## ----include = FALSE----------------------------------------------------------
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>",
  tidy = TRUE,
  fig.width = 11,
  tidy.opts = list(width.cutoff = 95),
  message = FALSE,
  warning = FALSE,
  time_it = TRUE
)
all_times <- list()  # store the time for each chunk
knitr::knit_hooks$set(time_it = local({
  now <- NULL
  function(before, options) {
    if (before) {
      now <<- Sys.time()
    } else {
      res <- difftime(Sys.time(), now, units = "secs")
      all_times[[options$label]] <<- res
    }
  }
}))

## ----eval=FALSE---------------------------------------------------------------
# githubURL <- "https://github.com/feiyoung/PRECAST/blob/main/vignettes_data/bc2.rda?raw=true"
# download.file(githubURL,"bc2.rda",mode='wb')

## ----eval =  FALSE------------------------------------------------------------
# load("bc2.rda")

## ----eval=  FALSE-------------------------------------------------------------
# dir.file <- "Section" ## the folders Section1 and Section2, and each includes two folders spatial and filtered_feature_bc_matrix
# seuList <- list()
# for (r in 1:2) {
#   message("r = ", r)
#   seuList[[r]] <- DR.SC::read10XVisium(paste0(dir.file, r))
# }
# bc2 <- seuList

## ----eval =  FALSE------------------------------------------------------------
# library(PRECAST)
# library(Seurat)

## ----eval =  FALSE------------------------------------------------------------
# bc2 ## a list including two Seurat object

## ----eval =  FALSE------------------------------------------------------------
# head(bc2[[1]])

## ----eval= FALSE--------------------------------------------------------------
# ## Get the gene-by-spot read count matrices
# ## countList <- lapply(bc2, function(x) x[["RNA"]]@counts)
# countList <- lapply(bc2, function(x){
#   assay <- DefaultAssay(x)
#   GetAssayData(x, assay = assay, slot='counts')
# 
# } )
# 
# M <- length(countList)
# ## Get the meta data of each spot for each data batch
# metadataList <- lapply(bc2, function(x) x@meta.data)
# 
# for(r in 1:M){
#   meta_data <- metadataList[[r]]
#   all(c("row", "col") %in% colnames(meta_data)) ## the names are correct!
#   head(meta_data[,c("row", "col")])
# }
# 
# 
# ## ensure the row.names of metadata in metaList are the same as that of colnames count matrix in countList
# 
# for(r in 1:M){
#   row.names(metadataList[[r]]) <- colnames(countList[[r]])
# }
# 
# 
# ## Create the Seurat list  object
# 
# seuList <- list()
# for(r in 1:M){
#   seuList[[r]] <- CreateSeuratObject(counts = countList[[r]], meta.data=metadataList[[r]], project = "BreastCancerPRECAST")
# }
# 
# bc2 <- seuList
# rm(seuList)
# head(meta_data[,c("row", "col")])

## ----eval =  FALSE------------------------------------------------------------
# ## Create PRECASTObject.
# set.seed(2022)
# PRECASTObj <- CreatePRECASTObject(bc2, project = 'BC2', gene.number = 2000, selectGenesMethod = 'SPARK-X', premin.spots = 20,  premin.features=20, postmin.spots = 1, postmin.features = 10)
# 
# ## User can retain the raw seuList by the following commond.
# ##  PRECASTObj <-  CreatePRECASTObject(seuList, customGenelist=row.names(seuList[[1]]), rawData.preserve = TRUE)

## ----eval =  FALSE------------------------------------------------------------
# ## check the number of genes/features after filtering step
# PRECASTObj@seulist
# 
# ## seuList is null since the default value `rawData.preserve` is FALSE.
# PRECASTObj@seuList
# 
# ## Add adjacency matrix list for a PRECASTObj object to prepare for PRECAST model fitting.
# PRECASTObj <-  AddAdjList(PRECASTObj, platform = "Visium")
# 
# ## Add a model setting in advance for a PRECASTObj object. verbose =TRUE helps outputing the information in the algorithm.
# PRECASTObj <- AddParSetting(PRECASTObj, Sigma_equal=FALSE, verbose=TRUE, maxIter=30)
# 

## ----eval =  FALSE------------------------------------------------------------
# ### Given K
# PRECASTObj <- PRECAST(PRECASTObj, K=14)
# 

## ----eval =  FALSE------------------------------------------------------------
# ## backup the fitting results in resList
# resList <- PRECASTObj@resList
# PRECASTObj <- SelectModel(PRECASTObj)
# 

## ----eval =  FALSE------------------------------------------------------------
# print(PRECASTObj@seuList)
# seuInt <- IntegrateSpaData(PRECASTObj, species='Human')
# seuInt
# ## The low-dimensional embeddings obtained by PRECAST are saved in PRECAST reduction slot.

