BCR pre-processing

[saramoein372]

Would you please help me to figure out the problem with BSR Pre_processing. For the step 4 I get this error:

ddl.pp.assign_isotypes(samples, blastdb = "/Users/saramoein/Downloads/dandelion-master/container/database/blast/human/human_BCR_C.fasta")

The error:

FileNotFoundError Traceback (most recent call last)
in
----> 1 ddl.pp.assign_isotypes(samples, blastdb = "/Users/saramoein/Downloads/dandelion-master/container/database/blast/human/human_BCR_C.fasta")
2
3
4

/opt/anaconda3/lib/python3.8/site-packages/dandelion/preprocessing/_preprocessing.py in assign_isotypes(fastas, fileformat, org, correct_c_call, correction_dict, plot, save_plot, show_plot, figsize, blastdb, allele, parallel, ncpu, filename_prefix, verbose)
1004
1005 for i in range(0, len(fastas)):
-> 1006 assign_isotype(fastas[i],
1007 fileformat=fileformat,
1008 org=org,

/opt/anaconda3/lib/python3.8/site-packages/dandelion/preprocessing/_preprocessing.py in assign_isotype(fasta, fileformat, org, correct_c_call, correction_dict, plot, save_plot, show_plot, figsize, blastdb, allele, parallel, ncpu, filename_prefix, verbose)
818 if verbose:
819 print('Running blastn \n')
--> 820 _run_blastn(filePath, blastdb, format_dict[fileformat], org, verbose)
821 # parsing output into a summary.txt file
822 if verbose:

/opt/anaconda3/lib/python3.8/site-packages/dandelion/preprocessing/_preprocessing.py in _run_blastn(fasta, blastdb, fileformat, org, verbose)
416 print('Running command: %s\n' % (' '.join(cmd)))
417 with open(blast_out, 'w') as out:
--> 418 run(cmd, stdout=out, env=env)
419
420 def _parse_BLAST(fasta, fileformat):

/opt/anaconda3/lib/python3.8/subprocess.py in run(input, capture_output, timeout, check, *popenargs, **kwargs)
491 kwargs['stderr'] = PIPE
492
--> 493 with Popen(*popenargs, **kwargs) as process:
494 try:
495 stdout, stderr = process.communicate(input, timeout=timeout)

/opt/anaconda3/lib/python3.8/subprocess.py in init(self, args, bufsize, executable, stdin, stdout, stderr, preexec_fn, close_fds, shell, cwd, env, universal_newlines, startupinfo, creationflags, restore_signals, start_new_session, pass_fds, encoding, errors, text)
856 encoding=encoding, errors=errors)
857
--> 858 self._execute_child(args, executable, preexec_fn, close_fds,
859 pass_fds, cwd, env,
860 startupinfo, creationflags, shell,

/opt/anaconda3/lib/python3.8/subprocess.py in _execute_child(self, args, executable, preexec_fn, close_fds, pass_fds, cwd, env, startupinfo, creationflags, shell, p2cread, p2cwrite, c2pread, c2pwrite, errread, errwrite, restore_signals, start_new_session)
1704 if errno_num != 0:
1705 err_msg = os.strerror(errno_num)
-> 1706 raise child_exception_type(errno_num, err_msg, err_filename)
1707 raise child_exception_type(err_msg)
1708

FileNotFoundError: [Errno 2] No such file or directory: 'blastn'

Thanks

[zktuong]

Hi @saramoein372, thank you for interest in this package.

Your error is saying that it cannot find blastn in your path. blastn comes with blast+ (and not with igblast) so you might want to check if you have it with:

which blastn

It should return with an output like this:

/Users/kt16/Softwares/ncbi-blast-2.12.0+/bin/blastn

if not, download it (manually or with conda), and add it to your path:

export PATH=/path/to/folder/where/blastn/is:$PATH

To add it permanently, you can consider exporting it to your ~/.bashrc or ~/.bash_profile or equivalent files.
You can refer to the external software section of https://sc-dandelion.readthedocs.io/en/latest/README.html#installation.

Hope that helps!

