Version Compatibility

Reference examples tested with: numpy 1.26+, scipy 1.12+, pandas 2.2+, anndata 0.10+, scanpy 1.10+, Matrix R package 1.6+, HDF5Array 1.30+ (Bioconductor), DelayedArray 0.28+, zellkonverter 1.12+, zarr-python 2.18+, dask 2024.1+

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.

Sparse Matrix Handling

"Store / compute on a single-cell matrix without blowing up memory" -> Pick the sparse format that matches the access pattern (CSC for column ops, CSR for row ops), respect the R/Python convention difference (Bioconductor stores cells in columns; AnnData stores cells in rows), and use HDF5/Zarr backed mode for matrices too large for RAM.

The Single Most Important Modern Insight -- The R/Python interop transpose is silent and catastrophic

R Bioconductor (Seurat, SingleCellExperiment) stores cells in COLUMNS and uses dgCMatrix (Column-Compressed Sparse). Python scverse (AnnData, scanpy) stores cells in ROWS and defaults to CSR (Compressed Sparse Row). Round-tripping with anndata2ri, zellkonverter, or rpy2 triggers a TRANSPOSE under the hood -- once per direction. Two flips silently cancel. A debugging session that converts back and forth multiple times can end up with mysteriously transposed data and no error.

| Conversion | Implicit transpose | |------------|-------------------| | Python CSR -> R dgCMatrix | YES (rows <-> cols) | | AnnData .X (cells x genes) -> SingleCellExperiment counts (genes x cells) | YES | | Seurat @assays$RNA@counts (genes x cells) -> AnnData .X (cells x genes) | YES | | scipy.sparse.csr_matrix(dense) | None (just format conversion) | | csr.tocsc() / csc.tocsr() | None (semantically same matrix, different layout) |

The safe pattern for one-shot conversions: file-based intermediate. adata.write('file.h5ad') then zellkonverter::readH5AD('file.h5ad') -- avoids the in-memory rpy2/reticulate gymnastics, and the file roundtrip makes orientation explicit.

For a 1M-cell single-cell matrix, the implicit transpose is non-trivial -- minutes of wall time and a temporary memory peak roughly equal to nnz x 12 bytes (CSC) or x 16 bytes (CSR with int64). Avoid unnecessary transposes by aligning the format to the consumer.

Two adjacent insights:

1. Sparse becomes inefficient above ~10-15% density. Sparse iteration has cache-unfriendly indirection; dense iteration is sequential. Above ~10-15% nonzero, dense is often faster for most operations even though it uses more memory.
2. AnnData backed mode quietly differs from in-memory in important ways. sc.read_h5ad('file.h5ad', backed='r') returns an AnnData where .X is a wrapped HDF5 dataset, read-only. Many scanpy functions silently load to memory; some functions error or hang on backed mode.

Algorithmic Taxonomy

| Format | Layout | Fast for | Slow for | |--------|--------|----------|----------| | dgCMatrix (CSC, R) | i (row indices), p (col pointers), x (values) | Column slicing; per-cell ops in single-cell (cells in cols); matrix-vector with col vector | Row slicing | | dgRMatrix (CSR, R) | j (col indices), p (row pointers), x (values) | Row slicing; per-gene ops when genes in rows | Column slicing | | dgTMatrix (COO, R triplet) | i, j, x | Random insertion when building; reading MTX | Most operations -- convert to dgCMatrix after build | | scipy csc_matrix | Same as dgCMatrix | Column ops in Python | Row ops | | scipy csr_matrix | Same as dgRMatrix | Row ops (NumPy convention); matmul; sklearn defaults | Column ops | | scipy coo_matrix | Triplet (i, j, data) | Construction; MTX I/O | Most ops -- convert after build | | HDF5 (h5ad, h5) | Single-file binary chunked | Random access via chunks; compression; widely supported | Cloud / parallel writes | | Zarr | Chunked array, per-chunk file (or S3 object) | Cloud-native; parallel writes; Dask integration | Single-file simplicity | | DelayedArray + HDF5Array | Bioc lazy evaluation over HDF5 | Out-of-memory bulk ops in R | Speed of in-memory |

