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Read 10X Xenium output as SpatialFeatureExperiment

Source: R/read.R
readXenium.Rd

This function reads the standard 10X Xenium output into an SFE object.

Usage

readXenium(
  data_dir,
  sample_id = "sample01",
  min_area = NULL,
  image = c("morphology_focus", "morphology_mip"),
  segmentations = c("cell", "nucleus"),
  row.names = c("id", "symbol"),
  flip = c("geometry", "image", "none"),
  max_flip = "50 MB",
  filter_counts = FALSE,
  add_molecules = FALSE,
  min_phred = 20,
  BPPARAM = SerialParam(),
  file_out = file.path(data_dir, "tx_spots.parquet")
)

Arguments

data_dir

Top level output directory.

sample_id

A character sample identifier, which matches the sample_id in imgData. The sample_id will also be stored in a new column in colData, if not already present. Default = sample01.

min_area

Minimum cell area in square microns or pixel units (eg for CosMX). Anything smaller will be considered artifact or debris and removed. Default to `NULL`, ie no filtering of polygons.

image

Which image(s) to load, can be "morphology_mip", "morphology_focus" or both. Note that in Xenium Onboarding Analysis (XOA) v2, there is no longer "morphology_mip" and "morphology_focus" is a directory with 4 images corresponding to 4 channels: DAPI, "Cadherin", 18S, and Vimentin. So this argument is ignored for XOA v2.

segmentations

Which segmentation outputs to read, can be "cell", "nucleus", or both.

row.names

String specifying whether to use Ensembl IDs ("id") or gene symbols ("symbol") as row names. If using symbols, the Ensembl ID will be appended to disambiguate in case the same symbol corresponds to multiple Ensembl IDs. Always "symbol" if `add_molecules = TRUE` because only gene symbols are used in the transcript spot files.

flip

To flip the image, geometry coordinates, or none. Because the image has the origin at the top left while the geometry has origin at the bottom left, one of them needs to be flipped for them to match. If one of them is already flipped, then use "none". The image will not be flipped if it's GeoTIFF.

max_flip

Maximum size of the image allowed to flip the image. Because the image will be loaded into memory to be flipped. If the image is larger than this size then the coordinates will be flipped instead.

filter_counts

Logical, whether to keep cells with counts > 0.

add_molecules

Logical, whether to add transcripts coordinates to an object.

min_phred

Minimum Phred score to keep spot. By default 20, the conventional threshold indicating "acceptable", meaning that there's 1 chance that the spot was decoded in error.

BPPARAM

A BiocParallelParam object specifying parallel processing backend and number of threads to use for parallelizable tasks:

  1. To load cell segmentation from HDF5 files from different fields of view (FOVs) with multiple cores. A progress bar can be configured in the BiocParallelParam object. When there are numerous FOVs, reading in the geometries can be time consuming, so we recommend using a server and larger number of threads. This argument is not used if use_cellpose = TRUE and the parquet file is present.

  2. To get the largest piece and see if it's larger than min_area when there are multiple pieces in the cell segmentation for one cell.

file_out

Name of file to save the geometry or raster to disk. Especially when the geometries are so large that it's unwieldy to load everything into memory. If this file (or directory for multiple files) already exists, then the existing file(s) will be read, skipping the processing. When writing the file, extensions supplied are ignored and extensions are determined based on `dest`.

Value

An SFE object. If reading segmentations, the cell or nuclei segmentation will be saved to `cell_boundaries_sf.parquet` and `nucleus_boundaries_sf.parquet` respectively in `data.dir` so next time the boundaries can be read much more quickly. If reading transcript spots (`add_molecules = TRUE`), then the reformatted transcript spots are saved to file specified in the `file_out` argument, which is by default `tx_spots.parquet` in the same directory as the rest of the data. If images are present, then the images will be of the BioFormatsImage class and not loaded into memory until necessary in later operations.

Note

Sometimes when reading images, you will see this error the first time: 'java.lang.NullPointerException: Cannot invoke "loci.formats.DimensionSwapper.setMetadataFiltered(boolean)" because "RBioFormats.reader" is null'. See this issue https://github.com/aoles/RBioFormats/issues/42 Rerun the code and it should work the second time.

Examples

library(SFEData)
library(RBioFormats)
fp <- tempfile()
dir_use <- XeniumOutput("v2", file_path = fp)
#> see ?SFEData and browseVignettes('SFEData') for documentation
#> loading from cache
#> The downloaded files are in /tmp/RtmpUGEtoo/file834d4fae360a/xenium2 
# RBioFormats issue
try(sfe <- readXenium(dir_use, add_molecules = TRUE))
#> >>> Must use gene symbols as row names when adding transcript spots.
#> >>> Cell segmentations are found in `.parquet` file(s)
#> >>> Reading cell and nucleus segmentations
#> >>> Making MULTIPOLYGON nuclei geometries
#> >>> Making POLYGON cell geometries
#> Sanity checks on cell segmentation polygons:
#> >>> ..found 132 cells with (nested) polygon lists
#> >>> ..applying filtering
#> >>> Checking polygon validity
#> >>> Saving geometries to parquet files
#> >>> Reading cell metadata -> `cells.parquet`
#> >>> Reading h5 gene count matrix
#> >>> filtering cellSeg geometries to match 6272 cells with counts > 0
#> >>> filtering nucSeg geometries to match 6158 cells with counts > 0
#> >>> Reading transcript coordinates
#> >>> Converting transcript spots to geometry
#> >>> Writing reformatted transcript spots to disk
#> >>> Total of 116 features/genes with no transcript detected or `min_phred` < 20 are removed from SFE object
#> >>> To keep all features -> set `min_phred = NULL`
sfe <- readXenium(dir_use, add_molecules = TRUE)
#> >>> Must use gene symbols as row names when adding transcript spots.
#> >>> Preprocessed sf segmentations found
#> >>> Reading cell and nucleus segmentations
#> >>> Reading cell metadata -> `cells.parquet`
#> >>> Reading h5 gene count matrix
#> >>> filtering cellSeg geometries to match 6272 cells with counts > 0
#> >>> filtering nucSeg geometries to match 6158 cells with counts > 0
#> >>> Reading transcript coordinates
#> >>> Total of 116 features/genes with no transcript detected or `min_phred` < 20 are removed from SFE object
#> >>> To keep all features -> set `min_phred = NULL`
unlink(dir_use, recursive = TRUE)