---
title: "Single Cell Analysis"
author:
- Max Mattessich
- Joaquin Reyna
- Edel Aron
- Anna Konstorum
date: "Compiled: `r format(Sys.time(), '%B %d, %Y')`"
output:
  BiocStyle::html_document:
    dev: 'jpeg'
    df_print: paged
    fig_retina: 1
    number_sections: FALSE
    toc_depth: 4
    toc_float: TRUE
vignette: >
  %\VignetteIndexEntry{Single Cell Analysis}
  %\VignetteEncoding{UTF-8}
  %\VignetteEngine{knitr::rmarkdown}
---

```{r setup, include = FALSE}
knitr::opts_chunk$set(echo = TRUE)
```

# Introduction

In this vignette, we will cover some of the possible sources for publicly
available single-cell data, how to format it for and process it with MCIA and
how to run a basic analysis in order to annotate the data and use the results
as metadata for exploring the decomposition results.

![Vignette 2 Pipelines by Major Section](https://github.com/Muunraker/nipalsMCIA/releases/download/v0.99.7/Vignette-2-Pipeline.png)

*The lines for the data_sc_sce.Rda are dotted because we do not provide this object below; however, the code describing how to generate it is still included.*

## Installation

```{r installation-github, eval = FALSE}
# install.packages("devtools")
devtools::install_github("Muunraker/nipalsMCIA", ref = "code-development",
                         force = TRUE, build_vignettes = TRUE) # devel version
```

```{r installation-bioconductor, eval = FALSE}
# after acceptance
# install.packages("BiocManager")
BiocManager::install("nipalsMCIA")
```

```{r load-packages, message = FALSE}
# note that the TENxPBMCData package is not included in this list as you may
# decide to pull data from another source or use our provided objects

library(dplyr)
library(ggplot2)
library(ggpubr)
library(nipalsMCIA)
library(piggyback)
library(Seurat)

# NIPALS starts with a random vector
set.seed(42)
```

```{r set-paths}
# if you would like to save any of the data loaded/created locally
path_data <- file.path("..", "data")

# recommended location for external data
path_inst <- tempdir()
```

# Data

We will be using the 10x Genomics "pbmc5k-CITEseq" dataset, which was published in May 2019 and processed using Cell Ranger 3.0.2. It contains 5,247 detected cells from PBMCs and 33,538 genes along with 32 cell surface markers. We chose this dataset due to it containing both gene expression (GEX) and cell surface protein (ADT) data as well as being relatively recent, publicly available and from a widely used platform.

The following code details several ways in which to load in this dataset. You may prefer to use data from other sources outside of 10x Genomics, in which case you will have to format it to work with nipalsMCIA.

## All Sources

We have the objects described below available in data files which you can download and load in here if you do not want to run the following sections. **These files includes the processed object that you will need in order to run MCIA (data_blocks_sc.Rda).**

```{r data-all-list}
# list all of the currently available files in the latest release
piggyback::pb_list(repo = "Muunraker/nipalsMCIA", tag = "latest")
```

```{r data-all-download}
# specify `tag = ` to use a different release other than latest

# files needed for running MCIA
piggyback::pb_download(file = "metadata_sc.csv",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")
piggyback::pb_download(file = "data_blocks_sc.Rda",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")

# MCIA results
piggyback::pb_download(file = "mcia_results_sc.Rds",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")

# marker genes for cell type annotation with Seurat
piggyback::pb_download(file = "marker_genes.csv",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")

# the Seurat data's metric summary file from 10x Genomics
piggyback::pb_download(file = "5k_pbmc_protein_v3_metrics_summary.csv",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")

# Seurat objects in different stages of processing
piggyback::pb_download(file = "tenx_pbmc5k_CITEseq_raw.rds",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")
piggyback::pb_download(file = "tenx_pbmc5k_CITEseq_annotated.rds",
                       dest = path_inst, repo = "Muunraker/nipalsMCIA")
```

## Bioconductor

There are several Bioconductor packages which provide single-cell data for users
as part of the package. The [TENxPBMCData](https://bioconductor.org/packages/3.15/data/experiment/html/TENxPBMCData.html) contains 9 different publicly available datasets from 10x Genomics (stored as [SingleCellExperiment](https://bioconductor.org/packages/release/bioc/html/SingleCellExperiment.html) classes), including the one that we will be analyzing.

