spacr.ml — spacr 0.5.0 documentation

# Source code for spacr.ml

    import os, shap, re
    import pandas as pd
    import numpy as np
    from scipy import stats
    from scipy.stats import shapiro
    from math import pi

    from sklearn.linear_model import Lasso, Ridge, LassoCV, RidgeCV
    from sklearn.metrics import mean_squared_error
    import numpy as np

    import matplotlib.pyplot as plt
    from IPython.display import display
    import scipy.stats as st
    import statsmodels.api as sm
    import statsmodels.formula.api as smf
    from statsmodels.tools import add_constant
    from statsmodels.regression.mixed_linear_model import MixedLM
    from statsmodels.tools.sm_exceptions import PerfectSeparationError
    from statsmodels.stats.outliers_influence import variance_inflation_factor
    from statsmodels.genmod.families import Binomial
    from statsmodels.genmod.families.links import logit
    from statsmodels.othermod.betareg import BetaModel
    from scipy.optimize import minimize
    from scipy.special import gammaln, psi, expit
    from sklearn.linear_model import Lasso, Ridge
    from sklearn.preprocessing import FunctionTransformer
    from patsy import dmatrices

    from sklearn.metrics import classification_report, accuracy_score
    from sklearn.model_selection import StratifiedKFold, cross_val_score
    from sklearn.feature_selection import SelectKBest, f_classif
    from sklearn.model_selection import train_test_split
    from sklearn.ensemble import RandomForestClassifier, HistGradientBoostingClassifier
    from sklearn.linear_model import LogisticRegression
    from sklearn.inspection import permutation_importance
    from sklearn.metrics import classification_report, precision_recall_curve
    from sklearn.preprocessing import StandardScaler
    from sklearn.preprocessing import MinMaxScaler
    from scipy.spatial.distance import cosine, euclidean, mahalanobis, cityblock, minkowski, chebyshev, hamming, jaccard, braycurtis
    from xgboost import XGBClassifier

    import numpy as np
    from scipy.stats import kstest, normaltest
    import statsmodels.api as sm

    import matplotlib
    matplotlib.use('Agg')

    import warnings
    warnings.filterwarnings("ignore", message="3D stack used, but stitch_threshold=0 and do_3D=False, so masks are made per plane only")

    class QuasiBinomial(Binomial):
        """Custom Quasi-Binomial family with adjustable variance."""
        def __init__(self, link=logit(), dispersion=1.0):
            super().__init__(link=link)

            self.dispersion = dispersion

        def variance(self, mu):
            """Adjust the variance with the dispersion parameter."""
            return self.dispersion * super().variance(mu)

    def calculate_p_values(X, y, model):
        # Predict y values
        y_pred = model.predict(X)
        # Calculate residuals
        residuals = y - y_pred
        # Calculate the standard error of the residuals
        dof = X.shape[0] - X.shape[1] - 1
        residual_std_error = np.sqrt(np.sum(residuals ** 2) / dof)
        # Calculate the standard error of the coefficients
        X_design = np.hstack((np.ones((X.shape[0], 1)), X))  # Add intercept
        # Use pseudoinverse instead of inverse to handle singular matrices
        coef_var_covar = residual_std_error ** 2 * np.linalg.pinv(X_design.T @ X_design)
        coef_standard_errors = np.sqrt(np.diag(coef_var_covar))
        # Calculate t-statistics
        t_stats = model.coef_ / coef_standard_errors[1:]  # Skip intercept error
        # Calculate p-values
        p_values = [2 * (1 - stats.t.cdf(np.abs(t), dof)) for t in t_stats]
        return np.array(p_values)  # Ensure p_values is a 1-dimensional array

    def perform_mixed_model(y, X, groups, alpha=1.0):
        # Ensure groups are defined correctly and check for multicollinearity
        if groups is None:
            raise ValueError("Groups must be defined for mixed model regression")

        # Check for multicollinearity by calculating the VIF for each feature
        X_np = X.values
        vif = [variance_inflation_factor(X_np, i) for i in range(X_np.shape[1])]
        print(f"VIF: {vif}")
        if any(v > 10 for v in vif):
            print(f"Multicollinearity detected with VIF: {vif}. Applying Ridge regression to the fixed effects.")
            ridge = Ridge(alpha=alpha)
            ridge.fit(X, y)
            X_ridge = ridge.coef_ * X  # Adjust X with Ridge coefficients
            model = MixedLM(y, X_ridge, groups=groups)
        else:
            model = MixedLM(y, X, groups=groups)

        result = model.fit()
        return result

    def create_volcano_filename(csv_path, regression_type, alpha, dst):
        """Create and return the volcano plot filename based on regression type and alpha."""
        volcano_filename = os.path.splitext(os.path.basename(csv_path))[0] + '_volcano_plot.pdf'
        volcano_filename = f"{regression_type}_{volcano_filename}" if regression_type != 'quantile' else f"{alpha}_{volcano_filename}"

        if dst:
            return os.path.join(dst, volcano_filename)
        return os.path.join(os.path.dirname(csv_path), volcano_filename)

    def scale_variables(X, y):
        """Scale independent (X) and dependent (y) variables using MinMaxScaler."""
        scaler_X = MinMaxScaler()
        scaler_y = MinMaxScaler()

        X_scaled = pd.DataFrame(scaler_X.fit_transform(X), columns=X.columns)
        y_scaled = scaler_y.fit_transform(y)

        return X_scaled, y_scaled

    def process_model_coefficients(model, regression_type, X, y, nc, pc, controls):
        """Return DataFrame of model coefficients and p-values."""
        if regression_type in ['ols', 'gls', 'wls', 'rlm', 'glm', 'mixed', 'quantile', 'logit', 'probit', 'poisson']:
            coefs = model.params
            p_values = model.pvalues

            coef_df = pd.DataFrame({
                'feature': coefs.index,
                'coefficient': coefs.values,
                'p_value': p_values.values
            })

        elif regression_type in ['ridge', 'lasso']:
            coefs = model.coef_.flatten()
            p_values = calculate_p_values(X, y, model)

            coef_df = pd.DataFrame({
                'feature': X.columns,
                'coefficient': coefs,
                'p_value': p_values
            })

        else:
            coefs = model.coef_
            intercept = model.intercept_
            feature_names = X.design_info.column_names

            coef_df = pd.DataFrame({
                'feature': feature_names,
                'coefficient': coefs
            })
            coef_df.loc[0, 'coefficient'] += intercept
            coef_df['p_value'] = np.nan  # Placeholder since sklearn doesn't provide p-values

        coef_df['-log10(p_value)'] = -np.log10(coef_df['p_value'])
        coef_df['grna'] = coef_df['feature'].str.extract(r'\[(.*?)\]')[0]
        coef_df['condition'] = coef_df.apply(lambda row: 'nc' if nc in row['feature'] else 'pc' if pc in row['feature'] else ('control' if row['grna'] in controls else 'other'),axis=1)
        return coef_df[~coef_df['feature'].str.contains('row|column')]

    def check_distribution(y):
        """Check the type of distribution to recommend a model."""
        if np.all((y == 0) | (y == 1)):
            print("Detected binary data.")
            return 'logit'
        elif (y > 0).all() and (y < 1).all():
            print("Detected continuous data between 0 and 1 (excluding 0 and 1).")
            return 'beta'
        elif (y >= 0).all() and (y <= 1).all():
            print("Detected continuous data between 0 and 1 (including 0 or 1).")
            # Consider quasi-binomial regression
            return 'quasi_binomial'
        else:
            print("Using OLS as a fallback.")
            return 'ols'

    def select_glm_family(y):
        """Select the appropriate GLM family based on the data."""
        if np.all((y == 0) | (y == 1)):
            print("Using Binomial family (for binary data).")
            return sm.families.Binomial()
        elif (y >= 0).all() and (y <= 1).all():
            print("Using Quasi-Binomial family (for proportion data including 0 and 1).")
            return QuasiBinomial()
        elif np.all(y.astype(int) == y) and (y >= 0).all():
            print("Using Poisson family (for count data).")
            return sm.families.Poisson()
        else:
            print("Using Gaussian family (for continuous data).")
            return sm.families.Gaussian()

    def prepare_formula(dependent_variable, random_row_column_effects=False):
        """Return the regression formula using random effects for plate, row, and column."""
        if random_row_column_effects:
            # Random effects for row and column + gene weighted by gene_fraction + grna weighted by fraction
            return f'{dependent_variable} ~ fraction:grna + gene_fraction:gene'
        return f'{dependent_variable} ~ fraction:grna + gene_fraction:gene + rowID + columnID'

    def fit_mixed_model(df, formula, dst):
        from .plot import plot_histogram

        """Fit the mixed model with plate, row_name, and columnID as random effects and return results."""
        # Specify random effects for plate, row, and column
        model = smf.mixedlm(formula,
                            data=df,
                            groups=df['plateID'],
                            re_formula="1 + rowID + columnID",
                            vc_formula={"rowID": "0 + rowID", "columnID": "0 + columnID"})

        mixed_model = model.fit()

