In LTLA/scRNAseq: Collection of Public Single-Cell RNA-Seq Datasets

library(BiocStyle)
knitr::opts_chunk$set(error=FALSE, message=FALSE, warning=FALSE)

Downloading the data

We obtain a single-cell RNA sequencing dataset of the mouse nervous system from Zeisel et al. (2018). Counts for endogenous genes are available from http://mousebrain.org/downloads.html as a loom file. We download and cache it using the r Biocpkg("BiocFileCache") package.

library(BiocFileCache)
bfc <- BiocFileCache("raw_data", ask = FALSE)
loom.url <- "https://storage.googleapis.com/linnarsson-lab-loom/l5_all.loom"
loom.path <- bfcrpath(bfc, loom.url)

We load this into our R session using the r Biocpkg("LoomExperiment") package.

library(LoomExperiment)
scle <- import(loom.path, type="SingleCellLoomExperiment")
scle 

Then it's just a matter of peeling apart the bits that we need. For starters, let's clean up the rowData by moving the gene IDs to the row names:

rd <- rowData(scle)
colnames(rd) <- sub("^X_", "", colnames(rd))
rownames(rd) <- rd$Accession
rd$Accession <- NULL
rd

Cleaning up column annotations

We also need to clean up the colData. This is, sadly, a gargantuan manual effort - so much for loom being a ready-to-use file format! We start by stripping out non-informative or redundant fields:

cd <- colData(scle)

# Useless pieces of information, or pieces that are obvious from the experimental design.
cd$Bucket <- NULL
cd$Species <- NULL
cd$AnalysisProject <- NULL
cd$Transcriptome <- NULL
cd$TimepointPool <- NULL
cd$NGI_PlateWell <- NULL
cd$PlugDate <- NULL
cd$Plug_Date <- NULL
cd$Project <- NULL

# Redundant fields.
cd$Cell_Conc <- NULL
cd$ngperul_cDNA <- NULL
cd$cDNA_Lib_Ok <- NULL
cd$Target_Num_Cells <- NULL
cd$Date_Captured <- NULL
cd$Num_Pooled_Animals <- NULL
cd$PCR_Cycles <- NULL
cd$Sample_Index <- NULL
cd$Seq_Comment <- NULL
cd$Seq_Lib_Date <- NULL
cd$Seq_Lib_Ok <- NULL

Converting various character fields into numbers, where applicable. On occassion, we have to get rid of some nonsense fields with quotation marks; something probably got corrupted when the file was saved.

options(warn=2)
for (i in colnames(cd)) {
    current <- cd[[i]]

    if (is.character(current)) {
        converted <- current
        is.bad <- converted=="" | grepl('"', converted)
        converted[is.bad] <- NA
        changed <- TRUE

        if (any(has.pct <- grepl("%$", current))) {
            converted[!has.pct] <- NA
            converted <- sub("%$", "", converted)
            converted <- as.numeric(converted)/100
            cd[[i]] <- converted
        } else if (any(has.comma <- grepl(",[0-9]{3}", current))) {
            converted[!has.comma] <- NA # these are probably corruptions of some sort.
            converted <- gsub(",([0-9]{3})", "\\1", converted)
            converted <- as.numeric(converted)
            cd[[i]] <- converted
        } else if (any(grepl("^[0-9]+,[0-9]+$", current))){
            converted <- sub(",", ".", converted)
            converted <- as.numeric(converted)
            cd[[i]] <- converted
        } else if (any(grepl("^[0-9]+(\\.[0-9]+)?$", current))) {
            cd[[i]] <- as.numeric(converted)
        } else {
            changed <- FALSE
        }

#        # For debugging purposes, to check that the corruptions are purged correctly.
#        if (changed) {
#            print(i)
#            print(names(table(current)))
#            print(names(table(converted)))
#        }
    }
}
options(warn=1)

We replace the variuos nan strings with NAs in the character columns.

for (i in colnames(cd)) {
    current <- cd[[i]]
    if (is.character(current) && any(lost <- current=="nan")) {
        current[lost] <- NA_character_
        cd[[i]] <- current
    }    
}

We shift the class probabilities into a nested data frame.

clcols <- grep("ClassProbability", colnames(cd))
clprobs <- cd[,clcols]
colnames(clprobs) <- sub("ClassProbability_", "", colnames(clprobs))
cd <- cd[,-clcols]
cd$ClassProbability <- clprobs

We also hack out the reduced dimensions.

pca.cols <- c("X_PC1", "X_PC2")
tsne.cols <- c("X_tSNE1", "X_tSNE2")
other.cols <- c("X_X", "X_Y")
reddim <- list(
    PCA=as.matrix(cd[,pca.cols]),
    tSNE=as.matrix(cd[,tsne.cols]),
    unnamed=as.matrix(cd[,other.cols])
)
cd <- cd[,!colnames(cd) %in% c(pca.cols, tsne.cols, other.cols)]

Finally, we get rid of the prefixing with X_. Don't know why it's there but, well, whatever.

colnames(cd) <- sub("X_", "", colnames(cd))
cd

Saving to file

We now create a new SingleCellExperiment object. I'm not storing the cell-cell graph, though, because I don't want to.

sce <- SingleCellExperiment(
    list(counts=as(assay(scle), 'dgCMatrix')),
    rowData=rd,
    colData=cd
)

We apply some polish to save disk space:

library(scRNAseq)
sce <- polishDataset(sce)
sce 

We then save it to disk in preparation for upload:

meta <- list(
    title="Molecular Architecture of the Mouse Nervous System",
    description="The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.",
    taxonomy_id="10090",
    genome="GRCm38",
    sources=list(
        list(provider="PubMed", id="30096314"),
        list(provider="URL", id=loom.url, version=as.character(Sys.Date()))
    ),
    maintainer_name="Aaron Lun",
    maintainer_email="[e-mail]"
)

saveDataset(sce, "2023-12-22_output", meta)

Session information {-}

sessionInfo()

LTLA/scRNAseq documentation built on Jan. 22, 2025, 7:43 p.m.