Test fails due to "No module named 'pysam'"

[SuhasSrinivasan]

Dear Authors,

Thank you for developing and maintaining IsoQuant.

OS: macOS 26.1
CPU: M2 Ultra
Conda: 25.7.0

I am trying to run the post-installation test, but that is failing due to pysam not being found.
However, pysam is present in the conda environment, please see below.

% isoquant.py --test                                                                                                                                                             
Traceback (most recent call last):                                                                                                                                                                           
  File "/Users/user1/miniconda3/envs/modkit/bin/isoquant.py", line 23, in <module>                                                                                                                           
    import pysam                                                                                                                                                                                             
ModuleNotFoundError: No module named 'pysam'

% conda list                                                                                                                                                                                                                                                                                                                                                                            
# Name                       Version          Build                Channel                                                                                                                                   
_openmp_mutex                4.5              7_kmp_llvm           conda-forge                                                                                                                               
aom                          3.9.1            h7bae524_0           conda-forge                                                                                                                               
argcomplete                  3.6.3            pyhd8ed1ab_0         conda-forge                                                                                                                               
argh                         0.31.3           pyhd8ed1ab_1         conda-forge                                                                                                                               
biopython                    1.85             py311h72d1ebd_0                                                                                                                                                
blas                         2.304            openblas             conda-forge
blas-devel                   3.11.0           4_h11c0a38_openblas  conda-forge
bottleneck                   1.4.2            py311h03d0aa3_1
brotli                       1.2.0            h7d5ae5b_1           conda-forge
brotli-bin                   1.2.0            hc919400_1           conda-forge
bzip2                        1.0.8            h80987f9_6
c-ares                       1.34.6           hc919400_0           conda-forge
ca-certificates              2025.12.2        hca03da5_0
cairo                        1.18.4           h6a3b0d2_0           conda-forge
contourpy                    1.3.3            py311h20e30b6_0
cycler                       0.12.1           pyhcf101f3_2         conda-forge
dav1d                        1.2.1            hb547adb_0           conda-forge
expat                        2.7.3            h50f4ffc_4
filelock                     3.20.0           pypi_0               pypi
font-ttf-dejavu-sans-mono    2.37             hab24e00_0           conda-forge
font-ttf-inconsolata         3.000            h77eed37_0           conda-forge
font-ttf-source-code-pro     2.038            h77eed37_0           conda-forge
font-ttf-ubuntu              0.83             h77eed37_3           conda-forge
fontconfig                   2.15.0           h1383a14_1           conda-forge
fonts-conda-ecosystem        1                0                    conda-forge
fonts-conda-forge            1                hc364b38_1           conda-forge
fonttools                    4.61.1           pyh7db6752_0         conda-forge
freetype                     2.14.1           hce30654_0           conda-forge
fribidi                      1.0.16           hc919400_0           conda-forge
fsspec                       2025.12.0        pypi_0               pypi
gffutils                     0.13             pyh7cba7a3_0         bioconda
graphite2                    1.3.14           hec049ff_2           conda-forge
harfbuzz                     10.4.0           hb72c1af_0           conda-forge
htslib                       1.22.1           hfcd771d_0           bioconda
icu                          75.1             hfee45f7_0           conda-forge
importlib-metadata           8.7.0            pyhe01879c_1         conda-forge
isoquant                     3.10.0           hdfd78af_0           bioconda
jinja2                       3.1.6            pypi_0               pypi
jpeg                         9e               h1a8c8d9_3           conda-forge
k8                           1.2              hda5e58c_6           bioconda
kiwisolver                   1.4.9            py311haeee614_0
krb5                         1.21.3           h237132a_0           conda-forge
lcms2                        2.17             h7418793_0
lerc                         4.0.0            hd64df32_1           conda-forge
libavif                      1.3.0            hb06b76e_2           conda-forge
libavif16                    1.3.0            hb06b76e_2           conda-forge
libblas                      3.11.0           4_h51639a9_openblas  conda-forge
libbrotlicommon              1.2.0            hc919400_1           conda-forge
libbrotlidec                 1.2.0            hc919400_1           conda-forge
