Welcome to CeleScope Discussions!

[zhouyiqi91]

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[mehranfazel]

Hi,

I use Telescope to analyze single cell RNA data generated by Singleron.
I want to extract the barcodes with following command but I receive this error.
$ celescope rna barcode --fq1 /prj/ssc-ssrna-seq/row_data/example_1.fastq.gz --fq2 /prj/ssc-ssrna-seq/row_data/Example_2.fastq.gz --outdir /prj/ssc-ssrna-seq/countTable --sample example

-- celescope.tools.step.get_slot_key - WARNING - Assay not found in sample_summary.metrics
Traceback (most recent call last):
File "/homes/mfazel/mambaforge/envs/celescope/bin/celescope", line 8, in
sys.exit(main())
...

i think its related with the --sample Attribute.
can you please help me with this error?

thanks!

[zhouyiqi91]

You need to run sample before barcode. The barcode step needs the .metrics.json file generated with the sample step.

[Acet221]

Hi,

I'm kind of new to bioinformatics and I wanted to try out CeleScope according to the user guide.
I have noticed that some packages in conda_pkgs.txt are only available for Linux/Mac, so I'm wondering if the whole workflow is designed to be done in Linux OS only, or if it might also be possible to run in Windows by replacing some packages for alternatives?

[zhouyiqi91]

CeleScope only runs on Linux systems.
For example, CeleScope relys heavily on STAR which can not be run on Windows. alexdobin/STAR#1428

[amarinderthind]

Hi, How to install it on public HPC system, with no conda command available?

[amarinderthind]

Thanks for that. Maenwhile, Wondering if you know something about this error that I encountered with standard installation (mamba;pip) on my desktop after pip installation command;

"..SOme_PATH/miniconda3/envs/celescope/lib/python3.9/subprocess.py", line 524, in run
          raise CalledProcessError(retcode, process.args,
      subprocess.CalledProcessError: Command '['make', '-s', 'print-config']' returned non-zero exit status 2.
      [end of output]
  
  note: This error originates from a subprocess, and is likely not a problem with pip.
error: subprocess-exited-with-error

× Getting requirements to build wheel did not run successfully.
│ exit code: 1
╰─> See above for output.
note: This error originates from a subprocess, and is likely not a problem with pip.

[zhouyiqi91]

This error message is incomplete and does not show the specific error. Please attach the command and the full log.

[amarinderthind]

Full log `pip install celescope
Collecting celescope
Using cached celescope-2.0.7-py3-none-any.whl.metadata (1.6 kB)
Collecting cutadapt==3.7 (from celescope)
Using cached cutadapt-3.7-cp39-cp39-manylinux_2_5_x86_64.manylinux1_x86_64.manylinux_2_12_x86_64.manylinux2010_x86_64.whl (166 kB)
Collecting pysam>=0.18.0 (from celescope)
Using cached pysam-0.22.0.tar.gz (4.6 MB)
Installing build dependencies ... done
Getting requirements to build wheel ... error
error: subprocess-exited-with-error

