---
title: "Lecture 1 -- Introduction to R / Bioconductor"
author: "Martin Morgan <Martin.Morgan@RoswellPark.org>"
output:
  BiocStyle::html_document:
    toc: true
    toc_depth: 2
vignette: >
  % \VignetteIndexEntry{Lecture 1 -- Introduction to R / Bioconductor}
  % \VignetteEngine{knitr::rmarkdown}
---

```{r style, echo = FALSE, results = 'asis'}
knitr::opts_chunk$set(
    eval=as.logical(Sys.getenv("KNITR_EVAL", "TRUE")),
    cache=as.logical(Sys.getenv("KNITR_CACHE", "TRUE")))
```

Author: [Martin Morgan][]</br>
Date: 22 July, 2019</br >

[Martin Morgan]: mailto: Martin.Morgan@RoswellPark.org

# _R_

## History of _R_ and _CRAN_

- Statistical programming language. Concieved 1992, initial version
  1996, stable beta version in 2000; an implementation of _S_. CRAN
  started in 1997.
- 'Free' software: no cost, open source, broad use.
- Extensible: packages (15,000 on [CRAN][], 1750 on [Bioconductor][])
- Key features
    - Intrinsic _statistical_ concepts
    - _Vectorized_ computation
    - 'Old-school' scripts rather than graphical user interface -- great
      for reproducibility!
    - (Advanced) _copy-on-change_ semanatics

## Vectors and data frames

```{r}
1 + 2
x = c(1, 2, 3)
1:3             # sequence of integers from 1 to 3
x + c(4, 5, 6)  # vectorized
x + 4           # recycling
```

Vectors

- `numeric()`, `character()`, `logical()`, `integer()`, `complex()`, ...
- `NA`: 'not available'
- `factor()`: values from restricted set of 'levels'.

Operations

- numeric: `==`, `<`, `<=`, `>`, `>=`, ...
- logical: `|` (or), `&` (and), `!` (not)
- subset: `[`, e.g., `x[c(2, 3)]`
- assignment: `[<-`, e.g., `x[c(1, 3)] = x[c(1, 3)]`
- other: `is.na()`

Functions

```{r}
x = rnorm(100)
y = x + rnorm(100)
plot(x, y)
```

- Many!

`data.frame`

```{r}
df <- data.frame(Independent = x, Dependent = y)
head(df)
df[1:5, 1:2]
df[1:5, ]
plot(Dependent ~ Independent, df)  # 'formula' interface
```

- List of equal-length vectors
- Vectors can be of different type
- Two-dimensional subset and assignment
- Column access: `df[, 1]`, `df[, "Indep"]`, `df[[1]]`,
  `df[["Indep"]]`, `df$Indep`

Exercise: plot only values with `Dependent > 0`, `Independent > 0`

1. Select rows

    ```{r}
    ridx <- (df$Dependent > 0) & (df$Independent > 0)
    ```

2. Plot subset

    ```{r}
    plot(Dependent ~ Independent, df[ridx, ])
    ```

3. Skin the cat another way

    ```{r}
    plot(
        Dependent ~ Independent, df,
        subset = (Dependent > 0) & (Independent > 0)
    )
    ```

## Analysis: functions, classes, methods

```{r}
fit <- lm(Dependent ~ Independent, df)  # linear model -- regression
anova(fit)                              # summary table
plot(Dependent ~ Independent, df)
abline(fit)
```

- `lm()`: plain-old function
- `fit`: an _object_ of class "lm"
- `anova()`: a _generic_ with a specific _method_ for class "lm"

```{r}
class(fit)
methods(class="lm")
```

## Help!

```{r, eval=FALSE}
?"plot"          # plain-old-function or generic
?"plot.formula"  # method
?"plot.lm"       # method for object of class 'lm', plot(fit)
```

## Packages


```{r}
library(ggplot2)
ggplot(df, aes(x = Independent, y = Dependent)) +
    geom_point() + geom_smooth(method = "lm")
```

- General purpose: >15,000 packages on [CRAN][]
- Gain contributor's domain expertise _and_ weird (or other) idiosyncracies
- _Installation_ (once only per computer) versus _load_ (via
  `library(ggplot2)`, once per session)

[CRAN]: https://cran.r-project.org
[Bioconductor]: https://bioconductor.org

# _Bioconductor_

Started 2002 as a platform for understanding analysis of microarray
data

## Packages

1,750 packages. Domains of expertise:

- Sequencing (RNASeq, ChIPSeq, single-cell, called variants, ...)
- Microarrays (methylation, expression, copy number, ...)
- flow cytometry
- proteomics
- ...

Important themes

- Reproducible research
- Interoperability between packages & work kflows
- Usability

Resources

- https://bioconductor.org
- https://bioconductor.org/packages -- software, annotation, experiment, workflow
- https://support.bioconductor.org
- Community [slack][] ([sign-up][slack-sign-up])

[slack]: https://community-bioc.slack.com
[slack-sign-up]: https://bioc-community.herokuapp.com/

## Objects

A distinctive feature of _Bioconductor_ -- use of _objects_ for
representing data

```{r, message = FALSE}
library(Biostrings)
dna <- DNAStringSet(c("AACTCC", "CTGCA"))
dna
reverseComplement(dna)
```

- [Biostrings][]: DNA, RNA, AA representation and manipulation
- [GenomicRanges][]: Coordinates in genome space
- [SummarizedExperiment][]: coordinating 'assay' data (e.g., counts
  from an RNASeq experiment) with row and column annotations (e.g.,
  information about samples and experimental treatments).
  
[Biostrings]: https://bioconductor.org/packages/Biostrings
[GenomicRanges]: https://bioconductor.org/packages/GenomicRanges
[SummarizedExperiment]: https://bioconductor.org/packages/SummarizedExperiment

## High-throughput sequence work flow

![](lecture-01-figures/SequencingEcosystem.png)

Web site, https://bioconductor.org

1750 'software' packages, https://bioconductor.org/packages

- Sequence analysis (RNASeq, ChIPSeq, called variants, copy number, 
  single cell)
- Microarrays (methylation, copy number, classical expression, ...)
- Annotation (more about annotations later this morning...)
- Flow cytometry
- Proteomics, image analysis, ...

Discovery and use, e.g., [DESeq2][]

- Landing pages: title, description (abstract), installation instructions,
  badges
- Vignettes!

Also:

- 'Annotation' packages
- 'Experiment data' packages
- Workflows
- [Course material][], ...

[DESeq2]: https://bioconductor.org/packages/DESeq2
[Course material]: https://bioconductor.org/help/course-materials/

# End matter

## Session Info

```{r}
sessionInfo()
```

## Acknowledgements

Research reported in this tutorial was supported by the National Human
Genome Research Institute and the National Cancer Institute of the
National Institutes of Health under award numbers U41HG004059 and
U24CA180996.

This project has received funding from the European Research Council
(ERC) under the European Union's Horizon 2020 research and innovation
programme (grant agreement number 633974)