libbrotlienc                 1.2.0            hc919400_1           conda-forge
libcblas                     3.11.0           4_hb0561ab_openblas  conda-forge
libcurl                      8.17.0           hdece5d2_1           conda-forge
libcxx                       20.1.8           hd7fd590_1
libdeflate                   1.22             hd74edd7_0           conda-forge
libedit                      3.1.20250104     pl5321hafb1f1b_0     conda-forge
libev                        4.33             h93a5062_2           conda-forge
libexpat                     2.7.3            h50f4ffc_4
libffi                       3.4.4            hca03da5_1
libfreetype                  2.14.1           hce30654_0           conda-forge
libfreetype6                 2.14.1           h6da58f4_0           conda-forge
libgcc                       15.2.0           hcbb3090_16          conda-forge
libgfortran                  15.2.0           h07b0088_16          conda-forge
libgfortran5                 15.2.0           hdae7583_16          conda-forge
libglib                      2.84.0           hdff4504_0           conda-forge
libiconv                     1.18             h23cfdf5_2           conda-forge
libintl                      0.25.1           h493aca8_0           conda-forge
liblapack                    3.11.0           4_hd9741b5_openblas  conda-forge
liblapacke                   3.11.0           4_h1b118fd_openblas  conda-forge
liblzma                      5.8.1            h39f12f2_2           conda-forge
liblzma-devel                5.8.1            h39f12f2_2           conda-forge
libnghttp2                   1.67.0           hc438710_0           conda-forge
libopenblas                  0.3.30           openmp_ha158390_3    conda-forge
libopenjpeg                  2.5.4            haa24f5a_1
libpng                       1.6.53           hfab5511_0           conda-forge
libssh2                      1.11.1           h1590b86_0           conda-forge
libtiff                      4.7.1            h367c460_0
libwebp-base                 1.6.0            h07db88b_0           conda-forge
libzlib                      1.3.1            h5f15de7_0
llvm-openmp                  21.1.7           h4a912ad_0           conda-forge
markupsafe                   3.0.3            pypi_0               pypi
matplotlib-base              3.10.7           py311haa222d5_0
minimap2                     2.30             hba9b596_0           bioconda
mpmath                       1.3.0            pypi_0               pypi
munkres                      1.0.7            py_1                 bioconda
ncurses                      6.5              hee39554_0
networkx                     3.6.1            pypi_0               pypi
numexpr                      2.14.1           py311h5fbe615_0
numpy                        2.3.5            py311h4bb6f22_0
numpy-base                   2.3.5            py311h23175f9_0
openblas                     0.3.30           openmp_hea878ba_3    conda-forge
openssl                      3.6.0            h5503f6c_0           conda-forge
packaging                    25.0             pyh29332c3_1         conda-forge
pandas                       2.3.3            py311h3f644e9_1
pcre2                        10.44            ha881caa_2           conda-forge
pillow                       12.0.0           py311he2d6fe4_1
pip                          25.3             pyhc872135_0
pixman                       0.46.4           h81086ad_1           conda-forge
pyfaidx                      0.9.0.3          pyhdfd78af_0         bioconda
pyparsing                    3.2.5            pyhcf101f3_0         conda-forge
pysam                        0.23.3           py311h0b322ec_1      bioconda
python                       3.11.14          hf701271_0
python-dateutil              2.9.0.post0      pyhe01879c_2         conda-forge
python-tzdata                2025.3           pyhd8ed1ab_0         conda-forge
python_abi                   3.11             2_cp311              conda-forge
pytz                         2025.2           pyhd8ed1ab_0         conda-forge
pyvcf3                       1.0.4            py311h9bd965f_0      bioconda
pyyaml                       6.0.3            pyh7db6752_0         conda-forge
rav1e                        0.7.1            h0716509_3           conda-forge
readline                     8.3              h0b18652_0
samtools                     1.22.1           h267f7b9_0           bioconda
scipy                        1.16.3           py311h08a1045_0
seaborn-base                 0.13.2           pyhd8ed1ab_3         conda-forge
setuptools                   80.9.0           py311hca03da5_0
simplejson                   3.20.2           py311haa24f5a_0
six                          1.17.0           pyhe01879c_1         conda-forge
sqlite                       3.51.0           hab6afd1_0
star                         2.7.11b          haf7d672_8           bioconda
svt-av1                      3.1.2            h12ba402_0           conda-forge
sympy                        1.13.1           pypi_0               pypi
tk                           8.6.15           hcd8a7d5_0
torch                        2.6.0            pypi_0               pypi
torchaudio                   2.6.0            pypi_0               pypi
torchvision                  0.21.0           pypi_0               pypi
typing-extensions            4.15.0           pypi_0               pypi
tzdata                       2025b            h04d1e81_0
unicodedata2                 16.0.0           py311haa24f5a_0
wheel                        0.45.1           py311hca03da5_0
xz                           5.8.1            h9a6d368_2           conda-forge
xz-gpl-tools                 5.8.1            h9a6d368_2           conda-forge
xz-tools                     5.8.1            h39f12f2_2           conda-forge
yaml                         0.2.5            h925e9cb_3           conda-forge
zipp                         3.23.0           pyhcf101f3_1         conda-forge
zlib                         1.3.1            h5f15de7_0
zstd                         1.5.7            hbf9d68e_6           conda-forge

