# An introduction to the SingleCellExperiment class

Davide Risso1 and Aaron Lun*

1Division of Biostatistics and Epidemiology, Weill Cornell Medicine

*infinite.monkeys.with.keyboards@gmail.com

#### 30 October 2019

#### Package

SingleCellExperiment 1.8.0

# 1 Motivation

The `SingleCellExperiment` class is a light-weight container for single-cell genomics data. It extends the `RangedSummarizedExperiment` class and follows similar conventions, i.e., rows should represent features (genes, transcripts, genomic regions) and columns should represent cells. It provides methods for storing dimensionality reduction results and data for alternative feature sets (e.g., synthetic spike-in transcripts, antibody tags). It is the central data structure for Bioconductor single-cell packages like scater and scran.

# 2 Creating SingleCellExperiment instances

`SingleCellExperiment` objects can be created via the constructor of the same name:

    library(SingleCellExperiment)
    counts <- matrix(rpois(100, lambda = 10), ncol=10, nrow=10)
    sce <- SingleCellExperiment(assays = list(counts = counts))
    sce

    ## class: SingleCellExperiment
    ## dim: 10 10
    ## metadata(0):
    ## assays(1): counts
    ## rownames: NULL
    ## rowData names(0):
    ## colnames: NULL
    ## colData names(0):
    ## reducedDimNames(0):
    ## spikeNames(0):
    ## altExpNames(0):

An alternative approach is via coercion from `SummarizedExperiment` objects.

    se <- SummarizedExperiment(list(counts=counts))
    as(se, "SingleCellExperiment")

    ## class: SingleCellExperiment
    ## dim: 10 10
    ## metadata(0):
    ## assays(1): counts
    ## rownames: NULL
    ## rowData names(0):
    ## colnames: NULL
    ## colData names(0):
    ## reducedDimNames(0):
    ## spikeNames(0):
    ## altExpNames(0):

To demonstrate the use of the class, we will the Allen data set from the scRNAseq package.

    library(scRNAseq)
    sce <- ReprocessedAllenData("tophat_counts")
    sce

    ## class: SingleCellExperiment
    ## dim: 20816 379
    ## metadata(2): SuppInfo which_qc
    ## assays(1): tophat_counts
    ## rownames(20816): 0610007P14Rik 0610009B22Rik ... Zzef1 Zzz3
    ## rowData names(0):
    ## colnames(379): SRR2140028 SRR2140022 ... SRR2139341 SRR2139336
    ## colData names(22): NREADS NALIGNED ... Animal.ID passes_qc_checks_s
    ## reducedDimNames(0):
    ## spikeNames(0):
    ## altExpNames(1): ERCC

The set of operations that can be applied to a `RangedSummarizedExperiment` are also applicable to any instance of a `SingleCellExperiment`. This includes access to assay data via `assay()`, column metadata with `colData()`, and so on.

# 3 Adding low-dimensional representations

We compute log-transformed normalized expression values from the count matrix. (We note that many of these steps can be performed as one-liners from the scater package, but we will show them here in full to demonstrate the capabilities of the `SingleCellExperiment` class.)

    counts <- assay(sce, "tophat_counts")
    libsizes <- colSums(counts)
    size.factors <- libsizes/mean(libsizes)
    logcounts(sce) <- log2(t(t(counts)/size.factors) + 1)
    assayNames(sce)

    ## [1] "tophat_counts" "logcounts"

We obtain the PCA and t-SNE representations of the data and add them to the object with the `reducedDims()<-` method.

    pca_data <- prcomp(t(logcounts(sce)), rank=50)

    library(Rtsne)
    set.seed(5252)
    tsne_data <- Rtsne(pca_data$x[,1:50], pca = FALSE)

    reducedDims(sce) <- list(PCA=pca_data$x, TSNE=tsne_data$Y)
    sce

    ## class: SingleCellExperiment
    ## dim: 20816 379
    ## metadata(2): SuppInfo which_qc
    ## assays(2): tophat_counts logcounts
    ## rownames(20816): 0610007P14Rik 0610009B22Rik ... Zzef1 Zzz3
    ## rowData names(0):
    ## colnames(379): SRR2140028 SRR2140022 ... SRR2139341 SRR2139336
    ## colData names(22): NREADS NALIGNED ... Animal.ID passes_qc_checks_s
    ## reducedDimNames(2): PCA TSNE
    ## spikeNames(0):
    ## altExpNames(1): ERCC

The stored coordinates can be retrieved by name or by numerical index. Each row of the coordinate matrix is assumed to correspond to a cell, while each column represents a dimension.

