Usage Tutorial

This tutorial provides detailed instructions for using mLLMCelltype for cell type annotation in single-cell RNA sequencing data. We’ll cover various usage scenarios, parameter configurations, and integration with Seurat.

Comprehensive Function Parameters

annotate_cell_types()

The main function for cell type annotation with a single model:

library(mLLMCelltype)

results <- annotate_cell_types(
  input,                # Marker gene data (data frame, list, or file path)
  tissue_name,          # Tissue name (e.g., "human PBMC", "mouse brain")
  model,                # LLM model to use
  api_key = NA,         # API key (if not set in environment, NA returns prompt only)
  top_gene_count = 10,  # Number of top genes per cluster to use
  debug = FALSE         # Whether to print debugging information
)

interactive_consensus_annotation()

Function for creating consensus annotations from multiple models through interactive discussion:

consensus_results <- interactive_consensus_annotation(
  input,                # Original marker gene data (Seurat FindAllMarkers result or list of genes)
  tissue_name = NULL,   # Optional tissue name
  models = c("claude-3-7-sonnet-20250219", "gpt-4o", "gemini-1.5-pro"),  # Models to use
  api_keys,             # Named list of API keys
  top_gene_count = 10,  # Number of top genes to use
  controversy_threshold = 0.7,  # Threshold for identifying controversial clusters
  entropy_threshold = 1.0,  # Entropy threshold for controversial clusters
  max_discussion_rounds = 3,  # Maximum discussion rounds
  consensus_check_model = NULL,  # Model to use for consensus checking (see recommendations below)
  log_dir = "logs",     # Directory for logs
  cache_dir = "consensus_cache",  # Directory for cache
  use_cache = TRUE      # Whether to use cache
)

Detailed Usage Scenarios

Scenario 1: Basic Annotation with a Single Model

For quick exploration or when API usage is a concern:

# Load example data
library(Seurat)
data("pbmc_small")

# Find markers
pbmc_markers <- FindAllMarkers(pbmc_small, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)

# Run annotation with a single model
results <- annotate_cell_types(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  model = "claude-3-7-sonnet-20250219",
  api_key = Sys.getenv("ANTHROPIC_API_KEY"),
  top_gene_count = 10
)

# Add annotations to Seurat object
pbmc_small$cell_type_claude <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = as.character(0:(length(results)-1)),
  to = results
)

# Visualize
DimPlot(pbmc_small, group.by = "cell_type_claude", label = TRUE)

Scenario 2: Multi-Model Consensus for High Accuracy

For publication-quality annotations with uncertainty quantification:

# Define multiple models to use
models <- c(
  "claude-3-7-sonnet-20250219",  # Anthropic
  "gpt-4o",                      # OpenAI
  "gemini-1.5-pro",              # Google
  "grok-3"                       # X.AI
)

# API keys for different providers
api_keys <- list(
  anthropic = Sys.getenv("ANTHROPIC_API_KEY"),
  openai = Sys.getenv("OPENAI_API_KEY"),
  gemini = Sys.getenv("GEMINI_API_KEY"),
  grok = Sys.getenv("GROK_API_KEY")
)

# Run annotation with multiple models
results <- list()
for (model in models) {
  provider <- get_provider(model)
  api_key <- api_keys[[provider]]

  results[[model]] <- annotate_cell_types(
    input = pbmc_markers,
    tissue_name = "human PBMC",
    model = model,
    api_key = api_key,
    top_gene_count = 10
  )
}

# Create consensus
consensus_results <- interactive_consensus_annotation(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  models = models,  # Use all the models defined above
  api_keys = api_keys,
  controversy_threshold = 0.7,
  entropy_threshold = 1.0,
  consensus_check_model = "claude-3-7-sonnet-20250219"
)

# View consensus results
# You can access the final annotations with consensus_results$final_annotations

# Add consensus annotations and metrics to Seurat object
pbmc_small$cell_type_consensus <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = as.character(0:(length(consensus_results$final_annotations)-1)),
  to = consensus_results$final_annotations
)

