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Some ISSUEs concerning about constructing REFERENCE on vdj sequencing and full length immune receptor sequencing #269

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Gethell opened this issue Nov 1, 2023 · 6 comments
Open

Some ISSUEs concerning about constructing REFERENCE on vdj sequencing and full length immune receptor sequencing #269

Gethell opened this issue Nov 1, 2023 · 6 comments

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@Gethell
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Gethell commented Nov 1, 2023

Description
When I did as the tutorial on official tweets, I met some issues as running the following codes:
celescope vdj mkref human TR
but encounted issue like :

Traceback (most recent call last):
File "/bios-store1/home/logcoin/.conda/envs/Celescope/bin/celescope", line 8, in
sys.exit(main())
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/celescope.py", line 54, in main
args.func(args)
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 81, in mkref
runner()
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 35, in call
self.combine_seq()
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/tools/utils.py", line 45, in wrapper
result = func(*args, **kwargs)
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 59, in combine_seq
assert len(imgt_files) == 7
AssertionError

And I run the code:
celescope vdj mkref human IG
I met another issue like:

Building a new DB, current time: 11/01/2023 15:27:28 New DB name: /bios-store1/home/logcoin/Reference/hs_vdj/Homo_sapiens/IG/human_cele_BR/IGV.fa New DB title: IGV.fa Sequence type: Nucleotide Deleted existing Nucleotide BLAST database named /bios-store1/home/logcoin/Reference/hs_vdj/Homo_sapiens/IG/human_cele_BR/IGV.fa Keep MBits: T Maximum file size: 3000000000B BLAST options error: File IGV.fa is empty Traceback (most recent call last): File "/bios-store1/home/logcoin/.conda/envs/Celescope/bin/celescope", line 8, in <module> sys.exit(main()) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/celescope.py", line 54, in main args.func(args) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 81, in mkref runner() File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 36, in __call__ self.build_index() File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/tools/utils.py", line 45, in wrapper result = func(*args, **kwargs) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 76, in build_index subprocess.check_call(f"makeblastdb -parse_seqids -dbtype nucl -in {out_file_name}.fa", shell=True) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/subprocess.py", line 373, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'makeblastdb -parse_seqids -dbtype nucl -in IGV.fa' returned non-zero exit status 1.

To Troubleshot
I think the isse NO.1 might cause by the wrong format but have no idea about another issue, so I did some troubleshots:

  1. Format of one of the IMGT reference files showed as following:
    head -n 20 TRAJ.fasta
    trouble1

head -n 20 IGHD.fasta
trouble3

  1. Check whether the output directory is null:
    ls -al ../IG/human_IG
    trouble2
    but the "TR" directory is null.
  2. And I finally check version of celescope
    Version
    celescope -v
    2.0.3

I sincerely expect some responses on resolution about these issues, or show me some normative format of IMGT data for constructing index file. Moreover, I hope that some detailed instance on how to construct index file about vdj or full length immune receptors sequencing.
Last question I would like to raise:
Whether the index files based on IMGT data between single cell vdj and full length immune receptor sequencing are coincident?
@Chenjunjie1996
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  1. Make sure celescope version>=1.15.1 and refer to https://github.com/singleron-RD/CeleScope/blob/master/doc/assay/multi_vdj.md.
  2. IMGT and full length vdj reference are different.

@Gethell
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Gethell commented Nov 3, 2023

Thanks for your response! I tried as tutorial you provided, but unfortunately I encunted another issue while running codes like:
wget https://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Homo_sapiens/TR/TR{A,B}{V,J}.fasta
and
wget http://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Mus_musculus/IG/IG{H,K,L}{V,J}.fasta
The issue descriped as:

--2023-11-03 10:47:00-- https://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Homo_sapiens/TR/TR%7BA,B%7D%7BV,J%7D.fasta
正在解析主机 www.imgt.org (www.imgt.org)... 195.83.84.12
正在连接 www.imgt.org (www.imgt.org)|195.83.84.12|:443... 已连接。
已发出 HTTP 请求，正在等待回应... 404 Not Found
2023-11-03 10:47:01 错误 404：Not Found。

trouble4

@Chenjunjie1996
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Remove character \
Correct link:

wget https://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Homo_sapiens/TR/TR{A,B}{V,J}.fasta

@Gethell
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Gethell commented Nov 29, 2023

Remove character \ Correct link:

wget https://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Homo_sapiens/TR/TR{A,B}{V,J}.fasta

Thanks for your response. And finally I succeed following your advise!