Decision Tree by Scenario

| Scenario | Recommended approach | |----------|---------------------| | Single-cell (>10k cells), per-cell ops | dgCMatrix in R (Bioconductor); CSR adata.X in Python (scanpy) | | Bulk RNA-seq (60-80% density) | Dense -- sparse overhead exceeds benefit | | Single-cell pseudobulk (after donor aggregation) | Dense -- now 60-80% density typically | | 1M+ cells, can't fit in RAM | scanpy backed mode OR HDF5SummarizedExperiment + DelayedArray | | TCGA + GTEx + recount3 scale (100k+ samples) | HDF5Array / Zarr + Dask | | 10X CellRanger 3.0+ output | sc.read_10x_h5() or Read10X_h5() -- the .h5 is faster than the .mtx triplet | | Cloud-native (anndata on S3, dask compute) | Zarr | | Local workstation, single-machine | HDF5 -- faster, more widely supported | | Building sparse matrix incrementally | COO (dgTMatrix / coo_matrix); convert to CSC/CSR after | | R <-> Python conversion | File-based intermediate (adata.write then zellkonverter::readH5AD); aware of the transpose |

Check Sparsity

Goal: Decide whether sparse is the right format given the actual data density.

Approach: Compute nonzero fraction; rule of thumb is sparse > ~85% (single-cell). Below that, dense often wins.

import numpy as np
import scipy.sparse as sp

def sparsity(m):
    if sp.issparse(m):
        return 1 - m.nnz / (m.shape[0] * m.shape[1])
    return (m == 0).mean()

s = sparsity(adata.X)
print(f'{s:.1%} sparse')

Memory math:

| Data | Format | Bytes | |------|--------|-------| | 30k x 100k single-cell matrix, 5% density | dgCMatrix | (5% * 3e9) * 12 bytes ~= 1.8 GB | | Same | Dense double | 24 GB | | 60k x 100k bulk, 70% density | dgCMatrix | 50 GB (worse than dense!) | | Same | Dense double | 48 GB |

For single-cell (typically 90-95% sparse), sparse is essential. For bulk RNA-seq (typically 60-80% density), dense is faster and not appreciably larger.

dgCMatrix / scipy CSC / CSR

Goal: Construct, query, and convert sparse matrices in the format matching the consumer's expected layout.

Approach: Matrix::sparseMatrix(i, j, x, dims=...) (R) or scipy.sparse.csr_matrix((data, (i, j))) (Python); preserve row/column names; convert layout (CSC <-> CSR) without changing semantics.

import scipy.sparse as sp
import pandas as pd

dense_df = pd.read_csv('counts.csv', index_col=0)
sparse_csr = sp.csr_matrix(dense_df.values)
sparse_csc = sp.csc_matrix(dense_df.values)

gene_names = dense_df.index.tolist()
sample_names = dense_df.columns.tolist()

sparse_csr.tocsc()
sparse_csc.tocsr()

library(Matrix)

dense_mat <- as.matrix(read.csv('counts.csv', row.names = 1))
sparse_dgc <- as(dense_mat, 'CsparseMatrix')

class(sparse_dgc)
rownames(sparse_dgc) <- rownames(dense_mat)
colnames(sparse_dgc) <- colnames(dense_mat)

dgTMatrix is best for building matrices incrementally (reading MTX, parsing per-row); convert to dgCMatrix for downstream ops:

mat_t <- as(triplet_data, 'TsparseMatrix')
mat_c <- as(mat_t, 'CsparseMatrix')

HDF5 vs Zarr -- The Cloud-Native Shift

HDF5 (Hierarchical Data Format 5): hierarchical, single-file binary. Random access via chunks; supports compression (gzip, blosc, lz4). On-disk format for AnnData .h5ad, MuData .h5mu, 10x Genomics .h5, HDF5SummarizedExperiment.

Zarr: cloud-native, chunked array storage. Each chunk is a separate file (or S3 object). Parallel-write friendly; splittable by Dask. Format used by recent AnnData (anndata.write_zarr), SpatialData, and large-cohort consortia.

| Criterion | HDF5 | Zarr | |-----------|------|------| | File structure | Single binary file | Directory of chunk files | | Parallel writes | Limited (process-level locks) | Native | | S3 / cloud object storage | Workarounds (h5cloud); often slow | Native; first-class | | Compression options | gzip, blosc, lz4, szip | gzip, blosc, lz4, zstd, custom | | Local workstation speed | Faster | Slightly slower (many small files) | | Wide ecosystem support | Yes (mature) | Growing; modern scverse |

For local workstation work, HDF5 is faster and more widely supported. For cloud-mounted analysis (anndata on S3 with dask-distributed compute), Zarr wins because of object-storage friendliness.

HDF5SummarizedExperiment + DelayedArray (Bioconductor)

Goal: Work with bulk SummarizedExperiment objects too large to fit in RAM by keeping the matrix on disk.