× Getting requirements to build wheel did not run successfully.
│ exit code: 1
╰─> [42 lines of output]
# pysam: cython is available - using cythonize if necessary
# pysam: htslib mode is shared
# pysam: HTSLIB_CONFIGURE_OPTIONS=None
# pysam: (sysconfig) CC=gcc -pthread -B /home/asthind/miniforge3/envs/celescope/compiler_compat -Wl,--sysroot=/
# pysam: (sysconfig) CFLAGS=-Wno-unused-result -Wsign-compare -DNDEBUG -fwrapv -O2 -Wall -fPIC -O2 -isystem /home/asthind/miniforge3/envs/celescope/include -fPIC -O2 -isystem /home/asthind/miniforge3/envs/celescope/include -fPIC
# pysam: (sysconfig) LDFLAGS=-Wl,-rpath,/home/asthind/miniforge3/envs/celescope/lib -Wl,-rpath-link,/home/asthind/miniforge3/envs/celescope/lib -L/home/asthind/miniforge3/envs/celescope/lib -Wl,-rpath,/home/asthind/miniforge3/envs/celescope/lib -Wl,-rpath-link,/home/asthind/miniforge3/envs/celescope/lib -L/home/asthind/miniforge3/envs/celescope/lib
checking for gcc... gcc -pthread -B /home/asthind/miniforge3/envs/celescope/compiler_compat -Wl,--sysroot=/
checking whether the C compiler works... no
configure: error: in /tmp/pip-install-4ks3wrcb/pysam_9da6d68ab7ce426fbd0a81f760f6963a/htslib': configure: error: C compiler cannot create executables See config.log' for more details
checking for gcc... gcc -pthread -B /home/asthind/miniforge3/envs/celescope/compiler_compat -Wl,--sysroot=/
checking whether the C compiler works... no
configure: error: in /tmp/pip-install-4ks3wrcb/pysam_9da6d68ab7ce426fbd0a81f760f6963a/htslib': configure: error: C compiler cannot create executables See config.log' for more details
Makefile:142: htscodecs.mk: No such file or directory
/bin/sh: 1: ./hts_probe_cc.sh: not found
make: *** [htscodecs.mk] Error 127
# pysam: htslib configure options: None
Traceback (most recent call last):
File "/home/asthind/miniforge3/envs/celescope/lib/python3.9/site-packages/pip/_vendor/pyproject_hooks/_in_process/_in_process.py", line 353, in
main()
File "/home/asthind/miniforge3/envs/celescope/lib/python3.9/site-packages/pip/_vendor/pyproject_hooks/_in_process/_in_process.py", line 335, in main
json_out['return_val'] = hook(**hook_input['kwargs'])
File "/home/asthind/miniforge3/envs/celescope/lib/python3.9/site-packages/pip/_vendor/pyproject_hooks/_in_process/_in_process.py", line 118, in get_requires_for_build_wheel
return hook(config_settings)
File "/tmp/pip-build-env-xuzqycgb/overlay/lib/python3.9/site-packages/setuptools/build_meta.py", line 325, in get_requires_for_build_wheel
return self._get_build_requires(config_settings, requirements=['wheel'])
File "/tmp/pip-build-env-xuzqycgb/overlay/lib/python3.9/site-packages/setuptools/build_meta.py", line 295, in _get_build_requires
self.run_setup()
File "/tmp/pip-build-env-xuzqycgb/overlay/lib/python3.9/site-packages/setuptools/build_meta.py", line 480, in run_setup
super(_BuildMetaLegacyBackend, self).run_setup(setup_script=setup_script)
File "/tmp/pip-build-env-xuzqycgb/overlay/lib/python3.9/site-packages/setuptools/build_meta.py", line 311, in run_setup
exec(code, locals())
File "", line 442, in
File "", line 82, in run_make_print_config
File "/home/asthind/miniforge3/envs/celescope/lib/python3.9/subprocess.py", line 420, in check_output
return run(*popenargs, stdout=PIPE, timeout=timeout, check=True,
File "/home/asthind/miniforge3/envs/celescope/lib/python3.9/subprocess.py", line 524, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['make', '-s', 'print-config']' returned non-zero exit status 2.
[end of output]

note: This error originates from a subprocess, and is likely not a problem with pip.
error: subprocess-exited-with-error

× Getting requirements to build wheel did not run successfully.
│ exit code: 1
╰─> See above for output.

note: This error originates from a subprocess, and is likely not a problem with pip.
`

[zhouyiqi91]

It may be related to the gcc you were using. velocyto-team/velocyto.py#88

I think it can be fixed by installing pysam with mamba instead of pip
mamba install -c bioconda pysam
then
pip install celescope

[amarinderthind]

Thanks, seems conda install numpy scipy cython numba matplotlib scikit-learn h5py click worked.

[amarinderthind]

Hi, I was looking for information about index files, I got for each sample in context to scRNAseq. Do I need to provide this as an input somewhere? I could not find this info here https://github.com/singleron-RD/CeleScope/blob/master/doc/assay/multi_rna.md

[zhouyiqi91]

If your data is already demultiplexed (you have separate R1.fastq and R2.fastq files for each sample), then you don't really need it.
https://bioinformatics.stackexchange.com/questions/5178/what-is-the-index-fastq-file-sample-i-fastq-gz-generated-when-demultiplexing

[amarinderthind]

Great Got you. Thanks

[amarinderthind]

Where can I find the complete documentation for Celescope? I've received the final report, which includes two folders in the output—'raw' and 'filtered.' I'm unsure about which input to use for Seurat (raw or filtered). I couldn't find any documentation that explains the output details comprehensively, including the default filters applied.

Additionally, I'm curious about the tool or method Celescope uses for clustering cell types. When I use filtered data as input in Seurat, I observe different clusters than those generated by Celescope. Can you provide insights into this discrepancy?"

