# Single-cell gene expression file contents

Single-cell or single-nuclei gene expression data (filtered or unfiltered) is provided for use with R as an RDS file containing a SingleCellExperiment object. This object contains the expression data, cell and gene metrics, associated metadata, and, in the case of multimodal data like CITE-seq, data from additional cell-based assays.

We highly encourage you to familiarize yourself with the general object structure and functions available as part of the SingleCellExperiment package from Bioconductor. Below we present some details about the specific contents of the objects we provide.

To begin, you will need to load the SingleCellExperiment package and read the RDS file:

library(SingleCellExperiment)


## Components of a SingleCellExperiment object

### Expression counts

The counts assay of the SingleCellExperiment object for single-cell and single-nuclei experiments (both unfiltered and filtered) contains the primary RNA-seq expression data as integer counts. The data is stored as a sparse matrix, and each column represents a cell or droplet, each row a gene. Column names are cell barcode sequences and row names are Ensembl gene IDs. The counts assay can be accessed with the following R code:

count_matrix <- counts(sce)


### Cell metrics

Cell metrics calculated from the RNA-seq expression data are stored as a DataFrame in the colData slot, with the cell barcodes as the names of the rows.

cell_metrics <- colData(sce)


The following per-cell data columns are included for each cell, calculated using the scuttle::addPerCellQCMetrics function.

Column name

Contents

sum

UMI count for RNA-seq data

detected

Number of genes detected (gene count > 0 )

subsets_mito_sum

UMI count of mitochondrial genes

subsets_mito_detected

Number of mitochondrial genes detected

subsets_mito_percent

Percent of all UMI counts assigned to mitochondrial genes

total

Total UMI count for RNA-seq data and any alternative experiments (i.e., CITE-seq)

The following are additional per-cell data columns included only in filtered objects. These metrics were calculated by using miQC, a package that jointly models proportion of reads belonging to mitochondrial genes and number of unique genes detected to predict low-quality cells.

Column name

Contents

prob_compromised

Probability that a cell is compromised (i.e., dead or damaged), as calculated by miQC

miQC_pass

Indicates whether the cell passed the default miQC filtering. TRUE is assigned to cells with a low probability of being compromised (prob_compromised < 0.75) or sufficiently low mitochondrial content.

### Gene information and metrics

Gene information and metrics calculated from the RNA-seq expression data are stored as a DataFrame in the rowData slot, with the Ensembl ID as the names of the rows.

gene_info <- rowData(sce)


The following columns are included for all genes. Metrics were calculated using the scuttle::addPerFeatureQCMetrics function.

Column name

Contents

gene_symbol

HUGO gene symbol, if defined

mean

Mean count across all cells/droplets

detected

Percent of cells in which the gene was detected (gene count > 0 )

Metadata associated with data processing is included in the metadata slot as a list.

expt_metadata <- metadata(sce)


Item name

Contents

salmon_version

Version of salmon used for initial mapping

reference_index

Transcriptome reference file used for mapping

total_reads

Total number of reads processed by salmon

mapped_reads

mapping_tool

Pipeline used for mapping and quantification (alevin-fry for all current data in ScPCA)

alevinfry_version

Version of alevin-fry used for mapping and quantification

af_permit_type

alevin-fry generate-permit-list method used for filtering cell barcodes

af_resolution

alevin-fry quant resolution mode used

usa_mode

Boolean indicating whether quantification was done using alevin-fry USA mode

af_num_cells

Number of cells reported by alevin-fry

tech_version

A string indicating the technology and version used for the single-cell library, such as 10Xv2, 10Xv3, or 10Xv3.1

transcript_type

Transcripts included in gene counts: spliced for single-cell samples and unspliced for single-nuclei

miQC_model

The model object that miQC fit to the data and was used to calculate prob_compromised. Only present for filtered objects

filtering_method

The method used for cell filtering. One of emptyDrops, emptyDropsCellRanger, or UMI cutoff. Only present for filtered objects

umi_cutoff

The minimum UMI count per cell used as a threshold for filtering. Only present for filtered objects where the filtering_method is UMI cutoff

