# FAST: single DLPFC section

#### Wei Liu

#### 2024-03-18

This vignette introduces the FAST workflow for the analysis of single-section rather than multi-secitons data, one humn dorsolateral prefrontal cortex (DLPFC) spatial transcriptomics dataset. In this vignette, the workflow of FAST consists of three steps

- Independent preprocessing
- Spatial dimension reduction using FAST
- Downstream analysis (i.e. , embedding alignment and spatial clustering using iSC-MEB, visualization of clusters and embeddings, remove unwanted variation, differential expression analysis)

We demonstrate the use of FAST to one DLPFC Visium data that are here, which can be downloaded to the current working path by the following command:

    githubURL <- "https://github.com/feiyoung/FAST/blob/main/vignettes_data/seulist2_ID9_10.RDS?raw=true"
    download.file(githubURL,"seulist2_ID9_10.RDS",mode='wb')

Then load to R. Here, we only focus one section.

    dlpfc2 <- readRDS("./seulist2_ID9_10.RDS")
    dlpfc <- dlpfc2[[1]]

The package can be loaded with the command:

    library(ProFAST) # load the package of FAST method
    library(PRECAST)
    library(Seurat)

## View the DLPFC data

First, we view the the spatial transcriptomics data with Visium platform. There are ~15000 genes and ~3600 spots.

    dlpfc ## a list including three Seurat object with default assay: RNA

We observed that the genes are Ensembl IDs. In the following, we will transfer the Ensembl IDs to gene symbols for matching the housekeeping genes in the downstream analysis for removing the unwanted variations.

    print(row.names(dlpfc)[1:10])
    count <- dlpfc[['RNA']]@counts
    row.names(count) <- unname(transferGeneNames(row.names(count), now_name = "ensembl",
                                                     to_name="symbol",
                                                     species="Human", Method='eg.db'))
    print(row.names(count)[1:10])
    seu <- CreateSeuratObject(counts = count, meta.data = dlpfc@meta.data)
    seu

## Preprocessing

We show how to preprocessing before fitting FAST, including log-normalization (if user use the gaussian version of FAST), and select highly variable genes.

- Note: the spatial coordinates must be contained in the meta data and named as `row` and `col`, which benefits the identification of spaital coordinates by FAST.

    ## Check the spatial coordinates: they are named as "row" and "col"!
    print(head(seu@meta.data))

    seu <- NormalizeData(seu)
    seu <- FindVariableFeatures(seu)
    print(seu)
    print(seu[['RNA']]@var.features[1:10])

Find spatially variable genes
Users can also use the spatially variable genes by the following command:

    set.seed(2023)
    seu <- DR.SC::FindSVGs(seu)
    ### Check the results
    print(seu[['RNA']]@var.features[1:10])

## Fit FAST for this data

For function `FAST_single`, users can specify the number of factors `q` and the fitted model `fit.model`. The `q` sets the number of spatial factors to be extracted, and a lareger one means more information to be extracted but higher computaional cost. The `fit.model` specifies the version of FAST to be fitted. The Gaussian version (`gaussian`) models the log-normalized matrix while the Poisson verion (`poisson`) models the count matrix; default as `poisson`. (Note: The computational time required to run the analysis on personal PCs is approximately ~0.5 minute on a personal PC.)

    Adj_sp <- AddAdj(as.matrix(seu@meta.data[,c("row", "col")]), platform = "Visium")
    ### set q= 15 here
    set.seed(2023)
    seu <- FAST_single(seu, Adj_sp=Adj_sp, q= 15, fit.model='poisson')
    seu

Run possion version
Users can also use the gaussian version by the following command:

    Adj_sp <- AddAdj(as.matrix(seu@meta.data[,c("row", "col")]), platform = "Visium")
    set.seed(2023)
    seu <- FAST_single(dlpfc, Adj_sp=Adj_sp, q= 15, fit.model='gaussian')
    ### Check the results
    seu

### Evaluate the adjusted McFadden’s pseudo R-square

Next, we investigate the performance of dimension reduction by calculating the adjusted McFadden’s pseudo R-square. The manual annotations are regarded as the ground truth in the `meta.data` of `seu`.

