SeuratMap2Ref Process Configuration

Purpose

Map query single-cell datasets to high-quality reference atlases using Seurat's reference mapping workflow. Performs label transfer, UMAP projection, and integration with reference annotations without modifying query expression data. Enables transfer learning from curated atlases like Azimuth PBMC or custom tissue-specific references.

When to Use

• Reference-based annotation: Well-curated reference atlas available for your tissue/cell type
• Transfer learning: Leverage existing annotations from large-scale atlases (Azimuth, HCA)
• UMAP projection: Visualize query cells on reference UMAP structure
• Label transfer: Propagate cell type labels, gene scores, metadata from reference

Configuration Structure

Process Enablement

[SeuratMap2Ref]
cache = true

Input Specification

[SeuratMap2Ref.in]
srtobj = ["SeuratClustering"]

Environment Variables

[SeuratMap2Ref.envs]
# REQUIRED
ref = "/path/to/reference.rds"  # Reference Seurat object (RDS/h5seurat)
use = "celltype"               # Reference metadata column to transfer
ident = "seurat_clusters"      # Name for transferred labels in query

# NORMALIZATION
refnorm = "auto"               # "LogNormalize", "SCTransform", "auto"
skip_if_normalized = true       # Skip if query already normalized

# PERFORMANCE
ncores = 1                     # Cores for parallelization
cache = "/tmp"                 # Cache intermediate results

# LARGE DATASETS
split_by = "sample"            # Split query by metadata column

# ANCHOR FINDING
[SeuratMap2Ref.envs.FindTransferAnchors]
normalization.method = "auto"
reference.reduction = "pca"
dims = "1:30"
k.anchor = 5

# MAPPING
[SeuratMap2Ref.envs.MapQuery]
reference.reduction = "pca"
reduction.model = "umap"
refdata = {}

# NORMALIZATION
[SeuratMap2Ref.envs.SCTransform]
do.center = true
[SeuratMap2Ref.envs.NormalizeData]
normalization.method = "LogNormalize"

External References

Seurat Functions

• FindTransferAnchors: https://satijalab.org/seurat/reference/findtransferanchors
• TransferData: https://satijalab.org/seurat/reference/transferdata
• MapQuery: https://satijalab.org/seurat/reference/mapquery
• Tutorial: https://satijalab.org/seurat/articles/integration_mapping

Configuration Examples

Minimal (Azimuth PBMC)

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
ref = "/path/to/pbmc_reference.rds"
use = "celltype.l1"

Custom with Multi-Level Transfer

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
ref = "/path/to/pancreas_atlas.rds"
use = "celltype.l2"
refdata = {celltype = "celltype", donor = "orig.ident"}
ident = "ref_celltype"

Large Dataset with Splitting

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
split_by = "sample"
ncores = 4
refnorm = "SCTransform"

Common Patterns

Pattern 1: Azimuth PBMC Reference

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
ref = "/data/azimuth/pbmc_reference.rds"
use = "celltype.l1"
refdata = {celltype = "celltype.l1", celltype.l2 = "celltype.l2"}

Pattern 2: Human Cell Atlas Reference

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
ref = "/data/hca/bone_marrow_reference.rds"
use = "cell_type"
refnorm = "SCTransform"
FindTransferAnchors.dims = "1:30"

Pattern 3: Multi-Label with Confidence Scores

[SeuratMap2Ref]
[SeuratMap2Ref.envs]
refdata = {
  "l1_celltype" = "celltype.l1",
  "l2_celltype" = "celltype.l2",
  "l1_score" = "celltype.l1.score",
  "l2_score" = "celltype.l2.score"
}

Available References

Azimuth References

• PBMC Reference: Human PBMC (celltype.l1, celltype.l2)
  • URL: https://azimuth.hubmapconsortium.org
• Bone Marrow Reference: Human BMNC cells
• Pan-human Reference: Cross-tissue reference (CloudAzimuth)

Human Cell Atlas (HCA)

• Tissue-specific atlases: Bone marrow, pancreas, lung
• Download: https://www.humancellatlas.org/data/

SeuratData References

library(SeuratData)
InstallData("pbmc3k")  # PBMC dataset
InstallData("bmcite")  # Bone marrow
InstallData("panc8")   # Pancreas islets

Dependencies

• Upstream: SeuratClustering
• Downstream: ClusterMarkers, ScFGSEA

Validation Rules

Reference Format Requirements

• File type: RDS (.rds, .RDS) or h5seurat (.h5seurat, .h5)
• Metadata: Must contain cell type column specified by use
• Reduction: Should have PCA or dimensional reduction computed
• Normalization: Must match query normalization (detected by refnorm)

Compatibility Checks

• Gene names: Query and reference must use same naming convention
• Species: Same species (unless ortholog conversion applied)
• Dimensionality: Reference PCs must exist for anchor finding

Troubleshooting

Dimension Mismatch

Problem: Reference has 30 PCs but query has 50 Solution: Set FindTransferAnchors.dims = "1:30"

Poor Anchor Quality

Problem: Low prediction scores or failed transfer Solutions:

• Increase FindTransferAnchors.k.anchor (5 to 10)
• Check normalization method consistency
• Verify gene names match
• Increase FindTransferAnchors.dims (e.g., "1:40")

Out-of-Memory Errors

Problem: Large query dataset causes memory overflow Solutions:

• Use split_by to process in chunks
• Reduce ncores to limit parallelization
• Reduce FindTransferAnchors.dims

UMAP Projection Failure

Problem: ref.umap not found in query object Solutions:

• Ensure reference has UMAP with return.model = TRUE
• Set MapQuery.reduction.model = "umap"
• Check MapQuery.reference.reduction matches reference

Cache Issues

Problem: Unexpected results after parameter changes Solution: Delete cached files <signature>.<kind>.RDS or set cache = false

Label Column Overwrites

Problem: Multiple annotation processes overwrite each other Solution: Set unique ident for each process

Normalization Method Mismatch

Problem: Query is SCTransform but reference is LogNormalize Solutions:

• Set refnorm = "LogNormalize" and re-normalize
• Convert reference to SCTransform
• Set skip_if_normalized = false

Output

Files Generated

• Seurat object: With mapped metadata and ref.umap reduction
• Plots: Mapped identity and mapping score visualizations
• Cache: Intermediate RDS files (if cache enabled)

Metadata Added

• Transferred labels: Column named by ident
• Prediction scores: {ident}.score
• Additional data: Fields specified in refdata

Visualization

DimPlot(seurat_obj, reduction = "ref.umap", group.by = "predicted_celltype")

Related Processes

• CellTypeAnnotation: Alternative automated annotation
• SeuratClustering: Upstream clustering for query
• ClusterMarkers: Downstream marker finding with transferred labels
• ScFGSEA: Pathway enrichment using reference-based annotations