Introducción

# <strong>Introducción</strong>
## <strong>Bioconductor</strong> para datos transcriptómicos de célula única (<strong>scRNA-seq</strong>) – <strong>CDSB2020</strong>
### <a href="http://lcolladotor.github.io/">Leonardo Collado-Torres</a>
### 2020-08-05

---

<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('comunidadbioinfo/cdsb2020')` or view online at [**comunidadbioinfo.github.io/cdsb2020**](http://comunidadbioinfo.github.io/cdsb2020).]

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---

# Orígenes del curso

* El libro [**Orchestrating Single Cell Analysis With Bioconductor**](ohttps://osca.bioconductor.org/) escrito por [Aaron Lun](https://www.linkedin.com/in/aaron-lun-869b5894/), [Robert Amezquita](https://robertamezquita.github.io/), [Stephanie Hicks](https://www.stephaniehicks.com/) y [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)

* [**Curso de scRNA-seq de WEHI**](https://drive.google.com/drive/folders/1cn5d-Ey7-kkMiex8-74qxvxtCQT6o72h) creado por [Peter Hickey](https://www.peterhickey.org/)

---

# Instructor

**Leonardo Collado-Torres**

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

---

---

---

---

---

# Pre-requisitos

Instala R 4.0.x desde [CRAN](https://cran.r-project.org/) y luego instala los siguientes paquetes de R:

```r
## Para instalar paquetes de Bioconductor
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

## Instala los paquetes de R que necesitamos
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',
        'spatialLIBD',
        'sessioninfo',
        'scPipe'
    )
)
```

Tabmién debes instalar [RStudio](https://rstudio.com/products/rstudio/download/#download) versión 1.3.1 o la más nueva de preferencia.

]

---

# Ayudantes del curso

* [Ana Betty Villaseñor Altamirano](https://twitter.com/AnaBetty2304)

* [Aurora Labastida](https://twitter.com/alabasti1)

* [Carlos Gabriel Aguilar Pérez](https://twitter.com/Carlos_Aplysia)

---

# Materiales del curso

* Descargalos con `usethis::use_course('comunidadbioinfo/cdsb2020')`

* Checalos en línea a través de [**comunidadbioinfo.github.io/cdsb2020**](http://comunidadbioinfo.github.io/cdsb2020)

* **Clona** el repositorio de GitHub, lo cual te hará más fácil el poder actualizar tus archivos con la versión más reciente usando *git pull*

```bash
## Si tienes las llaves de SSH configuradas
git clone git@github.com:comunidadbioinfo/cdsb2020.git

## o vía https
git clone https://github.com/comunidadbioinfo/cdsb2020.git
```

Desde R:

```r
git2r::clone('https://github.com/comunidadbioinfo/cdsb2020',
    'csdb2020')
```

---

# Crea tu propio proyecto

Te recomiendo que crees tu propio proyecto y actives el control de versiones

```r
usethis::create_project('~/Desktop/cdsb2020_leo')
```

```r
## Crea un archivo de setup
usethis::use_r('00-setup.R')
```

---

En este script de setup, guarda los siguientes comandos

```r
## Crea el repositorio de Git
usethis::use_git()

## Configura tu conexión a GitHub de ser necesario
usethis::browse_github_token()
usethis::edit_r_environ() ## y después reinicia R

## Utiliza GitHub
usethis::use_github() ## crea un commit, luego corre este comando

## Empieza tus notas sobre la introducción a scRNA-seq
usethis::use_r('01-introduction.R')
```

Ve el ejemplo en [**cdsb2020_leo**](https://github.com/lcolladotor/cdsb2020_leo)

---

# Pedir ayuda

* Usa la manita azul de Zoom: "raise your **hand**"

* Crea un **issue** en [cdsb2020](https://github.com/comunidadbioinfo/cdsb2020/issues). ¡Acuérdate de incluir un ejemplo reproducible!

* De forma más general, a través del [**Bioconductor Support Website**](https://support.bioconductor.org/) etiquetando el paquete de R relacionado.

