GeneActivity without Fragments file in Seurat for Integrating scRNA-seq and scATAC-seq

Question

[el24]

Hi all,

I am new to R and Seurat, and I am following Seurat tutorials to find anchors between RNA-seq and ATAC-seq data according to:

• https://satijalab.org/seurat/articles/atacseq_integration_vignette.html
• https://satijalab.org/seurat/articles/weighted_nearest_neighbor_analysis.html#wnn-analysis-of-10x-multiome-rna-atac-1

Combining the two tutorials is difficult for a cell line data set I am using for SNARE-seq Human here.

I managed to run the following code and get PCA and UMAP for both RNA-seq and ATAC-seq data, but when I run the following code I get an error:

gene.activities <- GeneActivity(atac, features = VariableFeatures(rna))

Error in GeneActivity(atac, features = VariableFeatures(rna)) : The requested assay is not a ChromatinAssay.

I am struggling to create a chromatin assay using the following code in the second tutorial because the fragments file (fragments structure) is not available in this dataset, I only have TSV matrix format of cells x genes (or peak regions in ATAC-seq)

chrom_assay <- CreateChromatinAssay( counts = atac_counts, sep = c(":", "-"), genome = 'hg38', fragments = frag.file, min.cells = 10, annotation = annotations )

I appreciate any solutions or workaround to fix this issue so that I can run the GeneActivity() function to identify anchors between RNAseq and ATACseq data. I have read tutorials on Seurat and Signac packages, but none helped me fix this issue.

p.n: In an article, I found sinto package is being used to create fragments files from bam files. I also found fastq files on SRA so I am creating fragments files.

Thank you very much!

Answer 1

[mk]

I modified the old Seurat::GeneActivityMatrix function which has since been removed from Seurat, in favor of the functionality provided by Signac. As has been mentioned elsewhere, this package, while excellent, prejudices the analysis of data for which fragment files are unavailable, which is unfortunately the case for lots of interesting published data (e.g. the required data accession contains a simple count matrix in the GEO supplement).

# peak.matrix is a data frame of counts (called peaks from atac raw data
# annotation file is a GFF3 file eg "Homo_sapiens.GRCh38.109.chr.gff3"
CreateGeneActivityMatrix <- function(
  peak.matrix,
  annotation.file,
  seq.levels = c(1:22, "X", "Y"),
  include.body = TRUE,
  upstream = 2000,
  downstream = 0,
  verbose = TRUE
) {
    plan(multisession, workers = 8)
    # convert peak matrix to GRanges object
    peak.df <- rownames(x = peak.matrix)
    peak.df <- do.call(what = rbind, args = strsplit(x = gsub(peak.df, pattern = ":", replacement = "-"), split = "-"))
    peak.df <- as.data.frame(x = peak.df)
    colnames(x = peak.df) <- c("chromosome", 'start', 'end')
    peaks.gr <- GenomicRanges::makeGRangesFromDataFrame(df = peak.df)

    # if any peaks start at 0, change to 1
    # otherwise GenomicRanges::distanceToNearest will not work
    BiocGenerics::start(peaks.gr[BiocGenerics::start(peaks.gr) == 0, ]) <- 1
    # get annotation file, select genes
    #   anno <- rtracklayer::import(con = annotation.file)
    # anno <- GenomeInfoDb::keepSeqlevels(x = anno, value = seq.levels, pruning.mode = 'coarse')

    gtf <- rtracklayer::import(con = annotation.file)
    gtf <- GenomeInfoDb::keepSeqlevels(x = gtf, value = seq.levels, pruning.mode = 'coarse')

    # change seqlevelsStyle if not the same
    if (!any(GenomeInfoDb::seqlevelsStyle(x = gtf) == GenomeInfoDb::seqlevelsStyle(x = peaks.gr))) {
        GenomeInfoDb::seqlevelsStyle(gtf) <- GenomeInfoDb::seqlevelsStyle(peaks.gr)
    }
    gtf.genes <- gtf[gtf$type == 'gene']

    # 
    # Extend definition up/downstream
    if (include.body) {
        gtf.body_prom <- Signac::Extend(x = gtf.genes, upstream = upstream, downstream = downstream)
    } else {
        gtf.body_prom <- SummarizedExperiment::promoters(x = gtf.genes, upstream = upstream, downstream = downstream)
    }
    gene.distances <- GenomicRanges::distanceToNearest(x = peaks.gr, subject = gtf.body_prom)
    keep.overlaps <- gene.distances[rtracklayer::mcols(x = gene.distances)$distance == 0]
    peak.ids <- peaks.gr[S4Vectors::queryHits(x = keep.overlaps)]
    gene.ids <- gtf.genes[S4Vectors::subjectHits(x = keep.overlaps)]

    # Some gtf rows will not have gene_name attribute
    # Replace it by gene_id attribute
    # 
    gene.ids$Name[is.na(gene.ids$Name)] <- gene.ids$gene_id[is.na(gene.ids$Name)]

    peak.ids$gene.name <- gene.ids$Name
    peak.ids <- as.data.frame(x = peak.ids)
    peak.ids$peak <- rownames(peak.matrix)[S4Vectors::queryHits(x = keep.overlaps)]
    annotations <- peak.ids[, c('peak', 'gene.name')]
    colnames(x = annotations) <- c('feature', 'new_feature')

    # collapse into expression matrix
    peak.matrix <- as(object = peak.matrix, Class = 'matrix')
    all.features <- unique(x = annotations$new_feature)

    if (future::nbrOfWorkers() > 1) {
        mysapply <- future.apply::future_sapply
    } else {
        mysapply <- ifelse(test = verbose, yes = pbapply::pbsapply, no = sapply)
    }
    newmat <- mysapply(X = 1:length(x = all.features), FUN = function(x){

    features.use <- annotations[annotations$new_feature == all.features[[x]], ]$feature

    submat <- peak.matrix[features.use, ]

    if (length(x = features.use) > 1) {
        return(Matrix::colSums(x = submat))
    } else {
        return(submat)
    }
    })

    newmat <- t(x = newmat)

    rownames(x = newmat) <- all.features

    colnames(x = newmat) <- colnames(x = peak.matrix)

    return(as(object = newmat, Class = 'dgCMatrix'))
}

Answer 2

[Min]

Did you solve this problem? I am also struggled in creating ChromatinAssay object without fragment files......