Introduction

# <strong>Introduction</strong>
## Analyzing <strong>scRNA-seq</strong> data with <strong>Bioconductor</strong> for <strong>LCG-EJ-UNAM</strong> March 2020
### <a href="http://lcolladotor.github.io/">Leonardo Collado-Torres</a>
### 2020-03-23

---

<a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/4.0/"><img alt="Creative Commons License" style="border-width:0" src="https://i.creativecommons.org/l/by-nc-sa/4.0/88x31.png" /></a><br />This work is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/4.0/">Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License</a>.

]

.footnote[ Download the materials for this course with `usethis::use_course('lcolladotor/osca_LIIGH_UNAM_2020')` or view online at [**lcolladotor.github.io/osca_LIIGH_UNAM_2020**](http://lcolladotor.github.io/osca_LIIGH_UNAM_2020).]

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---

# Course origins

* [**Orchestrating Single Cell Analysis With Bioconductor**](ohttps://osca.bioconductor.org/) book by [Aaron Lun](https://www.linkedin.com/in/aaron-lun-869b5894/), [Robert Amezquita](https://robertamezquita.github.io/), [Stephanie Hicks](https://www.stephaniehicks.com/) and [Raphael Gottardo](http://rglab.org)

Amezquita, R.A., Lun, A.T.L., Becht, E. et al. Orchestrating single-cell analysis with Bioconductor. _Nat Methods_ 17, 137–145 (2020). DOI: [10.1038/s41592-019-0654-x](https://doi.org/10.1038/s41592-019-0654-x)

* [**WEHI's scRNA-seq course**](https://drive.google.com/drive/folders/1cn5d-Ey7-kkMiex8-74qxvxtCQT6o72h) by [Peter Hickey](https://www.peterhickey.org/)

---

# Instructor

**Leonardo Collado-Torres**

* Website: [lcolladotor.github.io](http://lcolladotor.github.io)
* Twitter: [fellgernon](https://twitter.com/fellgernon)

---

---

---

---

---

# Course Prerequisites

Install R 3.6.x from [CRAN](https://cran.r-project.org/) then install the following R packages:

```r
## For installing Bioconductor packages
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

## Install required packages
BiocManager::install(
    c(
        'SingleCellExperiment',
        'usethis',
        'here',
        'scran',
        'scater',
        'scRNAseq',
        'org.Mm.eg.db',
        'AnnotationHub',
        'ExperimentHub',
        'BiocFileCache',
        'DropletUtils',
        'EnsDb.Hsapiens.v86',
        'TENxPBMCData',
        'BiocSingular',
        'batchelor',
        'uwot',
        'Rtsne',
        'pheatmap',
        'fossil',
        'ggplot2',
        'cowplot',
        'RColorBrewer',
        'plotly',
        'iSEE',
        'pryr',
        'LieberInstitute/spatialLIBD',
        'sessioninfo'
    )
)
```

You will also need to install [RStudio](https://rstudio.com/products/rstudio/download/#download) version 1.2.5 or newer.

]

---

# LIIGH Cluster DNA setup

If you add the following code to your `~/.Rprofile` at the DNA LIIGH-UNAM cluster, you'll be able to use the same R packages I installed.

```bash
## Log into the cluster

## Load the R 3.6.1 module
module load r/3.6.1

## Edit your ~/.Rprofile
vi ~/.Rprofile
```

```r
## Add this to your ~/.Rprofile file
if(R.home() == '/cm/shared/apps/r/3.6.1-studio/lib64/R') {
    if (interactive())
        message("Using the following library: /mnt/Genoma/amedina/lcollado/R/3.6.1")
    .libPaths(
        c(
            '/mnt/Genoma/amedina/lcollado/R/3.6.1',
            '/cm/shared/apps/r/3.6.1-studio/lib64/R/library'
        )
    )
}
```

If you are using RStudio through Cyberduck or something like that, you could use `usethis::edit_r_profile()`.