        # Plot residuals
        df['residuals'] = mixed_model.resid
        plot_histogram(df, 'residuals', dst=dst)

        # Return coefficients and p-values
        coefs = mixed_model.params
        p_values = mixed_model.pvalues

        coef_df = pd.DataFrame({
            'feature': coefs.index,
            'coefficient': coefs.values,
            'p_value': p_values.values
        })

        return mixed_model, coef_df

    def check_and_clean_data(df, dependent_variable):
        """Check for collinearity, missing values, or invalid types in relevant columns. Clean data accordingly."""

        def handle_missing_values(df, columns):
            """Handle missing values in specified columns."""
            missing_summary = df[columns].isnull().sum()
            print("Missing values summary:")
            print(missing_summary)

            # Drop rows with missing values in these fields
            df_cleaned = df.dropna(subset=columns)
            if df_cleaned.shape[0] < df.shape[0]:
                print(f"Dropped {df.shape[0] - df_cleaned.shape[0]} rows with missing values in {columns}.")
            return df_cleaned

        def ensure_valid_types(df, columns):
            """Ensure that specified columns are categorical."""
            for col in columns:
                if not pd.api.types.is_categorical_dtype(df[col]):
                    df[col] = pd.Categorical(df[col])
                    print(f"Converted {col} to categorical type.")
            return df

        def check_collinearity(df, columns):
            """Check for collinearity using VIF (Variance Inflation Factor)."""
            print("Checking for collinearity...")

            # Only include fraction and the dependent variable for collinearity check
            df_encoded = df[columns]

            # Ensure all data in df_encoded is numeric
            df_encoded = df_encoded.apply(pd.to_numeric, errors='coerce')

            # Check for perfect multicollinearity (i.e., rank deficiency)
            if np.linalg.matrix_rank(df_encoded.values) < df_encoded.shape[1]:
                print("Warning: Perfect multicollinearity detected! Dropping correlated columns.")
                df_encoded = df_encoded.loc[:, ~df_encoded.columns.duplicated()]

            # Calculate VIF for each feature
            vif_data = pd.DataFrame()
            vif_data["Feature"] = df_encoded.columns
            try:
                vif_data["VIF"] = [variance_inflation_factor(df_encoded.values, i) for i in range(df_encoded.shape[1])]
            except np.linalg.LinAlgError:
                print("LinAlgError: Unable to compute VIF due to matrix singularity.")
                return df_encoded

            print("Variance Inflation Factor (VIF) for each feature:")
            print(vif_data)

            # Drop columns with VIF > 10 (a common threshold to identify multicollinearity)
            high_vif_columns = vif_data[vif_data["VIF"] > 10]["Feature"].tolist()
            if high_vif_columns:
                print(f"Dropping columns with high VIF: {high_vif_columns}")
                df_encoded.drop(columns=high_vif_columns, inplace=True)

            return df_encoded

        # Step 1: Handle missing values in relevant fields
        df = handle_missing_values(df, ['fraction', dependent_variable])

        # Step 2: Ensure grna, gene, plate, row, column, and prc are categorical types
        df = ensure_valid_types(df, ['grna', 'gene', 'plateID', 'rowID', 'columnID', 'prc'])

        # Step 3: Check for multicollinearity in fraction and the dependent variable
        df_cleaned = check_collinearity(df, ['fraction', dependent_variable])

        # Ensure that the prc, plate, row, and column columns are still included for random effects
        df_cleaned['gene'] = df['gene']
        df_cleaned['grna'] = df['grna']
        df_cleaned['prc'] = df['prc']
        df_cleaned['plateID'] = df['plateID']
        df_cleaned['rowID'] = df['rowID']
        df_cleaned['columnID'] = df['columnID']

        # Create a new column 'gene_fraction' that sums the fractions by gene within the same well
        df_cleaned['gene_fraction'] = df_cleaned.groupby(['prc', 'gene'])['fraction'].transform('sum')

        print("Data is ready for model fitting.")
        return df_cleaned

    def check_normality(y, variable_name):
        """Check if the data is normally distributed using the Shapiro-Wilk test."""
        from scipy.stats import shapiro

        stat, p = shapiro(y)
        alpha = 0.05
        if p > alpha:
            print(f"{variable_name} is normally distributed (fail to reject H0)")
            return True
        else:
            print(f"{variable_name} is not normally distributed (reject H0)")
            return False

    def minimum_cell_simulation(settings, num_repeats=10, sample_size=100, tolerance=0.02, smoothing=10, increment=10):
        """
        Plot the mean absolute difference with standard deviation as shaded area vs. sample size.
        Detect and mark the elbow point (inflection) with smoothing and tolerance control.
        """

        from spacr.utils import correct_metadata_column_names

        # Load and process data
        if isinstance(settings['score_data'], str):
            settings['score_data'] = [settings['score_data']]

        dfs = []
        for i, score_data in enumerate(settings['score_data']):
            df = pd.read_csv(score_data)
            df = correct_metadata_column_names(df)
            df['plateID'] = f'plate{i + 1}'

            if 'prc' not in df.columns:
                df['prc'] = df['plateID'] + '_' + df['rowID'].astype(str) + '_' + df['columnID'].astype(str)

            dfs.append(df)

        df = pd.concat(dfs, axis=0)

        # Compute the number of cells per well and select the top 100 wells by cell count
        cell_counts = df.groupby('prc').size().reset_index(name='cell_count')
        top_wells = cell_counts.nlargest(sample_size, 'cell_count')['prc']

        # Filter the data to include only the top 100 wells
        df = df[df['prc'].isin(top_wells)]

        # Initialize storage for absolute difference data
        diff_data = []

        # Group by wells and iterate over them
        for i, (prc, group) in enumerate(df.groupby('prc')):
            original_mean = group[settings['score_column']].mean()  # Original full-well mean
            max_cells = len(group)
            sample_sizes = np.arange(2, max_cells + 1, increment)  # Sample sizes from 2 to max cells

            # Iterate over sample sizes and compute absolute difference
            for sample_size in sample_sizes:
                abs_diffs = []

                # Perform multiple random samples to reduce noise
                for _ in range(num_repeats):
                    sample = group.sample(n=sample_size, replace=False)
                    sampled_mean = sample[settings['score_column']].mean()
                    abs_diff = abs(sampled_mean - original_mean)  # Absolute difference
                    abs_diffs.append(abs_diff)

                # Compute the average absolute difference across all repeats
                avg_abs_diff = np.mean(abs_diffs)

                # Store the result for plotting
                diff_data.append((sample_size, avg_abs_diff))

        # Convert absolute difference data to DataFrame for plotting
        diff_df = pd.DataFrame(diff_data, columns=['sample_size', 'avg_abs_diff'])

        # Group by sample size to calculate mean and standard deviation
        summary_df = diff_df.groupby('sample_size').agg(
            mean_abs_diff=('avg_abs_diff', 'mean'),
            std_abs_diff=('avg_abs_diff', 'std')
        ).reset_index()

        # Apply smoothing using a rolling window
        summary_df['smoothed_mean_abs_diff'] = summary_df['mean_abs_diff'].rolling(window=smoothing, min_periods=1).mean()

        # Convert percentage to fraction
        if isinstance(settings['tolerance'], int):
            tolerance_fraction = settings['tolerance'] / 100  # Convert 2% to 0.02
        elif isinstance(settings['tolerance'], float):
            tolerance_fraction = settings['tolerance']
        else:
            raise ValueError("Tolerance must be an integer 0 - 100 or float 0.0 - 1.0.")

