spSeudoMap: cell type mapping of spatial transcriptomics using unmatched single-cell RNA-seq data

Number of marker genes per cluster: 40 (>20)
m/k ratio = 2 (> 1)
pseudo_frac_m = average fraction of negative non-sorted population (literature evidence or cell sorting experiment)
pseudo_frac_std = 0.1 (> 0.05)
Number of pseudospots = 5 to 10 times the number of real spots (20,000~40,000 per Visium slide)
Number of sampled cells in a pseudospot (virtual mixture of single-cell data) = 8 (brain), 10 (breast cancer)
Iteration number = 3,000
Mini-batch size = 512
Loss weights between source and domain classifier (alpha) = 0.6
Learning rate = 0.001 * alpha_lr = 0.005

Python example: spSeudoMap_example.ipynb
R example: Refer to /vignettes/introduction.Rmd

spatial data: 'V1_Adult_Mouse_Brain_Coronal_Section_1' from 10X Genomics Repository

conda create -n spSeudoMap python=3.7.12 -c conda-forge  
conda activate spSeudoMap  
pip install git+https://github.com/bsungwoo/spSeudoMap.git  
python -m ipykernel install --user --name spSeudoMap --display-name spSeudoMap  

from spSeudoMap.pred_cellf_spSeudoMap import pred_cellf_spSeudoMap  
adata_sp = pred_cellf_spSeudoMap(adata_sp=None, adata_sc=None, count_from_raw=False,   
                                 gpu=True, celltype='cluster', num_markers=40,  
                                 mixture_mode='pseudotype', seed_num=0,  
                                 mk_ratio_fix=False, mk_ratio=2, pseudo_num_genes=40,  
                                 pseudo_frac_m=0.5, pseudo_frac_std=0.1, num_top_genes=20,  
                                 nmix=10, npseudo=20000, alpha=0.6, alpha_lr=5, emb_dim=64, 
                                 batch_size=512, n_iterations=3000, init_train_epoch=10, 
                                 outdir='./output', return_format='anndata')  

(1) adata_sp: spatial data (AnnData object) with raw count matrix to be used in predicting cell fraction (default: None)
(2) adata_sc: single-cell data (AnnData object) with raw count matrix to be used in making pseudospots (default: None)
(3) count_from_raw: whether to extract count matrix frow .raw of AnnData (default: False)
-> non-normalized raw count matrix should be contained in the AnnData .raw file
-> if False, then utilize the count matrices saved in adata_sp and adata_sc directly
(4) gpu: check whether to use gpu (True) or not (False) (default = True)
(5) celltype: column name for single-cell annotation data in .obs (default: 'celltype')
(6) num_markers: number of selected marker genes in each celltype (default = 40)
(7) mixture_mode: mode of the pseudospot generation ('default': same as CellDART, 'pseudotype': assuming exclusive cell type in spatial data)
(8) seed_num: seed to be used in random sampling (default = 0)
(9) mk_ratio_fix: whether to fix the mk_ratio when selecting the pseudotype markers (default = True)
(10) mk_ratio: ratio of number of single-cell markers to virtual pseudotype markers (default = 2)
(11) pseudo_frac_m: average of presumed fraction of the pseudotype (cell types exclusively present in spatial data) across all spatial spots (default = 0.5)
-> determined by cell sorting study or by literature based evidence that enables speculation of average negative non-sorted cell fraction in the tissue
(12) pseudo_frac_std: standard deviation of the distribution of presumed pseudotype fraction across all spatial spots (default = 0.1)
(13) num_top_genes: number of top genes having highest log fold change between spatial and single-cell normalized pseudobulk counts (spatial/single-cell) (default = 20)
(14) nmix: sampling number of cells in pseudospot (default = 10)
(15) npseudo: a total number of pseudospots (default = 20,000); approximately 5~10 times the number of pseudospots

