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bioconductor-singler

Performs unbiased cell type recognition from single-cell RNA sequencing data, by leveraging reference transcriptomic datasets of pure cell types to infer the cell of origin of each single cell independently.

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SingleR

Dependencies & Environment

Package-intrinsic requirements from the Bioconductor landing page — reproduce in any R environment.

  • Version: 2.14.0 · Bioconductor: 3.23 · R: ≥ 4.6
  • Depends: SummarizedExperiment
  • Imports: Matrix, BiocGenerics, S4Vectors, DelayedArray, Rcpp, beachmat
  • System requirements: C++17
  • Install: BiocManager::install("SingleR")

When to Use

  • Annotating unlabelled single-cell datasets using pre-labelled reference datasets (e.g., HumanPrimaryCellAtlasData()).
  • Propagating biological knowledge to new datasets in an automated manner without manually interpreting clusters.
  • Resolving closely related cell type labels using fine-tuning.

When NOT to Use

  • For unsupervised clustering without a reference (use scran instead because SingleR requires known labels).
  • For manual marker gene definition and cluster interpretation (use Seurat instead because SingleR automates this using a reference).

Data Requirements

  • Test dataset: Unlogged counts are acceptable, as only ranks are used by SingleR().
  • Reference dataset: Must be normalized and log-transformed (e.g., using normalizeRnaCounts.se()).

Key Parameters

  • test: The unlabelled single-cell dataset to be annotated.
  • ref: The pre-labelled reference dataset.
  • labels: The cell type labels corresponding to the reference dataset.
  • assay.type.test: The assay index or name to use from the test dataset.
  • de.method: The marker detection mode (e.g., "wilcox" for single-cell references).

Best Practices

  • Perform cell-based quality control before running SingleR().
  • Normalize and log-transform reference datasets using normalizeRnaCounts.se().
  • Inspect assignment confidence using plotScoreHeatmap() and plotDeltaDistribution().
  • Remove poor-quality or ambiguous assignments using pruneScores().
  • Examine the expression of marker genes for each label in the test dataset using plotMarkerHeatmap().

Common Pitfalls

  • Using raw counts for the reference (fix: log-transform and normalize the reference first using normalizeRnaCounts.se()).
  • Default marker detection failing on low-coverage data (fix: use de.method="wilcox" to consider variance of expression across cells).
  • Uncertain assignments for cells whose true label does not exist in the reference (fix: inspect per-cell deltas and use pruneScores() to replace low-quality assignments with NA).

Alternatives

  • Seurat (for manual marker gene definition and cluster interpretation).
  • scmap (for projection-based single-cell annotation).

Citations

  • Aran, D., et al. 2019. "Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage." Nat. Immunol. 20 (2): 163–72.
  • Grun, D., et al. 2016. "De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data." Cell Stem Cell 19 (2): 266–77.

References

  • Homepage: bioconductor.org/packages/SingleR
  • Vignette: vignette_0_9645ffcc.txt
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