* Algunos blog posts relacionados: [**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), y [**Learning from our search history**](http://lcolladotor.github.io/2020/02/12/learning-from-our-search-history/)

* La plática de [Jenny Bryan](https://twitter.com/JennyBryan) para `rstudio::conf(2020)`: [**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://raw.githubusercontent.com/Bioconductor/OrchestratingSingleCellAnalysis-release/master/images/cover.png)
background-size: contain

---

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

---

background-image: url(https://raw.githubusercontent.com/Bioconductor/OrchestratingSingleCellAnalysis-release/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

---

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

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

---

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

```
## snapshotDate(): 2020-04-27
```

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

```
## loading from cache
```

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

```
## loading from cache
```

```r
## ¿Qué tan grandes son los datos?
pryr::object_size(sce)
```

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

```
## 461 MB
```

```r
## ¿Cómo es el objeto?
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):
## altExpNames(0):
```

]

---

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

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

# Selección de genes.
dec <- scran::modelGeneVar(sce)
hvg <- scran::getTopHVGs(dec, prop = 0.1)

# Reducción de dimensiones.
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
# Visualización.
scater::plotUMAP(sce, colour_by = "clusters")
```

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

---

```r
# Visualización interactiva.
p <- scater::plotUMAP(sce, colour_by = "clusters")
plotly::ggplotly(p)
```

---

# ¡Gracias!

Las diapositivias fueron hechas con el paquete de R [**xaringan**](https://github.com/yihui/xaringan) y configuradas con [**xaringanthemer**](https://github.com/gadenbuie/xaringanthemer).

Este curso está basado en el libro [**Orchestrating Single Cell Analysis with Bioconductor**](https://osca.bioconductor.org/) de [Aaron Lun](https://www.linkedin.com/in/aaron-lun-869b5894/), [Robert Amezquita](https://robertamezquita.github.io/), [Stephanie Hicks](https://www.stephaniehicks.com/) y [Raphael Gottardo](http://rglab.org), además del [**curso de scRNA-seq para WEHI**](https://drive.google.com/drive/folders/1cn5d-Ey7-kkMiex8-74qxvxtCQT6o72h) creado por [Peter Hickey](https://www.peterhickey.org/).

Puedes encontrar los archivos para este taller en  [comunidadbioinfo/cdsb2020](https://github.com/comunidadbioinfo/cdsb2020).

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

]

.footnote[Descarga los materiales con `usethis::use_course('comunidadbioinfo/cdsb2020')` o revisalos en línea vía [**comunidadbioinfo.github.io/cdsb2020**](http://comunidadbioinfo.github.io/cdsb2020).]

---

# Detalles de la sesión de R

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