Approach: HDF5Array wraps an HDF5 dataset as a DelayedArray. Subsetting builds a delayed operation tree -- no I/O until realization. DelayedMatrixStats provides delayed-friendly stat functions.

library(HDF5Array)
library(SummarizedExperiment)
library(DelayedMatrixStats)

se <- loadHDF5SummarizedExperiment('saved_se_dir')

s <- se[1:1000, 1:50]
row_means <- rowMeans2(assay(se))

saveHDF5SummarizedExperiment(se, 'saved_se_dir', replace = TRUE)

The assay(se) returns a DelayedMatrix backed by HDF5. DESeq2, edgeR, limma have varying levels of DelayedArray support; consult package docs before assuming all ops work in delayed mode.

For TCGA + GTEx + recount3 scale (100k+ samples, 60k genes), a dense matrix is ~48 GB (double); dgCMatrix at 70% density is ~50 GB (sparse loses). HDF5Array + chunk-aware ops keeps memory at whatever-fits-in-RAM.

scanpy Backed Mode

Goal: Work with h5ad files too large for memory by loading only accessed slices on demand.

Approach: sc.read_h5ad(..., backed='r') returns an AnnData with .X as a wrapped HDF5 dataset. Subset operations are lazy; .to_memory() realizes.

import scanpy as sc

adata = sc.read_h5ad('large_dataset.h5ad', backed='r')
print(f'Shape: {adata.shape}, X type: {type(adata.X)}')

t_cells = adata[adata.obs['cell_type'] == 'T_cell', :].to_memory()

Limitations:

• .X is read-only in backed='r'. Use backed='r+' for in-place updates, but only .X updates supported.
• .obs and .var are fully loaded -- only .X supports backed access.
• Very large sparse h5ad (>35 GB) can still cause memory issues even in backed mode (anndata library overhead).
• Many scanpy functions internally load to memory; check ?function docs for backed compatibility.
• Functions like sc.tl.pca, sc.pp.neighbors typically require in-memory; subset first with .to_memory().

For datasets too large for backed mode, process in chunks:

import anndata as ad

def process_in_chunks(h5ad_path, chunk_size=10000, func=None):
    adata = sc.read_h5ad(h5ad_path, backed='r')
    n_cells = adata.shape[0]
    results = []
    for start in range(0, n_cells, chunk_size):
        end = min(start + chunk_size, n_cells)
        chunk = adata[start:end].to_memory()
        if func:
            chunk = func(chunk)
        results.append(chunk)
    return ad.concat(results)

10X Genomics Format Proliferation

| Format | Files | Notes | |--------|-------|-------| | MTX (pre-CellRanger 3.0) | matrix.mtx + barcodes.tsv + features.tsv (or genes.tsv) | Triplet format; slow to read for large matrices | | H5 (CellRanger 3.0+) | filtered_feature_bc_matrix.h5 | HDF5 with /matrix/data, /matrix/indices, /matrix/indptr, /matrix/shape; single file, fast | | H5AD | data.h5ad | AnnData; convert on import | | kallisto|bustools output | output.bus + barcode and gene mappings | BUSpaRse / kb-python |

import scanpy as sc

adata = sc.read_10x_h5('filtered_feature_bc_matrix.h5')
adata = sc.read_10x_mtx('filtered_feature_bc_matrix/')

library(Seurat)
mat <- Read10X_h5('filtered_feature_bc_matrix.h5')
mat <- Read10X(data.dir = 'filtered_feature_bc_matrix/')

library(DropletUtils)
sce <- read10xCounts('filtered_feature_bc_matrix/')

For 10X output, prefer the .h5 over the MTX triplet -- typically 5-10x faster for large matrices.

Dense Conversion -- The Memory Blow-Up

adata.X.toarray() (Python) or as.matrix(seurat_obj@assays$RNA@counts) (R) on a 30k x 100k single-cell matrix instantiates a ~24 GB dense double array. Common triggers:

• Passing sparse to a function that internally calls as.matrix() (older R cor() implementations).
• Heatmap functions (pheatmap, ComplexHeatmap) that require dense.
• ML libraries with no sparse support (some sklearn models; XGBoost requires specific sparse API).
• Plotting functions (plot(), ggplot2) called on the full matrix.

Defensive pattern: subset to a manageable gene/cell set BEFORE dense conversion.

For per-cell PCA-style ops, use sparse-aware solvers:

from scipy.sparse.linalg import svds
U, s, Vt = svds(adata.X, k=50)

library(irlba)
svd_res <- irlba(sparse_mat, nv = 50)

irlba (R) and scipy.sparse.linalg.svds (Python) compute truncated SVD without densifying.