(1) alpha: loss weights of the domain classifier to the source classifier (default = 0.6)
(2) alpha_lr: learning rate for the domain classifier (alpha_lr*0.001, default = 5)
(3) emb_dim: output size of dimensions for feature extractor (default = 64)
(4) batch_size: minibatch size for pseudospots and spatial data during the training (default = 512)
(5) n_iterations: iteration number for the adversarial learning (default = 3,000)
(6) init_train_epoch: iteration number for the pre-training process (default = 10)
(7) outdir: the directory to save output files (models and results)
(8) return_format: whether to return spatial AnnData file with predicted cell fraction in .obs (default: False)

devtools::install_github("bsungwoo/spSeudoMap", build_vignettes = T, force = T)  
library(spSeudoMap)  
help(pred_cellf_spSeudoMap) # Explanation for the parameters and short examples  
browseVignettes("spSeudoMap")  # Browse for the vignettes (/vignettes/introduction.Rmd)  

Linux distributions: The environment will be automatically installed by running the function
Windows: Install conda environment first and then run the function with env.select = 'conda' and python.install=F

Using conda environment (environment will be automatically installed in Linux distributions)
If using Windows, then install conda environment first and then run the function below with python.install = F
pseudo_frac_m <- 0.5 (average presumed pseudotype fraction in the tissue)
npseudo <- dim(sp_data)[2]*5
sp_data_cellf <- pred_cellf_spSeudoMap(sp_data, sc_data,
                                       outdir=output_folder_name,
                                       sp_subset=F, spot.cluster.name='seurat_clusters',
                                       spot.cluster.of.interest=NULL,
                                       env.select='conda', python.install=T,
                                       python_path=NULL, env.name='spSeudoMap',
                                       gpu=TRUE, metadata_celltype='annotation_1',
                                       num_markers=40, mixture_mode='pseudotype',
                                       seed_num=0,
                                       mk_ratio_fix=T, mk_ratio=4,
                                       pseudo_frac_m=pseudo_frac_m, pseudo_frac_std=0.1,
                                       nmix=8, npseudo=nspeudo, alpha=0.6, alpha_lr=5,
                                       emb_dim=64, batch_size=512, n_iterations=3000, init_train_epoch=10)

 Using virtual environment (environment will be automatically installed in Linux distributions)
 Not recommended for Windows
 pseudo_frac_m <- 0.5 (average presumed pseudotype fraction in the tissue)
 npseudo <- dim(sp_data)[2]*5
 sp_data_cellf <- pred_cellf_spSeudoMap(sp_data, sc_data,
                                        outdir=output_folder_name,
                                        sp_subset=F, spot.cluster.name='seurat_clusters',
                                        spot.cluster.of.interest=NULL,
                                        env.select='virtual', python.install=T,
                                        python_path=NULL, env.name='spSeudoMap',
                                        gpu=TRUE, metadata_celltype='annotation_1',
                                        num_markers=40, mixture_mode='pseudotype',
                                        seed_num=0,
                                        mk_ratio_fix=T, mk_ratio=4,
                                        pseudo_frac_m=pseudo_frac_m, pseudo_frac_std=0.1,
                                        nmix=8, npseudo=nspeudo, alpha=0.6, alpha_lr=5,
                                        emb_dim=64, batch_size=512, n_iterations=3000, init_train_epoch=10)

(1) outdir: the directory to save output files (models and results) (default = '.')
(2) sp_subset: whether to subset spatial data and calculate for specific spot cluster (default = FALSE)
(3) spot.cluster.name: group name of the cluster used for subsetting spatial data (default = 'seurat_clusters')
(4) spot.cluster.of.interest: name of each spot clusters to be used (default = NULL)
(5) env.select: select between using reticulate virtual environment or conda environment (default = 'conda')
-> either of the selection will search the already installed environment
-> if environment is not found, then it will automatically install the new environment
(6) python.install: whether to automatically install python version 3.7.12 (default = F)
-> For Windows, set python.install = F
(7) python_path: path for the python 3.7.12 (default = NULL)
(8) env.name: name of the virtual or conda environment to use for the analysis (default = 'spSeudoMap')
(9) metadata_celltype: column name for single-cell annotation data in metadata (default = 'celltype')

GLIBCXX_3.4.26 should be already installed
Check if it is installed: strings /usr/lib/x86_64-linux-gnu/libstdc++.so.6 | grep GLIBCXX
reference: https://stackoverflow.com/questions/63190229/glibcxx-3-4-26-not-found-running-cross-complied-program-on-beaglebone

sudo apt install wget gcc-8 unzip libssl1.0.0 software-properties-common  
sudo add-apt-repository ppa:ubuntu-toolchain-r/test  
sudo apt-get install --only-upgrade libstdc++6