@Gethell
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Gethell commented Nov 29, 2023

Description When I did as the tutorial on official tweets, I met some issues as running the following codes: celescope vdj mkref human TR but encounted issue like :

Traceback (most recent call last):
File "/bios-store1/home/logcoin/.conda/envs/Celescope/bin/celescope", line 8, in
sys.exit(main())
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/celescope.py", line 54, in main
args.func(args)
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 81, in mkref
runner()
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 35, in call
self.combine_seq()
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/tools/utils.py", line 45, in wrapper
result = func(*args, **kwargs)
File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 59, in combine_seq
assert len(imgt_files) == 7
AssertionError

And I run the code: celescope vdj mkref human IG I met another issue like:

Building a new DB, current time: 11/01/2023 15:27:28 New DB name: /bios-store1/home/logcoin/Reference/hs_vdj/Homo_sapiens/IG/human_cele_BR/IGV.fa New DB title: IGV.fa Sequence type: Nucleotide Deleted existing Nucleotide BLAST database named /bios-store1/home/logcoin/Reference/hs_vdj/Homo_sapiens/IG/human_cele_BR/IGV.fa Keep MBits: T Maximum file size: 3000000000B BLAST options error: File IGV.fa is empty Traceback (most recent call last): File "/bios-store1/home/logcoin/.conda/envs/Celescope/bin/celescope", line 8, in <module> sys.exit(main()) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/celescope.py", line 54, in main args.func(args) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 81, in mkref runner() File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 36, in __call__ self.build_index() File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/tools/utils.py", line 45, in wrapper result = func(*args, **kwargs) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/site-packages/celescope/vdj/mkref.py", line 76, in build_index subprocess.check_call(f"makeblastdb -parse_seqids -dbtype nucl -in {out_file_name}.fa", shell=True) File "/bios-store1/home/logcoin/.conda/envs/Celescope/lib/python3.9/subprocess.py", line 373, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'makeblastdb -parse_seqids -dbtype nucl -in IGV.fa' returned non-zero exit status 1.

To Troubleshot I think the isse NO.1 might cause by the wrong format but have no idea about another issue, so I did some troubleshots:

  1. Format of one of the IMGT reference files showed as following:
    head -n 20 TRAJ.fasta
    trouble1

head -n 20 IGHD.fasta trouble3

  1. Check whether the output directory is null:
    ls -al ../IG/human_IG
    trouble2
    but the "TR" directory is null.
  2. And I finally check version of celescope
    Version
    celescope -v
    2.0.3

I sincerely expect some responses on resolution about these issues, or show me some normative format of IMGT data for constructing index file. Moreover, I hope that some detailed instance on how to construct index file about vdj or full length immune receptors sequencing. Last question I would like to raise: Whether the index files based on IMGT data between single cell vdj and full length immune receptor sequencing are coincident?

I found out the key of issue that there remained some undesired files when I mannually downloaded data from IMGT. For instance, redundant file named "TRGJ" in the directory Path/TR/ made constructing reference failure.
solution1

@Chenjunjie1996
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Our vdj pipeline focuses on alpha/beta TCR.
Using following command to download only alpha/beta TCR sequence from IMGT to avoid error when running celescope vdj mkref.

wget https://www.imgt.org/download/V-QUEST/IMGT_V-QUEST_reference_directory/Homo_sapiens/TR/TR{A,B}{V,J}.fasta

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@Chenjunjie1996 @Gethell