Read, write, create, and convert single-cell objects across AnnData (Python), Seurat (R), and SingleCellExperiment (R). Use when loading 10X Cell Ranger output (raw vs filtered), importing or exporting h5ad/RDS/h5mu/zarr, building AnnData or Seurat objects from matrices, moving objects between Python and R, or debugging lost layers, transposed matrices, or mangled gene names during conversion.
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Read, write, create, and convert single-cell objects across AnnData (Python), Seurat (R), and SingleCellExperiment (R). Use when loading 10X Cell Ranger output (raw vs filtered), importing or exporting h5ad/RDS/h5mu/zarr, building AnnData or Seurat objects from matrices, moving objects between Python and R, or debugging lost layers, transposed matrices, or mangled gene names during conversion.
Before using code patterns, verify installed versions match. If versions differ:
Python: pip show <package> then help(module.function) to check signatures
R: packageVersion('<pkg>') then ?function_name to verify parameters
If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.
Single-Cell Data I/O
"Load my 10X data" -> Parse a Cell Ranger matrix into an annotated object (cells, genes, counts, metadata).
The dominant failure in single-cell I/O is not a crash; it is a silent semantic change to the matrix during read or conversion. Three traps drive almost every lost-data bug.
Orientation is opposite across ecosystems. AnnData is cells x genes (observations on rows, obs indexes rows, var indexes columns); Seurat and SingleCellExperiment are genes x cells (features on rows, cells on columns). So adata.X is the transpose of LayerData(seu) and assay(sce). A faithful conversion must transpose AND swap which axis the metadata annotates; getting the transpose right but the metadata axis wrong is the single most common silent conversion bug.
Sparse storage compounds the transpose. R Matrix::dgCMatrix is CSC; scanpy conventionally stores X as CSR. Transposing a CSR matrix yields CSC for free, so a correct AnnData->Seurat hop involves both a logical transpose and a CSR<->CSC change. Forcing dense (.toarray(), as_dense= on write) on a 500k-cell x 30k-gene float32 matrix materializes ~60 GB; keep X and layers sparse and check with scipy.sparse.issparse(adata.X).
Conversion is lossy by default, and the loss is silent. Cross-ecosystem hops drop layers, obsp/varp, nested uns, and coerce categoricals to character/NA. adata.raw has its own var (its purpose is to survive HVG subsetting) and tools disagree on whether to read X or .raw.X (use_raw=), so a mismatched expectation silently uses the wrong matrix. Always diff slot inventories before and after any cross-ecosystem conversion, and keep the original file.
One more governing fact: the Cell Ranger filtered matrix is cell-CALLED, not ambient-corrected. The widespread claim that the filtered matrix is "decontaminated" is false. Keep the RAW (unfiltered) matrix, because EmptyDrops, SoupX, CellBender, and DecontX all require it and filtered-only storage is irreversible.
Choosing a Storage Format
Format
Backing
Use when
Fails / weak when
h5ad (HDF5)
single file
Default single-machine Python I/O and sharing
Not cloud-native; concurrent/partial reads limited
Many small files awkward on local FS; v2/v3 version skew breaks old readers
RDS
single R binary
Seurat-only workflow, full object fidelity in R
R-only; not portable to Python; version-tied
h5mu (MuData)
HDF5
Multimodal (RNA + ADT + ATAC), one AnnData per modality
Less tool support than h5ad; needs mdata.update() discipline
Loom (HDF5)
single file
Legacy interchange (velocyto, older Seurat)
write_loom(write_obsm_varm=False) DROPS obsm/varm by default; aging
Methods and tool maturity move fast here. Before committing a conversion route, verify the chosen package is still maintained and matches installed versions (packageVersion, pip show).
Loading 10X Cell Ranger Output
Goal: Read a Cell Ranger matrix correctly, keeping the raw matrix and non-GEX features when present.
