Joint Trace Analysis

This example demonstrates how to analyze joint trace data from multiple reporters (e.g., MS2 and PP7) in live cell imaging experiments.

Setup

First, let's set up our project directory and load the package:

using StochasticGene

# Create project directory
rna_setup("joint_trace_example")
cd("joint_trace_example")

Data Preparation

Place your joint trace data in the data/ directory. The data should be organized as follows:

data/
├── gene_name/
│   ├── condition1/
│   │   ├── ms2_traces.csv
│   │   ├── pp7_traces.csv
│   │   └── metadata.csv
│   └── condition2/
│       ├── ms2_traces.csv
│       ├── pp7_traces.csv
│       └── metadata.csv

Each trace CSV file should contain:

Time points
Fluorescence intensity values
Optional background values

Model Definition

We'll fit a joint model with:

2 gene states (G=2) - ON and OFF
2 pre-RNA steps (R=2)
Simple transitions between states
Reporter positions for MS2 and PP7

# Define model parameters
G = 2  # Number of gene states (ON and OFF)
R = 2  # Number of pre-RNA steps

# Define state transitions
# Format: (from_states, to_states)
transitions = (
    [1, 2],  # From states
    [2, 1]   # To states
)

# Define transcription rates for each state
transcription_rates = [0.0, 1.0]  # No transcription in OFF state

# Define reporter positions
reporter_positions = (
    ms2 = 1,  # MS2 reporter at first step
    pp7 = 2   # PP7 reporter at second step
)

Fitting the Model

Now we can fit the joint model to our trace data:

# Fit the model
fits, stats, measures, data, model, options = fit(
    G = G,
    R = R,
    transitions = transitions,
    transcription_rates = transcription_rates,
    datatype = "joint_trace",
    datapath = "data/",
    gene = "MYC",
    datacond = "CONTROL",
    reporter_positions = reporter_positions
)

Analyzing Results

Basic Analysis

# Print basic statistics
println(stats)

# Plot the results
using Plots
plot(fits)

# Save results
save_results(fits, "results/")

Reporter-Specific Analysis

# Analyze MS2 reporter
ms2_analysis = analyze_reporter(fits, "ms2")
plot_reporter(ms2_analysis, "results/ms2/")

# Analyze PP7 reporter
pp7_analysis = analyze_reporter(fits, "pp7")
plot_reporter(pp7_analysis, "results/pp7/")

Cross-Correlation Analysis

# Calculate cross-correlation
cross_corr = calculate_cross_correlation(fits)
plot_cross_correlation(cross_corr, "results/cross_correlation/")

# Analyze time lag
time_lag = analyze_time_lag(fits)
plot_time_lag(time_lag, "results/time_lag/")

Advanced Analysis

Burst Analysis

# Analyze transcriptional bursts
burst_stats = analyze_bursts(fits)
println(burst_stats)

# Plot burst statistics
plot_bursts(fits, "results/bursts/")

Multiple Conditions

# Compare different conditions
conditions = ["CONTROL", "TREATMENT"]
condition_fits = []

for cond in conditions
    fits, stats, measures, data, model, options = fit(
        G = G,
        R = R,
        transitions = transitions,
        transcription_rates = transcription_rates,
        datatype = "joint_trace",
        datapath = "data/",
        gene = "MYC",
        datacond = cond,
        reporter_positions = reporter_positions
    )
    push!(condition_fits, (fits, stats))
end

# Compare conditions
compare_conditions(condition_fits, conditions, "results/condition_comparison/")

Best Practices

Data Quality
- Check for photobleaching
- Verify signal-to-noise ratio
- Consider background correction
Model Selection
- Start with simple models
- Use model selection criteria
- Validate assumptions
Analysis Pipeline
- Document preprocessing steps
- Save intermediate results
- Version control your analysis

Common Issues and Solutions

Photobleaching

# Check for photobleaching
bleaching = check_photobleaching(fits)
plot_photobleaching(bleaching, "results/photobleaching/")

# Correct for photobleaching
corrected_fits = correct_photobleaching(fits)

Background Noise

# Analyze background noise
noise = analyze_background_noise(fits)
plot_background_noise(noise, "results/background_noise/")

# Apply background correction
corrected_fits = correct_background(fits)

Next Steps

Try different reporter configurations
Experiment with preprocessing steps
Compare results across different genes

For more advanced examples, see: