TypeError: unsupported operand type(s) for -: 'NoneType' and 'int'

[evantroendle]

Hello,

I am attempting to use cojac for a research project and have created a conda environment (conda version 4.13.0) for this, but when supplying *.bam files to test the package I have encountered an error.

First I will list my conda environment for your reference in case you notice important versioning differences (cojac and pysam in bold ):

packages in environment at /home/evan/anaconda3/envs/cojac:

Name Version Build Channel

_libgcc_mutex 0.1 conda_forge conda-forge
_openmp_mutex 4.5 2_gnu conda-forge
brotli 1.0.9 h5eee18b_7
brotli-bin 1.0.9 h5eee18b_7
brotlipy 0.7.0 py39h27cfd23_1003
bzip2 1.0.8 h7b6447c_0
c-ares 1.18.1 h7f8727e_0
ca-certificates 2022.07.19 h06a4308_0
certifi 2022.6.15 py39h06a4308_0
cffi 1.15.1 py39h74dc2b5_0
charset-normalizer 2.0.4 pyhd3eb1b0_0
cojac 0.2 hdfd78af_0 bioconda
colorama 0.4.5 py39h06a4308_0
cryptography 37.0.1 py39h9ce1e76_0
cycler 0.11.0 pyhd3eb1b0_0
dbus 1.13.18 hb2f20db_0
expat 2.4.8 h27087fc_0 conda-forge
fontconfig 2.14.0 h8e229c2_0 conda-forge
fonttools 4.25.0 pyhd3eb1b0_0
freetype 2.11.0 h70c0345_0
gettext 0.21.0 hf68c758_0
giflib 5.2.1 h7b6447c_0
glib 2.72.1 h6239696_0 conda-forge
glib-tools 2.72.1 h6239696_0 conda-forge
gst-plugins-base 1.14.0 h8213a91_2
gstreamer 1.14.0 h28cd5cc_2
icu 58.2 he6710b0_3
idna 3.3 pyhd3eb1b0_0
jpeg 9e h7f8727e_0
kiwisolver 1.4.2 py39h295c915_0
krb5 1.19.2 hac12032_0
lcms2 2.12 h3be6417_0
ld_impl_linux-64 2.38 h1181459_1
libblas 3.9.0 15_linux64_openblas conda-forge
libbrotlicommon 1.0.9 h5eee18b_7
libbrotlidec 1.0.9 h5eee18b_7
libbrotlienc 1.0.9 h5eee18b_7
libcblas 3.9.0 15_linux64_openblas conda-forge
libclang 10.0.1 default_hb85057a_2
libcurl 7.84.0 h91b91d3_0
libdeflate 1.10 h7f98852_0 conda-forge
libedit 3.1.20210910 h7f8727e_0
libev 4.33 h7f8727e_1
libevent 2.1.12 h8f2d780_0
libffi 3.4.2 h295c915_4
libgcc-ng 12.1.0 h8d9b700_16 conda-forge
libgfortran-ng 11.2.0 h00389a5_1
libgfortran5 11.2.0 h1234567_1
libglib 2.72.1 h2d90d5f_0 conda-forge
libgomp 12.1.0 h8d9b700_16 conda-forge
libiconv 1.16 h7f8727e_2
liblapack 3.9.0 15_linux64_openblas conda-forge
libllvm10 10.0.1 hbcb73fb_5
libnghttp2 1.46.0 hce63b2e_0
libnsl 2.0.0 h7f98852_0 conda-forge
libopenblas 0.3.20 h043d6bf_1
libpng 1.6.37 hbc83047_0
libpq 12.9 h16c4e8d_3
libssh2 1.10.0 h8f2d780_0
libstdcxx-ng 12.1.0 ha89aaad_16 conda-forge
libtiff 4.2.0 h2818925_1
libuuid 2.32.1 h7f98852_1000 conda-forge
libwebp 1.2.2 h55f646e_0
libwebp-base 1.2.2 h7f8727e_0
libxcb 1.15 h7f8727e_0
libxkbcommon 1.0.1 hfa300c1_0
libxml2 2.9.14 h74e7548_0
libxslt 1.1.35 h4e12654_0
libzlib 1.2.12 h166bdaf_2 conda-forge
lz4-c 1.9.3 h295c915_1
matplotlib 3.5.2 py39hf3d152e_1 conda-forge
matplotlib-base 3.5.2 py39h700656a_1 conda-forge
munkres 1.1.4 py_0
ncurses 6.3 h5eee18b_3
nspr 4.33 h295c915_0
nss 3.74 h0370c37_0
numpy 1.23.1 py39hba7629e_0 conda-forge
openssl 1.1.1q h7f8727e_0
packaging 21.3 pyhd3eb1b0_0
pandas 1.4.3 py39h1832856_0 conda-forge
pcre 8.45 h295c915_0
pillow 9.2.0 py39hace64e9_1
pip 22.1.2 py39h06a4308_0
ply 3.11 py39h06a4308_0
pycparser 2.21 pyhd3eb1b0_0
pyopenssl 22.0.0 pyhd3eb1b0_0
pyparsing 3.0.4 pyhd3eb1b0_0
pyqt 5.15.7 py39h6a678d5_1
pyqt5-sip 12.11.0 py39h6a678d5_1
pysam 0.19.1 py39h5030a8b_0 bioconda
pysocks 1.7.1 py39h06a4308_0
python 3.9.13 h9a8a25e_0_cpython conda-forge
python-dateutil 2.8.2 pyhd3eb1b0_0
python_abi 3.9 2_cp39 conda-forge
pytz 2022.1 py39h06a4308_0
pyyaml 6.0 py39hb9d737c_4 conda-forge
qt-main 5.15.2 h327a75a_6
qt-webengine 5.15.9 hd2b0992_4
qtwebkit 5.212 h4eab89a_4
readline 8.1.2 h7f8727e_1
requests 2.28.1 pyhd8ed1ab_0 conda-forge
ruamel.yaml 0.17.4 py39h3811e60_0 conda-forge
ruamel.yaml.clib 0.2.6 py39h7f8727e_0
setuptools 61.2.0 py39h06a4308_0
sip 6.6.2 py39h6a678d5_0
six 1.16.0 pyhd3eb1b0_1
sqlite 3.39.0 h5082296_0
strictyaml 1.6.1 pyhd8ed1ab_0 conda-forge
tk 8.6.12 h1ccaba5_0
toml 0.10.2 pyhd3eb1b0_0
tornado 6.1 py39h27cfd23_0
tqdm 4.64.0 pyhd8ed1ab_0 conda-forge
tzdata 2022a hda174b7_0
urllib3 1.26.11 py39h06a4308_0
wheel 0.37.1 pyhd3eb1b0_0
xz 5.2.5 h7f8727e_1
yaml 0.2.5 h7b6447c_0
zlib 1.2.12 h7f8727e_2
zstd 1.5.2 ha4553b6_0