[saramoein372]

Hi Kelvin,

Thank you for the help! I could solve the problem in step4.
I just have one question left for the step 3. When I run the step3, it give me this error:

ddl.pp.reassign_alleles(samples[0:1], combined_folder = '/users/saramoein/downloads/BCR/tutorial_scgp2', germline = "/Users/saramoein/Downloads/dandelion-master/container/database/germlines/imgt/human/vdj")


Running tigger-genotype with novel allele discovery.
      Novel allele discovery execution halted.
      Attempting to run tigger-genotype without novel allele discovery.
     Insufficient contigs for running tigger-genotype. Defaulting to original heavy chain v_calls.
            Reconstructing heavy chain dmask germline sequences with v_call.
            Reconstructing light chain dmask germline sequences with v_call.
      For convenience, entries for heavy chain in `v_call` are copied to `v_call_genotyped`.
---------------------------------------------------------------------------
TypeError                                 Traceback (most recent call last)
<ipython-input-16-21002daefa5a> in <module>
----> 1 ddl.pp.reassign_alleles(samples[0:1], combined_folder = '/users/saramoein/downloads/BCR/tutorial_scgp2', germline = "/Users/saramoein/Downloads/dandelion-master/container/database/germlines/imgt/human/vdj")
      2 
      3 
      4 
      5 

/opt/anaconda3/lib/python3.8/site-packages/dandelion/preprocessing/_preprocessing.py in reassign_alleles(data, combined_folder, v_germline, germline, org, v_field, germ_types, novel, cloned, plot, save_plot, show_plot, figsize, sample_id_dictionary, filename_prefix, verbose)
   1388             '      For convenience, entries for heavy chain in `v_call` are copied to `v_call_genotyped`.'
   1389         )
-> 1390         heavy = load_data(outDir + '/' + outDir + '_heavy' +
   1391                           germpass_dict[fileformat])
   1392         heavy['v_call_genotyped'] = heavy['v_call']

/opt/anaconda3/lib/python3.8/site-packages/dandelion/utilities/_core.py in load_data(obj)
   1035         obj_ = obj.copy()
   1036     else:
-> 1037         raise TypeError(
   1038             "Either input is not of <class 'pandas.core.frame.DataFrame'> or file does not exist."
   1039         )

TypeError: Either input is not of <class 'pandas.core.frame.DataFrame'> or file does not exist.

[saramoein372]

Sorry Kelvin, for unknown reason my last message got bolded and editing could not fix it.

[zktuong]

hi @saramoein372, for combined_folder, can you try and see if just specifying combined_folder = 'tutorial_scgp2' works?

[saramoein372]

Cool! it worked Kelvin. Thank you!

But it does not generates the plots. Is it because of the "Insufficient contigs for running tigger-genotype"?
These are the messages during running:

Processing data file(s) : 100%|██████████| 1/1 [00:01<00:00, 1.06s/it]
Running tigger-genotype with novel allele discovery.
Novel allele discovery execution halted.
Attempting to run tigger-genotype without novel allele discovery.
Insufficient contigs for running tigger-genotype. Defaulting to original heavy chain v_calls.
Reconstructing heavy chain dmask germline sequences with v_call.
Reconstructing light chain dmask germline sequences with v_call.
For convenience, entries for heavy chain in v_call are copied to v_call_genotyped.
For convenience, entries for light chain v_call are copied to v_call_genotyped.
Writing out to individual folders : 100%|██████████| 1/1 [00:00<00:00, 7.71it/s]
Although tigger-genotype was not run successfully, file will still be saved with _genotyped.tsvextension for convenience.

[zktuong]

But it does not generates the plots. Is it because of the "Insufficient contigs for running tigger-genotype"?

That's correct. If tigger doesn't manage to work, it will just make a copy of whatever's in the v_call column and there will be no further outputs related to tigger. You can also manually remove the v_call_genotyped column and it shouldn't impact on subsequent analysis.

If this is all, I will close this issue now. Feel free to reopen or open a new issue if required though!

[saramoein372]

Sure. These were all my questions about preprocessing step. Thank you.