge/osx-64::zstd-1.5.2-ha9df2e0_1

Proceed ([y]/n)? y

Downloading and Extracting Packages
pandas-1.3.4 | 11.3 MB | ##################################### | 100%
typing-extensions-4. | 8 KB | ##################################### | 100%
statsmodels-0.13.2 | 10.6 MB | ##################################### | 100%
fonttools-4.33.3 | 1.6 MB | ##################################### | 100%
pysam-0.19.1 | 2.3 MB | ##################################### | 100%
python-3.7.12 | 24.3 MB | ##################################### | 100%
unicodedata2-14.0.0 | 497 KB | ##################################### | 100%
brotlipy-0.7.0 | 366 KB | ##################################### | 100%
cryptography-37.0.2 | 1.2 MB | ##################################### | 100%
importlib-metadata-4 | 33 KB | ##################################### | 100%
bx-python-0.8.13 | 931 KB | ##################################### | 100%
future-0.18.2 | 709 KB | ##################################### | 100%
biopython-1.79 | 2.6 MB | ##################################### | 100%
h5py-3.6.0 | 1.1 MB | ##################################### | 100%
typing_extensions-4. | 27 KB | ##################################### | 100%
biom-format-2.1.12 | 10.4 MB | ##################################### | 100%
certifi-2022.5.18.1 | 150 KB | ##################################### | 100%
scipy-1.7.3 | 19.7 MB | ##################################### | 100%
click-8.1.3 | 145 KB | ##################################### | 100%
numpy-1.21.6 | 6.0 MB | ##################################### | 100%
bowtie2-2.4.5 | 1.4 MB | ##################################### | 100%
cffi-1.15.0 | 218 KB | ##################################### | 100%
kiwisolver-1.4.2 | 61 KB | ##################################### | 100%
metaphlan-3.0.14 | 237 KB | ##################################### | 100%
setuptools-59.8.0 | 1.0 MB | ##################################### | 100%
matplotlib-base-3.5. | 7.4 MB | ##################################### | 100%
zipp-3.8.0 | 12 KB | ##################################### | 100%
python-lzo-1.14 | 18 KB | ##################################### | 100%
pillow-9.1.1 | 44.5 MB | ##################################### | 100%
pysocks-1.7.1 | 28 KB | ##################################### | 100%
python_abi-3.7 | 4 KB | ##################################### | 100%
Preparing transaction: done
Verifying transaction: done
Executing transaction: done

To activate this environment, use

$ conda activate mpa

To deactivate an active environment, use

$ conda deactivate

(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % cd
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % mkdir -p db/metaphlan_databases
(base) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % conda activate mpa
(mpa) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % metaphlan --install --bowtie2db ~/db/metaphlan_databases

Downloading http://cmprod1.cibio.unitn.it/biobakery3/metaphlan_databases/mpa_latest
Downloading file of size: 0.00 MB
0.01 MB 31507.69 % 53.19 MB/sec 0 min -0 sec
Downloading MetaPhlAn database
Please note due to the size this might take a few minutes

Downloading http://cmprod1.cibio.unitn.it/biobakery3/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.tar
Downloading file of size: 366.62 MB
366.62 MB 100.00 % 7.71 MB/sec 0 min -0 sec
Downloading http://cmprod1.cibio.unitn.it/biobakery3/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.md5
Downloading file of size: 0.00 MB
0.01 MB 12800.00 % 35.01 MB/sec 0 min -0 sec

Decompressing /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.fna.bz2 into /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.fna

Building Bowtie2 indexes
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.3.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.3.bt2
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.4.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.4.bt2
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.1.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.1.bt2
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.2.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.2.bt2
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.rev.1.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.rev.1.bt2
Renaming /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.rev.2.bt2.tmp to /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.rev.2.bt2
Removing uncompress database /Users/stephaniegetto/db/metaphlan_databases/mpa_v30_CHOCOPhlAn_201901.fna

Download complete
The database is installed
(mpa) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % mkdir metaphlan_analysis
(mpa) stephaniegetto@Stephanies-MacBook-Pro-3 ~ % cd metaphlan_analysis
(mpa) stephaniegetto@Stephanies-MacBook-Pro-3 metaphlan_analysis % curl -LO https://github.com/biobakery/biobakery/raw/master/demos/biobakery_demos/data/metaphlan3/input/SRS014476-Supragingival_plaque.fasta.gz
% Total % Received % Xferd Average Speed Time Time Time Current
Dload Upload Total Spent Left Speed
100 192k 0 192k 0 0 360k 0 --:–:-- --:–:-- --:–:-- 365k

I have run this sample in other programs and gotten some results, so I don’t think it’s bad data… any ideas?

Hi @sgetto
From the results you are showing here it seems that metaphlan was not able to map any read against the markers database. From the bowtie2out file, you can see that metaphlan was only able to use 175 reads from your fastq file. Is the 9-8_R1.fastq file containing only 175 reads? and in case it contains more reads, are the reads short in length (<70nt)?