Freyja variants invalid option -- 'r'

[jacqgg]

Hi, I'm trying to run Freyja on bam file (aligned reads to covid reference, with trimmed primers - bam file after artic-ncov2019 pipeline):

freyja variants --variants "var_output" --depths "var_depth_output" --ref "nCoV-2019.reference.fasta" "primertrimmed.rg.sorted.bam"

and I always get error info "ivar: invalid option -- 'r' ". The same error is when running freyja variants without options like --variants, --depths or even --ref, so it is probably not the freyja arguments problem...

this topic: https://unix.stackexchange.com/questions/555360/bash-cat-invalid-option-r resolving similar problem with cat command, where cat is interpreting -rsomething like an additional argument '-r'. The problem was resolved by ending arguments section with standalone " -- ". Maybe here I have similar problem, but how to edit iVar command running by Freya? [Freyja version 1.3.8, iVar version 1.0]

[joshuailevy]

Hi @jacqgg! I can't say I've seen that error before. Are those quotation marks just for the purposes of this issue or are those actually in your command? If so, that could be part of your problem. If you try running this command in line with the README

freyja variants primertrimmed.rg.sorted.bam --variants var_output.tsv --depths depth_output.depth --ref nCoV-2019.reference.fasta

does that work? Or still the same error?

[jacqgg]

Hi @joshuailevy, i was trying to run it with and without quotations and still getting the same info. Being more precise, exactly:

ivar: invalid option -- 'r'
[mpileup] 1 samples in 1 input files

[joshuailevy]

I'm not sure what's going on here, but there shouldn't be quotation marks around filenames used in your command. Can you post the whole command as well as any additional outputs/errors that are produced when you run it? Will help me diagnose what's going on. Thanks!

[jacqgg]

that's all, unfortunately. The only output is above and the command looks the same as in my first post (but without quotations). The environment is from clear conda installation

[joshuailevy]

Unfortunately I haven't been able to reproduce this error. What sort of operating system are you using? I've tested extensively on mac/linux machines, but it's possible that things could be behaving weirdly on windows, for example.

[jacqgg]

Linux Debian 4.19.194-3. Conda env packages below:

# Name                    Version                   Build  Channel
_libgcc_mutex             0.1                 conda_forge    conda-forge
_openmp_mutex             4.5                       2_gnu    conda-forge
boost-cpp                 1.74.0               h75c5d50_8    conda-forge
brotli                    1.0.9                h166bdaf_7    conda-forge
brotli-bin                1.0.9                h166bdaf_7    conda-forge
bzip2                     1.0.8                h7f98852_4    conda-forge
ca-certificates           2022.6.15            ha878542_0    conda-forge
certifi                   2022.6.15        py39hf3d152e_0    conda-forge
click                     8.1.3            py39hf3d152e_0    conda-forge
colorama                  0.4.5              pyhd8ed1ab_0    conda-forge
curl                      7.71.1               he644dc0_3    conda-forge
cvxpy                     1.2.1            py39hf3d152e_0    conda-forge
cvxpy-base                1.2.1            py39h1832856_0    conda-forge
cycler                    0.11.0             pyhd8ed1ab_0    conda-forge
ecos                      2.0.10           py39hce5d2b2_0    conda-forge
fonttools                 4.34.4           py39hb9d737c_0    conda-forge
freetype                  2.10.4               h0708190_1    conda-forge
freyja                    1.3.8              pyhdfd78af_0    bioconda
htslib                    1.9                  ha228f0b_7    bioconda
icu                       70.1                 h27087fc_0    conda-forge
isa-l                     2.30.0               ha770c72_4    conda-forge
ivar                      1.0                  hddee448_1    bioconda
joblib                    1.1.0              pyhd8ed1ab_0    conda-forge
jpeg                      9e                   h166bdaf_2    conda-forge
kiwisolver                1.4.3            py39hf939315_0    conda-forge
krb5                      1.17.2               h926e7f8_0    conda-forge
lcms2                     2.12                 hddcbb42_0    conda-forge
ld_impl_linux-64          2.36.1               hea4e1c9_2    conda-forge
libblas                   3.9.0           15_linux64_openblas    conda-forge
libbrotlicommon           1.0.9                h166bdaf_7    conda-forge
libbrotlidec              1.0.9                h166bdaf_7    conda-forge
libbrotlienc              1.0.9                h166bdaf_7    conda-forge
libcblas                  3.9.0           15_linux64_openblas    conda-forge
libcurl                   7.71.1               hcdd3856_3    conda-forge
libdeflate                1.0                  h14c3975_1    bioconda
libedit                   3.1.20191231         he28a2e2_2    conda-forge
libffi                    3.4.2                h7f98852_5    conda-forge
libgcc-ng                 12.1.0              h8d9b700_16    conda-forge
libgfortran-ng            12.1.0              h69a702a_16    conda-forge
libgfortran5              12.1.0              hdcd56e2_16    conda-forge
libgomp                   12.1.0              h8d9b700_16    conda-forge
liblapack                 3.9.0           15_linux64_openblas    conda-forge
libnsl                    2.0.0                h7f98852_0    conda-forge
libopenblas               0.3.20          pthreads_h78a6416_0    conda-forge
libpng                    1.6.37               h753d276_3    conda-forge
libprotobuf               3.18.1               h780b84a_0    conda-forge
libssh2                   1.10.0               ha56f1ee_2    conda-forge
libstdcxx-ng              12.1.0              ha89aaad_16    conda-forge
libtiff                   4.2.0                hf544144_3    conda-forge
libuuid                   2.32.1            h7f98852_1000    conda-forge
libwebp-base              1.2.2                h7f98852_1    conda-forge
libzlib                   1.2.12               h166bdaf_1    conda-forge
lz4-c                     1.9.3                h9c3ff4c_1    conda-forge
mafft                     7.505                hec16e2b_0    bioconda
matplotlib-base           3.5.2            py39h700656a_0    conda-forge
mpi                       1.0                     openmpi    conda-forge
munkres                   1.0.7                      py_1    bioconda
mysql-connector-c         6.1.11            h6eb9d5d_1007    conda-forge
ncurses                   6.3                  h27087fc_1    conda-forge
numpy                     1.23.1           py39hba7629e_0    conda-forge
olefile                   0.46               pyh9f0ad1d_1    conda-forge
openjpeg                  2.4.0                hb52868f_1    conda-forge
openmpi                   4.1.2                hbfc84c5_0    conda-forge
openssh                   8.6p1                h7271633_0    conda-forge
openssl                   1.1.1q               h166bdaf_0    conda-forge
osqp                      0.6.2.post0      py39hde0f152_3    conda-forge
packaging                 21.3               pyhd8ed1ab_0    conda-forge
pandas                    1.4.3            py39h1832856_0    conda-forge
pillow                    8.2.0            py39hf95b381_1    conda-forge
pip                       22.1.2             pyhd8ed1ab_0    conda-forge
plotly                    5.9.0              pyhd8ed1ab_0    conda-forge
pyparsing                 3.0.9              pyhd8ed1ab_0    conda-forge
python                    3.9.13          h9a8a25e_0_cpython    conda-forge
python-dateutil           2.8.2              pyhd8ed1ab_0    conda-forge
python_abi                3.9                      2_cp39    conda-forge
pytz                      2022.1             pyhd8ed1ab_0    conda-forge
qdldl-python              0.1.5            py39hde0f152_2    conda-forge
readline                  8.1.2                h0f457ee_0    conda-forge
samtools                  1.6                  h9dace67_6    bioconda
scipy                     1.8.1            py39he49c0e8_0    conda-forge
scs                       3.2.0            py39h0ec8588_0    conda-forge
setuptools                63.1.0           py39hf3d152e_0    conda-forge
six                       1.16.0             pyh6c4a22f_0    conda-forge
sqlite                    3.39.0               h4ff8645_0    conda-forge
tenacity                  8.0.1              pyhd8ed1ab_0    conda-forge
tk                        8.6.12               h27826a3_0    conda-forge
tqdm                      4.64.0             pyhd8ed1ab_0    conda-forge
tzdata                    2022a                h191b570_0    conda-forge
ucsc-fatovcf              426                  h954228d_0    bioconda
unicodedata2              14.0.0           py39hb9d737c_1    conda-forge
usher                     0.5.6                hbc2f876_0    bioconda
wheel                     0.37.1             pyhd8ed1ab_0    conda-forge
xz                        5.2.5                h516909a_1    conda-forge
zlib                      1.2.12               h166bdaf_1    conda-forge
zstd                      1.5.2                h8a70e8d_2    conda-forge

maybe it's the problem with the bam file? Bam file comes from ARTIC sars-cov-2 pipeline (nanopore reads aligned to Wuhan reference with trimmed primers)

[joshuailevy]

Hm... I'd be happy to take a look at your bam file to try to investigate further if you're up for sharing it.

[jacqgg]

Sure:

• bam file https://drive.google.com/file/d/1snC_vUT-8FSQDkEeQ46roRBzMYDaDuzg/view?usp=sharing
• reference https://drive.google.com/file/d/1fJJLw-tW7HZ9ZKThZv6lLYH3sAJllw8I/view?usp=sharing

[joshuailevy]

Sorry for the delay. I just ran the following command it seems to have worked just fine: freyja variants BC22.primertrimmed.rg.sorted.bam --variants test.tsv --depths test.depth --ref nCoV-2019.reference.fasta

Perhaps there's an issue with your installation of freyja and/or ivar?

[joshuailevy]

Any luck getting freyja to run on your samples? Going to close this since there hasn't been much activity here and it seems to be more of a freyja/ivar installation issue.