Resolved - R Shiny - Navigation containers expect a collection of

An error has occurred!

Navigation containers expect a collection of `bslib::nav_panel()`/`shiny::tabPanel()`s and/or `bslib::nav_menu()`/`shiny::navbarMenu()`s. Consider using `header` or `footer` if you wish to place content above (or below) every panel's contents.

Solution:

My current shiny version was 1.7.X, which gave me the above error. 

Downgrading shiny version to 1.6 solves the problem. I delete the shiny version 1.7.X version from the packages and installed 1.6 in R console using the below command:

remotes::install_version("shiny", "1.6.0")

When you deploy your Shiny app to a server or cloud service (e.g., shinyapps.io), the server environment typically uses the version of Shiny that's installed in that environment.

You don't explicitly specify the Shiny version when deploying; instead, the server environment determines which version of Shiny to use based on its package setup.

Resolved - Souporcell installation issues

 $ 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

Resolved - Issues with running Monocle 3

I converted my adata to rds format using sceasy first since I cannot use the anndata object directly in R, specifically monocle 3.

Issue 1: 

cds <- as.cell_data_set(sce)
Error in UseMethod(generic = "as.cell_data_set", object = x) : 
  no applicable method for 'as.cell_data_set' applied to an object of class "c('SingleCellExperiment', 'RangedSummarizedExperiment', 'SummarizedExperiment', 'RectangularData', 'Vector', 'Annotated', 'vector_OR_Vector')"

Solution : 

This was because my sce object was: 

> class(sce)
[1] "SingleCellExperiment"
attr(,"package")
[1] "SingleCellExperiment"

When I changed sce object as seurat object, this error was resolved: 

> class(sce)
[1] "Seurat"
attr(,"package")
[1] "SeuratObject"

Now, cds object looks like this:

> class(cds)

[1] "cell_data_set"
attr(,"package")
[1] "monocle3"

Issue 2: 

cds <- preprocess_cds(cds, num_dim = 100)
Error in names(sf) <- colnames(SingleCellExperiment::counts(cds)) : 
  attempt to set an attribute on NULL

Solution : source 

> ## Calculate size factors using built-in function in monocle3
> cds <- estimate_size_factors(cds)

QA on in Single cell data analysis

Question 1:

I have adata object which does not have layers with spliced and unspliced counts information. Is it even possible one of the kernels from cellrank2 without this information to find the cell fate transition? If yes for above, could you kindly advice what could be used alternative to the spl/unspl count layers?

Yes, we can use pseudotime instead of RNAvelocity to calculate cellfates and transitions.

In cases, where I am not sure of the root celltype - is it possible to use cellrank2?

Yes, using the following command we can:
adata.obsm['X_diffmap'][:, 3].argmax()

Single cell analysis - My short summaries collection

1. In single-cell RNA sequencing, mitochondrial (mt), ribosomal (ribo), and hemoglobin (Hb) gene expression levels serve as quality indicators:

• mt Genes: High levels indicate cell stress or damage. (Should be less 5-10%)
• ribo Genes: Elevated levels suggest RNA isolation or library preparation issues. (no standard cutoff)  
  
  
• Hb Genes: High expression in non-blood cells flags potential red blood cell contamination. (maybe 50% in RBC but no standard cutoff)

2. The choice between RNA velocity and pseudotime them depends on the specific research context and data quality, with RNA velocity requiring data on both spliced and unspliced transcripts, while pseudotime focuses on overall gene expression changes.