        # Compute the relative threshold for each well
        relative_thresholds = {
            prc: tolerance_fraction * group[settings['score_column']].mean()  # Compute % of original mean
            for prc, group in df.groupby('prc')
        }

        # Detect the elbow point when mean absolute difference is below the relative threshold
        summary_df['relative_threshold'] = summary_df['sample_size'].map(
            lambda size: np.mean([relative_thresholds[prc] for prc in top_wells])  # Average across selected wells
        )

        elbow_df = summary_df[summary_df['smoothed_mean_abs_diff'] <= summary_df['relative_threshold']]

        # Select the first occurrence if it exists; otherwise, use the last point
        if not elbow_df.empty:
            elbow_point = elbow_df.iloc[0]  # First point where condition is met
        else:
            elbow_point = summary_df.iloc[-1]  # Fallback to last point

        # Plot the mean absolute difference with standard deviation as shaded area
        fig, ax = plt.subplots(figsize=(10, 10))
        ax.plot(
            summary_df['sample_size'], summary_df['smoothed_mean_abs_diff'], color='teal', label='Smoothed Mean Absolute Difference'
        )
        ax.fill_between(
            summary_df['sample_size'],
            summary_df['smoothed_mean_abs_diff'] - summary_df['std_abs_diff'],
            summary_df['smoothed_mean_abs_diff'] + summary_df['std_abs_diff'],
            color='teal', alpha=0.3, label='±1 Std. Dev.'
        )

        if settings['min_cell_count'] is None:
            # Mark the elbow point (inflection) on the plot
            ax.axvline(elbow_point['sample_size'], color='black', linestyle='--', label='Elbow Point')
        else:
            ax.axvline(settings['min_cell_count'], color='black', linestyle='--', label='Elbow Point')

        # Formatting the plot
        ax.set_xlabel('Sample Size')
        ax.set_ylabel('Mean Absolute Difference')
        ax.set_title('Mean Absolute Difference vs. Sample Size with Standard Deviation')
        ax.legend().remove()

        # Save the plot if a destination is provided
        dst = os.path.dirname(settings['count_data'][0])
        if dst is not None:
            fig_path = os.path.join(dst, 'results')
            os.makedirs(fig_path, exist_ok=True)
            fig_file_path = os.path.join(fig_path, 'cell_min_threshold.pdf')
            fig.savefig(fig_file_path, format='pdf', dpi=600, bbox_inches='tight')
            print(f"Saved {fig_file_path}")

        plt.show()
        return elbow_point['sample_size']

    def process_model_coefficients(model, regression_type, X, y, nc, pc, controls):
        """Return DataFrame of model coefficients, standard errors, and p-values."""

        if regression_type == 'beta':
            # Extract coefficients and standard errors
            coefs = model.params
            std_err = model.bse

            # Compute Wald test (coefficient / standard error)
            wald_stats = coefs / std_err

            # Calculate two-tailed p-values
            p_values = 2 * (1 - st.norm.cdf(np.abs(wald_stats)))

            coef_df = pd.DataFrame({
                'feature': coefs.index,
                'coefficient': coefs.values,
                'std_err': std_err.values,
                'wald_stat': wald_stats.values,
                'p_value': p_values
            })

        elif regression_type in ['ols', 'glm', 'logit', 'probit', 'quasi_binomial']:
            coefs = model.params
            p_values = model.pvalues

            coef_df = pd.DataFrame({
                'feature': coefs.index,
                'coefficient': coefs.values,
                'p_value': p_values.values
            })

        elif regression_type in ['ridge', 'lasso']:
            coefs = model.coef_.flatten()
            p_values = calculate_p_values(X, y, model)

            coef_df = pd.DataFrame({
                'feature': X.columns,
                'coefficient': coefs,
                'p_value': p_values
            })

        else:
            raise ValueError(f"Unsupported regression type: {regression_type}")

        # Additional formatting
        coef_df['-log10(p_value)'] = -np.log10(coef_df['p_value'])
        coef_df['grna'] = coef_df['feature'].str.extract(r'\[(.*?)\]')[0]
        coef_df['condition'] = coef_df.apply(
            lambda row: 'nc' if nc in row['feature'] else
                        'pc' if pc in row['feature'] else
                        ('control' if row['grna'] in controls else 'other'),
            axis=1
        )

        return coef_df[~coef_df['feature'].str.contains('row|column')]

    def check_distribution(y, epsilon=1e-6):
        """Check the distribution of y and recommend an appropriate model."""

        # Check if the dependent variable is binary (only 0 and 1)
        if np.all((y == 0) | (y == 1)):
            print("Detected binary data.")
            return 'logit'

        # Continuous data between 0 and 1 (excluding exact 0 and 1)
        elif (y > 0).all() and (y < 1).all():
            # Check if the data is close to 0 or 1 (boundary issues)
            if np.any((y < epsilon) | (y > 1 - epsilon)):
                print("Detected continuous data near 0 or 1. Using quasi-binomial.")
                return 'quasi_binomial'
            else:
                print("Detected continuous data between 0 and 1 (no boundary issues). Using beta regression.")
                return 'beta'

        # Continuous data between 0 and 1 (including exact 0 or 1)
        elif (y >= 0).all() and (y <= 1).all():
            print("Detected continuous data with boundary values (0 or 1). Using quasi-binomial.")
            return 'quasi_binomial'

        # Check if the data is normally distributed for OLS suitability
        stat, p_value = stats.normaltest(y)  # D’Agostino and Pearson’s test for normality
        print(f"Normality test p-value: {p_value:.4f}")

        if p_value > 0.05:
            print("Detected normally distributed data. Using OLS.")
            return 'ols'

        # Check if the data fits a Beta distribution
        if stats.kstest(y, 'beta', args=(2, 2)).pvalue > 0.05:
            # Check if the data is close to 0 or 1 (boundary issues)
            if np.any((y < epsilon) | (y > 1 - epsilon)):
                print("Detected continuous data near 0 or 1. Using quasi-binomial.")
                return 'quasi_binomial'
            else:
                print("Detected continuous data between 0 and 1 (no boundary issues). Using beta regression.")
                return 'beta'

        print("Detected non-normally distributed data. Using GLM.")
        return 'glm'

    def pick_glm_family_and_link(y):
        """Select the appropriate GLM family and link function based on data."""
        if np.all((y == 0) | (y == 1)):
            print("Binary data detected. Using Binomial family with Logit link.")
            return sm.families.Binomial(link=sm.families.links.Logit())

        elif (y > 0).all() and (y < 1).all():
            print("Data strictly between 0 and 1. Beta regression recommended.")
            raise ValueError("Use BetaModel for this data; GLM is not applicable.")

        elif (y >= 0).all() and (y <= 1).all():
            print("Data between 0 and 1 (including boundaries). Using Quasi-Binomial.")
            return sm.families.Binomial(link=sm.families.links.Logit())

        stat, p_value = normaltest(y)
        print(f"Normality test p-value: {p_value:.4f}")
        if p_value > 0.05:
            print("Normally distributed data detected. Using Gaussian with Identity link.")
            return sm.families.Gaussian(link=sm.families.links.Identity())

        if (y >= 0).all() and np.all(y.astype(int) == y):
            print("Count data detected. Using Poisson with Log link.")
            return sm.families.Poisson(link=sm.families.links.Log())

        if (y > 0).all() and kstest(y, 'invgauss', args=(1,)).pvalue > 0.05:
            print("Inverse Gaussian distribution detected. Using InverseGaussian with Log link.")
            return sm.families.InverseGaussian(link=sm.families.links.Log())

        if (y >= 0).all():
            print("Overdispersed count data detected. Using Negative Binomial with Log link.")
            return sm.families.NegativeBinomial(link=sm.families.links.Log())

        print("Using default Gaussian family with Identity link.")
        return sm.families.Gaussian(link=sm.families.links.Identity())

    def regression_model(X, y, regression_type='ols', groups=None, alpha=1.0, cov_type=None):
        def plot_regression_line(X, y, model):
            """Helper to plot regression line for lasso and ridge models."""
            y_pred = model.predict(X)
            plt.scatter(X.iloc[:, 1], y, color='blue', label='Data')
            plt.plot(X.iloc[:, 1], y_pred, color='red', label='Regression line')
            plt.xlabel('Features')
            plt.ylabel('Dependent Variable')
            plt.legend()
            plt.show()

        def find_best_alpha(model_cls):
            """Find optimal alpha using cross-validation."""
            alphas = np.logspace(-5, 5, 100)  # Search over a range of alphas
            if model_cls == 'lasso':
                model_cv = LassoCV(alphas=alphas, cv=5).fit(X, y)
            elif model_cls == 'ridge':
                model_cv = RidgeCV(alphas=alphas, cv=5).fit(X, y)
            print(f"Optimal alpha for {model_cls}: {model_cv.alpha_}")
            return model_cv