SCE vs AnnData vs MuData -- Where Bulk Fits

| Container | Library | Cells/samples | Multi-modal | Bulk fit | |-----------|---------|---------------|-------------|----------| | SummarizedExperiment / RangedSummarizedExperiment | Bioconductor | n/a; bulk | No | YES -- standard for DESeq2/edgeR/limma bulk | | SingleCellExperiment (Amezquita 2020) | Bioconductor | cells in cols | Via altExps | scRNA-seq with spike-ins, ADT | | AnnData | scverse/Python | cells in rows | Via layers | scRNA-seq; bulk is unusual | | MuData | scverse/Python | cells in rows | Yes, multiple AnnData | Multi-modal scRNA + ATAC + protein | | MultiAssayExperiment | Bioconductor | samples | Yes | R-side multi-modal analog |

Bulk RNA-seq rarely uses AnnData -- it shines on the single-cell dimensionality reduction / neighbors / clustering machinery. For bulk in R, use SummarizedExperiment. For bulk in Python, a tidy DataFrame + numpy array is usually sufficient.

Dask + Zarr for Consortium-Scale Matrices

For TCGA + GTEx + recount3 (Wilks 2021 Genome Biol 22:323) or pancancer assemblies:

import zarr
import dask.array as da

z = zarr.open('counts.zarr', mode='r')
da_arr = da.from_zarr(z)

col_sums = da_arr.sum(axis=0).compute()
filtered = da_arr[da_arr.sum(axis=1) > 100, :]

import anndata as ad
adata_disk = ad.read_zarr('large_data.zarr')

For R: HDF5Array + DelayedArray is the equivalent, but R doesn't have a true Dask analog. BiocParallel can parallelize chunks, but lazy planning is more manual.

Sparse Operations

import numpy as np
import scipy.sparse as sp

row_sums = np.array(sparse_matrix.sum(axis=1)).flatten()
col_sums = np.array(sparse_matrix.sum(axis=0)).flatten()

keep_rows = row_sums > 10
sparse_filt = sparse_matrix[keep_rows, :]

sparse_log = sparse_matrix.copy()
sparse_log.data = np.log1p(sparse_log.data)

Subsetting: select genes (rows) or samples (cols) by index:

gene_idx = [gene_names.index(g) for g in ['TP53', 'BRCA1', 'MYC'] if g in gene_names]
subset = sparse_matrix[gene_idx, :]

CPM Normalization on Sparse

Goal: Apply CPM normalization without densifying.

Approach: Compute library sizes from column sums; broadcast scaling factors with sparse multiply for CPM; transform only the nonzero data array in-place with log1p.

import numpy as np
import scipy.sparse as sp

def normalize_sparse_cpm(sparse_matrix):
    lib_sizes = np.array(sparse_matrix.sum(axis=0)).flatten()
    scaling = 1e6 / lib_sizes
    return sparse_matrix.multiply(scaling)

def log1p_inplace(sparse_matrix):
    out = sparse_matrix.copy()
    out.data = np.log1p(out.data)
    return out

cpm = normalize_sparse_cpm(adata.X)
log_cpm = log1p_inplace(cpm)

After log-transformation, sparsity is PRESERVED (log1p(0) = 0). After CPM with pseudocount, zeros become nonzero -- check sparsity and convert to dense if density drops below ~15%.

Save / Load Sparse Matrices

import scipy.sparse as sp
import numpy as np

sp.save_npz('counts_sparse.npz', sparse_matrix)
loaded = sp.load_npz('counts_sparse.npz')

np.savez('counts_with_meta.npz',
    data    = sparse_matrix.data,
    indices = sparse_matrix.indices,
    indptr  = sparse_matrix.indptr,
    shape   = sparse_matrix.shape,
    genes   = np.array(gene_names),
    samples = np.array(sample_names))

For interop and durable storage, prefer h5ad or zarr:

adata.write_h5ad('counts.h5ad')
adata.write_zarr('counts.zarr')

Per-Method Failure Modes

Implicit transpose in R/Python conversion

Trigger: AnnData with cells in rows passed to a SingleCellExperiment workflow that expects cells in cols; downstream colSums returns gene-level totals.

Mechanism: AnnData stores cells in rows; SCE in cols. The conversion auto-transposes ONCE per direction; two roundtrips silently restore.

Symptom: Per-cell stats look like per-gene stats; QC plots have wrong axes.