    reducedDims(sce)

    ## List of length 2
    ## names(2): PCA TSNE

    reducedDimNames(sce)

    ## [1] "PCA"  "TSNE"

    head(reducedDim(sce, "PCA")[,1:2])

    ##                   PC1       PC2
    ## SRR2140028  -70.23670  33.78880
    ## SRR2140022  -41.00366  49.76633
    ## SRR2140055 -133.70763   7.68870
    ## SRR2140083  -36.26099 113.18806
    ## SRR2139991 -100.86439  15.94740
    ## SRR2140067  -97.71210  35.77881

    head(reducedDim(sce, "TSNE")[,1:2])

    ##                 [,1]       [,2]
    ## SRR2140028  5.496253 -3.0603142
    ## SRR2140022  6.028424 -1.2858942
    ## SRR2140055 -2.105741 -2.6448354
    ## SRR2140083  5.755633  6.3093892
    ## SRR2139991  4.929426 -0.5097958
    ## SRR2140067  4.898115 -5.2522140

Any subsetting by column of `sce_sub` will also lead to subsetting of the dimensionality reduction results by cell.

    dim(reducedDim(sce, "PCA"))

    ## [1] 379  50

    dim(reducedDim(sce[,1:10], "PCA"))

    ## [1] 10 50

# 4 Convenient access to named assays

In the `SingleCellExperiment`, users can assign arbitrary names to entries of `assays`. To assist interoperability between packages, we provide some suggestions for what the names should be for particular types of data:

- `counts`: Raw count data, e.g., number of reads or transcripts for a particular gene.
- `normcounts`: Normalized values on the same scale as the original counts. For example, counts divided by cell-specific size factors that are centred at unity.
- `logcounts`: Log-transformed counts or count-like values. In most cases, this will be defined as log-transformed `normcounts`, e.g., using log base 2 and a pseudo-count of 1.
- `cpm`: Counts-per-million. This is the read count for each gene in each cell, divided by the library size of each cell in millions.
- `tpm`: Transcripts-per-million. This is the number of transcripts for each gene in each cell, divided by the total number of transcripts in that cell (in millions).

Each of these suggested names has an appropriate getter/setter method for convenient manipulation of the `SingleCellExperiment`. For example, we can take the (very specifically named) `tophat_counts` name and assign it to `counts` instead:

    counts(sce) <- assay(sce, "tophat_counts")
    sce

    ## class: SingleCellExperiment
    ## dim: 20816 379
    ## metadata(2): SuppInfo which_qc
    ## assays(3): tophat_counts logcounts counts
    ## rownames(20816): 0610007P14Rik 0610009B22Rik ... Zzef1 Zzz3
    ## rowData names(0):
    ## colnames(379): SRR2140028 SRR2140022 ... SRR2139341 SRR2139336
    ## colData names(22): NREADS NALIGNED ... Animal.ID passes_qc_checks_s
    ## reducedDimNames(2): PCA TSNE
    ## spikeNames(0):
    ## altExpNames(1): ERCC

    dim(counts(sce))

    ## [1] 20816   379

This means that functions expecting count data can simply call `counts()` without worrying about package-specific naming conventions.

# 5 Adding alternative feature sets

Many scRNA-seq experiments contain sequencing data for multiple feature types beyond the endogenous genes:

- Externally added spike-in transcripts for plate-based experiments.
- Antibody tags for CITE-seq experiments.
- CRISPR tags for CRISPR-seq experiments.
- Allele information for experiments involving multiple genotypes.

Such features can be stored inside the `SingleCellExperiment` via the concept of “alternative Experiments”. These are nested `SummarizedExperiment` instances that are guaranteed to have the same number and ordering of columns as the `SingleCellExperiment` itself. Data for endogenous genes and other features can thus be kept separate, which is often desirable as they need to be processed differently.

To illustrate, consider the case of the spike-in transcripts in the Allen data. We move the corresponding rows out of the assays of the main `SingleCellExperiment` and into a nested alternative Experiment.

    is.spike <- grepl("^ERCC-", rownames(sce))
    sce <- splitAltExps(sce, ifelse(is.spike, "ERCC", "gene"))
    altExpNames(sce)

    ## [1] "ERCC"

The `altExp()` method returns a self-contained `SingleCellExperiment` instance containing only the spike-in transcripts.

    altExp(sce)

    ## class: SingleCellExperiment
    ## dim: 92 379
    ## metadata(2): SuppInfo which_qc
    ## assays(1): tophat_counts
    ## rownames(92): ERCC-00002 ERCC-00003 ... ERCC-00170 ERCC-00171
    ## rowData names(0):
    ## colnames(379): SRR2140028 SRR2140022 ... SRR2139341 SRR2139336
    ## colData names(0):
    ## reducedDimNames(0):
    ## spikeNames(0):
    ## altExpNames(0):