        # Dictionary of models
        model_map = {
            'ols': lambda: sm.OLS(y, X).fit(cov_type=cov_type) if cov_type else sm.OLS(y, X).fit(),
            'glm': lambda: sm.GLM(y, X, family=pick_glm_family_and_link(y)).fit(),
            'beta': lambda: BetaModel(endog=y, exog=X).fit(),
            'logit': lambda: sm.Logit(y, X).fit(),
            'probit': lambda: sm.Probit(y, X).fit(),
            'lasso': lambda: find_best_alpha('lasso') if alpha in [0, None] else Lasso(alpha=alpha).fit(X, y),
            'ridge': lambda: find_best_alpha('ridge') if alpha in [0, None] else Ridge(alpha=alpha).fit(X, y)
        }

        # Select the model based on regression_type
        if regression_type in model_map:
            model = model_map[regression_type]()
        elif regression_type == 'mixed':
            model = perform_mixed_model(y, X, groups, alpha=alpha)
        else:
            raise ValueError(f"Unsupported regression type {regression_type}")

        # Plot regression line for Lasso and Ridge
        if regression_type in ['lasso', 'ridge']:
            plot_regression_line(X, y, model)

        # Handle GLM-specific statistics
        if regression_type == 'glm':
            llf_model = model.llf  # Log-likelihood of the fitted model
            llf_null = model.null_deviance / -2  # Log-likelihood of the null model
            mcfadden_r2 = 1 - (llf_model / llf_null)
            print(f"McFadden's R²: {mcfadden_r2:.4f}")
            print(model.summary())

        if regression_type in ['lasso', 'ridge']:
            # Calculate the Mean Squared Error (MSE)
            mse = mean_squared_error(y, model.predict(X))
            print(f"{regression_type.capitalize()} Regression MSE: {mse:.4f}")

            # Display coefficients
            coef_df = pd.DataFrame({
                'Feature': X.columns,
                'Coefficient': model.coef_
            })
            print(coef_df)

        return model

    def regression(df, csv_path, dependent_variable='predictions', regression_type=None, alpha=1.0,
                   random_row_column_effects=False, nc='233460', pc='220950', controls=[''],
                   dst=None, cov_type=None, plot=False):

        from .plot import volcano_plot, plot_histogram
        #from .ml import create_volcano_filename, check_and_clean_data, prepare_formula, scale_variables

        # Generate the volcano filename
        volcano_path = create_volcano_filename(csv_path, regression_type, alpha, dst)

        # Determine regression type if not specified
        if regression_type is None:
            regression_type = check_distribution(df[dependent_variable])

        print(f"Using regression type: {regression_type}")

        df = check_and_clean_data(df, dependent_variable)

        # Handle mixed effects if row/column effect is treated as random
        if random_row_column_effects:
            regression_type = 'mixed'
            formula = prepare_formula(dependent_variable, random_row_column_effects=True)
            mixed_model, coef_df = fit_mixed_model(df, formula, dst)
            model = mixed_model
        else:
            # Prepare the formula
            formula = prepare_formula(dependent_variable, random_row_column_effects=False)
            y, X = dmatrices(formula, data=df, return_type='dataframe')

            # Plot histogram of the dependent variable
            plot_histogram(y, dependent_variable, dst=dst)
            plot_histogram(df, 'fraction', dst=dst)

            # Scale the independent variables and dependent variable
            if regression_type in ['beta', 'quasi_binomial', 'logit']:
                print('Data will not be scaled')
            else:
                X, y = scale_variables(X, y)

            # Perform the regression
            groups = df['prc'] if regression_type == 'mixed' else None
            print(f'Performing {regression_type} regression')

            model = regression_model(X, y, regression_type=regression_type, groups=groups, alpha=alpha, cov_type=cov_type)

            # Process the model coefficients
            coef_df = process_model_coefficients(model, regression_type, X, y, nc, pc, controls)
            display(coef_df)
        if plot:
            volcano_plot(coef_df, volcano_path)

        return model, coef_df, regression_type

    def save_summary_to_file(model, file_path='summary.csv'):
        """
        Save the model's summary output to a CSV or text file.
        """
        # Get the summary as a string
        summary_str = model.summary().as_text()

        # Save it as a plain text file or CSV
        with open(file_path, 'w') as f:
            f.write(summary_str)

    def perform_regression(settings):

        from .plot import plot_plates, plot_data_from_csv
        from .utils import merge_regression_res_with_metadata, save_settings, calculate_shortest_distance, correct_metadata
        from .settings import get_perform_regression_default_settings
        from .toxo import go_term_enrichment_by_column, custom_volcano_plot, plot_gene_phenotypes, plot_gene_heatmaps
        from .sequencing import graph_sequencing_stats

        def _perform_regression_read_data(settings):

            if not isinstance(settings['score_data'], list):
                settings['score_data'] = [settings['score_data']]
            if not isinstance(settings['count_data'], list):
                settings['count_data'] = [settings['count_data']]

            score_data_df = pd.DataFrame()
            for i, score_data in enumerate(settings['score_data']):
                df = pd.read_csv(score_data)
                df = correct_metadata(df)
                if not 'plateID' in df.columns:
                    df['plateID'] = f'plate{i+1}'

                score_data_df = pd.concat([score_data_df, df])
                print('Score data:', len(score_data_df))

            count_data_df = pd.DataFrame()
            for i, count_data in enumerate(settings['count_data']):
                df = pd.read_csv(count_data)
                df = correct_metadata(df)
                if not 'plateID' in df.columns:
                    df['plateID'] = f'plate{i+1}'

                count_data_df = pd.concat([count_data_df, df])
                print('Count data:', len(count_data_df))

            print(f"Dependent variable: {len(score_data_df)}")
            print(f"Independent variable: {len(count_data_df)}")

            if settings['dependent_variable'] not in score_data_df.columns:
                print(f'Columns in DataFrame:')
                for col in score_data_df.columns:
                    print(col)
                if not settings['dependent_variable'] == 'pathogen_nucleus_shortest_distance':
                    raise ValueError(f"Dependent variable {settings['dependent_variable']} not found in the DataFrame")

            reg_types = ['ols','gls','wls','rlm','glm','mixed','quantile','logit','probit','poisson','lasso','ridge', None]
            if settings['regression_type'] not in reg_types:
                print(f'Possible regression types: {reg_types}')
                raise ValueError(f"Unsupported regression type {settings['regression_type']}")

            return count_data_df, score_data_df

        def _perform_regression_set_paths(settings):

            if isinstance(settings['score_data'], list):
                score_data = settings['score_data'][0]
            else:
                score_data = settings['score_data']

            score_source = os.path.splitext(os.path.basename(score_data))[0]

            if isinstance(settings['count_data'], list):
                src = os.path.dirname(settings['count_data'][0])
                csv_path = settings['count_data'][0]
            else:
                src = os.path.dirname(settings['count_data'])
                csv_path = settings['count_data']

            settings['src'] = src

            if settings['regression_type'] is None:
                res_folder = os.path.join(src, 'results', score_source, 'auto')
            else:
                res_folder = os.path.join(src, 'results', score_source, settings['regression_type'])

            if isinstance(settings['count_data'], list):
                res_folder = os.path.join(res_folder, 'list')

            os.makedirs(res_folder, exist_ok=True)
            results_filename = 'results.csv'
            results_filename_gene = 'results_gene.csv'
            results_filename_grna = 'results_grna.csv'
            hits_filename = 'results_significant.csv'
            results_path=os.path.join(res_folder, results_filename)
            results_path_gene=os.path.join(res_folder, results_filename_gene)
            results_path_grna=os.path.join(res_folder, results_filename_grna)
            hits_path=os.path.join(res_folder, hits_filename)

            return results_path, results_path_gene, results_path_grna, hits_path, res_folder, csv_path

        def _count_variable_instances(df, column_1, column_2):
            if column_1 is not None:
                n_grna = df[column_1].value_counts().reset_index()
                n_grna.columns = [column_1, f'n_{column_1}']
            if column_2 is not None:
                n_gene = df[column_2].value_counts().reset_index()
                n_gene.columns = [column_2, f'n_{column_2}']
            if column_1 is not None and column_2 is not None:
                return df, n_grna, n_gene
            elif column_1 is not None:
                return df, n_grna
            elif column_2 is not None:
                return df, n_gene
            else:
                return df

        def grna_metricks(df):
            df[['plateID', 'rowID', 'columnID']] = df['prc'].str.split('_', expand=True)

            # --- 2) Compute GRNA-level Well Counts ---
            # For each (grna, plate), count the number of unique prc (wells)
            grna_well_counts = (df.groupby(['grna', 'plateID'])['prc'].nunique().reset_index(name='grna_well_count'))

            # --- 3) Compute Gene-level Well Counts ---
            # For each (gene, plate), count the number of unique prc
            gene_well_counts = (df.groupby(['gene', 'plateID'])['prc'].nunique().reset_index(name='gene_well_count'))