To test that it works, run:

```bash
qrsh
module load r/3.6.1
Rscript -e "packageVersion('spatialLIBD')"
```

]

---

# Course Materials

* Download them with `usethis::use_course('lcolladotor/osca_LIIGH_UNAM_2020')`

* View online at [**lcolladotor.github.io/osca_LIIGH_UNAM_2020**](http://lcolladotor.github.io/osca_LIIGH_UNAM_2020)

* **Clone** the GitHub repository, which will make it easy for you to download the latest version with *git pull*

```bash
## If you have SSH keys enabled
git clone git@github.com:lcolladotor/osca_LIIGH_UNAM_2020.git

## or
git clone https://github.com/lcolladotor/osca_LIIGH_UNAM_2020.git
```

From R:

```r
git2r::clone('https://github.com/lcolladotor/osca_LIIGH_UNAM_2020',
    'osca_LIIGH_UNAM_2020')
```

---

# Create your own project

I recommend that you create your own project and version control it

```r
usethis::create_project('~/Desktop/osca_playground_leo')
```

```r
## Start a setup file
usethis::use_r('00-setup.R')
```

Inside the setup script, save the commands to

```r
## Start git repo
usethis::use_git()

## Use GitHub
usethis::browse_github_token()
usethis::edit_r_environ() ## then restart R
usethis::use_github() ## commit first, then run this command

## Start 01-intro notes
usethis::use_r('01-introduction.R')
```

Example at [**osca_playground_leo**](https://github.com/lcolladotor/osca_playground_leo/blob/master/R/00-setup.R)

---

# Class schedule

| Time | Activity |
| ---: | :--- |
| 9:00-9:50 | class |
| 9:50 - 10:00 | break |
| 10:00-10:50 | class |
| 10:50 - 11:30 | lunch break |
| 11:30 - 12:20 | class |
| 12:20 - 12:30 | break |
| 12:30 - 13:20 | class |
| 13:20 - 13:30 | break |
| 13:30 - 14:00 | class wrap up |

* Timezone: Central Mexico
* Days: Tuesday March 24 to Friday March 27

---

# Asking for help

* Use the "raise your **hand**" feature in Zoom

* Create an **issue** at [osca_LIIGH_UNAM_2020](https://github.com/lcolladotor/osca_LIIGH_UNAM_2020/issues). Remember to include a reproducible example!

* More generally, through the [**Bioconductor Support Website**](https://support.bioconductor.org/) tagging the appropriate package.

* Related blog posts: [**How to ask for help for Bioconductor packages**](http://lcolladotor.github.io/2017/03/06/how-to-ask-for-help-for-bioconductor-packages/#.XnjLRNNKh0s), [**Asking for help is challenging but is typically worth it**](http://lcolladotor.github.io/2018/11/12/asking-for-help-is-challenging-but-is-typically-worth-it/#.XnjLf9NKh0s), and [**Learning from our search history**](http://lcolladotor.github.io/2020/02/12/learning-from-our-search-history/)

* Related `rstudio::conf(2020)` keynote by [Jenny Bryan](https://twitter.com/JennyBryan): [**Object of type ‘closure’ is not subsettable**](https://resources.rstudio.com/rstudio-conf-2020/object-of-type-closure-is-not-subsettable-jenny-bryan)

---

background-image: url(https://github.com/Bioconductor/OrchestratingSingleCellAnalysis/raw/master/images/cover.png)
background-size: contain

---

background-image: url(https://github.com/Bioconductor/OrchestratingSingleCellAnalysis/raw/master/images/SingleCellExperiment.png)
background-size: contain

---

background-image: url(https://github.com/Bioconductor/OrchestratingSingleCellAnalysis/raw/master/images/Workflow.png)
background-size: contain

---

background-image: url(http://research.libd.org/spatialLIBD/reference/figures/README-access_data-1.png)
background-size: contain

---

# Quick Introduction: [OSCA](https://osca.bioconductor.org/overview.html#quick-start)

```r
library('scRNAseq')
library('scater')
library('scran')
library('plotly')
```

---

```r
sce <- scRNAseq::MacoskoRetinaData()
```

```
## snapshotDate(): 2019-10-22
```

```
## see ?scRNAseq and browseVignettes('scRNAseq') for documentation
```

```
## loading from cache
```

```
## see ?scRNAseq and browseVignettes('scRNAseq') for documentation
```

```
## loading from cache
```

```r
## How big is the data?
pryr::object_size(sce)
```

```
## Registered S3 method overwritten by 'pryr':
##   method      from
##   print.bytes Rcpp
```

```
## 461 MB
```

```r
## How does it look?
sce
```

```
## class: SingleCellExperiment 
## dim: 24658 49300 
## metadata(0):
## assays(1): counts
## rownames(24658): KITL TMTC3 ... 1110059M19RIK GM20861
## rowData names(0):
## colnames(49300): r1_GGCCGCAGTCCG r1_CTTGTGCGGGAA ... p1_TAACGCGCTCCT
##   p1_ATTCTTGTTCTT
## colData names(2): cell.id cluster
## reducedDimNames(0):
## spikeNames(0):
## altExpNames(0):
```