Note that the CITE-Seq information is being stored as an "[alternative experiment](https://bioconductor.org/books/3.16/OSCA.intro/the-singlecellexperiment-class.html#alternative-experiments)" within the SingleCellExperiment object. Both the GEX (`"mainExpName: Gene Expression"`) and ADT (`"altExpNames(1): Antibody Capture"`) data were stored in the DelayedMatrix format since they are so large. In this object, the rows represent the features and the columns represent the cells.

```{r data-bioconductor-load, eval = FALSE}
# read in the data as a SingleCellExperiment object
tenx_pbmc3k <- TENxPBMCData::TENxPBMCData(dataset = "pbmc5k-CITEseq")

# examine the data
tenx_pbmc3k
## class: SingleCellExperiment
## dim: 33538 5247
## metadata(0):
## assays(1): counts
## rownames(33538): ENSG00000243485 ENSG00000237613 ... ENSG00000277475 ENSG00000268674
## rowData names(4): ENSEMBL_ID Symbol_TENx Type Symbol
## colnames: NULL
## colData names(11): Sample Barcode ... Individual Date_published
## reducedDimNames(0):
## mainExpName: Gene Expression
## altExpNames(1): Antibody Capture

counts(tenx_pbmc3k)
## <33538 x 5247> sparse matrix of class DelayedMatrix and type "integer":
##                    [, 1]    [, 2]    [, 3]    [, 4] ... [, 5244] [, 5245] [, 5246] [, 5247]
## ENSG00000243485       0       0       0       0   .       0       0       0       0
## ENSG00000237613       0       0       0       0   .       0       0       0       0
## ENSG00000186092       0       0       0       0   .       0       0       0       0
## ENSG00000238009       0       0       0       0   .       0       0       0       0
## ENSG00000239945       0       0       0       0   .       0       0       0       0
##             ...       .       .       .       .   .       .       .       .       .
## ENSG00000277856       0       0       0       0   .       0       0       0       0
## ENSG00000275063       0       0       0       0   .       0       0       0       0
## ENSG00000271254       0       0       0       0   .       0       0       0       0
## ENSG00000277475       0       0       0       0   .       0       0       0       0
## ENSG00000268674       0       0       0       0   .       0       0       0       0

counts(altExp(tenx_pbmc3k))
## <32 x 5247> sparse matrix of class DelayedMatrix and type "integer":
##           [, 1]    [, 2]    [, 3]    [, 4] ... [, 5244] [, 5245] [, 5246] [, 5247]
##    CD3      25     959     942     802   .     402     401       6    1773
##    CD4     164     720    1647    1666   .    1417       1      46    1903
##   CD8a      16       8      21       5   .       8     222       3       9
##  CD11b    3011      12      11      11   .      15       7    1027       9
##   CD14     696      12      13       9   .       9      17     382       8
##    ...       .       .       .       .   .       .       .       .       .
## HLA-DR     573      15      11      19   .       6      40     184      32
##  TIGIT      10       3       3       3   .       2      15       1      12
##   IgG1       4       4       2       4   .       1       0       2       4
##  IgG2a       1       3       0       6   .       4       0       4       2
##  IgG2b       6       2       4       8   .       0       0       2       5

# examine the metadata:
head(colData(tenx_pbmc3k), n = 3)
## DataFrame with 6 rows and 11 columns
##           Sample            Barcode         Sequence   Library Cell_ranger_version Tissue_status Barcode_type
##      <character>        <character>      <character> <integer>         <character>   <character>  <character>
## 1 pbmc5k-CITEseq AAACCCAAGAGACAAG-1 AAACCCAAGAGACAAG         1              v3.0.2            NA     Chromium
## 2 pbmc5k-CITEseq AAACCCAAGGCCTAGA-1 AAACCCAAGGCCTAGA         1              v3.0.2            NA     Chromium
## 3 pbmc5k-CITEseq AAACCCAGTCGTGCCA-1 AAACCCAGTCGTGCCA         1              v3.0.2            NA     Chromium
##     Chemistry Sequence_platform   Individual Date_published
##   <character>       <character>  <character>    <character>
## 1 Chromium_v3           NovaSeq HealthyDonor     2019-05-29
## 2 Chromium_v3           NovaSeq HealthyDonor     2019-05-29
## 3 Chromium_v3           NovaSeq HealthyDonor     2019-05-29

head(rowData(tenx_pbmc3k), n = 3)
## DataFrame with 6 rows and 4 columns
##                      ENSEMBL_ID Symbol_TENx            Type       Symbol
##                     <character> <character>     <character>  <character>
## ENSG00000243485 ENSG00000243485 MIR1302-2HG Gene Expression           NA
## ENSG00000237613 ENSG00000237613     FAM138A Gene Expression      FAM138A
## ENSG00000186092 ENSG00000186092       OR4F5 Gene Expression        OR4F5

metadata(tenx_pbmc3k)
## list()

# change the gene names from Ensembl IDs to the 10x genes
rownames(tenx_pbmc3k) <- rowData(tenx_pbmc3k)$Symbol_TENx
```

In order to run MCIA, the format of this data must be slightly modified:

```{r data-bioconductor-formatting, eval = FALSE}
# set up the list
data_blocks_sc_sce <- list()
data_blocks_sc_sce$mrna <- data.frame(as.matrix(counts(tenx_pbmc3k)))
data_blocks_sc_sce$adt <- data.frame(as.matrix(counts(altExp(tenx_pbmc3k))))

summary(data_blocks_sc_sce)
##      Length Class      Mode
## mrna 5247   data.frame list
## adt  5247   data.frame list

# convert to a Seurat object (using `as.Seurat` won't work here)
obj_sce <- CreateSeuratObject(counts = data_blocks_sc_sce$mrna, # assay = "RNA"
                              project = "pbmc5k_CITEseq")
obj_sce[["ADT"]] <- CreateAssayObject(counts = data_blocks_sc_sce$adt)

# name the cells with their barcodes
obj_sce <- RenameCells(object = obj_sce,
                       new.names = colData(tenx_pbmc3k)$Sequence)

# add metadata from the SingleCellExperiment object
obj_sce <- AddMetaData(object = obj_sce,
                       metadata = as.data.frame(colData(tenx_pbmc3k),
                                                row.names = Cells(obj_sce)))

# this object will be slightly different than from the Seurat one down below
# e.g. 5297 rows vs. 4193 rows (since QC wasn't done) and different metadata

head(obj_sce[[]], n = 3)
##                     orig.ident nCount_RNA nFeature_RNA nCount_ADT nFeature_ADT         Sample            Barcode
## AAACCCAAGAGACAAG SeuratProject       7375         2363       5178           31 pbmc5k-CITEseq AAACCCAAGAGACAAG-1
## AAACCCAAGGCCTAGA SeuratProject       3772         1259       2893           29 pbmc5k-CITEseq AAACCCAAGGCCTAGA-1
## AAACCCAGTCGTGCCA SeuratProject       4902         1578       3635           29 pbmc5k-CITEseq AAACCCAGTCGTGCCA-1
##                          Sequence Library Cell_ranger_version Tissue_status Barcode_type   Chemistry Sequence_platform
## AAACCCAAGAGACAAG AAACCCAAGAGACAAG       1              v3.0.2          <NA>     Chromium Chromium_v3           NovaSeq
## AAACCCAAGGCCTAGA AAACCCAAGGCCTAGA       1              v3.0.2          <NA>     Chromium Chromium_v3           NovaSeq
## AAACCCAGTCGTGCCA AAACCCAGTCGTGCCA       1              v3.0.2          <NA>     Chromium Chromium_v3           NovaSeq
##                    Individual Date_published
## AAACCCAAGAGACAAG HealthyDonor     2019-05-29
## AAACCCAAGGCCTAGA HealthyDonor     2019-05-29
## AAACCCAGTCGTGCCA HealthyDonor     2019-05-29

# save the data locally if desired
save(data_blocks_sc_sce, obj_sce,
     file = file.path(path_data, "data_sc_sce.Rda"))
```

