# scater: single-cell analysis toolkit for expression with R [](https://semaphoreci.com/davismcc/scater) [](https://codecov.io/github/davismcc/scater?branch=master) [](https://ci.appveyor.com/project/davismcc/scater) <!--- [](https://travis-ci.org/davismcc/scater) ---> <img src=inst/scater_sticker_300dpi.png height="200"> This package contains useful tools for the analysis of single-cell gene expression data using the statistical software R. The package places an emphasis on tools for quality control, visualisation and pre-processing of data before further downstream analysis. We hope that `scater` fills a useful niche between raw RNA-sequencing count or transcripts-per-million data and more focused downstream modelling tools such as [monocle](http://www.bioconductor.org/packages/release/bioc/html/monocle.html), [scLVM](http://github.com/PMBio/scLVM), [SCDE](http://pklab.med.harvard.edu/scde/index.html), [edgeR](http://www.bioconductor.org/packages/release/bioc/html/edgeR.html), [limma](http://www.bioconductor.org/packages/release/bioc/html/limma.html) and so on. Briefly, `scater` enables the following: 1. Automated computation of QC metrics 1. Transcript quantification from read data with pseudo-alignment 2. Data format standardisation 3. Rich visualisations for exploratory analysis 4. Seamless integration into the Bioconductor universe 5. Simple normalisation methods See below for information about installation, getting started and highlights of the package. ## Installation ### Installation from Bioconductor (recommended) The `scater` package has been accepted into Bioconductor! Thus, the most reliable way to install the package is to use the usual Bioconductor method: ```{R} ## try http:// if https:// URLs are not supported source("https://bioconductor.org/biocLite.R") biocLite("scater") ``` The `scater` package has been available as a "release" version in the Bioconductor since April 2016. The release version of `scater` works with the release version of R and Bioconductor, and development will continue in the devel version of the package on Bioconductor. Future releases will follow the regular Bioconductor release cycle. ## Installation from Github Alternatively, `scater` can be installed directly from GitHub as described below. In this case, package that `scater` uses ("depends on" in R parlance) may not be automatically installed, so you may have to install the required packages as shown below. I recommend using Hadley Wickham's `devtools` package to install `scater` directly from GitHub. If you don't have `devtools` installed, then install that from CRAN (as shown below) and then run the call to install `scater`: **If you are using R version 3.3:** ```{r} install.packages("devtools") devtools::install_github("davismcc/scater", build_vignettes = TRUE) ``` **If you are using R version 3.2.3:** ```{r} devtools::install_github("davismcc/scater", ref = "release-R-3.2", build_vignettes = TRUE) ``` If you find that the above will not install on Linux systems, please try with the option `build_vignettes = FALSE`. This is a known issue that we are working to resolve. Using the most recent version of R is strongly recommended (R 3.3.2 at the time of writing). Effort has been made to ensure the package works with R >3.0, but the package has not been tested with R <3.1.1. There are several other packages from CRAN and Bioconductor that `scater` uses, so you will need to have these packages installed as well. The CRAN packages should install automatically when `scater` is installed, but you will need to install the Bioconductor packages manually. Not all of the following are strictly necessary, but they enhance the functionality of `scater` and are good packages in their own right. The commands below should help with package installations. CRAN packages: ```{r} install.packages(c("data.table", "ggplot2", "knitr", "matrixStats", "MASS", "plyr", "reshape2", "rjson", "testthat", "viridis")) ``` Bioconductor packages: ```{r} source("http://bioconductor.org/biocLite.R") biocLite(c("Biobase", "biomaRt", "edgeR", "limma", "rhdf5")) ``` Optional packages that are not strictly required but enhance the functionality of `scater`: ```{r} install.packages(c("cowplot", "cluster", "mvoutlier", "parallel", "Rtsne")) biocLite(c("destiny", "monocle")) ``` You might also like to install `dplyr` for convenient data manipulation: ```{r} install.packages("dplyr") ``` ## Getting started <!