Each alternative Experiment can have a different set of assays from the main `SingleCellExperiment`. This is useful in cases where the other feature types must be normalized or transformed differently. Similarly, the alternative Experiments can have different `rowData()` from the main object.

    rowData(altExp(sce))$concentration <- runif(nrow(altExp(sce)))
    rowData(altExp(sce))

    ## DataFrame with 92 rows and 1 column
    ##                 concentration
    ##                     <numeric>
    ## ERCC-00002  0.034970022039488
    ## ERCC-00003  0.979286155896261
    ## ERCC-00004  0.171510147629306
    ## ERCC-00009  0.577605556230992
    ## ERCC-00012  0.965110752964392
    ## ...                       ...
    ## ERCC-00164  0.294522685930133
    ## ERCC-00165  0.444945453200489
    ## ERCC-00168  0.282102683791891
    ## ERCC-00170 0.0817114380188286
    ## ERCC-00171  0.417851015692577

    rowData(sce)

    ## DataFrame with 20816 rows and 0 columns

# 6 Session Info

    sessionInfo()

    ## R version 3.6.1 (2019-07-05)
    ## Platform: x86_64-w64-mingw32/x64 (64-bit)
    ## Running under: Windows Server 2012 R2 x64 (build 9600)
    ##
    ## Matrix products: default
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.1252
    ## [3] LC_MONETARY=English_United States.1252
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.1252
    ##
    ## attached base packages:
    ## [1] parallel  stats4    stats     graphics  grDevices utils     datasets
    ## [8] methods   base
    ##
    ## other attached packages:
    ##  [1] Rtsne_0.15                  scRNAseq_1.99.8
    ##  [3] SingleCellExperiment_1.8.0  SummarizedExperiment_1.16.0
    ##  [5] DelayedArray_0.12.0         BiocParallel_1.20.0
    ##  [7] matrixStats_0.55.0          Biobase_2.46.0
    ##  [9] GenomicRanges_1.38.0        GenomeInfoDb_1.22.0
    ## [11] IRanges_2.20.0              S4Vectors_0.24.0
    ## [13] BiocGenerics_0.32.0         BiocStyle_2.14.0
    ##
    ## loaded via a namespace (and not attached):
    ##  [1] Rcpp_1.0.2                    lattice_0.20-38
    ##  [3] assertthat_0.2.1              zeallot_0.1.0
    ##  [5] digest_0.6.22                 mime_0.7
    ##  [7] BiocFileCache_1.10.0          R6_2.4.0
    ##  [9] backports_1.1.5               RSQLite_2.1.2
    ## [11] evaluate_0.14                 httr_1.4.1
    ## [13] pillar_1.4.2                  zlibbioc_1.32.0
    ## [15] rlang_0.4.1                   curl_4.2
    ## [17] blob_1.2.0                    Matrix_1.2-17
    ## [19] rmarkdown_1.16                AnnotationHub_2.18.0
    ## [21] stringr_1.4.0                 RCurl_1.95-4.12
    ## [23] bit_1.1-14                    shiny_1.4.0
    ## [25] httpuv_1.5.2                  compiler_3.6.1
    ## [27] xfun_0.10                     pkgconfig_2.0.3
    ## [29] htmltools_0.4.0               tidyselect_0.2.5
    ## [31] tibble_2.1.3                  GenomeInfoDbData_1.2.2
    ## [33] interactiveDisplayBase_1.24.0 bookdown_0.14
    ## [35] later_1.0.0                   crayon_1.3.4
    ## [37] dplyr_0.8.3                   dbplyr_1.4.2
    ## [39] bitops_1.0-6                  rappdirs_0.3.1
    ## [41] grid_3.6.1                    xtable_1.8-4
    ## [43] DBI_1.0.0                     magrittr_1.5
    ## [45] stringi_1.4.3                 XVector_0.26.0
    ## [47] promises_1.1.0                vctrs_0.2.0
    ## [49] tools_3.6.1                   bit64_0.9-7
    ## [51] glue_1.3.1                    BiocVersion_3.10.1
    ## [53] purrr_0.3.3                   fastmap_1.0.1
    ## [55] yaml_2.2.0                    AnnotationDbi_1.48.0
    ## [57] BiocManager_1.30.9            ExperimentHub_1.12.0
    ## [59] memoise_1.1.0                 knitr_1.25