            # --- 4) Merge These Counts into a Single DataFrame ---
            # Because each grna is typically associated with one gene, we bring them together.
            # First, create a unique (grna, gene, plate) reference from the original df
            unique_triplets = df[['grna', 'gene', 'plateID']].drop_duplicates()

            # Merge the grna_well_count
            merged_df = pd.merge(unique_triplets, grna_well_counts, on=['grna', 'plateID'], how='left')

            # Merge the gene_well_count
            merged_df = pd.merge(merged_df, gene_well_counts, on=['gene', 'plateID'], how='left')

            # Keep only the columns needed (if you want to keep 'gene', remove the drop below)
            final_grna_df = merged_df[['grna', 'plateID', 'grna_well_count', 'gene_well_count']]

            # --- 5) Compute gene_count per prc ---
            # For each prc (well), how many distinct genes are there?
            prc_gene_count_df = (df.groupby('prc')['gene'].nunique().reset_index(name='gene_count'))
            prc_gene_count_df[['plateID', 'rowID', 'columnID']] = prc_gene_count_df['prc'].str.split('_', expand=True)

            return final_grna_df, prc_gene_count_df

        def get_outlier_reference_values(df, outlier_col, return_col):
            """
            Detect outliers in 'outlier_col' of 'df' using the 1.5 × IQR rule,
            and return values from 'return_col' that correspond to those outliers.

            Parameters:
            -----------
            df : pd.DataFrame
                Input DataFrame.
            outlier_col : str
                Column in which to check for outliers.
            return_col : str
                Column whose values to return for rows that are outliers in 'outlier_col'.

            Returns:
            --------
            pd.Series
                A Series containing values from 'return_col' for the outlier rows.
            """
            # Calculate Q1, Q3, and IQR for the outlier_col
            Q1 = df[outlier_col].quantile(0.05)
            Q3 = df[outlier_col].quantile(0.95)
            IQR = Q3 - Q1

            # Determine the outlier cutoffs
            lower_bound = Q1 - 1.5 * IQR
            upper_bound = Q3 + 1.5 * IQR

            # Create a mask for outliers
            outlier_mask = (df[outlier_col] < lower_bound) | (df[outlier_col] > upper_bound)

            outliers = df.loc[outlier_mask, return_col]

            outliers_ls = outliers.unique().tolist()

            return outliers_ls

        settings = get_perform_regression_default_settings(settings)
        count_data_df, score_data_df = _perform_regression_read_data(settings)

        if "rowID" in count_data_df.columns:
            num_parts = len(count_data_df['rowID'].iloc[0].split('_'))
            if num_parts == 2:
                split = count_data_df['rowID'].str.split('_', expand=True)
                count_data_df['rowID'] = split[1]

        if "prc" in score_data_df.columns:
            num_parts = len(score_data_df['prc'].iloc[0].split('_'))
            if num_parts == 3:
                split = score_data_df['prc'].str.split('_', expand=True)
                score_data_df['plateID'] = settings['plateID']
                score_data_df['prc'] = score_data_df['plateID'] + '_' + split[1] + '_' + split[2]

        results_path, results_path_gene, results_path_grna, hits_path, res_folder, csv_path = _perform_regression_set_paths(settings)
        save_settings(settings, name='regression', show=True)

        count_source = os.path.dirname(settings['count_data'][0])
        volcano_path = os.path.join(count_source, 'volcano_plot.pdf')

        if isinstance(settings['filter_value'], list):
            filter_value = settings['filter_value']
        else:
            filter_value = []
        if isinstance(settings['filter_column'], str):
            filter_column = settings['filter_column']

        score_data_df = clean_controls(score_data_df, settings['filter_value'], settings['filter_column'])

        if settings['verbose']:
            print(f"Dependent variable after clean_controls: {len(score_data_df)}")

        sim_min_count = minimum_cell_simulation(settings, tolerance=settings['tolerance'])

        if settings['min_cell_count'] is None:
            settings['min_cell_count'] = sim_min_count

        if settings['verbose']:
            print(f"Minimum cell count: {settings['min_cell_count']}")
            print(f"Dependent variable after minimum cell count filter: {len(score_data_df)}")
            display(score_data_df)

        orig_dv = settings['dependent_variable']

        dependent_df, dependent_variable = process_scores(score_data_df, settings['dependent_variable'], settings['plateID'], settings['min_cell_count'], settings['agg_type'], settings['transform'])

        if settings['verbose']:
            print(f"Dependent variable after process_scores: {len(dependent_df)}")
            display(dependent_df)

        if settings['fraction_threshold'] is None:
            settings['fraction_threshold'] = graph_sequencing_stats(settings)

        independent_df = process_reads(count_data_df, settings['fraction_threshold'], settings['plateID'], filter_column=filter_column, filter_value=filter_value)

        independent_df, n_grna, n_gene = _count_variable_instances(independent_df, column_1='grna', column_2='gene')

        if settings['verbose']:
            print(f"Independent variable after process_reads: {len(independent_df)}")

        merged_df = pd.merge(independent_df, dependent_df, on='prc')

        if settings['verbose']:
            display(independent_df)
            display(dependent_df)
            display(merged_df)

        merged_df[['plateID', 'rowID', 'columnID']] = merged_df['prc'].str.split('_', expand=True)

        try:
            os.makedirs(res_folder, exist_ok=True)
            data_path = os.path.join(res_folder, 'regression_data.csv')
            merged_df.to_csv(data_path, index=False)
            print(f"Saved regression data to {data_path}")

            cell_settings = {'src':data_path,
                            'graph_name':'cell_count',
                            'data_column':['cell_count'],
                            'grouping_column':'plateID',
                            'graph_type':'jitter_bar',
                            'theme':'bright',
                            'save':True,
                            'y_lim':[None,None],
                            'log_y':False,
                            'log_x':False,
                            'representation':'well',
                            'verbose':False}

            _, _ = plot_data_from_csv(settings=cell_settings)

            final_grna_df, prc_gene_count_df = grna_metricks(merged_df)

            if settings['outlier_detection']:
                outliers_grna = get_outlier_reference_values(final_grna_df,outlier_col='grna_well_count',return_col='grna')
                if len (outliers_grna) > 0:
                    merged_df = merged_df[~merged_df['grna'].isin(outliers_grna)]
                    final_grna_df, prc_gene_count_df = grna_metricks(merged_df)
                    merged_df.to_csv(data_path, index=False)
                    print(f"Saved regression data to {data_path}")

            grna_data_path = os.path.join(res_folder, 'grna_well.csv')
            final_grna_df.to_csv(grna_data_path, index=False)
            print(f"Saved grna per well data to {grna_data_path}")

            wells_per_gene_settings = {'src':grna_data_path,
                        'graph_name':'wells_per_gene',
                        'data_column':['grna_well_count'],
                        'grouping_column':'plateID',
                        'graph_type':'jitter_bar',
                        'theme':'bright',
                        'save':True,
                        'y_lim':[None,None],
                        'log_y':False,
                        'log_x':False,
                        'representation':'object',
                        'verbose':True}

            _, _ = plot_data_from_csv(settings=wells_per_gene_settings)

            grna_well_data_path = os.path.join(res_folder, 'well_grna.csv')
            prc_gene_count_df.to_csv(grna_well_data_path, index=False)
            print(f"Saved well per grna data to {grna_well_data_path}")

            grna_per_well_settings = {'src':grna_well_data_path,
                                    'graph_name':'gene_per_well',
                                    'data_column':['gene_count'],
                                    'grouping_column':'plateID',
                                    'graph_type':'jitter_bar',
                                    'theme':'bright',
                                    'save':True,
                                    'y_lim':[None,None],
                                    'log_y':False,
                                    'log_x':False,
                                    'representation':'well',
                                    'verbose':False}

            _, _ = plot_data_from_csv(settings=grna_per_well_settings)

        except Exception as e:
            print(e)