```
## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value                       
##  version  R version 4.0.2 (2020-06-22)
##  os       macOS Catalina 10.15.5      
##  system   x86_64, darwin17.0          
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  ctype    en_US.UTF-8                 
##  tz       America/New_York            
##  date     2020-08-02                  
## 
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────
##  package                * version  date       lib source                            
##  AnnotationDbi            1.50.3   2020-07-25 [1] Bioconductor                      
##  AnnotationHub            2.20.0   2020-04-27 [1] Bioconductor                      
##  assertthat               0.2.1    2019-03-21 [1] CRAN (R 4.0.0)                    
##  beeswarm                 0.2.3    2016-04-25 [1] CRAN (R 4.0.0)                    
##  Biobase                * 2.48.0   2020-04-27 [1] Bioconductor                      
##  BiocFileCache            1.12.0   2020-04-27 [1] Bioconductor                      
##  BiocGenerics           * 0.34.0   2020-04-27 [1] Bioconductor                      
##  BiocManager              1.30.10  2019-11-16 [1] CRAN (R 4.0.0)                    
##  BiocNeighbors            1.6.0    2020-04-27 [1] Bioconductor                      
##  BiocParallel             1.22.0   2020-04-27 [1] Bioconductor                      
##  BiocSingular             1.4.0    2020-04-27 [1] Bioconductor                      
##  BiocVersion              3.11.1   2020-04-07 [1] Bioconductor                      
##  bit                      1.1-15.2 2020-02-10 [1] CRAN (R 4.0.0)                    
##  bit64                    0.9-7.1  2020-07-15 [1] CRAN (R 4.0.2)                    
##  bitops                   1.0-6    2013-08-17 [1] CRAN (R 4.0.0)                    
##  blob                     1.2.1    2020-01-20 [1] CRAN (R 4.0.0)                    
##  cli                      2.0.2    2020-02-28 [1] CRAN (R 4.0.0)                    
##  codetools                0.2-16   2018-12-24 [1] CRAN (R 4.0.2)                    
##  colorout               * 1.2-2    2020-03-16 [1] Github (jalvesaq/colorout@726d681)
##  colorspace               1.4-1    2019-03-18 [1] CRAN (R 4.0.0)                    
##  cowplot                  1.0.0    2019-07-11 [1] CRAN (R 4.0.0)                    
##  crayon                   1.3.4    2017-09-16 [1] CRAN (R 4.0.0)                    
##  curl                     4.3      2019-12-02 [1] CRAN (R 4.0.0)                    
##  data.table               1.13.0   2020-07-24 [1] CRAN (R 4.0.2)                    
##  DBI                      1.1.0    2019-12-15 [1] CRAN (R 4.0.0)                    
##  dbplyr                   1.4.4    2020-05-27 [1] CRAN (R 4.0.2)                    
##  DelayedArray           * 0.14.1   2020-07-14 [1] Bioconductor                      
##  DelayedMatrixStats       1.10.1   2020-07-03 [1] Bioconductor                      
##  digest                   0.6.25   2020-02-23 [1] CRAN (R 4.0.0)                    
##  dplyr                    1.0.0    2020-05-29 [1] CRAN (R 4.0.2)                    
##  dqrng                    0.2.1    2019-05-17 [1] CRAN (R 4.0.0)                    
##  edgeR                    3.30.3   2020-06-02 [1] Bioconductor                      
##  ellipsis                 0.3.1    2020-05-15 [1] CRAN (R 4.0.0)                    
##  evaluate                 0.14     2019-05-28 [1] CRAN (R 4.0.0)                    
##  ExperimentHub            1.14.0   2020-04-27 [1] Bioconductor                      
##  fansi                    0.4.1    2020-01-08 [1] CRAN (R 4.0.0)                    
##  farver                   2.0.3    2020-01-16 [1] CRAN (R 4.0.0)                    
##  fastmap                  1.0.1    2019-10-08 [1] CRAN (R 4.0.0)                    
##  generics                 0.0.2    2018-11-29 [1] CRAN (R 4.0.0)                    
##  GenomeInfoDb           * 1.24.2   2020-06-15 [1] Bioconductor                      
##  GenomeInfoDbData         1.2.3    2020-04-16 [1] Bioconductor                      
##  GenomicRanges          * 1.40.0   2020-04-27 [1] Bioconductor                      
##  ggbeeswarm               0.6.0    2017-08-07 [1] CRAN (R 4.0.0)                    
##  ggplot2                * 3.3.2    2020-06-19 [1] CRAN (R 4.0.2)                    
##  glue                     1.4.1    2020-05-13 [1] CRAN (R 4.0.0)                    
##  gridExtra                2.3      2017-09-09 [1] CRAN (R 4.0.0)                    
##  gtable                   0.3.0    2019-03-25 [1] CRAN (R 4.0.0)                    
##  htmltools                0.5.0    2020-06-16 [1] CRAN (R 4.0.2)                    
##  htmlwidgets              1.5.1    2019-10-08 [1] CRAN (R 4.0.0)                    
##  httpuv                   1.5.4    2020-06-06 [1] CRAN (R 4.0.2)                    
##  httr                     1.4.2    2020-07-20 [1] CRAN (R 4.0.2)                    
##  igraph                   1.2.5    2020-03-19 [1] CRAN (R 4.0.0)                    
##  interactiveDisplayBase   1.26.3   2020-06-02 [1] Bioconductor                      
##  IRanges                * 2.22.2   2020-05-21 [1] Bioconductor                      
##  irlba                    2.3.3    2019-02-05 [1] CRAN (R 4.0.0)                    
##  jsonlite                 1.7.0    2020-06-25 [1] CRAN (R 4.0.0)                    