# Extract consensus metrics from the consensus results
consensus_metrics <- lapply(names(consensus_results$initial_results$consensus_results), function(cluster_id) {
  metrics <- consensus_results$initial_results$consensus_results[[cluster_id]]
  return(list(
    cluster = cluster_id,
    consensus_proportion = metrics$consensus_proportion,
    entropy = metrics$entropy
  ))
})

# Convert to data frame for easier handling
metrics_df <- do.call(rbind, lapply(consensus_metrics, data.frame))

# Add consensus proportion to Seurat object
pbmc_small$consensus_proportion <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = metrics_df$cluster,
  to = metrics_df$consensus_proportion
)

# Add entropy to Seurat object
pbmc_small$shannon_entropy <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = metrics_df$cluster,
  to = metrics_df$entropy
)

Scenario 2b: Using Free OpenRouter Models

For users with limited API credits or budget constraints:

# Set OpenRouter API key
openrouter_api_key <- Sys.getenv("OPENROUTER_API_KEY")

# Define free OpenRouter models to use
free_models <- c(
  "meta-llama/llama-4-maverick:free",                # Meta Llama 4 Maverick (free)
  "nvidia/llama-3.1-nemotron-ultra-253b-v1:free",    # NVIDIA Nemotron Ultra 253B (free)
  "deepseek/deepseek-chat-v3-0324:free",             # DeepSeek Chat v3 (free)
  "microsoft/mai-ds-r1:free"                         # Microsoft MAI-DS-R1 (free)
)

# Run annotation with free OpenRouter models
free_results <- list()
for (model in free_models) {
  free_results[[model]] <- annotate_cell_types(
    input = pbmc_markers,
    tissue_name = "human PBMC",
    model = model,  # OpenRouter models are automatically detected by format: 'provider/model-name:free'
    api_key = openrouter_api_key,
    top_gene_count = 10
  )
}

# Create consensus with free models
free_consensus_results <- interactive_consensus_annotation(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  models = free_models,  # Use all the free models defined above
  api_keys = list("openrouter" = openrouter_api_key),
  controversy_threshold = 0.7,
  entropy_threshold = 1.0,
  consensus_check_model = "meta-llama/llama-4-maverick:free"  # Use a free model for consensus checking
)

# View free model consensus results
# You can access the final annotations with free_consensus_results$final_annotations

# Add free model consensus annotations to Seurat object
pbmc_small$free_model_consensus <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = as.character(0:(length(free_consensus_results$final_annotations)-1)),
  to = free_consensus_results$final_annotations
)

# Compare paid vs. free model results
comparison <- data.frame(
  cluster = as.character(0:(length(consensus_results$final_annotations)-1)),
  paid_models = consensus_results$final_annotations,
  free_models = free_consensus_results$final_annotations,
  agreement = consensus_results$final_annotations == free_consensus_results$final_annotations
)
print(comparison)

Scenario 3: Working with CSV Files

For users who prefer working with files:

# Save markers to CSV
write.csv(pbmc_markers, "pbmc_markers.csv", row.names = FALSE)

# Run annotation using the CSV file
results <- annotate_cell_types(
  input = "pbmc_markers.csv",
  tissue_name = "human PBMC",
  model = "claude-3-7-sonnet-20250219",
  api_key = Sys.getenv("ANTHROPIC_API_KEY")
)

Scenario 4: Custom Caching

For better control over caching behavior:

# Note: The annotate_cell_types function does not have built-in caching.
# If you need caching, you can implement it separately.