## 10x Genomics and Seurat

The original dataset can be found on the [10x Genomics Datasets website](https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.2/5k_pbmc_protein_v3) and an explanation of the file types for this version can be found [here](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/3.0/output/overview). You can download them all to a directory of your choosing with their suggested terminal commands:

```{bash data-10x-download, eval = FALSE}
# Input Files
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_fastqs.tar
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_feature_ref.csv

# Output Files
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_possorted_genome_bam.bam
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_possorted_genome_bam.bam.bai
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_molecule_info.h5
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_filtered_feature_bc_matrix.tar.gz
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_raw_feature_bc_matrix.h5
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_raw_feature_bc_matrix.tar.gz
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_analysis.tar.gz
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_metrics_summary.csv
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_web_summary.html
curl -O https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_cloupe.cloupe
```

If you would like to view some basic information about the data, you can open the *web_summary.html* in your favorite web browser. It will show the estimated number of cells, mean reads per cell, median genes per cell and a variety of other sample metrics. These metrics are also available in the *metrics_summary.csv* file.

You will need the *filtered_feature_bc_matrix* directory for the following analysis; it can be extracted with `tar -xvzf 5k_pbmc_protein_v3_filtered_feature_bc_matrix.tar.gz` from within the relevant data directory.

```{r data-10x-object, eval = FALSE}
# load the data (change the file path as needed)
data <- Seurat::Read10X(data.dir = file.path(path_data, "tenx_pbmc5k_CITEseq",
                                             "filtered_feature_bc_matrix"),
                        strip.suffix = TRUE) # remove the "-1"s from barcodes
## 10X data contains more than one type and is being returned as a list
## containing matrices of each type.

# set minimum cells and/or features here if you'd like
obj <- Seurat::CreateSeuratObject(counts = data$`Gene Expression`,
                                  project = "pbmc5k_CITEseq")
obj[["ADT"]] <- Seurat::CreateAssayObject(counts = data$`Antibody Capture`)
## Warning: Feature names cannot have underscores ('_'), replacing with dashes ('-')

# check the assays
Seurat::Assays(object = obj)
## "RNA" "ADT"

# list out the CITE-Seq surface protein markers
rownames(obj[["ADT"]])
## [1] "CD3-TotalSeqB"            "CD4-TotalSeqB"           "CD8a-TotalSeqB"
## [4] "CD11b-TotalSeqB"          "CD14-TotalSeqB"          "CD15-TotalSeqB"
## [7] "CD16-TotalSeqB"           "CD19-TotalSeqB"          "CD20-TotalSeqB"
## [10] "CD25-TotalSeqB"          "CD27-TotalSeqB"          "CD28-TotalSeqB"
## [13] "CD34-TotalSeqB"          "CD45RA-TotalSeqB"        "CD45RO-TotalSeqB"
## [16] "CD56-TotalSeqB"          "CD62L-TotalSeqB"         "CD69-TotalSeqB"
## [19] "CD80-TotalSeqB"          "CD86-TotalSeqB"          "CD127-TotalSeqB"
## [22] "CD137-TotalSeqB"         "CD197-TotalSeqB"         "CD274-TotalSeqB"
## [25] "CD278-TotalSeqB"         "CD335-TotalSeqB"         "PD-1-TotalSeqB"
## [28] "HLA-DR-TotalSeqB"        "TIGIT-TotalSeqB"         "IgG1-control-TotalSeqB"
## [31] "IgG2a-control-TotalSeqB" "IgG2b-control-TotalSeqB"

# save the data locally if desired
saveRDS(obj, file.path(path_data, "tenx_pbmc5k_CITEseq_raw.rds"))
```

You can also load the Seurat object directly, such as in the [Read in and process the data] subsection in the [Deep dive: Seurat analysis] section later in the vignette.