        _ = plot_plates(merged_df, variable=orig_dv, grouping='mean', min_max='allq', cmap='viridis', min_count=None, dst=res_folder)

        model, coef_df, regression_type = regression(merged_df, csv_path, dependent_variable, settings['regression_type'], settings['alpha'], settings['random_row_column_effects'], nc=settings['negative_control'], pc=settings['positive_control'], controls=settings['controls'], dst=res_folder, cov_type=settings['cov_type'])

        coef_df['grna'] = coef_df['feature'].apply(lambda x: re.search(r'grna\[(.*?)\]', x).group(1) if 'grna' in x else None)
        coef_df['gene'] = coef_df['feature'].apply(lambda x: re.search(r'gene\[(.*?)\]', x).group(1) if 'gene' in x else None)

        coef_df = coef_df.merge(n_grna, how='left', on='grna')
        coef_df = coef_df.merge(n_gene, how='left', on='gene')

        gene_coef_df = coef_df[coef_df['n_gene'] != None]
        grna_coef_df = coef_df[coef_df['n_grna'] != None]
        gene_coef_df = gene_coef_df.dropna(subset=['n_gene'])
        grna_coef_df = grna_coef_df.dropna(subset=['n_grna'])

        if settings['controls'] is not None:

            control_coef_df = grna_coef_df[grna_coef_df['grna'].isin(settings['controls'])]
            mean_coef = control_coef_df['coefficient'].mean()
            significant_c = control_coef_df[control_coef_df['p_value']<= 0.05]
            mean_coef_c = significant_c['coefficient'].mean()

            if settings['verbose']:
                print(mean_coef, mean_coef_c)

            if settings['threshold_method'] in ['var','variance']:
                coef_mes = control_coef_df['coefficient'].var()
            elif settings['threshold_method'] in ['std', 'standard_deveation']:
                coef_mes = control_coef_df['coefficient'].std()
            else:
                raise ValueError(f"Unsupported threshold method {settings['threshold_method']}. Supported methods: ['var','variance','std','standard_deveation']")

            reg_threshold = mean_coef + (settings['threshold_multiplier'] * coef_mes)

        coef_df.to_csv(results_path, index=False)
        gene_coef_df.to_csv(results_path_gene, index=False)
        grna_coef_df.to_csv(results_path_grna, index=False)

        if regression_type == 'lasso':
            significant = coef_df[coef_df['coefficient'] > 0]

        else:
            significant = coef_df[coef_df['p_value']<= 0.05]
            if settings['controls'] is not None:
                significant_high = significant[significant['coefficient'] >= reg_threshold]
                significant_low = significant[significant['coefficient'] <= reg_threshold]
                significant = pd.concat([significant_high, significant_low])
            significant.sort_values(by='coefficient', ascending=False, inplace=True)
            significant = significant[~significant['feature'].str.contains('row|column')]

        if regression_type in ['ols', 'beta']:
            if settings['verbose']:
                print(model.summary())
                save_summary_to_file(model, file_path=f'{res_folder}/mode_summary.csv')

        significant.to_csv(hits_path, index=False)
        significant_grna_filtered = significant[significant['n_grna'] > settings['min_n']]
        significant_gene_filtered = significant[significant['n_gene'] > settings['min_n']]
        significant_filtered = pd.concat([significant_grna_filtered, significant_gene_filtered])
        filtered_hit_path = os.path.join(os.path.dirname(hits_path), 'results_significant_filtered.csv')
        significant_filtered.to_csv(filtered_hit_path, index=False)

        if isinstance(settings['metadata_files'], str):
            settings['metadata_files'] = [settings['metadata_files']]

        for metadata_file in settings['metadata_files']:
            file = os.path.basename(metadata_file)
            filename, _ = os.path.splitext(file)
            _ = merge_regression_res_with_metadata(hits_path, metadata_file, name=filename)
            merged_df = merge_regression_res_with_metadata(results_path, metadata_file, name=filename)
            gene_merged_df = merge_regression_res_with_metadata(results_path_gene, metadata_file, name=filename)
            grna_merged_df = merge_regression_res_with_metadata(results_path_grna, metadata_file, name=filename)

        if settings['toxo']:
            data_path = merged_df
            data_path_gene = gene_merged_df
            data_path_grna = grna_merged_df
            base_dir = os.path.dirname(os.path.abspath(__file__))
            metadata_path = os.path.join(base_dir, 'resources', 'data', 'lopit.csv')

            if settings['volcano'] == 'all':
                print('all')
                gene_list = custom_volcano_plot(data_path, metadata_path, metadata_column='tagm_location', point_size=600, figsize=20, threshold=reg_threshold, save_path=volcano_path, x_lim=settings['x_lim'],y_lims=settings['y_lims'])
                display(gene_list)
            elif settings['volcano'] == 'gene':
                print('gene')
                gene_list = custom_volcano_plot(data_path_gene, metadata_path, metadata_column='tagm_location', point_size=600, figsize=20, threshold=reg_threshold, save_path=volcano_path, x_lim=settings['x_lim'],y_lims=settings['y_lims'])
                display(gene_list)
            elif settings['volcano'] == 'grna':
                print('grna')
                gene_list = custom_volcano_plot(data_path_grna, metadata_path, metadata_column='tagm_location', point_size=600, figsize=20, threshold=reg_threshold, save_path=volcano_path, x_lim=settings['x_lim'],y_lims=settings['y_lims'])
                display(gene_list)

            phenotype_plot = os.path.join(res_folder,'phenotype_plot.pdf')
            transcription_heatmap = os.path.join(res_folder,'transcription_heatmap.pdf')
            data_GT1 = pd.read_csv(settings['metadata_files'][1], low_memory=False)
            data_ME49 = pd.read_csv(settings['metadata_files'][0], low_memory=False)

            columns = ['sense - Tachyzoites', 'sense - Tissue cysts', 'sense - EES1', 'sense - EES2', 'sense - EES3', 'sense - EES4', 'sense - EES5']

            print('Plotting gene phenotypes and heatmaps')
            print(gene_list)

            plot_gene_phenotypes(data=data_GT1, gene_list=gene_list, save_path=phenotype_plot)
            plot_gene_heatmaps(data=data_ME49, gene_list=gene_list, columns=columns, x_column='Gene ID', normalize=True, save_path=transcription_heatmap)

            #if len(significant) > 2:
            #    metadata_path = os.path.join(base_dir, 'resources', 'data', 'toxoplasma_metadata.csv')
            #    go_term_enrichment_by_column(significant, metadata_path)

        print('Significant Genes')
        grnas = significant['grna'].unique().tolist()
        genes = significant['gene'].unique().tolist()
        print(f"Found p<0.05 coedfficients for {len(grnas)} gRNAs and {len(genes)} genes")
        display(significant)

        output = {'results':coef_df,
                  'significant':significant}

        return output

    def process_reads(csv_path, fraction_threshold, plate, filter_column=None, filter_value=None):

        from .utils import correct_metadata

        if isinstance(csv_path, pd.DataFrame):
            csv_df = csv_path
        else:
            # Read the CSV file into a DataFrame
            csv_df = pd.read_csv(csv_path)

        csv_df = correct_metadata(csv_df)

        if 'grna_name' in csv_df.columns:
            csv_df = csv_df.rename(columns={'grna_name': 'grna'})
        if 'plate_row' in csv_df.columns:
            csv_df[['plateID', 'rowID']] = csv_df['plate_row'].str.split('_', expand=True)

        if not 'plateID' in csv_df.columns:
            if not plate is None:
                csv_df['plateID'] = plate
            else:
                csv_df['plateID'] = 'plate1'

        if 'prcfo' in csv_df.columns:
            #csv_df = csv_df.loc[:, ~csv_df.columns.duplicated()].copy()
            csv_df[['plateID', 'rowID', 'columnID', 'fieldID', 'objectID']] = csv_df['prcfo'].str.split('_', expand=True)
            csv_df['prc'] = csv_df['plateID'].astype(str) + '_' + csv_df['rowID'].astype(str) + '_' + csv_df['columnID'].astype(str)

        if isinstance(filter_column, str):
            filter_column = [filter_column]

        if isinstance(filter_value, str):
            filter_value = [filter_value]

        if isinstance(filter_column, list):
            for filter_col in filter_column:
                for value in filter_value:
                    csv_df = csv_df[csv_df[filter_col] != value]

        # Ensure the necessary columns are present
        if not all(col in csv_df.columns for col in ['rowID','columnID','grna','count']):
            raise ValueError("The CSV file must contain 'grna', 'count', 'rowID', and 'columnID' columns.")