# Run annotation
results <- annotate_cell_types(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  model = "claude-3-7-sonnet-20250219",
  api_key = Sys.getenv("ANTHROPIC_API_KEY"),
  top_gene_count = 10,
  debug = FALSE
)

# If you need custom caching, you can implement it using your own cache manager
# This is just a conceptual example and not part of the actual package
# cache_manager <- YourCacheManager$new(cache_dir = "path/to/cache")
# cache_manager$clear_cache()

Model Selection Guide

mLLMCelltype supports a wide range of LLM models. Here’s a guide to help you choose:

High Performance Models

For the most accurate annotations:

Anthropic Claude 3.7 Sonnet (claude-3-7-sonnet-20250219): Excellent biological knowledge, best for discussion
OpenAI GPT-4o (gpt-4o): Strong overall performance, good biological knowledge
Google Gemini 1.5 Pro (gemini-1.5-pro): Good performance with detailed reasoning

Balanced Performance/Cost Models

For good results with lower API costs:

Anthropic Claude 3.5 Sonnet (claude-3-5-sonnet-20240620): Good balance of performance and cost
X.AI Grok-3 (grok-3): Competitive performance at lower cost
DeepSeek V3 (deepseek-v3): Good performance for specialized tissues

Economy Models

For preliminary exploration or large datasets:

Qwen 2.5 (qwen-max-2025-01-25): Good performance for the cost
Zhipu GLM-4 (glm-4): Economical option with decent performance
MiniMax (minimax): Cost-effective for initial exploration

Free Models via OpenRouter

For users with limited API credits or budget constraints:

Meta Llama 4 Maverick (meta-llama/llama-4-maverick:free): Most reliable and fast, recommended for consensus checking
NVIDIA Nemotron Ultra 253B (nvidia/llama-3.1-nemotron-ultra-253b-v1:free): Good performance with consistent formatting
Microsoft MAI-DS-R1 (microsoft/mai-ds-r1:free): Reliable with good response time
DeepSeek Chat v3 (deepseek/deepseek-chat-v3-0324:free): Free model with 163K context window (may occasionally return empty results)

Based on our testing, we recommend the following free models:

meta-llama/llama-4-maverick:free: Most reliable and fast, recommended for consensus checking
nvidia/llama-3.1-nemotron-ultra-253b-v1:free: Good performance with consistent formatting
microsoft/mai-ds-r1:free: Reliable with good response time

Some models may have limitations:

deepseek/deepseek-chat-v3-0324:free: May occasionally return empty results
thudm/glm-z1-9b:free: May return Chinese error messages (“非字符参数”) when used for consensus checking

These free models are accessed through OpenRouter and don’t consume credits, but may have limitations compared to paid models. Use the :free suffix in the model name to access them.

# Example of using a free model via OpenRouter
# First, set your OpenRouter API key
Sys.setenv(OPENROUTER_API_KEY = "your-openrouter-api-key")

# Then use a free model with the :free suffix
free_model_results <- annotate_cell_types(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  model = "meta-llama/llama-4-maverick:free",  # Note the :free suffix
  api_key = Sys.getenv("OPENROUTER_API_KEY")
  # No need to specify provider - it's automatically detected from the model name format
)

Integration with Seurat Workflow

Here’s a complete example of integrating mLLMCelltype with a Seurat workflow:

library(Seurat)
library(mLLMCelltype)
library(ggplot2)

# Load data
data("pbmc_small")

# Standard Seurat preprocessing
pbmc_small <- NormalizeData(pbmc_small)
pbmc_small <- FindVariableFeatures(pbmc_small)
pbmc_small <- ScaleData(pbmc_small)
pbmc_small <- RunPCA(pbmc_small)
pbmc_small <- FindNeighbors(pbmc_small)
pbmc_small <- FindClusters(pbmc_small, resolution = 0.5)
pbmc_small <- RunUMAP(pbmc_small, dims = 1:10)

# Find markers for each cluster
pbmc_markers <- FindAllMarkers(pbmc_small, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)

# Define models to use
models <- c(
  "claude-3-7-sonnet-20250219",
  "gpt-4o",
  "gemini-1.5-pro"
)

# API keys
api_keys <- list(
  anthropic = Sys.getenv("ANTHROPIC_API_KEY"),
  openai = Sys.getenv("OPENAI_API_KEY"),
  gemini = Sys.getenv("GEMINI_API_KEY")
)