Approach: Read the raw (unfiltered) MEX/HDF5 matrix; select stable Ensembl IDs for reproducible joins; retain Antibody/CRISPR features by disabling gex_only.
import scanpy as sc
# raw_feature_bc_matrix has every barcode (needed by EmptyDrops/SoupX/CellBender); filtered_feature_bc_matrix has only called cells
adata = sc.read_10x_mtx('raw_feature_bc_matrix/', var_names='gene_ids', gex_only=False)
# gene_ids (Ensembl) is stable across annotation releases; gene_symbols (default) is ambiguous and non-unique# gex_only=False keeps Antibody Capture / CRISPR Guide; split later by adata.var['feature_types']
adata.var_names_make_unique()
Read functions return symbols by default. sc.read_10x_h5 has no var_names argument (symbols by default; Ensembl IDs land in var['gene_ids']). make_unique appends -1/-2 to duplicate symbols, which can mask distinct paralog/PAR loci, so prefer IDs when joining datasets.
AnnData Object Structure
Goal: Place counts, normalized values, metadata, and embeddings in the conventional slots so downstream tools find them.
Approach: Keep integer counts in layers['counts'], log-normalized values in X, and a frozen full-gene snapshot in .raw before HVG subsetting.
import anndata as ad
# X is (n_obs, n_vars) = cells x genes; obs indexes rows, var indexes columns
adata.layers['counts'] = adata.X.copy() # integer UMIs, kept to recompute or feed count models (scVI, DESeq2)# ... normalize_total + log1p populate X ...
adata.raw = adata # frozen log-normalized full-gene snapshot; survives later var-subsetting
adata = adata[:, adata.var['highly_variable']].copy()
Slot roles: X/layers align to both axes (each exactly cells x genes); obs/obsm align to cells; var/varm align to genes; obsp/varp are square pairwise graphs; uns is unstructured. adata.raw.to_adata() reconstitutes the snapshot. Slicing the parent by obs also slices raw on obs, but var-slicing does NOT shrink raw.
Seurat v5 Object Structure
Goal: Read and write the right assay layer under the v5 layers API.
Approach: Use LayerData()/$-accessors; rejoin split layers after merge() before any function expecting one layer.
counts <- LayerData(seurat_obj, layer ='counts')# v5; GetAssayData(slot=) is the superseded v4 form
counts <- seurat_obj[['RNA']]$counts # shorthand
merged <- merge(obj1, y =c(obj2, obj3), add.cell.ids =c('S1','S2','S3'))
merged <- JoinLayers(merged)# merge() splits layers (counts.1, counts.2); rejoin first
Seurat v5 stores counts/data/scale.data as layers in an Assay5; v4 used fixed slots via GetAssayData(slot=). After merge(), layers split per object until JoinLayers().
Converting Between Python and R
Goal: Move an object across ecosystems without dropping layers, embeddings, or raw.
Approach: Prefer a maintained pure-R or Python-pinned converter; transpose and remap metadata; diff slots before and after.
Tool
Direction
Maintained 2026
Use when
anndataR
AnnData <-> SCE <-> Seurat; h5ad+zarr R/W
Yes (v1.2.0, pure R, no Python)
First choice for R-native, Python-free h5ad/zarr I/O and conversion
zellkonverter
AnnData <-> SCE
Yes (Bioc 3.23)
Mature SCE<->AnnData; robust Python reader with pinned anndata
schard
h5ad -> Seurat/SCE (read-only)
Yes
Robust pure-R READING of h5ad (SeuratDisk replacement)
anndata2ri
AnnData <-> SCE (rpy2)
Yes
Live mixed Python+R sessions / Jupyter %%R
sceasy
everything -> AnnData hub
Aging
Quick one-call conversion (mind drop_single_values data loss)
SeuratDisk
AnnData <-> h5Seurat
NO (last commit 2023, broken on Seurat v5)
Avoid for new work; legacy only
# Preferred R-native read of an h5ad written in Python (no reticulate)
library(anndataR)
adata <- read_h5ad('data.h5ad')
seurat_obj <- adata$to_Seurat()# Or via Bioconductor with a pinned Python anndata:# library(zellkonverter); sce <- readH5AD('data.h5ad'); writeH5AD(sce, 'out.h5ad')
zellkonverter maps asymmetrically: obsm->reducedDims, varm->a rowData matrix column (NOT reducedDims), obsp/varp->colPairs/rowPairs, uns->metadata() (lossy), and raw->altExp(sce,'raw') only when raw=TRUE (default FALSE). sceasy's drop_single_values=TRUE silently deletes every obs/var column with one unique value (a one-sample object loses its constant batch/condition label), so set FALSE.