Fix: Use file-based intermediate (adata.write('file.h5ad'); zellkonverter::readH5AD('file.h5ad')). Always verify dimensions and orientation after conversion.

Dense conversion blew up memory

Trigger: as.matrix(seurat_obj@assays$RNA@counts) on a 100k-cell dataset; R session crashes with OOM.

Mechanism: 100k cells x 30k genes = 3e9 entries; double precision = 24 GB.

Symptom: R session killed; "cannot allocate vector of size N GB".

Fix: Don't densify the full matrix. Subset to genes/cells of interest first. For dimensionality reduction, use irlba::irlba() (sparse SVD).

scanpy backed mode silently loaded to memory

Trigger: adata = sc.read_h5ad(path, backed='r') then sc.tl.pca(adata); memory spikes to dense-equivalent.

Mechanism: Many scanpy functions internally call .to_memory() because they cannot operate on backed mode. sc.tl.pca, sc.pp.neighbors, sc.tl.umap all materialize.

Symptom: OOM despite backed mode.

Fix: Subset first (adata[mask].to_memory()), then operate. Or use a streaming-aware alternative (Dask + Zarr).

Sparse stored where dense would be faster

Trigger: Bulk RNA-seq with 70% density stored as dgCMatrix; per-gene rowVars is slow.

Mechanism: Sparse iteration has cache-unfriendly indirection; above ~10-15% density, dense wins.

Symptom: Operations notably slower than expected; profiler shows time in sparse indexing.

Fix: Convert to dense for the hot path: as.matrix(sparse_mat) (R) or sparse_matrix.toarray() (Python). Memory may go up but speed improves substantially.

10X MTX read is slow

Trigger: Reading a 100k-cell 10X dataset via the MTX three-file format; takes 10+ minutes.

Mechanism: MTX is a text format; parsing is slow for large matrices.

Symptom: Long load times; user kills the process before completion.

Fix: Use the CellRanger H5 (.h5) instead -- typically 5-10x faster.

Common errors

| Error / symptom | Cause | Fix | |-----------------|-------|-----| | cannot allocate vector of size N GB | Implicit dense conversion | Subset first; use sparse-aware solver (irlba) | | Sparse-dense arithmetic returns numpy.matrix | Deprecated NumPy type from sparse+dense | np.asarray(sparse + dense) to force ndarray | | KeyError: '_index' reading h5ad | anndata version mismatch | Update anndata; or sc.read_h5ad(..., backed=None) | | Empty rows/cols after sparse subset | Subset removed all data | Verify the index list; cross-check sample/gene names | | Backed mode AnnData crash on sc.tl.umap | Function not backed-compatible | .to_memory() on the subset first | | Per-cell totals look wrong after R<->Python conversion | Implicit transpose | Verify dimensions; use file-based intermediate | | CSR (Python) <-> dgCMatrix (R) treated as same | Convention difference | They're transposes of each other; verify shape and a known cell-gene pair |

References

• Amezquita RA, Lun ATL, Becht E et al. 2020. Orchestrating single-cell analysis with Bioconductor. Nat Methods 17:137-145. doi:10.1038/s41592-019-0654-x
• Wolf FA, Angerer P, Theis FJ. 2018. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19:15. doi:10.1186/s13059-017-1382-0
• Wilks C et al. 2021. recount3: summaries and queries for large-scale RNA-seq expression and splicing. Genome Biol 22:323. doi:10.1186/s13059-021-02533-6
• Bates D, Maechler M. 2023. Matrix: Sparse and Dense Matrix Classes and Methods. R package version 1.6-x.
• Pages H et al. 2020. HDF5Array: HDF5 backend for DelayedArray objects. Bioconductor package.
• Miles A et al. 2020. zarr-python. Python package documentation.
• Rocklin M. 2015. Dask: Parallel Computation with Blocked algorithms and Task Scheduling. Proc Python Sci Conf. (canonical Dask reference)
• Lachmann A et al. 2018. Massive mining of publicly available RNA-seq data from human and mouse. Nat Commun 9:1366. doi:10.1038/s41467-018-03751-6

Related Skills

• counts-ingest - Reading 10X formats; building sparse matrices from quantification output
• gene-id-mapping - Var (gene) metadata in AnnData
• metadata-joins - Obs (sample) metadata in AnnData
• normalization - log1p and CPM patterns on sparse
• differential-expression/deseq2-basics - Pseudobulk aggregation makes dense
• single-cell/data-io - Single-cell file format ecosystem
• single-cell/preprocessing - Standard single-cell sparse pipeline