# MCIA

## Metadata

Note that the "-1"s have been removed from the cell barcodes.

```{r mcia-metadata}
# read in the annotated cells
metadata_sc <- read.csv(file = file.path(path_inst, "metadata_sc.csv"),
                        header = TRUE, row.names = 1)

# examples
metadata_sc %>% slice_sample(n = 5)
```

## Running the decomposition

This will run on cells with the top 2000 variable features (as defined in the later analysis).

```{r mcia-decomp-load-data, eval = FALSE}
# load the object setup for running MCIA [10x Genomics & Seurat]
load(file = file.path(path_inst, "data_blocks_sc.Rda"))
```

```{r mcia-decomp-run, eval = FALSE}
# "largest_sv" results in a more balanced contribution
# from the blocks than the default "unit_var"
set.seed(42)

# convert data_blocks_sc to an MAE object using the SingleCellExperiment class
data_blocks_sc_mae <-
  MultiAssayExperiment::MultiAssayExperiment(lapply(data_blocks_sc, function(x)
    SingleCellExperiment::SingleCellExperiment(t(as.matrix(x)))),
    colData = metadata_sc)
mcia_results_sc <- nipals_multiblock(data_blocks = data_blocks_sc_mae,
                                     col_preproc_method = "colprofile",
                                     block_preproc_method = "largest_sv",
                                     num_PCs = 10, tol = 1e-9,
                                     deflationMethod = "global",
                                     plots = "none")
## Performing column-level pre-processing...
## Column pre-processing completed.
## Performing block-level preprocessing...
## Block pre-processing completed.
## Computing order 1 scores
## Computing order 2 scores
## Computing order 3 scores
## Computing order 4 scores
## Computing order 5 scores
## Computing order 6 scores
## Computing order 7 scores
## Computing order 8 scores
## Computing order 9 scores
## Computing order 10 scores

# saveRDS(mcia_results_sc, file = file.path(path_data, "mcia_results_sc.Rds"))
```

```{r mcia-decomp-load}
# load the results of the previous block (if already run and saved)
mcia_results_sc <- readRDS(file = file.path(path_inst, "mcia_results_sc.Rds"))
mcia_results_sc
```

## Visualization

This data comes from only one subject, so we will use the annotated cell types for
the metadata (see the [Deep dive: Seurat analysis] section for details on their origin).

### Define colors

```{r mcia-plots-colors}
# for the projection plot
# technically you could just do color_pal_params = list(option = "D"), but saving
# the colors is useful for other plots like in the Seurat section
meta_colors_sc <- get_metadata_colors(mcia_results = mcia_results_sc,
                                      color_col = "CellType",
                                      color_pal = scales::viridis_pal,
                                      color_pal_params = list(option = "D"))

# for other plots
colors_omics_sc <- get_colors(mcia_results = mcia_results_sc)
```

### Eigenvalue scree plot

```{r mcia-plots-eigenvalue-scree, fig.dim = c(5, 4)}
global_scores_eigenvalues_plot(mcia_results = mcia_results_sc)
```

### Projection plot

```{r mcia-plots-projection, fig.dim = c(5, 5)}
projection_plot(mcia_results = mcia_results_sc,
                projection = "global", orders = c(1, 2),
                color_col = "CellType", color_pal = meta_colors_sc,
                legend_loc = "bottomright")
```

### Global scores heatmap

```{r mcia-plots-heatmap-global, fig.dim = c(7, 5)}
suppressMessages(global_scores_heatmap(mcia_results = mcia_results_sc,
                                       color_col = "CellType",
                                       color_pal = meta_colors_sc))
```

### Block weights heatmap

The block weights heatmap shows the distribution of the different block score
weights among the factors.

```{r mcia-plots-heatmap-block, fig.dim = c(4, 2.5)}
block_weights_heatmap(mcia_results = mcia_results_sc)
```

### Loadings

```{r mcia-plots-loadings, fig.dim = c(6, 4.5)}
vis_load_plot(mcia_out = mcia_results_sc,
              axes = c(1, 4), colors_omics = colors_omics_sc)
```

### Top features

In a few factors of interest:

#### Factor 1

```{r mcia-plots-top-features-factor-1, fig.dim = c(10, 4)}
# define the loadings
all_pos_1 <- ord_loadings(mcia_out = mcia_results_sc, omic = "all",
                          absolute = FALSE, descending = TRUE, factor = 1)
mrna_pos_1 <- ord_loadings(mcia_out = mcia_results_sc, omic = "mrna",
                           absolute = FALSE, descending = TRUE, factor = 1)

# visualization
all_pos_1_vis <- vis_load_ord(gl_f_ord = all_pos_1, omic_name = "all",
                              colors_omics = colors_omics_sc)
mrna_pos_1_vis <- vis_load_ord(gl_f_ord = mrna_pos_1, omic_name = "mrna",
                               colors_omics = colors_omics_sc)

ggpubr::ggarrange(all_pos_1_vis, mrna_pos_1_vis, widths = c(1, 1))
```

#### Factor 4

If you don't want to rank by the absolute value and see a large number of features:

```{r mcia-plots-top-features-factor-4, fig.dim = c(10, 5)}
# define the loadings
all_4 <- ord_loadings(mcia_results_sc, omic = "all",
                      absolute = FALSE, descending = TRUE, factor = 4)

# visualization
vis_load_ord(gl_f_ord = all_4, omic_name = "all",
             colors_omics = colors_omics_sc, n_feat = 60)
```

As we saw in the [Block weights heatmap], factor 4 is dominated by the mRNA data
and not the ADT data.