API Defaults That Surprise
Call
Surprising default
Consequence
sc.read_10x_mtx(gex_only=True)
drops Antibody/CRISPR/Custom features
CITE-seq ADT and guides silently vanish; set gex_only=False
sc.read_10x_mtx(var_names='gene_symbols')
non-unique, release-dependent symbols
Use 'gene_ids' for reproducible cross-dataset joins
AnnData.write_h5ad(compression=None)
no compression (gzip default removed after v0.6.16)
Larger files; pass compression='gzip'
write_loom(write_obsm_varm=False)
obsm/varm dropped
Embeddings lost on Loom write
read_h5ad(backed='r')
only X edits persist
obs/var/obsm edits in backed mode are NOT written; re-.write() to a new file
sceasy convertFormat(drop_single_values=TRUE)
constant columns deleted
Single-value batch/condition labels lost; set FALSE
sc.read_10x_mtx(cache=True)
cache keyed by path only
Re-reading a path with different var_names returns the STALE object; delete the .h5ad cache or omit cache
Common Errors
Symptom
Cause
Fix
Converted object has genes and cells swapped
Transpose not applied (or applied without swapping metadata axis)
Transpose the matrix AND move obs<->col-meta, var<->row-meta
Layers / embeddings / raw missing after conversion
Lossy converter dropped non-X slots
Diff slot inventories; use anndataR/zellkonverter; re-attach manually
Cannot run EmptyDrops/SoupX/CellBender
Only the filtered matrix was kept
Re-obtain and store the RAW (unfiltered) Cell Ranger matrix
ADT/guide counts absent after loading 10X
gex_only=True (default) dropped non-GEX features
Reload with gex_only=False, split by var['feature_types']
Kernel/session dies reading a large object
Dense materialization of a sparse matrix
Keep sparse; use backed='r' (Python) or BPCells/on-disk layers (Seurat v5)
Downstream tool uses wrong values
Tool read X vs .raw.X against expectation
Set use_raw= explicitly; confirm which matrix holds counts vs lognorm
Duplicate gene symbols collapsed or suffixed oddly
make_unique appended -1/-2 to distinct loci
Load with var_names='gene_ids' for stable identifiers
Related Skills
single-cell/preprocessing - QC, normalization, and HVG selection after loading
single-cell/doublet-detection - per-sample doublet calling on raw counts after loading
single-cell/clustering - dimensionality reduction and clustering on the loaded object
single-cell/multimodal-integration - MuData/h5mu handling for CITE-seq and Multiome
spatial-transcriptomics/spatial-data-io - SpatialData/zarr I/O for spatial omics
workflows/scrnaseq-pipeline - end-to-end scRNA-seq pipeline that starts from data loading
References
Virshup I, et al. (2023) The scverse project provides a computational ecosystem for single-cell omics. Nature Biotechnology 41:604-606. DOI 10.1038/s41587-023-01733-8
Virshup I, Rybakov S, Theis FJ, Angerer P, Wolf FA (2024) anndata: Access and store annotated data matrices. Journal of Open Source Software 9(101):4371. DOI 10.21105/joss.04371
Wolf FA, Angerer P, Theis FJ (2018) SCANPY: large-scale single-cell gene expression data analysis. Genome Biology 19:15. DOI 10.1186/s13059-017-1382-0
Hao Y, et al. (2024) Dictionary learning for integrative, multimodal and scalable single-cell analysis (Seurat v5). Nature Biotechnology 42(2):293-304. DOI 10.1038/s41587-023-01767-y
Amezquita RA, Lun ATL, Becht E, et al. (2020) Orchestrating single-cell analysis with Bioconductor. Nature Methods 17(2):137-145. DOI 10.1038/s41592-019-0654-x
Bredikhin D, Kats I, Stegle O (2022) MUON: multimodal omics analysis framework. Genome Biology 23:42. DOI 10.1186/s13059-021-02577-8
Lun ATL, Riesenfeld S, Andrews T, et al. (2019) EmptyDrops: distinguishing cells from empty droplets. Genome Biology 20:63. DOI 10.1186/s13059-019-1662-y