]

---

```r
# Quality control.
is.mito <- grepl("^MT-", rownames(sce))
qcstats <-
    scater::perCellQCMetrics(sce, subsets = list(Mito = is.mito))
filtered <-
    scater::quickPerCellQC(qcstats, percent_subsets = "subsets_Mito_percent")
sce <- sce[, !filtered$discard]

# Normalization.
sce <- scater::logNormCounts(sce)

# Feature selection.
dec <- scran::modelGeneVar(sce)
hvg <- scran::getTopHVGs(dec, prop = 0.1)

# Dimensionality reduction.
set.seed(1234)
sce <- scater::runPCA(sce, ncomponents = 25, subset_row = hvg)
sce <- scater::runUMAP(sce, dimred = 'PCA', external_neighbors = TRUE)

# Clustering.
g <- scran::buildSNNGraph(sce, use.dimred = 'PCA')
sce$clusters <- factor(igraph::cluster_louvain(g)$membership)
```

]

---

```r
# Visualization.
scater::plotUMAP(sce, colour_by = "clusters")
```

![](01-introduction_files/figure-html/quick_intro_04-1.png)

---

```r
# Interactive visualization
p <- scater::plotUMAP(sce, colour_by = "clusters")
plotly::ggplotly(p)
```

---

# Thanks!

Slides created via the R package [**xaringan**](https://github.com/yihui/xaringan) and themed with [**xaringanthemer**](https://github.com/gadenbuie/xaringanthemer).

This course is based on the book [**Orchestrating Single Cell Analysis with Bioconductor**](https://osca.bioconductor.org/) by [Aaron Lun](https://www.linkedin.com/in/aaron-lun-869b5894/), [Robert Amezquita](https://robertamezquita.github.io/), [Stephanie Hicks](https://www.stephaniehicks.com/) and [Raphael Gottardo](http://rglab.org), plus [**WEHI's scRNA-seq course**](https://drive.google.com/drive/folders/1cn5d-Ey7-kkMiex8-74qxvxtCQT6o72h) by [Peter Hickey](https://www.peterhickey.org/).

You can find the files for this course at [lcolladotor/osca_LIIGH_UNAM_2020](https://github.com/lcolladotor/osca_LIIGH_UNAM_2020).

Instructor: [**Leonardo Collado-Torres**](http://lcolladotor.github.io/).

]