# Run annotation with multiple models
results <- list()
for (model in models) {
  provider <- get_provider(model)
  api_key <- api_keys[[provider]]

  results[[model]] <- annotate_cell_types(
    input = pbmc_markers,
    tissue_name = "human PBMC",
    model = model,
    api_key = api_key,
    top_gene_count = 10
  )

  # Add individual model results to Seurat object
  column_name <- paste0("cell_type_", gsub("[^a-zA-Z0-9]", "_", model))
  pbmc_small[[column_name]] <- plyr::mapvalues(
    x = as.character(Idents(pbmc_small)),
    from = as.character(0:(length(results[[model]])-1)),
    to = results[[model]]
  )
}

# Create consensus
consensus_results <- interactive_consensus_annotation(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  models = models,  # Use all the models defined above
  api_keys = api_keys,
  controversy_threshold = 0.7,
  entropy_threshold = 1.0,
  consensus_check_model = "claude-3-7-sonnet-20250219"
)

# Add consensus results to Seurat object
pbmc_small$cell_type_consensus <- plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = as.character(0:(length(consensus_results$final_annotations)-1)),
  to = consensus_results$final_annotations
)

# Extract consensus metrics from the consensus results
consensus_metrics <- lapply(names(consensus_results$initial_results$consensus_results), function(cluster_id) {
  metrics <- consensus_results$initial_results$consensus_results[[cluster_id]]
  return(list(
    cluster = cluster_id,
    consensus_proportion = metrics$consensus_proportion,
    entropy = metrics$entropy
  ))
})

# Convert to data frame for easier handling
metrics_df <- do.call(rbind, lapply(consensus_metrics, data.frame))

# Add consensus proportion to Seurat object
pbmc_small$consensus_proportion <- as.numeric(plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = metrics_df$cluster,
  to = metrics_df$consensus_proportion
))

# Add entropy to Seurat object
pbmc_small$shannon_entropy <- as.numeric(plyr::mapvalues(
  x = as.character(Idents(pbmc_small)),
  from = metrics_df$cluster,
  to = metrics_df$entropy
))

# Visualize results
p1 <- DimPlot(pbmc_small, group.by = "cell_type_consensus", label = TRUE, repel = TRUE) +
  ggtitle("Cell Type Annotations") +
  theme(plot.title = element_text(hjust = 0.5))

p2 <- FeaturePlot(pbmc_small, features = "consensus_proportion", cols = c("yellow", "green", "blue")) +
  ggtitle("Consensus Proportion") +
  theme(plot.title = element_text(hjust = 0.5))

p3 <- FeaturePlot(pbmc_small, features = "shannon_entropy", cols = c("red", "orange")) +
  ggtitle("Shannon Entropy") +
  theme(plot.title = element_text(hjust = 0.5))

# Combine plots
p1 | p2 | p3

Advanced Parameter Tuning

Adjusting top_gene_count

The top_gene_count parameter controls how many top marker genes per cluster are used for annotation:

# Using more genes (better for well-characterized tissues)
results_more_genes <- annotate_cell_types(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  model = "claude-3-7-sonnet-20250219",
  api_key = Sys.getenv("ANTHROPIC_API_KEY"),
  top_gene_count = 20  # Using more genes
)

# Using fewer genes (better for noisy data)
results_fewer_genes <- annotate_cell_types(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  model = "claude-3-7-sonnet-20250219",
  api_key = Sys.getenv("ANTHROPIC_API_KEY"),
  top_gene_count = 5   # Using fewer genes
)

Adjusting controversy_threshold

The controversy_threshold parameter in the interactive_consensus_annotation function controls which clusters are considered controversial and require discussion:

# Example of using interactive_consensus_annotation with different controversy thresholds
# Lower threshold (more clusters will be discussed)
consensus_results_low_threshold <- interactive_consensus_annotation(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  models = c("claude-3-7-sonnet-20250219", "gpt-4o", "gemini-2.0-flash"),
  api_keys = list(
    "anthropic" = Sys.getenv("ANTHROPIC_API_KEY"),
    "openai" = Sys.getenv("OPENAI_API_KEY"),
    "gemini" = Sys.getenv("GEMINI_API_KEY")
  ),
  controversy_threshold = 0.3  # Lower threshold - more clusters will be discussed
)

# Higher threshold (fewer clusters will be discussed)
consensus_results_high_threshold <- interactive_consensus_annotation(
  input = pbmc_markers,
  tissue_name = "human PBMC",
  models = c("claude-3-7-sonnet-20250219", "gpt-4o", "gemini-2.0-flash"),
  api_keys = list(
    "anthropic" = Sys.getenv("ANTHROPIC_API_KEY"),
    "openai" = Sys.getenv("OPENAI_API_KEY"),
    "gemini" = Sys.getenv("GEMINI_API_KEY")
  ),
  controversy_threshold = 0.7  # Higher threshold - fewer clusters will be discussed
)

Performance Considerations

API Rate Limits and Costs

Different LLM providers have different rate limits and pricing:

OpenAI: Higher rate limits but can be more expensive
Anthropic: Good balance of rate limits and cost
Google: Competitive pricing with good rate limits
Others: Generally lower cost but may have stricter rate limits

To manage costs and rate limits:

Use caching to avoid redundant API calls
Start with a single model for exploration
Use more economical models for initial testing
Reserve multi-model consensus for final analysis

Execution Time

Typical execution times:

Single model annotation: 5-30 seconds per cluster
Multi-model consensus: 1-5 minutes for a typical dataset
Discussion process: Additional 1-3 minutes per controversial cluster

To improve performance:

Use a smaller top_gene_count for faster execution
Enable caching to reuse results
Use a higher controversy_threshold to reduce the number of clusters that require discussion

Troubleshooting

Common Issues with OpenRouter

If you encounter “No auth credentials found” errors with OpenRouter:

Verify your API key is correct and active
Ensure you’re using the correct format for the model name (e.g., provider/model-name:free)
Try a different free model, as some models may have access restrictions

Chinese Error Messages

If you see “非字符参数” (non-character parameter) errors:

This typically occurs when using Chinese models like thudm/glm-z1-9b:free for consensus checking
Switch to an English model like meta-llama/llama-4-maverick:free for consensus checking

Empty Results

If a model returns empty results:

Try increasing the timeout or retry with the same model
Some models like deepseek/deepseek-chat-v3-0324:free may occasionally return empty results
Switch to a more reliable model if the problem persists

Next Steps

Now that you understand the detailed usage of mLLMCelltype, you can explore:

Consensus Annotation Principles: Learn about the technical principles
Visualization Guide: Create publication-ready visualizations
FAQ: Find answers to common questions
Advanced Features: Explore hierarchical annotation and other advanced features

Usage Tutorial

Chen Yang

2025-05-10

Usage Tutorial

Comprehensive Function Parameters

annotate_cell_types()

interactive_consensus_annotation()

Detailed Usage Scenarios

Scenario 1: Basic Annotation with a Single Model

Scenario 2: Multi-Model Consensus for High Accuracy

Scenario 2b: Using Free OpenRouter Models

Scenario 3: Working with CSV Files

Scenario 4: Custom Caching

Model Selection Guide

High Performance Models

Balanced Performance/Cost Models

Economy Models

Free Models via OpenRouter

Integration with Seurat Workflow

Advanced Parameter Tuning

Adjusting top_gene_count

Adjusting controversy_threshold

Performance Considerations

API Rate Limits and Costs

Execution Time

Troubleshooting

Common Issues with OpenRouter

Chinese Error Messages

Empty Results

Next Steps