# Deep dive: Seurat analysis

This section demonstrates how to take in raw data (in this case, the output of Cell Ranger v3.0) and go through a popular analysis pipeline to ultimately cluster and annotate the data. Some people prefer to use Cell Ranger's built-in dimension reduction and clustering analysis and to view the results with [Loupe Cell Browser](https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser).

*Credit to Seurat ([RNA](https://satijalab.org/seurat/articles/pbmc3k_tutorial.html) and [multi-modal](https://satijalab.org/seurat/articles/multimodal_vignette.html#setup-a-seurat-object-add-the-rna-and-protein-data)) for the general steps.*

## Read in and process the data

```{r seurat-load-data, message = FALSE}
# load the data
obj <- readRDS(file = file.path(path_inst, "tenx_pbmc5k_CITEseq_raw.rds"))

# add useful metadata
obj[["percent.mt"]] <- PercentageFeatureSet(object = obj, pattern = "^MT-")
```

## Quality control

### Metrics summary

```{r seurat-qc-metrics-summary}
# read in and display the summary table
(metrics_summary <- read.csv(
    file = file.path(path_inst, "5k_pbmc_protein_v3_metrics_summary.csv")
))
```

### GEX QC metrics

#### Before filtering

```{r seurat-qc-pre-filter, fig.dim = c(8, 5)}
VlnPlot(object = obj,
        features = c("nFeature_RNA", "nCount_RNA", "percent.mt"),
        pt.size = 0.01, ncol = 3) &
  theme(axis.text.x = element_text(angle = 0, hjust = 0.5),
        axis.title.x = element_blank())
```

#### After filtering

Based on the previous plots, a minimum of 200 features and a maximum of 20% mitochondrial seemed like good cutoffs.

```{r seurat-qc-post-filter, fig.dim = c(8, 5)}
# adjust cutoffs as desired
obj <- subset(x = obj, subset = nFeature_RNA > 200 & percent.mt < 20)

VlnPlot(object = obj,
        features = c("nFeature_RNA", "nCount_RNA", "percent.mt"),
        pt.size = 0.01, ncol = 3) &
  theme(axis.text.x = element_text(angle = 0, hjust = 0.5),
        axis.title.x = element_blank())
```

## Standard Seurat pipeline

You can set `verbose = FALSE` for many of these commands if you don't want to
see outputs.

