$ bash run_Donor_Deconvolution.sh
checking modules
imports done
checking bam for expected tags
[W::hts_idx_load2] The index file is older than the data file: /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/1_raw_data/human/FC_12h/bamfile/possorted_genome_bam.bam.bai
checking fasta
restarting pipeline in existing directory /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution/
running souporcell clustering
/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell/target/release/souporcell -k 4 -a /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//alt.mtx -r /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//ref.mtx --restarts 100 -b /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/1_raw_data/human/FC_12h/count_matrices/filtered_feature_bc_matrix/barcodes.tsv --min_ref 10 --min_alt 10 --threads 56
running souporcell doublet detection
running co inference of ambient RNA and cluster genotypes
INFO:pystan:COMPILING THE C++ CODE FOR MODEL anon_model_ca32e407e94c33afe8f72cdc7357f09f NOW.
Traceback (most recent call last):
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/consensus.py", line 158, in <module>
sm = pystan.StanModel(model_code=cell_genotype_consensus)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/site-packages/pystan/model.py", line 384, in __init__
self.module = load_module(self.module_name, lib_dir)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/site-packages/pystan/model.py", line 50, in load_module
return __import__(module_name)
ImportError: /tmp/pystan_rk7jnbr7/stanfit4anon_model_ca32e407e94c33afe8f72cdc7357f09f_6509297500926240442.cpython-36m-x86_64-linux-gnu.so: undefined symbol: _ZSt28__throw_bad_array_new_lengthv
Traceback (most recent call last):
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 600, in <module>
consensus(args, ref_mtx, alt_mtx, doublet_file)
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 550, in consensus
"--output_dir",args.out_dir,"--soup_out", args.out_dir + "/ambient_rna.txt", "--vcf_out", args.out_dir + "/cluster_genotypes.vcf", "--vcf", final_vcf])
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/subprocess.py", line 291, in check_call
raise CalledProcessError(retcode, cmd)
subprocess.CalledProcessError: Command '['/home/ramadatta/sw/souporcell/hardinstall/souporcell/consensus.py', '-c', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//clusters.tsv', '-a', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//alt.mtx', '-r', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//ref.mtx', '-p', '2', '--output_dir', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution/', '--soup_out', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//ambient_rna.txt', '--vcf_out', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//cluster_genotypes.vcf', '--vcf', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//souporcell_merged_sorted_vcf.vcf.gz']' returned non-zero exit status 1.
110 mamba install -c conda-forge c-compiler
111 mamba install -c conda-forge cxx-compiler
112 bash run_Donor_Deconvolution.sh
checking modules
imports done
checking bam for expected tags
[W::hts_idx_load2] The index file is older than the data file: /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/1_raw_data/human/FC_12h/bamfile/possorted_genome_bam.bam.bai
checking fasta
restarting pipeline in existing directory /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution/
running co inference of ambient RNA and cluster genotypes
INFO:pystan:COMPILING THE C++ CODE FOR MODEL anon_model_ca32e407e94c33afe8f72cdc7357f09f NOW.