## Additional SingleCellExperiment components for CITE-seq libraries

CITE-seq data, when present, is included within the SingleCellExperiment as an “Alternative Experiment” named "CITEseq" , which can be accessed with the following command:

altExp(sce, "CITEseq")


Within this, the main expression matrix is again found in the counts assay, with each column corresponding to a cell or droplet (in the same order as the parent SingleCellExperiment) and each row corresponding to an antibody derived tag (ADT). Column names are again cell barcode sequences and row names the antibody targets for each ADT.

The following additional per-cell data columns for the CITE-seq data can be found in the main colData data frame (accessed with colData(sce) as above).

Column name

Contents

altexps_CITEseq_sum

altexps_CITEseq_detected

altexps_CITEseq_percent

Percent of total UMI count from ADT reads

Metrics for each of the ADTs assayed can be found as a DataFrame stored as rowData within the alternative experiment:

adt_info <- rowData(altExp(sce, "CITEseq"))


This data frame contains the following columns with statistics for each ADT:

Column name

Contents

mean

Mean ADT count across all cells/droplets

detected

Percent of cells in which the ADT was detected (ADT count > 0 )

Finally, additional metadata for the CITE-seq data processing can be found in the metadata slot of the alternative experiment, with the same contents as the parent experiment metadata

citeseq_metadata <- metadata(altExp(sce, "CITEseq"))


## Additional SingleCellExperiment components for multiplexed libraries

Multiplexed libraries will contain a number of additional components and fields.

Hashtag oligo (HTO) quantification for each cell is included within the SingleCellExperiment as an “Alternative Experiment” named "cellhash" , which can be accessed with the following command:

altExp(sce, "cellhash")


Within this, the main data matrix is again found in the counts assay, with each column corresponding to a cell or droplet (in the same order as the parent SingleCellExperiment) and each row corresponding to a hashtag oligo (HTO). Column names are again cell barcode sequences and row names the HTO ids for all assayed HTOs.

The following additional per-cell data columns for the cellhash data can be found in the main colData data frame (accessed with colData(sce) as above).

Column name

Contents

altexps_cellhash_sum

UMI count for cellhash HTOs

altexps_cellhash_detected

Number of HTOs detected per cell (HTO count > 0 )

altexps_cellhash_percent

Percent of total UMI count from HTO reads

Metrics for each of the HTOs assayed can be found as a DataFrame stored as rowData within the alternative experiment:

hto_info <- rowData(altExp(sce, "cellhash"))


This data frame contains the following columns with statistics for each HTO:

Column name

Contents

mean

Mean HTO count across all cells/droplets

detected

Percent of cells in which the HTO was detected (HTO count > 0 )

sample_id

Sample ID for this library that corresponds to the HTO (only present in _filtered.rds files)

Note that in the unfiltered SingleCellExperiment objects, this may include hashtag oligos that do not correspond to any included sample, but were part of the reference set used for mapping.

### Demultiplexing results

Demultiplexing results are included only in the _filtered.rds files. The demultiplexing methods applied for these files are as described in the multiplex data processing section.

Demultiplexing analysis adds the following additional fields to the colData(sce) data frame:

Column name

Contents

hashedDrops_sampleid

Most likely sample as called be DropletUtils::hashedDrops

HTODemux_sampleid

Most likely sample as called be Seurat::HTODemux

vireo_sampleid

Most likely sample as called by vireo (genetic demultiplexing)

For methods that rely on the HTO data, these statistics are found in the colData(altExp(sce, "cellhash")) data frame; DropletUtils::hashedDrops statistics have the prefix hashedDrops_ and Seurat::HTODemux statistics have the prefix HTODemux.
Genetic demultiplexing statistics are found in the main colData(sce) data frame, with the prefix vireo_.