[zhouyiqi91]

The filter matrix is used for downstream analysis:
https://github.com/singleron-RD/CeleScope/blob/master/doc/assay/multi_rna.md#main-output
Since CeleScope uses Starsolo to generate matrices, you can refer to the Starsolo documentation to learn about the filtering algorithm:
https://github.com/alexdobin/STAR/blob/master/docs/STARsolo.md#emptydrop-like-filtering

CeleScope uses Scanpy to do the downstream analysis which may use different paramaters as what you use in Seurat. For example, the default resolution used in CeleScope(Scanpy) is 1.2.

[amarinderthind]

Thanks

[daniquebax]

I am running some DynaSCOPE samples and I wanted to get familiar with the celescope analysis.
So I tried to run the test data, but I accounted some problems.

I first downloaded and installed the conda packages like on this site
https://github.com/singleron-RD/CeleScope/blob/master/doc/user_guide.md

Then I downloaded all the data and scripts like this

1. Get test data and scripts.
mkdir test_dir
cd test_dir
git clone https://github.com/singleron-RD/celescope_test_data.git
git clone https://github.com/singleron-RD/celescope_test_script.git

Then I generated the mouse genome like this:

Mus musculus
mkdir mmu_ensembl_99
cd mmu_ensembl_99

wget ftp://ftp.ensembl.org/pub/release-99/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
wget ftp://ftp.ensembl.org/pub/release-99/gtf/mus_musculus/Mus_musculus.GRCm38.99.gtf.gz

gunzip Mus_musculus.GRCm38.dna.primary_assembly.fa.gz 
gunzip Mus_musculus.GRCm38.99.gtf.gz

conda activate celescope
celescope utils mkgtf Mus_musculus.GRCm38.99.gtf Mus_musculus.GRCm38.99.filtered.gtf

celescope rna mkref \
--genome_name Mus_musculus_ensembl_99_filtered \
--fasta Mus_musculus.GRCm38.dna.primary_assembly.fa \
--gtf Mus_musculus.GRCm38.99.filtered.gtf \
--mt_gene_list mt_gene_list.txt

Then I modified arguments in the dynaseq script to my mouse genomeDir path like this

multi_dynaseq \
    --mapfile ./case.mapfile \
    --genomeDir /scratch/dbax/CeleScope/mmu_ensembl_99 \
    --mod shell

Then I ran the test like this

cd /scratch/dbax/CeleScope/test_dir/celescope_test_script
(celescope)dbax@cn106:/scratch/dbax/CeleScope/test_dir/celescope_test_script$ python -m pytest -s test_multi.py --assays dynaseq

Sadly this is the point where I get an error message. I looked at the log file (attached 1) and it seems to be a problem that it cannot find the mt_gene_list and gtf files. However, when I go to my genomeDIR I do think that I see these files (attached 2)
dynaseq.log

Do you know how I can fix this problem? I have limited bioinformatics experience, so it is difficult for me to logically describe my problem to you. I hope you understand my question.

[daniquebax]

Thanks for the quick reply!

The celescope_genome.config does indeed not include that data:

[files]
fasta = EBV_genome.fasta

[meta]
genome_name = EBV
genome_type = virus
celescope_version = 2.0.7
STAR_param =

[daniquebax]

I tried it again from scratch and now it works! :)

[zhouyiqi91]

Good to know. The previous celescope_genome.config corresponds to the EBV virus genome. By default there is no mt_gene_list and gtf.

[daniquebax]

Hi!

I hope you don't mind, but I have another question. I tried to run the pipeline now with my own data. I sequenced my DynaSCOPE libraries wich looked good on bioanalyzer and concentration. Multiple samples were pooled on the sequencing flowcell. So afterwards I demultiplexed the data with the adater sequences from the DynaSCOPE manual (I used cellranger which is uses on the bcl2fastq method from illumina).
However, I when I try to run the dynaseq pipeline I get an error and I think the main problem is that it says that my R1 is too short: GNGGATTGACGTTGATGTTGGTCTCTGAGTAGTCGACTTCGTATTAGCTTTGTAGGGCT. But its around 55 bp which is expected from the 2x 50 bp illumina kit?
Do you know a way to make this work?
error DynaSCOPE.txt

[zhouyiqi91]

DynaSCOPE requires at least 72bp in R1 read to work. Below is the library structure:
5' - read1(adapter) - bc1 - linker1 - bc2 - linker2 - bc3 - C - UMI - polyT - insert - TSO - 3'
bc1=bc2=bc3=9bp
linker1=linker2=16bp
UMI=12bp
9 * 3+16 * 2+12+1 = 72