[SuhasSrinivasan]

Hi @andrewprzh ,
Just wanted to bring this to your attention, since you had looked at a similar issue earlier this year
#300

Troubleshooting yielded this

head -1 $(which isoquant.py)
#!/usr/bin/env python3

It is using the OS Python binary and not from the conda environment.

After activating the conda environment I had to wrap calls as

python $(which isoquant.py)

[andrewprzh]

Dear @SuhasSrinivasan

I tried running IsoQuant with conda environment activated and it did use the right python version (see below).
Shebang #!/usr/bin/env python3 does not point directly to the system python3, but indicates a python3 in the current environment. I guess you need to check your conda installation or install IsoQuant directly via conda.

With conda:

$ ./isoquant.py --test                                                                                                                                                                                                              
=== Running in test mode ===                                                                                                                                                                                                                                                       
Any other option is ignored                                                                                                                                                                                                                                                        
2025-12-15 18:41:11,485 - INFO - Running IsoQuant version 3.10.0                                                                                                                                                                                                                   
2025-12-15 18:41:11,486 - INFO - Novel unspliced transcripts will not be reported, set --report_novel_unspliced true to discover them                                                                                                                                              
2025-12-15 18:41:11,486 - INFO -  === IsoQuant pipeline started ===                                                                                                                                                                                                                
2025-12-15 18:41:11,486 - INFO - Python version: 3.12.2 | packaged by Anaconda, Inc. | (main, Feb 27 2024, 17:35:02) [GCC 11.2.0]                                                                                                                                                 
2025-12-15 18:41:11,486 - INFO - gffutils version: 0.13                                                                                                                                                                                                                            
2025-12-15 18:41:11,486 - INFO - pysam version: 0.23.3                                                                                                                                                                                                                             
2025-12-15 18:41:11,486 - INFO - pyfaidx version: 0.9.0.3

Without conda:

$ ./isoquant.py --test
=== Running in test mode === 
Any other option is ignored 
2025-12-15 18:45:20,690 - INFO - Running IsoQuant version 3.10.0
2025-12-15 18:45:20,692 - INFO - Novel unspliced transcripts will not be reported, set --report_novel_unspliced true to discover them
2025-12-15 18:45:20,692 - INFO -  === IsoQuant pipeline started === 
2025-12-15 18:45:20,692 - INFO - Python version: 3.10.12 (main, Nov  4 2025, 08:48:33) [GCC 11.4.0]
2025-12-15 18:45:20,692 - INFO - gffutils version: 0.10.1
2025-12-15 18:45:20,692 - INFO - pysam version: 0.17.0

[SuhasSrinivasan]

Hi @andrewprzh ,

Thank you for reviewing and testing and for the clarification.
IsoQuant was indeed installed through conda as in the GitHub README.

isoquant                     3.10.0           hdfd78af_0           bioconda

At least for me, for some reason, #!/usr/bin/env python3 resolution seems to be calling the Python interpreter outside the activated environment.
Because after wrapping as python $(which isoquant.py) it solves the issue.

[andrewprzh]

@SuhasSrinivasan

This is weird indeed, I will try testing conda installation once again.
Could it be the case that shebang line leads to python3, but your conda installation uses python executable?

[SuhasSrinivasan]

Thank you.
Here is more info that I could check. This is the conda environment where I have IsoQuant.

(modkit) % which python3
/Users/user1/miniconda3/envs/modkit/bin/python3

(modkit) % which python
/Users/user1/miniconda3/envs/modkit/bin/python