        # Create the prc column
        csv_df['prc'] = csv_df['plateID'] + '_' + csv_df['rowID'] + '_' + csv_df['columnID']

        # Group by prc and calculate the sum of counts
        grouped_df = csv_df.groupby('prc')['count'].sum().reset_index()
        grouped_df = grouped_df.rename(columns={'count': 'total_counts'})
        merged_df = pd.merge(csv_df, grouped_df, on='prc')
        merged_df['fraction'] = merged_df['count'] / merged_df['total_counts']

        # Filter rows with fraction under the threshold
        if fraction_threshold is not None:
            observations_before = len(merged_df)
            merged_df = merged_df[merged_df['fraction'] >= fraction_threshold]
            observations_after = len(merged_df)
            removed = observations_before - observations_after
            print(f'Removed {removed} observation below fraction threshold: {fraction_threshold}')

        merged_df = merged_df[['prc', 'grna', 'fraction']]

        if not all(col in merged_df.columns for col in ['grna', 'gene']):
            try:
                merged_df[['org', 'gene', 'grna']] = merged_df['grna'].str.split('_', expand=True)
                merged_df = merged_df.drop(columns=['org'])
                merged_df['grna'] = merged_df['gene'] + '_' + merged_df['grna']
            except:
                print('Error splitting grna into org, gene, grna.')

        return merged_df

    def apply_transformation(X, transform):
        if transform == 'log':
            transformer = FunctionTransformer(np.log1p, validate=True)
        elif transform == 'sqrt':
            transformer = FunctionTransformer(np.sqrt, validate=True)
        elif transform == 'square':
            transformer = FunctionTransformer(np.square, validate=True)
        else:
            transformer = None
        return transformer

    def check_normality(data, variable_name, verbose=False):
        """Check if the data is normally distributed using the Shapiro-Wilk test."""
        stat, p_value = shapiro(data)
        if verbose:
            print(f"Shapiro-Wilk Test for {variable_name}:\nStatistic: {stat}, P-value: {p_value}")
        if p_value > 0.05:
            if verbose:
                print(f"Normal distribution: The data for {variable_name} is normally distributed.")
            return True
        else:
            if verbose:
                print(f"Normal distribution: The data for {variable_name} is not normally distributed.")
            return False

    def clean_controls(df,values, column):
        if column in df.columns:
            if isinstance(values, list):
                for value in values:
                    df = df[~df[column].isin([value])]
                    print(f'Removed data from {value}')
        return df

    def process_scores(df, dependent_variable, plate, min_cell_count=25, agg_type='mean', transform=None, regression_type='ols'):
        from .utils import calculate_shortest_distance, correct_metadata
        df = df.reset_index(drop=True)
        if 'prcfo' in df.columns:
            df = df.loc[:, ~df.columns.duplicated()].copy()
            if not all(col in df.columns for col in ['plateID', 'rowID', 'columnID']):
                df[['plateID', 'rowID', 'columnID', 'fieldID', 'objectID']] = df['prcfo'].str.split('_', expand=True)
            if all(col in df.columns for col in ['plateID', 'rowID', 'columnID']):
                df['prc'] = df['plateID'].astype(str) + '_' + df['rowID'].astype(str) + '_' + df['columnID'].astype(str)
        else:

            df = correct_metadata(df)

            if not plate is None:
                df['plateID'] = plate

            df = df.loc[:, ~df.columns.duplicated()].copy()

            if all(col in df.columns for col in ['plateID', 'rowID', 'columnID']):
                df['prc'] = df['plateID'].astype(str) + '_' + df['rowID'].astype(str) + '_' + df['columnID'].astype(str)
            else:
                raise ValueError("The DataFrame must contain 'plateID', 'rowID', and 'columnID' columns.")

        df = df[['prc', dependent_variable]]
        # Group by prc and calculate the mean and count of the dependent_variable
        grouped = df.groupby('prc')[dependent_variable]

        if regression_type != 'poisson':

            print(f'Using agg_type: {agg_type}')

            if agg_type == 'median':
                dependent_df = grouped.median().reset_index()
            elif agg_type == 'mean':
                dependent_df = grouped.mean().reset_index()
            elif agg_type == 'quantile':
                dependent_df = grouped.quantile(0.75).reset_index()
            elif agg_type == None:
                dependent_df = df.reset_index()
                if 'prcfo' in dependent_df.columns:
                    dependent_df = dependent_df.drop(columns=['prcfo'])
            else:
                raise ValueError(f"Unsupported aggregation type {agg_type}")

        if regression_type == 'poisson':
            agg_type = 'count'
            print(f'Using agg_type: {agg_type} for poisson regression')
            dependent_df = grouped.sum().reset_index()

        # Calculate cell_count for all cases
        cell_count = grouped.size().reset_index(name='cell_count')

        if agg_type is None:
            dependent_df = pd.merge(dependent_df, cell_count, on='prc')
        else:
            dependent_df['cell_count'] = cell_count['cell_count']

        dependent_df = dependent_df[dependent_df['cell_count'] >= min_cell_count]

        is_normal = check_normality(dependent_df[dependent_variable], dependent_variable)

        if not transform is None:
            transformer = apply_transformation(dependent_df[dependent_variable], transform=transform)
            transformed_var = f'{transform}_{dependent_variable}'
            dependent_df[transformed_var] = transformer.fit_transform(dependent_df[[dependent_variable]])
            dependent_variable = transformed_var
            is_normal = check_normality(dependent_df[transformed_var], transformed_var)

        if not is_normal:
            print(f'{dependent_variable} is not normally distributed')
        else:
            print(f'{dependent_variable} is normally distributed')

        return dependent_df, dependent_variable

    def generate_ml_scores(settings):

        from .io import _read_and_merge_data, _read_db
        from .plot import plot_plates
        from .utils import get_ml_results_paths, add_column_to_database, calculate_shortest_distance
        from .settings import set_default_analyze_screen

        settings = set_default_analyze_screen(settings)

        srcs = settings['src']

        settings_df = pd.DataFrame(list(settings.items()), columns=['Key', 'Value'])
        display(settings_df)

        if isinstance(srcs, str):
            srcs = [srcs]

        df = pd.DataFrame()
        for idx, src in enumerate(srcs):

            if idx == 0:
                src1 = src

            db_loc = [src+'/measurements/measurements.db']
            tables = ['cell', 'nucleus', 'pathogen','cytoplasm']

            dft, _ = _read_and_merge_data(db_loc,
                                        tables,
                                        settings['verbose'],
                                        nuclei_limit=settings['nuclei_limit'],
                                        pathogen_limit=settings['pathogen_limit'])
            df = pd.concat([df, dft])

        try:
            df = calculate_shortest_distance(df, 'pathogen', 'nucleus')
        except Exception as e:
            print(e)

        if settings['annotation_column'] is not None:

            settings['location_column'] = settings['annotation_column']

            png_list_df = _read_db(db_loc[0], tables=['png_list'])[0]
            if not {'prcfo', settings['annotation_column']}.issubset(png_list_df.columns):
                raise ValueError("The 'png_list_df' DataFrame must contain 'prcfo' and 'test' columns.")
            annotated_df = png_list_df[['prcfo', settings['annotation_column']]].set_index('prcfo')
            df = annotated_df.merge(df, left_index=True, right_index=True)
            unique_values = df[settings['annotation_column']].dropna().unique()

            if len(unique_values) == 1:
                unannotated_rows = df[df[settings['annotation_column']].isna()].index
                existing_value = unique_values[0]
                next_value = existing_value + 1

                settings['positive_control'] = str(existing_value)
                settings['negative_control'] = str(next_value)

                existing_count = df[df[settings['annotation_column']] == existing_value].shape[0]
                num_to_select = min(existing_count, len(unannotated_rows))
                selected_rows = np.random.choice(unannotated_rows, size=num_to_select, replace=False)
                df.loc[selected_rows, settings['annotation_column']] = next_value