**Most of these are run on the RNA**.

```{r seurat-pipeline-1, eval = FALSE}
# standard log normalization for RNA and centered log for ADT
obj <- NormalizeData(object = obj, normalization.method = "LogNormalize",
                     scale.factor = 10000, assay = "RNA")
## Performing log-normalization
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|

obj <- NormalizeData(object = obj, normalization.method = "CLR",
                     margin = 2, assay = "ADT") # go across cells, not features
## Normalizing across cells
##   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=00s

# highly variable features
obj <- FindVariableFeatures(object = obj, selection.method = "vst",
                            nfeatures = 2000)
## Calculating gene variances
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## Calculating feature variances of standardized and clipped values
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|

# scaling so the average expression is 0 and the variance is 1
obj <- ScaleData(object = obj, features = rownames(obj))
## Centering and scaling data matrix
##   |===============================================================================================| 100%

# dimensionality reduction
obj <- RunPCA(object = obj)
## PC_ 1
## Positive:  LYZ, FCN1, CST3, MNDA, CTSS, PSAP, S100A9, FGL2, AIF1, GRN
##     NCF2, LST1, CD68, TYMP, SERPINA1, CYBB, CLEC12A, CSTA, SPI1, TNFAIP2
##     CPVL, VCAN, MPEG1, TYROBP, KLF4, FTL, S100A8, IGSF6, CD14, MS4A6A
## Negative:  CD3D, TRAC, LTB, TRBC2, IL32, CD3G, IL7R, CD69, CD247, TRBC1
##     CD2, CD7, CD27, ARL4C, ISG20, HIST1H4C, SYNE2, GZMM, ITM2A, CCR7
##     RORA, MAL, CXCR4, LEF1, TRAT1, CTSW, GZMA, KLRB1, TRABD2A, CCL5
## PC_ 2
## Positive:  CD79A, MS4A1, IGHM, BANK1, HLA-DQA1, CD79B, IGKC, LINC00926, RALGPS2, TNFRSF13C
##     VPREB3, IGHD, SPIB, CD22, FCRL1, HLA-DQB1, BLK, FAM129C, FCRLA, TCL1A
##     GNG7, TCF4, COBLL1, PAX5, SWAP70, CD40, BCL11A, P2RX5, TSPAN13, ADAM28
## Negative:  NKG7, CST7, GZMA, PRF1, KLRD1, CTSW, FGFBP2, GNLY, GZMH, CCL5
##     GZMM, CD247, KLRF1, HOPX, SPON2, ADGRG1, TRDC, MATK, GZMB, FCGR3A
##     S100A4, CCL4, CLIC3, KLRB1, IL2RB, TBX21, TTC38, ANXA1, PTGDR, PLEKHF1
## PC_ 3
## Positive:  GZMB, NKG7, GNLY, CLIC3, PRF1, KLRD1, FGFBP2, KLRF1, SPON2, CST7
##     GZMH, FCGR3A, ADGRG1, GZMA, HOPX, CTSW, TRDC, CCL4, HLA-DPB1, C12orf75
##     PLAC8, TTC38, PLEK, APOBEC3G, TBX21, PRSS23, CYBA, MATK, SYNGR1, CXXC5
## Negative:  IL7R, MAL, LEF1, TRABD2A, TRAC, CCR7, LTB, CD27, FOS, LRRN3
##     FHIT, TRAT1, RGCC, CAMK4, CD3D, RGS10, CD40LG, FOSB, AQP3, SOCS3
##     FLT3LG, CD3G, SLC2A3, TSHZ2, VIM, S100A12, S100A8, CD28, PLK3, VCAN
## PC_ 4
## Positive:  FCER1A, PLD4, SERPINF1, IL3RA, CLEC10A, GAS6, LILRA4, TPM2, CLEC4C, ENHO
##     FLT3, SMPD3, ITM2C, LGMN, CD1C, P2RY14, PPP1R14B, SCT, PROC, LAMP5
##     RUNX2, AC119428.2, PACSIN1, DNASE1L3, PTCRA, RGS10, UGCG, CLIC2, PPM1J, P2RY6
## Negative:  MS4A1, CD79A, LINC00926, BANK1, TNFRSF13C, VPREB3, CD79B, RALGPS2, IGHD, FCRL1
##     BLK, IGHM, CD22, PAX5, ARHGAP24, CD24, P2RX5, NCF1, S100A12, CD19
##     SWAP70, FCRLA, VNN2, TNFRSF13B, FCER2, IGKC, FCRL2, RBP7, CD40, S100A8
## PC_ 5
## Positive:  BATF3, C1QA, TCF7L2, CTSL, CDKN1C, HLA-DQB1, HES4, SIGLEC10, CLEC10A, ABI3
##     HLA-DQA1, RHOC, CSF1R, ENHO, CAMK1, MTSS1, IFITM3, CD1C, LY6E, FCGR3A
##     HLA-DPA1, CLIC2, HLA-DPB1, YBX1, RRAS, AC064805.1, NR4A1, GBP1, ZNF703, CXCL16
## Negative:  LILRA4, TPM2, CLEC4C, SMPD3, IL3RA, SERPINF1, DERL3, MZB1, SCT, JCHAIN
##     PACSIN1, PROC, S100A12, PTCRA, LINC00996, PADI4, ASIP, KCNK17, ITM2C, EPHB1
##     ALOX5AP, LAMP5, DNASE1L3, MAP1A, S100A8, APP, CYP1B1, VNN2, UGCG, ZFAT

# clustering (adjust dimensions and resolutions as desired)
obj <- FindNeighbors(object = obj, reduction = "pca", dims = 1:20)
## Computing nearest neighbor graph
## Computing SNN

obj <- FindClusters(object = obj, resolution = 0.4)
## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
##
## Number of nodes: 4193
## Number of edges: 154360
##
## Running Louvain algorithm...
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## Maximum modularity in 10 random starts: 0.9097
## Number of communities: 10
## Elapsed time: 0 seconds

obj <- RunUMAP(object = obj, reduction = "pca", dims = 1:20)
## 14:44:35 UMAP embedding parameters a = 0.9922 b = 1.112
## 14:44:35 Read 4193 rows and found 20 numeric columns
## 14:44:35 Using Annoy for neighbor search, n_neighbors = 30
## 14:44:35 Building Annoy index with metric = cosine, n_trees = 50
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## 14:44:36 Writing NN index file to temp file /tmp/RtmpHhoQAU/filec346711dd9f93
## 14:44:36 Searching Annoy index using 1 thread, search_k = 3000
## 14:44:37 Annoy recall = 100%
## 14:44:38 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
## 14:44:39 Initializing from normalized Laplacian + noise (using RSpectra)
## 14:44:39 Commencing optimization for 500 epochs, with 174318 positive edges
## Using method 'umap'
## 0%   10   20   30   40   50   60   70   80   90   100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## 14:44:44 Optimization finished
```

## Dimensionality reduction

### Load in the processed object

To avoid having to save multiple large objects, this file already includes the
annotations defined in a later section, so we will clear them out here to
proceed as normal.

```{r seurat-load-processed-object}
obj <- readRDS(file = file.path(path_inst, "tenx_pbmc5k_CITEseq_annotated.rds"))

# reset to baseline
Idents(object = obj) <- "seurat_clusters"
obj$annotated_clusters <- c()
```

### PCA

**Typically this section would go in between the `RunPCA()` and `FindNeighbors()` steps above**.
It has been included here because of how we save and load the objects separately
for processing speed.

Now that we've run PCA, we can examine an elbow plot as a simple method for
selecting how many PCs to choose when identifying neighbors/clusters.

```{r seurat-elbow, fig.dim = c(6, 3)}
ElbowPlot(object = obj, ndims = 30)
```

From the plot, it looked like 20 works as a cut-off for the number of PCs to
include. We could have also chosen to use 10, but decided to go a little higher.

You can also examine other information which was used for the PCA, such as the
top twenty most variable features:

```{r seurat-top-features}
head(VariableFeatures(object = obj), 20)
```

```{r seurat-top-features-plot, fig.dim = c(6, 6), warning = FALSE, eval = FALSE}
# these can be plotted if desired
LabelPoints(plot = VariableFeaturePlot(object = obj),
            points = head(VariableFeatures(object = obj), 20),
            repel = TRUE, xnudge = 0, ynudge = 0) +
  labs(title = "Top Twenty Variable Features") +
  theme(legend.position = "bottom")
```


### UMAP

```{r seurat-umap-initial, fig.dim = c(6, 6), message = FALSE}
# you can also use the LabelClusters function to help label individual clusters
plot_seurat_clusters <- UMAPPlot(object = obj, label = TRUE, label.size = 4,
                                 label.box = TRUE) +
                          labs(title = "Initial Clusters") +
                          scale_fill_manual(
                               values = rep("white",
                                            n_distinct(obj$seurat_clusters))) +
                          theme(plot.title = element_text(hjust = 0.5),
                                axis.text = element_blank(),
                                axis.ticks = element_blank())
plot_seurat_clusters
```

## Marker overlays

Note that the following marker genes **are not meant to be an exhaustive list.**
They are included as examples of some of the cell types you could look for.

