Failed reading 10x h5 file - CellBender v2.1

Last week I ran CellBender v1 over a CellRanger V3 library I got, and then successfully loaded the h5 output into a Seurat object.

CreateSeuratObject(Read10X_h5("the_cell_bender_outout_filtered.h5"))

Now, when I tried using CellBender V2.1 instead of V1 over the same CellRanger library, I got the following exception:

Error in `[[.H5File`(infile, paste0(genome, "/", feature_slot)) :
  An object with name matrix/gene_names does not exist in this group

Comparing the output h5 files of both versions they seem to have the same format with the difference that the V2.1 output has the "PYTABLES_FORMAT_VERSION". Is it flagged like that on purpose? This is the flag that causes the Seurat Read10X_h5 function to fail: (part of Read10X_h5 code)

  infile <- hdf5r::H5File$new(filename = filename, mode = "r")
  genomes <- names(x = infile)
  output <- list()
  if (!infile$attr_exists("PYTABLES_FORMAT_VERSION")) {
    if (use.names) {
      feature_slot <- "features/name"
    }
    else {
      feature_slot <- "features/id"
    }
  }
  else {
    if (use.names) {
      feature_slot <- "gene_names"
    }
    else {
      feature_slot <- "genes"
    }
  }

[sjfleming]

https://github.com/satijalab/seurat/issues/3653

[sjfleming]

Okay, so the if statement that @GreenGilad mentioned above is indeed the problem. Since we create a file using PyTables, there will be a PYTABLES_FORMAT_VERSION attribute.

My proposal is to substitute their current if statement https://github.com/satijalab/seurat/blob/fe93b05745e55ec2f66e3f0b4c4196aad9f4d5a7/R/preprocessing.R#L1155

with

if (hdf5r::existsGroup(infile, 'matrix'))

For now, I think the code below is a potential workaround. I may try to submit a pull request to Seurat to incorporate this, as I think it makes more sense than relying on a version attribute from PyTables.

library(Matrix)

ReadCB_h5 <- function(filename, use.names = TRUE, unique.features = TRUE) {
  if (!requireNamespace('hdf5r', quietly = TRUE)) {
    stop("Please install hdf5r to read HDF5 files")
  }
  if (!file.exists(filename)) {
    stop("File not found")
  }
  infile <- hdf5r::H5File$new(filename = filename, mode = 'r')
  genomes <- names(x = infile)
  output <- list()
  if (hdf5r::existsGroup(infile, 'matrix')) {
    # cellranger version 3
    message('CellRanger version 3+ format H5')
    if (use.names) {
      feature_slot <- 'features/name'
    } else {
      feature_slot <- 'features/id'
    }
  } else {
    message('CellRanger version 2 format H5')
    if (use.names) {
      feature_slot <- 'gene_names'
    } else {
      feature_slot <- 'genes'
    }
  }
  for (genome in genomes) {
    counts <- infile[[paste0(genome, '/data')]]
    indices <- infile[[paste0(genome, '/indices')]]
    indptr <- infile[[paste0(genome, '/indptr')]]
    shp <- infile[[paste0(genome, '/shape')]]
    features <- infile[[paste0(genome, '/', feature_slot)]][]
    barcodes <- infile[[paste0(genome, '/barcodes')]]
    sparse.mat <- sparseMatrix(
      i = indices[] + 1,
      p = indptr[],
      x = as.numeric(x = counts[]),
      dims = shp[],
      giveCsparse = FALSE
    )
    if (unique.features) {
      features <- make.unique(names = features)
    }
    rownames(x = sparse.mat) <- features
    colnames(x = sparse.mat) <- barcodes[]
    sparse.mat <- as(object = sparse.mat, Class = 'dgCMatrix')
    # Split v3 multimodal
    if (infile$exists(name = paste0(genome, '/features'))) {
      types <- infile[[paste0(genome, '/features/feature_type')]][]
      types.unique <- unique(x = types)
      if (length(x = types.unique) > 1) {
        message("Genome ", genome, " has multiple modalities, returning a list of matrices for this genome")
        sparse.mat <- sapply(
          X = types.unique,
          FUN = function(x) {
            return(sparse.mat[which(x = types == x), ])
          },
          simplify = FALSE,
          USE.NAMES = TRUE
        )
      }
    }
    output[[genome]] <- sparse.mat
  }
  infile$close_all()
  if (length(x = output) == 1) {
    return(output[[genome]])
  } else{
    return(output)
  }
}

Loading a CellBender remove-background output file in a legacy CellRanger v2 format:

Loading a CellBender remove-background output file in the newer CellRanger v3+ format: (I had to subset to “Gene Expression” to successfully use CreateSeuratObject)