## ----eval =FALSE--------------------------------------------------------------
# ## assign the raw Seurat list object to it
# ## For illustration, we generate a new seuList with more genes;
# ## For integrating all genes, users can set `seuList <- bc2`.
# genes <- c(row.names(PRECASTObj@seulist[[1]]), row.names(bc2[[1]])[1:10])
# seuList <- lapply(bc2, function(x) x[genes,])
# PRECASTObj@seuList <- seuList #
# seuInt <- IntegrateSpaData(PRECASTObj, species='Human')
# seuInt

## ----eval =FALSE--------------------------------------------------------------
# PRECASTObj@seuList <- NULL
# ## At the same time, we can set subsampling to speed up the computation.
# seuInt <- IntegrateSpaData(PRECASTObj, species='Human', seuList=seuList, subsample_rate = 0.5)
# seuInt

## ----eval =  FALSE------------------------------------------------------------
# cols_cluster <- chooseColors(palettes_name = 'Classic 20', n_colors=14, plot_colors = FALSE)

## ----eval =  FALSE, fig.height = 4, fig.width=9-------------------------------
# 
# p12 <- SpaPlot(seuInt, item='cluster', batch=NULL,point_size=1, cols=cols_cluster, combine=TRUE, nrow.legend=7)
# p12
# # users can plot each sample by setting combine=FALSE

## ----eval =  FALSE, fig.height = 4, fig.width=7.5-----------------------------
# pList <- SpaPlot(seuInt, item='cluster', batch=NULL,point_size=1, cols=cols_cluster, combine=FALSE, nrow.legend=7)
# drawFigs(pList, layout.dim = c(1,2), common.legend = TRUE, legend.position = 'right', align='hv')
# 

## ----eval =  FALSE, fig.height = 4, fig.width=5.7-----------------------------
# seuInt <- AddUMAP(seuInt)
# p13 <- SpaPlot(seuInt, batch=NULL,item='RGB_UMAP',point_size=2, combine=TRUE, text_size=15)
# p13
# #seuInt <- AddTSNE(seuInt)
# #SpaPlot(seuInt, batch=NULL,item='RGB_TSNE',point_size=2, combine=T, text_size=15)

## ----eval =  FALSE, fig.height = 4.5, fig.width=12----------------------------
# seuInt <- AddTSNE(seuInt, n_comp = 2)
# p1 <- dimPlot(seuInt, item='cluster', point_size = 0.5, font_family='serif', cols=cols_cluster,border_col="gray10", nrow.legend=14, legend_pos='right') # Times New Roman
# p2 <- dimPlot(seuInt, item='batch', point_size = 0.5,  font_family='serif', legend_pos='right')
# 
# drawFigs(list(p1, p2), layout.dim = c(1,2), legend.position = 'right', align='hv')
# 

## ----eval =  FALSE------------------------------------------------------------
# library(Seurat)
# dat_deg <- FindAllMarkers(seuInt)
# library(dplyr)
# n <- 10
# dat_deg %>%
#   group_by(cluster) %>%
#   top_n(n = n, wt = avg_log2FC) -> top10
# 
# seuInt <- ScaleData(seuInt)
# seus <- subset(seuInt, downsample = 400)
# 
# 

## ----eval =  FALSE, fig.height = 8, fig.width=9-------------------------------
# color_id <- as.numeric(levels(Idents(seus)))
# library(ggplot2)
# ## HeatMap
# p1 <- doHeatmap(seus, features = top10$gene, cell_label= "Domain",
#                 grp_label = F, grp_color = cols_cluster[color_id],
#                 pt_size=6,slot = 'scale.data') +
#   theme(legend.text = element_text(size=10),
#         legend.title = element_text(size=13, face='bold'),
#         axis.text.y = element_text(size=5, face= "italic", family='serif'))
# p1

## -----------------------------------------------------------------------------
sessionInfo()