### Load marker genes

```{r seurat-markers-load}
# sort by Cell_Type and Marker if not already sorted
markers_all <- read.csv(file = file.path(path_inst, "marker_genes.csv"))
markers_all %>% slice_sample(n = 10) # example markers
```

### Dot plots

#### GEX

Here we only demonstrate a few of the cell types included within the provided
markers database. We plot those cell types on top of the dot plot to make it
easier to see which markers they correspond with, but you can comment that code
out if you would rather have a simple dot plot.

```{r seurat-markers-dot-gex, fig.dim = c(16, 6), warning = FALSE}
select_cell_types <- c("B", "Macrophages", "mDC", "NK", "T")

# do features = unique(markers_all$Marker) to use all possible features
p <- DotPlot(object = obj,
             features = markers_all %>%
                          dplyr::filter(Cell_Type %in% select_cell_types) %>%
                          distinct(Marker) %>% pull(),
             cols = "RdBu", col.min = -1, dot.scale = 3, cluster.idents = TRUE)

# add in the cell type information
# if desired, you could rename the "Cell_Type"s in the original database to be
# more informative e.g. Natural_Killers instead of NK
p$data <- left_join(p$data,
                    markers_all %>%
                      dplyr::filter(Cell_Type %in% select_cell_types) %>%
                      dplyr::rename(features.plot = "Marker"),
                    by = "features.plot", multiple = "all")
# depending on your version of dplyr, you can set `relationship = "many-to-many"`
# to surpress the warning

# plot
p +
  facet_grid(cols = vars(Cell_Type), scales = "free_x", space = "free") +
  theme(strip.text.x = element_text(size = 10)) + RotatedAxis()
```

#### ADT

The average expression was set to a minimum of zero to better see the
up-regulated features.

```{r seurat-markers-dot-adt, fig.dim = c(16, 6)}
# you have to change the default assay for the dot plot
DefaultAssay(object = obj) <- "ADT"

DotPlot(object = obj,
        features = rownames(GetAssayData(object = obj, assay = "ADT",
                                         slot = "counts")),
        cols = "RdBu", col.min = -1, dot.scale = 3, cluster.idents = TRUE) +
  RotatedAxis()

# reset the default
DefaultAssay(object = obj) <- "RNA"
```

### Feature plots

If you would like to generate feature plots for a cell type within your markers
database (if you are using one) e.g. for B cells, you could do `features = (dplyr::filter(markers_all, Cell_Type == "B"))$Marker` in the following command.
Here we just show a few characteristic markers for different cell types for
simplicity.

```{r seurat-markers-feature, fig.dim = c(12, 8)}
FeaturePlot(object = obj,
            features = c("MS4A1", "CD14", # MS4A1 is another name for CD20
                         "NKG7", "IL3RA", "CD3D", "IL7R"),
            min.cutoff = 0, label = TRUE, label.size = 4,
            ncol = 3, raster = FALSE)
```

### Violin plots

Here the point size is set to zero so that you can just see the violins. We only
show a few of the cell surface markers here, but you could plot them all with
`features = rownames(obj[["ADT"]])`. You can also plot the standard GEX markers
just like for the feature plots (just make sure to remove `assay = "ADT"`).

```{r seurat-markers-violin-adt, fig.dim = c(8, 8)}
VlnPlot(object = obj,
        features = c("CD3-TotalSeqB", "CD14-TotalSeqB",
                     "CD20-TotalSeqB", "CD335-TotalSeqB"),
        pt.size = 0, assay = "ADT", ncol = 2) &
  theme(plot.title = element_text(size = 10),
        axis.text.x = element_text(angle = 0, hjust = 0.5),
        axis.title.x = element_blank())
```

## Annotate cell clusters

These annotations are chosen here more as a proof of general concept instead of a more
highly refined and verified approach that you would typically see within a single-cell
publication. There are also [automated methods for annotation](https://www.sciencedirect.com/science/article/pii/S2001037021000192).