##  knitr                    1.29     2020-06-23 [1] CRAN (R 4.0.0)                    
##  labeling                 0.3      2014-08-23 [1] CRAN (R 4.0.0)                    
##  later                    1.1.0.1  2020-06-05 [1] CRAN (R 4.0.2)                    
##  lattice                  0.20-41  2020-04-02 [1] CRAN (R 4.0.2)                    
##  lazyeval                 0.2.2    2019-03-15 [1] CRAN (R 4.0.0)                    
##  lifecycle                0.2.0    2020-03-06 [1] CRAN (R 4.0.0)                    
##  limma                    3.44.3   2020-06-12 [1] Bioconductor                      
##  locfit                   1.5-9.4  2020-03-25 [1] CRAN (R 4.0.0)                    
##  magrittr                 1.5      2014-11-22 [1] CRAN (R 4.0.0)                    
##  Matrix                   1.2-18   2019-11-27 [1] CRAN (R 4.0.2)                    
##  matrixStats            * 0.56.0   2020-03-13 [1] CRAN (R 4.0.0)                    
##  memoise                  1.1.0    2017-04-21 [1] CRAN (R 4.0.0)                    
##  mime                     0.9      2020-02-04 [1] CRAN (R 4.0.0)                    
##  munsell                  0.5.0    2018-06-12 [1] CRAN (R 4.0.0)                    
##  pillar                   1.4.6    2020-07-10 [1] CRAN (R 4.0.2)                    
##  pkgconfig                2.0.3    2019-09-22 [1] CRAN (R 4.0.0)                    
##  plotly                 * 4.9.2.1  2020-04-04 [1] CRAN (R 4.0.0)                    
##  promises                 1.1.1    2020-06-09 [1] CRAN (R 4.0.2)                    
##  pryr                     0.1.4    2018-02-18 [1] CRAN (R 4.0.0)                    
##  purrr                    0.3.4    2020-04-17 [1] CRAN (R 4.0.0)                    
##  R6                       2.4.1    2019-11-12 [1] CRAN (R 4.0.0)                    
##  rappdirs                 0.3.1    2016-03-28 [1] CRAN (R 4.0.0)                    
##  Rcpp                     1.0.5    2020-07-06 [1] CRAN (R 4.0.2)                    
##  RCurl                    1.98-1.2 2020-04-18 [1] CRAN (R 4.0.0)                    
##  rlang                    0.4.7    2020-07-09 [1] CRAN (R 4.0.2)                    
##  rmarkdown                2.3      2020-06-18 [1] CRAN (R 4.0.0)                    
##  RSpectra                 0.16-0   2019-12-01 [1] CRAN (R 4.0.0)                    
##  RSQLite                  2.2.0    2020-01-07 [1] CRAN (R 4.0.0)                    
##  rsvd                     1.0.3    2020-02-17 [1] CRAN (R 4.0.0)                    
##  S4Vectors              * 0.26.1   2020-05-16 [1] Bioconductor                      
##  scales                   1.1.1    2020-05-11 [1] CRAN (R 4.0.0)                    
##  scater                 * 1.16.2   2020-06-26 [1] Bioconductor                      
##  scran                  * 1.16.0   2020-04-27 [1] Bioconductor                      
##  scRNAseq               * 2.2.0    2020-05-07 [1] Bioconductor                      
##  sessioninfo              1.1.1    2018-11-05 [1] CRAN (R 4.0.0)                    
##  shiny                    1.5.0    2020-06-23 [1] CRAN (R 4.0.2)                    
##  showtext                 0.8-1    2020-05-25 [1] CRAN (R 4.0.2)                    
##  showtextdb               3.0      2020-06-04 [1] CRAN (R 4.0.2)                    
##  SingleCellExperiment   * 1.10.1   2020-04-28 [1] Bioconductor                      
##  statmod                  1.4.34   2020-02-17 [1] CRAN (R 4.0.0)                    
##  stringi                  1.4.6    2020-02-17 [1] CRAN (R 4.0.0)                    
##  stringr                  1.4.0    2019-02-10 [1] CRAN (R 4.0.0)                    
##  SummarizedExperiment   * 1.18.2   2020-07-14 [1] Bioconductor                      
##  sysfonts                 0.8.1    2020-05-08 [1] CRAN (R 4.0.0)                    
##  tibble                   3.0.3    2020-07-10 [1] CRAN (R 4.0.2)                    
##  tidyr                    1.1.0    2020-05-20 [1] CRAN (R 4.0.2)                    
##  tidyselect               1.1.0    2020-05-11 [1] CRAN (R 4.0.2)                    
##  uwot                     0.1.8    2020-03-16 [1] CRAN (R 4.0.0)                    
##  vctrs                    0.3.2    2020-07-15 [1] CRAN (R 4.0.2)                    
##  vipor                    0.4.5    2017-03-22 [1] CRAN (R 4.0.0)                    
##  viridis                  0.5.1    2018-03-29 [1] CRAN (R 4.0.0)                    
##  viridisLite              0.3.0    2018-02-01 [1] CRAN (R 4.0.0)                    
##  whisker                  0.4      2019-08-28 [1] CRAN (R 4.0.0)                    
##  withr                    2.2.0    2020-04-20 [1] CRAN (R 4.0.0)                    
##  xaringan                 0.16     2020-03-31 [1] CRAN (R 4.0.0)                    
##  xaringanthemer         * 0.3.0    2020-05-04 [1] CRAN (R 4.0.0)                    
##  xfun                     0.16     2020-07-24 [1] CRAN (R 4.0.2)                    
##  xtable                   1.8-4    2019-04-21 [1] CRAN (R 4.0.0)                    
##  XVector                  0.28.0   2020-04-27 [1] Bioconductor                      
##  yaml                     2.2.1    2020-02-01 [1] CRAN (R 4.0.0)                    
##  zlibbioc                 1.34.0   2020-04-27 [1] Bioconductor                      
## 
## [1] /Library/Frameworks/R.framework/Versions/4.0/Resources/library
```

]]