So firstly, the command I used was:

cooc-mutbamscan -b ../SARS-CoV-2.insert.V4.txt -m ../voc/ -a NIRE-002f80_S267.sorted.bam NIRE-002f81_S268.sorted.bam -j ../out/cooc-test.json

The bams and bam indices are attached as .txt files for your testing.

NIRE-002f81_S268.sorted.bam.bai.txt
NIRE-002f81_S268.sorted.bam.txt
NIRE-002f80_S267.sorted.bam.bai.txt
NIRE-002f80_S267.sorted.bam.txt

I used the SARS-CoV-2.insert.V4.txt as from GitHub (and also tested V3 in case I was wrong about the primer version used).
The voc/ folder is mirrored from GitHub.

The output was:
insertions not supported (yet): +AACA : AACA
insertions not supported (yet): +ACT : ACT
insertions not supported (yet): +GAGCCAGAA : GAGCCAGAA
76_B16173 is identical to 76_ka
77_ga is identical to 77_be
76_de is identical to 76_AY42
92_de is identical to 92_AY42
78_BA1 is identical to 78_BA2
79_BA1 is identical to 79_BA2
82_BA1 is identical to 82_om1
88_BA1 is identical to 88_om1
94_BA1 is identical to 94_BA2
95_BA1 is identical to 95_BA2
autodecting reference as MN908947.3
amplicon_31_om2 9336 9467 {9424: 'G', 9534: 'T'} amplion: 410
Traceback (most recent call last):
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 479, in
main()
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 451, in main
table[sample]=scanbam(alnfname, amplicons,rq_chr)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 184, in scanbam
amp_results[amp_name] = scanamplicon(amplicon_iter, mut_dict)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 134, in scanamplicon
(t_pos, t_read) = test_read(R, mut_dict)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 82, in test_read
found_site = [p for p,m in mut_dict.items() if read.reference_start <= (p-1) <= (read.reference_end-len(m))]
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 82, in
found_site = [p for p,m in mut_dict.items() if read.reference_start <= (p-1) <= (read.reference_end-len(m))]
TypeError: unsupported operand type(s) for -: 'NoneType' and 'int'

The program execution issue may be resolved by rolling back some to previous versions of some packages/using an earlier version of python, so if you could please advise me as to this, I would appreciate it. Alternatively if you can discern something about my input files that makes cojac unsuitable to analyse them, this would also be useful information.

I also get a similar output when running the V3 primer:
cooc-mutbamscan -b ../nCoV-2019.insert.V3.bed -m ../voc/ -a NIRE-002f80_S267.sorted.bam NIRE-002f81_S268.sorted.bam -j ../out/cooc-test.json
insertions not supported (yet): +AACA : AACA
insertions not supported (yet): +ACT : ACT
insertions not supported (yet): +GAGCCAGAA : GAGCCAGAA
76_B16173 is identical to 76_ka
76_ga is identical to 76_be
76_de is identical to 76_AY42
91_de is identical to 91_AY42
78_BA1 is identical to 78_BA2
93_BA1 is identical to 93_BA2
autodecting reference as MN908947.3
amplicon_31_om2 9275 9472 {9424: 'G', 9534: 'T'} amplion: 460
Traceback (most recent call last):
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 479, in
main()
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 451, in main
table[sample]=scanbam(alnfname, amplicons,rq_chr)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 184, in scanbam
amp_results[amp_name] = scanamplicon(amplicon_iter, mut_dict)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 134, in scanamplicon
(t_pos, t_read) = test_read(R, mut_dict)
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 82, in test_read
found_site = [p for p,m in mut_dict.items() if read.reference_start <= (p-1) <= (read.reference_end-len(m))]
File "/home/evan/anaconda3/envs/cojac/bin/cooc-mutbamscan", line 82, in
found_site = [p for p,m in mut_dict.items() if read.reference_start <= (p-1) <= (read.reference_end-len(m))]
TypeError: unsupported operand type(s) for -: 'NoneType' and 'int'

All the best,
Evan

[pratibha-kadam]

@evantroendle I am encountered with the same error. Did you find any solution?