--- The best place to start is the [vignette](http://htmlpreview.github.io/?http://github.com/davismcc/scater/blob/master/vignettes/vignette.html). --> The best place to start is the vignette. From inside an R session, load `scater` and then browse the vignettes: ```{r} library(scater) browseVignettes("scater") ``` There is a detailed HTML document available that introduces the main features and functionality of `scater`. ## `scater` workflow The diagram below provised an overview of the pre-processing and QC workflow possible in `scater`, listing the functions that can be used at various stages. A first place to start is with the `newSCESet` function, which will allow you to create a data object for use with `scater`.  ## Highlights The `scater` package allows you to do some neat things relatively quickly. Some highlights are shown below with example code and screenshots. 1. Automated computation of QC metrics 1. Transcript quantification from read data with pseudo-alignment approaches 2. Data format standardisation 3. Rich visualisations for QC and exploratory analysis 4. Seamless integration into the Bioconductor universe 5. Simple normalisation methods For details of how to use these functions, please consult the **vignette** and **package documentation**. The plots shown use the example data included with the package (for which there is no interesting structure) and as shown require only one or two lines of code to generate. ### Automatic computation of QC metrics Use the `calculateQCMetrics` function to compute many metrics useful for gene/transcript-level and cell-level QC. Metrics computed include number of genes expressed per cell, percentage of expression from control genes (e.g. ERCC spike-ins) and many more. ### Transcript quantification with `kallisto` or `Salmon` The `runKallisto` and `runSalmon` functions provides wrappers to the [`kallisto`](http://pachterlab.github.io/kallisto) and ['Salmon'](https://combine-lab.github.io/salmon/) software for quantifying transcript abundance from FASTQ files using a "pseudo-alignment"" or "lightweight alignment" approaches. These new approaches are extremely fast while retaining accuracy. With `readKallistoResults` and `readSalmonResults`, transcript quantities can be read into a data object in `R`. ### Plotting functions Default `plot` for an SCESet object gives cumulative expression for the most-expressed features (genes or transcripts) The `plotTSNE` function produces a t-distributed stochastic neighbour embedding plot for the cells. The `plotPCA` function produces a principal components analysis plot for the cells. The `plotDiffusionMap` function produces a diffusion map plot for the cells. The `plotExpression` function plots the expression values for a selection of features. The `plotQC` function produces a variety of QC plots useful for diagnostics and feature and cell filtering. It can be used to plot the most highly-expressed genes (or features) in the data set or create density plots to assess the relative importance of explanatory variables, as well as many other visualisations useful for QC. The `plotPhenoData` function plots two phenotype metadata variables (such as QC metrics). See also `plotFeatureData` to plot feature (gene) metadata variables, including QC metrics. Plus many, many more possibilities. Please consult the vignette and documentation for details. ## Acknowledgements and disclaimer The package leans heavily on previously published work and packages, namely [edgeR](http://bioconductor.org/packages/release/bioc/html/edgeR.html) and [limma](http://bioconductor.org/packages/release/bioc/html/limma.html). The `SCESet` class is inspired by the `CellDataSet` class from [monocle](http://www.bioconductor.org/packages/release/bioc/html/monocle.html), and `SCESet` objects in `scater` can be easily converted to and from `monocle's` `CellDataSet` objects. The `scater` sticker is licensed under Creative Commons Attribution [CC-BY](https://creativecommons.org/licenses/by/2.0/). Feel free to share and adapt, but don't forget to credit the author. Skateboard icon made by [Nikita Golubev](http://www.flaticon.com/authors/nikita-golubev) from [Flaticon](http://www.flaticon.com) is licensed by [Creative Commons BY 3.0](http://creativecommons.org/licenses/by/3.0/). We hope the `scater` package makes your life easier when analysing single-cell RNA_seq data. Please do try it and contact me with bug reports, feedback, feature requests, questions and suggestions to improve the package. Davis McCarthy, April 2017