### Annotations

If you would like to further break down these clusters, you can increase the clustering resolution
in the standard pipeline in order to increase the number of clusters.

```{r seurat-annotations}
# mDC = myeloid dendritic cells
# pDC = plasmacytoid dendritic cells
obj_annotations <- rbind(c("0", "Macrophages/mDCs"), # difficult to separate
                         c("1", "T Cells"),
                         c("2", "T Cells"),
                         c("3", "T Cells"),
                         c("4", "Natural Killers"),
                         c("5", "B Cells"),
                         c("6", "Macrophages/mDCs"), # difficult to separate
                         c("7", "Macrophages/mDCs"), # difficult to separate
                         c("8", "T Cells"), # faint signal
                         c("9", "pDCs"))
colnames(obj_annotations) <- c("Cluster", "CellType")
obj_annotations <- data.frame(obj_annotations)

# save the annotations as a csv if you'd like
# write.csv(obj_annotations, file = file.path(path_data, "obj_annotations.csv"))

# prepare the annotation information
annotations <- setNames(obj_annotations$CellType, obj_annotations$Cluster)

# relabel the Seurat clusters
# Idents(obj) <- "seurat_clusters"
obj <- RenameIdents(object = obj, annotations)

# alphabetize the cell types
Idents(obj) <- factor(Idents(object = obj), levels = sort(levels(obj)))

# useful metadata (e.g. if you want to have multiple annotation sets)
obj[["annotated_clusters"]] <- Idents(object = obj)

# save the processed and annotated Seurat object
# saveRDS(obj, file = file.path(path_data, "tenx_pbmc5k_CITEseq_annotated.rds"))

# info about the clusters
obj_annotations %>%
  group_by(CellType) %>%
  transmute(Clusters = paste0(Cluster, collapse = ", ")) %>%
  distinct() %>%
  arrange(CellType)

# metadata file for MCIA
meta <- data.frame("CellType" = Idents(object = obj))

# save the metadata file for MCIA
# write.csv(meta, file = file.path(path_data, "metadata_sc.csv"))
```

### UMAPs

Here we use the selected metadata colors from the [MCIA] section, so be sure to
change that (or just remove the `cols = ...` to use the default) if you are running
this section first. If you don't want the cluster labels to have the white backgrounds,
remove `label.box` and `scale_fill_manual()`.

```{r seurat-umap-both, fig.dim = c(16, 8), message = FALSE}
# change colors and themes as desired
plot_annotated_clusters <- UMAPPlot(object = obj,
                                    label = TRUE, label.size = 4,
                                    label.box = TRUE,
                                    cols = meta_colors_sc) +
                             labs(title = "Annotated Clusters") +
                             scale_fill_manual(
                               values = rep("white", length(meta_colors_sc))) +
                             theme(plot.title = element_text(hjust = 0.5),
                                   axis.text = element_blank(),
                                   axis.ticks = element_blank()) +
                             NoLegend() # a legend is not needed here

ggpubr::ggarrange(plot_seurat_clusters + NoLegend(), plot_annotated_clusters,
                  heights = c(1, 1), widths = c(1, 1))
```

You should change the `DefaultAssay` to/from `ADT` like in [ADT] if you don't
want to specify "adt_" before the feature name.

```{r seurat-umap-adt, fig.dim = c(6, 6)}
# plot ADT information on top as an example
FeaturePlot(object = obj, features = "adt_CD19-TotalSeqB",
            label = TRUE, label.size = 4, ncol = 1, raster = FALSE)
```

### Check the annotations

For example, if you want to visually check your T cell cluster annotations (just
like before, you can also do `features = (dplyr::filter(markers_all, Cell_Type == "T"))$Marker`
to use all of the T cell markers in the markers database):

```{r seurat-check-annotations-violin, fig.dim = c(8, 8)}
VlnPlot(object = obj,
        features = c("CD3D", "CD4", "IL7R", "TRAC"),
        cols = meta_colors_sc, pt.size = 0.01, ncol = 2) &
  theme(plot.title = element_text(size = 10),
        axis.title.x = element_blank())
```

You can also obtain the following bar plot using the output of `nipals_multiblock` e.g. with `ggplot(data.frame(table(nmb_get_metadata(mcia_results_sc))), aes(x = CellType, y = Freq, fill = CellType)) + geom_bar(stat = "identity")` (for the base plot).

```{r seurat-check-annotations-bar, fig.dim = c(8, 6)}
# examine the total counts
ggplot(obj[[]], aes(x = annotated_clusters, fill = annotated_clusters)) +
  geom_bar(color = "black", linewidth = 0.2) +
  labs(title = "Cell Type Counts", x = "Cell Type", y = "Count") +
  scale_fill_manual(values = meta_colors_sc) +
  theme_bw() +
  theme(plot.title = element_text(size = 14, hjust = 0.5),
        axis.text.x = element_text(angle = 45, hjust = 1),
        legend.position = "none")
```

## Save for MCIA

The file has to be structured in the form of a (large) list, with each omic
saved as an element within it. If you would like to append the type of omic to
the end of your features, uncomment the commented lines.

```{r seurat-save-mcia, eval = FALSE}
# mrna
data_rna <- GetAssayData(object = obj, slot = "data",
                         assay = "RNA")[VariableFeatures(object = obj), ]
data_rna <- as.matrix(data_rna)
data_rna <- t(data_rna) # switch the rows
# colnames(data_rna) <- paste(colnames(data_rna), "mrna", sep = "_")
data_rna <- log2(data_rna + 1) # log-transform because the data is heavy tailed

# adt
data_adt <- GetAssayData(object = obj, slot = "data", assay = "ADT")
data_adt <- as.matrix(data_adt)
data_adt <- t(data_adt) # switch the rows
# colnames(data_adt) <- paste(colnames(data_adt), "adt", sep = "_")
data_adt <- log2(data_adt + 1) # log-transform because the data is heavy tailed

# combined
data_blocks_sc <- list(mrna = data_rna, adt = data_adt)

# examine the contents
data.frame(data_blocks_sc$mrna[1:5, 1:5])
data.frame(data_blocks_sc$adt[1:5, 1:5])

# save(data_blocks_sc, file = file.path(path_data, "data_blocks_sc.Rda"))
```

# Session Info

<details>
  <summary>**Session Info**</summary>
  
```{r session-info}
sessionInfo()
```

</details>