    ## Obtain the true labels
    y <- seu$layer_guess_reordered
    ### Evaluate the MacR2
    Mac <-  get_r2_mcfadden(Embeddings(seu, reduction='fast'), y)
    ### output them
    print(paste0("MacFadden's R-square of FAST is ", round(Mac, 3)))

## Clustering analysis based on FAST embedding

Based on the embeddings from FAST, we use `Louvain` to perform clustering. In this downstream analysis, other methods for clustering can be also used.

    seu <- FindNeighbors(seu, reduction = 'fast')
    seu <- FindClusters(seu, resolution = 0.4)
    seu$fast.cluster <- seu$seurat_clusters
    ARI.fast <- mclust::adjustedRandIndex(y, seu$fast.cluster)
    print(paste0("ARI of PCA is ", round(ARI.fast, 3)))

For comparison, we also run PCA to obtain PCA embeddings, and then conduct louvain clustering.

    seu <- ScaleData(seu)
    seu <- RunPCA(seu, npcs=15, verbose=FALSE)
    Mac.pca <- get_r2_mcfadden(Embeddings(seu, reduction='pca'), y)
    print(paste0("MacFadden's R-square of PCA is ", round(Mac.pca, 3)))
    set.seed(1)
    seu <- FindNeighbors(seu, reduction = 'pca', graph.name ="pca.graph")
    seu <- FindClusters(seu, resolution = 0.8,graph.name = 'pca.graph')
    seu$pca.cluster <- seu$seurat_clusters
    ARI.pca <- mclust::adjustedRandIndex(y, seu$pca.cluster)
    print(paste0("ARI of PCA is ", round(ARI.pca, 3)))

## Visualization

First, user can choose a beautiful color schema using `chooseColors()` in the R package `PRECAST`.

    cols_cluster <- chooseColors(palettes_name = "Nature 10", n_colors = 8, plot_colors = TRUE)

Then, we plot the spatial scatter plot for clusters using the function `DimPlot()` in the R package `Seurat`. We observe that the clusters from PCA are more messy while the clusters from FAST are more smoothing in spatial coordinates.

    seu <- PRECAST::Add_embed(embed = as.matrix(seu@meta.data[,c("row", "col")]), seu, embed_name = 'Spatial')
    seu
    p1 <- DimPlot(seu, reduction = 'Spatial', group.by = 'pca.cluster',cols = cols_cluster, pt.size = 1.5)
    p2 <- DimPlot(seu, reduction = 'Spatial', group.by = 'fast.cluster',cols = cols_cluster, pt.size = 1.5)
    drawFigs(list(p1, p2),layout.dim = c(1,2) )

Next, we visualize the clusters from `FAST` on the UMAP space, and observe the clusters are well separated in general.

    seu <- RunUMAP(seu, reduction = "fast", dims=1:15)
    seu
    DimPlot(seu, reduction='umap', group.by = "fast.cluster")

## Differential epxression analysis

Finally, we condut the differential expression (DE) analysis. The function `FindAllMarkers()` in the `Seurat` R package is ued to achieve this analysis. And we extract the top five DE genes.

    Idents(seu) <- seu$fast.cluster
    dat_deg <- FindAllMarkers(seu)
    library(dplyr)
    n <- 5
    dat_deg %>%
        group_by(cluster) %>%
        top_n(n = n, wt = avg_log2FC) -> top5
    top5

Session Info

    sessionInfo()
    #> R version 4.2.1 (2022-06-23 ucrt)
    #> Platform: x86_64-w64-mingw32/x64 (64-bit)
    #> Running under: Windows 10 x64 (build 22621)
    #>
    #> Matrix products: default
    #>
    #> locale:
    #> [1] LC_COLLATE=C
    #> [2] LC_CTYPE=Chinese (Simplified)_China.utf8
    #> [3] LC_MONETARY=Chinese (Simplified)_China.utf8
    #> [4] LC_NUMERIC=C
    #> [5] LC_TIME=Chinese (Simplified)_China.utf8
    #>
    #> attached base packages:
    #> [1] stats     graphics  grDevices utils     datasets  methods   base
    #>
    #> loaded via a namespace (and not attached):
    #>  [1] digest_0.6.33   R6_2.5.1        jsonlite_1.8.7  evaluate_0.21
    #>  [5] cachem_1.0.8    rlang_1.1.1     cli_3.4.1       rstudioapi_0.14
    #>  [9] jquerylib_0.1.4 bslib_0.5.0     rmarkdown_2.23  tools_4.2.1
    #> [13] xfun_0.39       yaml_2.3.7      fastmap_1.1.1   compiler_4.2.1
    #> [17] htmltools_0.5.5 knitr_1.43      sass_0.4.7