Traceback (most recent call last):
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/unixccompiler.py", line 197, in link
self.spawn(linker + ld_args)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/ccompiler.py", line 909, in spawn
spawn(cmd, dry_run=self.dry_run)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/spawn.py", line 36, in spawn
_spawn_posix(cmd, search_path, dry_run=dry_run)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/spawn.py", line 159, in _spawn_posix
% (cmd, exit_status))
distutils.errors.DistutilsExecError: command 'g++' failed with exit status 1
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/consensus.py", line 158, in <module>
sm = pystan.StanModel(model_code=cell_genotype_consensus)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/site-packages/pystan/model.py", line 378, in __init__
build_extension.run()
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/command/build_ext.py", line 339, in run
self.build_extensions()
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/command/build_ext.py", line 448, in build_extensions
self._build_extensions_serial()
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/command/build_ext.py", line 473, in _build_extensions_serial
self.build_extension(ext)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/command/build_ext.py", line 558, in build_extension
target_lang=language)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/ccompiler.py", line 717, in link_shared_object
extra_preargs, extra_postargs, build_temp, target_lang)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/distutils/unixccompiler.py", line 199, in link
raise LinkError(msg)
distutils.errors.LinkError: command 'g++' failed with exit status 1
Traceback (most recent call last):
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 600, in <module>
consensus(args, ref_mtx, alt_mtx, doublet_file)
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 550, in consensus
"--output_dir",args.out_dir,"--soup_out", args.out_dir + "/ambient_rna.txt", "--vcf_out", args.out_dir + "/cluster_genotypes.vcf", "--vcf", final_vcf])
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/subprocess.py", line 291, in check_call
raise CalledProcessError(retcode, cmd)
subprocess.CalledProcessError: Command '['/home/ramadatta/sw/souporcell/hardinstall/souporcell/consensus.py', '-c', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//clusters.tsv', '-a', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//alt.mtx', '-r', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//ref.mtx', '-p', '2', '--output_dir', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution/', '--soup_out', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//ambient_rna.txt', '--vcf_out', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//cluster_genotypes.vcf', '--vcf', '/home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution//souporcell_merged_sorted_vcf.vcf.gz']' returned non-zero exit status 1.
real 0m38,518s
user 0m39,721s
sys 0m8,153s
$ sudo apt-get install gcc
$ sudo apt-get install build-essentials
$ sudo apt-get install python-dev
$ sudo apt-get install python3-dev
No success!
$ ls ~/anaconda3/envs/souporcell/bin/ | grep clang gave nothing
$ clang --version
Command 'clang' not found, but can be installed with:
sudo apt install clang
distutils.errors.CompileError: command 'clang_osx' failed with exit status 1
conda deactivate souporcell
conda activate souporcell
Started working now...
$ bash run_Donor_Deconvolution.sh
checking modules
imports done
checking bam for expected tags
[W::hts_idx_load2] The index file is older than the data file: /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/1_raw_data/human/FC_12h/bamfile/possorted_genome_bam.bam.bai
checking fasta
restarting pipeline in existing directory /home/ramadatta/Analysis/1_Schiller_Lab/Projects/1_scGenomics_hPCLS/3_donor_deconvolution/
running co inference of ambient RNA and cluster genotypes
INFO:pystan:COMPILING THE C++ CODE FOR MODEL anon_model_ca32e407e94c33afe8f72cdc7357f09f NOW.
95804 excluded for potential RNA editing
1090 doublets excluded from genotype and ambient RNA estimation
924 not used for soup calculation due to possible RNA edit
Initial log joint probability = -13978.7
Iter log prob ||dx|| ||grad|| alpha alpha0 # evals Notes
3 -13973.8 0.00109013 0.0953795 0.9474 0.9474 4
Optimization terminated normally:
Convergence detected: relative gradient magnitude is below tolerance
====================================
freebayes -f /home/ramadatta/sw/souporcell/refdata-cellranger-GRCh38-3.0.0/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r 1:0-55352692
Traceback (most recent call last):
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 585, in <module>
final_vcf = freebayes(args, bam, fasta)
File "/home/ramadatta/sw/souporcell/hardinstall/souporcell/souporcell_pipeline.py", line 455, in freebayes
p = subprocess.Popen(cmd, stdout = filehandle, stderr = errhandle)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/subprocess.py", line 709, in __init__
restore_signals, start_new_session)
File "/home/ramadatta/anaconda3/envs/souporcell/lib/python3.6/subprocess.py", line 1344, in _execute_child
raise child_exception_type(errno_num, err_msg, err_filename)
FileNotFoundError: [Errno 2] No such file or directory: 'freebayes': 'freebayes'
Finally I installed freebayes, which worked without issue
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