---

# R session information

```r
options(width = 120)
sessioninfo::session_info()
```

```
## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value                       
##  version  R version 3.6.3 (2020-02-29)
##  os       macOS Catalina 10.15.3      
##  system   x86_64, darwin15.6.0        
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  ctype    en_US.UTF-8                 
##  tz       America/New_York            
##  date     2020-03-23                  
## 
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────
##  package                * version  date       lib source                                   
##  AnnotationDbi            1.48.0   2019-10-29 [1] Bioconductor                             
##  AnnotationHub            2.18.0   2019-10-29 [1] Bioconductor                             
##  assertthat               0.2.1    2019-03-21 [1] CRAN (R 3.6.0)                           
##  beeswarm                 0.2.3    2016-04-25 [1] CRAN (R 3.6.0)                           
##  Biobase                * 2.46.0   2019-10-29 [1] Bioconductor                             
##  BiocFileCache            1.10.2   2019-11-08 [1] Bioconductor                             
##  BiocGenerics           * 0.32.0   2019-10-29 [1] Bioconductor                             
##  BiocManager              1.30.10  2019-11-16 [1] CRAN (R 3.6.1)                           
##  BiocNeighbors            1.4.2    2020-02-29 [1] Bioconductor                             
##  BiocParallel           * 1.20.1   2019-12-21 [1] Bioconductor                             
##  BiocSingular             1.2.2    2020-02-14 [1] Bioconductor                             
##  BiocVersion              3.10.1   2019-06-06 [1] Bioconductor                             
##  bit                      1.1-15.2 2020-02-10 [1] CRAN (R 3.6.0)                           
##  bit64                    0.9-7    2017-05-08 [1] CRAN (R 3.6.0)                           
##  bitops                   1.0-6    2013-08-17 [1] CRAN (R 3.6.0)                           
##  blob                     1.2.1    2020-01-20 [1] CRAN (R 3.6.0)                           
##  cli                      2.0.2    2020-02-28 [1] CRAN (R 3.6.0)                           
##  colorout               * 1.2-1    2019-05-07 [1] Github (jalvesaq/colorout@7ea9440)       
##  colorspace               1.4-1    2019-03-18 [1] CRAN (R 3.6.0)                           
##  cowplot                  1.0.0    2019-07-11 [1] CRAN (R 3.6.0)                           
##  crayon                   1.3.4    2017-09-16 [1] CRAN (R 3.6.0)                           
##  curl                     4.3      2019-12-02 [1] CRAN (R 3.6.0)                           
##  data.table               1.12.8   2019-12-09 [1] CRAN (R 3.6.1)                           
##  DBI                      1.1.0    2019-12-15 [1] CRAN (R 3.6.0)                           
##  dbplyr                   1.4.2    2019-06-17 [1] CRAN (R 3.6.0)                           
##  DelayedArray           * 0.12.2   2020-01-06 [1] Bioconductor                             
##  DelayedMatrixStats       1.8.0    2019-10-29 [1] Bioconductor                             
##  digest                   0.6.25   2020-02-23 [1] CRAN (R 3.6.0)                           
##  dplyr                    0.8.5    2020-03-07 [1] CRAN (R 3.6.0)                           
##  dqrng                    0.2.1    2019-05-17 [1] CRAN (R 3.6.0)                           
##  edgeR                    3.28.1   2020-02-26 [1] Bioconductor                             
##  evaluate                 0.14     2019-05-28 [1] CRAN (R 3.6.0)                           
##  ExperimentHub            1.12.0   2019-10-29 [1] Bioconductor                             
##  fansi                    0.4.1    2020-01-08 [1] CRAN (R 3.6.0)                           
##  farver                   2.0.3    2020-01-16 [1] CRAN (R 3.6.0)                           
##  fastmap                  1.0.1    2019-10-08 [1] CRAN (R 3.6.0)                           
##  GenomeInfoDb           * 1.22.0   2019-10-29 [1] Bioconductor                             
##  GenomeInfoDbData         1.2.2    2019-10-31 [1] Bioconductor                             
##  GenomicRanges          * 1.38.0   2019-10-29 [1] Bioconductor                             
##  ggbeeswarm               0.6.0    2017-08-07 [1] CRAN (R 3.6.0)                           
##  ggplot2                * 3.3.0    2020-03-05 [1] CRAN (R 3.6.0)                           
##  glue                     1.3.2    2020-03-12 [1] CRAN (R 3.6.0)                           
##  gridExtra                2.3      2017-09-09 [1] CRAN (R 3.6.0)                           
##  gtable                   0.3.0    2019-03-25 [1] CRAN (R 3.6.0)                           
##  htmltools                0.4.0    2019-10-04 [1] CRAN (R 3.6.0)                           
##  htmlwidgets              1.5.1    2019-10-08 [1] CRAN (R 3.6.0)                           
##  httpuv                   1.5.2    2019-09-11 [1] CRAN (R 3.6.0)                           
##  httr                     1.4.1    2019-08-05 [1] CRAN (R 3.6.0)                           
##  igraph                   1.2.5    2020-03-19 [1] CRAN (R 3.6.0)                           
##  interactiveDisplayBase   1.24.0   2019-10-29 [1] Bioconductor                             
##  IRanges                * 2.20.2   2020-01-13 [1] Bioconductor                             
##  irlba                    2.3.3    2019-02-05 [1] CRAN (R 3.6.0)                           
##  jsonlite                 1.6.1    2020-02-02 [1] CRAN (R 3.6.0)                           
##  knitr                    1.28     2020-02-06 [1] CRAN (R 3.6.0)                           
##  labeling                 0.3      2014-08-23 [1] CRAN (R 3.6.0)                           
##  later                    1.0.0    2019-10-04 [1] CRAN (R 3.6.0)                           
##  lattice                  0.20-40  2020-02-19 [1] CRAN (R 3.6.0)                           
##  lazyeval                 0.2.2    2019-03-15 [1] CRAN (R 3.6.0)                           
##  lifecycle                0.2.0    2020-03-06 [1] CRAN (R 3.6.0)                           
##  limma                    3.42.2   2020-02-03 [1] Bioconductor                             
##  locfit                   1.5-9.1  2013-04-20 [1] CRAN (R 3.6.0)                           
##  magrittr                 1.5      2014-11-22 [1] CRAN (R 3.6.0)                           
##  Matrix                   1.2-18   2019-11-27 [1] CRAN (R 3.6.3)                           
##  matrixStats            * 0.56.0   2020-03-13 [1] CRAN (R 3.6.0)                           
##  memoise                  1.1.0    2017-04-21 [1] CRAN (R 3.6.0)                           
##  mime                     0.9      2020-02-04 [1] CRAN (R 3.6.0)                           
##  munsell                  0.5.0    2018-06-12 [1] CRAN (R 3.6.0)                           
##  pillar                   1.4.3    2019-12-20 [1] CRAN (R 3.6.0)                           
##  pkgconfig                2.0.3    2019-09-22 [1] CRAN (R 3.6.1)                           
##  plotly                 * 4.9.2    2020-02-12 [1] CRAN (R 3.6.0)                           
##  promises                 1.1.0    2019-10-04 [1] CRAN (R 3.6.0)                           
##  purrr                    0.3.3    2019-10-18 [1] CRAN (R 3.6.0)                           
##  R6                       2.4.1    2019-11-12 [1] CRAN (R 3.6.1)                           
##  rappdirs                 0.3.1    2016-03-28 [1] CRAN (R 3.6.0)                           
##  Rcpp                     1.0.4    2020-03-17 [1] CRAN (R 3.6.0)                           
##  RCurl                    1.98-1.1 2020-01-19 [1] CRAN (R 3.6.0)                           
##  rlang                    0.4.5    2020-03-01 [1] CRAN (R 3.6.0)                           
##  rmarkdown                2.1      2020-01-20 [1] CRAN (R 3.6.0)                           
##  RSQLite                  2.2.0    2020-01-07 [1] CRAN (R 3.6.0)                           
##  rsvd                     1.0.3    2020-02-17 [1] CRAN (R 3.6.0)                           
##  S4Vectors              * 0.24.3   2020-01-18 [1] Bioconductor                             
##  scales                   1.1.0    2019-11-18 [1] CRAN (R 3.6.1)                           
##  scater                 * 1.14.6   2019-12-16 [1] Bioconductor                             
##  scran                  * 1.14.6   2020-02-03 [1] Bioconductor                             
##  scRNAseq               * 2.0.2    2019-11-12 [1] Bioconductor                             
##  sessioninfo              1.1.1    2018-11-05 [1] CRAN (R 3.6.0)                           
##  shiny                    1.4.0.2  2020-03-13 [1] CRAN (R 3.6.0)                           
##  SingleCellExperiment   * 1.8.0    2019-10-29 [1] Bioconductor                             
##  statmod                  1.4.34   2020-02-17 [1] CRAN (R 3.6.0)                           
##  stringi                  1.4.6    2020-02-17 [1] CRAN (R 3.6.0)                           
##  stringr                  1.4.0    2019-02-10 [1] CRAN (R 3.6.0)                           
##  SummarizedExperiment   * 1.16.1   2019-12-19 [1] Bioconductor                             
##  tibble                   2.1.3    2019-06-06 [1] CRAN (R 3.6.0)                           
##  tidyr                    1.0.2    2020-01-24 [1] CRAN (R 3.6.2)                           
##  tidyselect               1.0.0    2020-01-27 [1] CRAN (R 3.6.0)                           
##  vctrs                    0.2.4    2020-03-10 [1] CRAN (R 3.6.0)                           
##  vipor                    0.4.5    2017-03-22 [1] CRAN (R 3.6.0)                           
##  viridis                  0.5.1    2018-03-29 [1] CRAN (R 3.6.0)                           
##  viridisLite              0.3.0    2018-02-01 [1] CRAN (R 3.6.0)                           
##  withr                    2.1.2    2018-03-15 [1] CRAN (R 3.6.0)                           
##  xaringan                 0.15     2020-03-04 [1] CRAN (R 3.6.3)                           
##  xaringanthemer         * 0.2.0    2020-03-22 [1] Github (gadenbuie/xaringanthemer@460f441)
##  xfun                     0.12     2020-01-13 [1] CRAN (R 3.6.0)                           
##  xtable                   1.8-4    2019-04-21 [1] CRAN (R 3.6.0)                           
##  XVector                  0.26.0   2019-10-29 [1] Bioconductor                             
##  yaml                     2.2.1    2020-02-01 [1] CRAN (R 3.6.0)                           
##  zlibbioc                 1.32.0   2019-10-29 [1] Bioconductor                             
## 
## [1] /Library/Frameworks/R.framework/Versions/3.6/Resources/library
```

]]