                # Print the counts for existing_value and next_value
                existing_count_final = df[df[settings['annotation_column']] == existing_value].shape[0]
                next_count_final = df[df[settings['annotation_column']] == next_value].shape[0]

                print(f"Number of rows with value {existing_value}: {existing_count_final}")
                print(f"Number of rows with value {next_value}: {next_count_final}")
                df[settings['annotation_column']] = df[settings['annotation_column']].apply(str)

        if settings['channel_of_interest'] in [0,1,2,3]:
            if f"pathogen_channel_{settings['channel_of_interest']}_mean_intensity" and f"cytoplasm_channel_{settings['channel_of_interest']}_mean_intensity" in df.columns:
                df['recruitment'] = df[f"pathogen_channel_{settings['channel_of_interest']}_mean_intensity"]/df[f"cytoplasm_channel_{settings['channel_of_interest']}_mean_intensity"]

        output, figs = ml_analysis(df,
                                   settings['channel_of_interest'],
                                   settings['location_column'],
                                   settings['positive_control'],
                                   settings['negative_control'],
                                   settings['exclude'],
                                   settings['n_repeats'],
                                   settings['top_features'],
                                   settings['reg_alpha'],
                                   settings['reg_lambda'],
                                   settings['learning_rate'],
                                   settings['n_estimators'],
                                   settings['test_size'],
                                   settings['model_type_ml'],
                                   settings['n_jobs'],
                                   settings['remove_low_variance_features'],
                                   settings['remove_highly_correlated_features'],
                                   settings['prune_features'],
                                   settings['cross_validation'],
                                   settings['verbose'])

        shap_fig = shap_analysis(output[3], output[4], output[5])

        features = output[0].select_dtypes(include=[np.number]).columns.tolist()
        train_features_df = pd.DataFrame(output[9], columns=['feature'])

        if not settings['heatmap_feature'] in features:
            raise ValueError(f"Variable {settings['heatmap_feature']} not found in the dataframe. Please choose one of the following: {features}")

        plate_heatmap = plot_plates(df=output[0],
                                    variable=settings['heatmap_feature'],
                                    grouping=settings['grouping'],
                                    min_max=settings['min_max'],
                                    cmap=settings['cmap'],
                                    min_count=settings['minimum_cell_count'],
                                    verbose=settings['verbose'])

        data_path, permutation_path, feature_importance_path, model_metricks_path, permutation_fig_path, feature_importance_fig_path, shap_fig_path, plate_heatmap_path, settings_csv, ml_features = get_ml_results_paths(src1, settings['model_type_ml'], settings['channel_of_interest'])
        df, permutation_df, feature_importance_df, _, _, _, _, _, metrics_df, _ = output

        settings_df.to_csv(settings_csv, index=False)
        df.to_csv(data_path, mode='w', encoding='utf-8')
        permutation_df.to_csv(permutation_path, mode='w', encoding='utf-8')
        feature_importance_df.to_csv(feature_importance_path, mode='w', encoding='utf-8')
        train_features_df.to_csv(ml_features, mode='w', encoding='utf-8')
        metrics_df.to_csv(model_metricks_path, mode='w', encoding='utf-8')

        plate_heatmap.savefig(plate_heatmap_path, format='pdf')
        figs[0].savefig(permutation_fig_path, format='pdf')
        figs[1].savefig(feature_importance_fig_path, format='pdf')
        shap_fig.savefig(shap_fig_path, format='pdf')

        if settings['save_to_db']:
            settings['csv_path'] = data_path
            settings['db_path'] = os.path.join(src1, 'measurements', 'measurements.db')
            settings['table_name'] = 'png_list'
            settings['update_column'] = 'predictions'
            settings['match_column'] = 'prcfo'
            add_column_to_database(settings)

        return [output, plate_heatmap]

    def ml_analysis(df, channel_of_interest=3, location_column='columnID', positive_control='c2', negative_control='c1', exclude=None, n_repeats=10, top_features=30, reg_alpha=0.1, reg_lambda=1.0, learning_rate=0.00001, n_estimators=1000, test_size=0.2, model_type='xgboost', n_jobs=-1, remove_low_variance_features=True, remove_highly_correlated_features=True, prune_features=False, cross_validation=False, verbose=False):

        """
        Calculates permutation importance for numerical features in the dataframe,
        comparing groups based on specified column values and uses the model to predict
        the class for all other rows in the dataframe.

        Args:
        df (pandas.DataFrame): The DataFrame containing the data.
        feature_string (str): String to filter features that contain this substring.
        location_column (str): Column name to use for comparing groups.
        positive_control, negative_control (str): Values in location_column to create subsets for comparison.
        exclude (list or str, optional): Columns to exclude from features.
        n_repeats (int): Number of repeats for permutation importance.
        top_features (int): Number of top features to plot based on permutation importance.
        n_estimators (int): Number of trees in the random forest, gradient boosting, or XGBoost model.
        test_size (float): Proportion of the dataset to include in the test split.
        random_state (int): Random seed for reproducibility.
        model_type (str): Type of model to use ('random_forest', 'logistic_regression', 'gradient_boosting', 'xgboost').
        n_jobs (int): Number of jobs to run in parallel for applicable models.

        Returns:
        pandas.DataFrame: The original dataframe with added prediction and data usage columns.
        pandas.DataFrame: DataFrame containing the importances and standard deviations.
        """

        from .utils import filter_dataframe_features
        from .plot import plot_permutation, plot_feature_importance

        random_state = 42

        if 'cells_per_well' in df.columns:
            df = df.drop(columns=['cells_per_well'])

        df_metadata = df[[location_column]].copy()

        df, features = filter_dataframe_features(df, channel_of_interest, exclude, remove_low_variance_features, remove_highly_correlated_features, verbose)
        print('After filtration:', len(df))

        if verbose:
            print(f'Found {len(features)} numerical features in the dataframe')
            print(f'Features used in training: {features}')
            print(f'Features: {features}')

        df = pd.concat([df, df_metadata[location_column]], axis=1)

        # Subset the dataframe based on specified column values
        if isinstance(negative_control, str):
            df1 = df[df[location_column] == negative_control].copy()
        elif isinstance(negative_control, list):
            df1 = df[df[location_column].isin(negative_control)].copy()

        if isinstance(positive_control, str):
            df2 = df[df[location_column] == positive_control].copy()
        elif isinstance(positive_control, list):
            df2 = df[df[location_column].isin(positive_control)].copy()

        # Create target variable
        df1['target'] = 0 # Negative control
        df2['target'] = 1 # Positive control

        # Combine the subsets for analysis
        combined_df = pd.concat([df1, df2])
        combined_df = combined_df.drop(columns=[location_column])

        if verbose:
            print(f'Found {len(df1)} samples for {negative_control} and {len(df2)} samples for {positive_control}. Total: {len(combined_df)}')

        X = combined_df[features]
        y = combined_df['target']

        if prune_features:
            before_pruning = len(X.columns)
            selector = SelectKBest(score_func=f_classif, k=top_features)
            X_selected = selector.fit_transform(X, y)

            # Get the selected feature names
            selected_features = X.columns[selector.get_support()]
            X = pd.DataFrame(X_selected, columns=selected_features, index=X.index)

            features = selected_features.tolist()

            after_pruning = len(X.columns)
            print(f"Removed {before_pruning - after_pruning} features using SelectKBest")

        # Split the data into training and testing sets
        X_train, X_test, y_train, y_test = train_test_split(X, y, test_size=test_size, random_state=random_state)

        # Add data usage labels
        combined_df['data_usage'] = 'train'
        combined_df.loc[X_test.index, 'data_usage'] = 'test'
        df['data_usage'] = 'not_used'
        df.loc[combined_df.index, 'data_usage'] = combined_df['data_usage']

        # Initialize the model based on model_type
        if model_type == 'random_forest':
            model = RandomForestClassifier(n_estimators=n_estimators, random_state=random_state, n_jobs=n_jobs)
        elif model_type == 'logistic_regression':
            model = LogisticRegression(max_iter=1000, random_state=random_state, n_jobs=n_jobs)
        elif model_type == 'gradient_boosting':
            model = HistGradientBoostingClassifier(max_iter=n_estimators, random_state=random_state)  # Supports n_jobs internally
        elif model_type == 'xgboost':
            model = XGBClassifier(reg_alpha=reg_alpha, reg_lambda=reg_lambda, learning_rate=learning_rate, n_estimators=n_estimators, random_state=random_state, nthread=n_jobs, use_label_encoder=False, eval_metric='logloss')

        else:
            raise ValueError(f"Unsupported model_type: {model_type}")

        # Perform k-fold cross-validation
        if cross_validation:

            # Cross-validation setup
            kfold = StratifiedKFold(n_splits=5, shuffle=True, random_state=random_state)
            fold_metrics = []

            for fold_idx, (train_index, test_index) in enumerate(kfold.split(X, y), start=1):
                X_train, X_test = X.iloc[train_index], X.iloc[test_index]
                y_train, y_test = y.iloc[train_index], y.iloc[test_index]

                # Train the model
                model.fit(X_train, y_train)

                # Predict for the current test set
                predictions_test = model.predict(X_test)
                combined_df.loc[X_test.index, 'predictions'] = predictions_test

                # Get prediction probabilities for the test set
                prediction_probabilities_test = model.predict_proba(X_test)

                # Find the optimal threshold
                optimal_threshold = find_optimal_threshold(y_test, prediction_probabilities_test[:, 1])
                if verbose:
                    print(f'Fold {fold_idx} - Optimal threshold: {optimal_threshold}')

                # Assign predictions and probabilities to the test set in the DataFrame
                df.loc[X_test.index, 'predictions'] = predictions_test
                for i in range(prediction_probabilities_test.shape[1]):
                    df.loc[X_test.index, f'prediction_probability_class_{i}'] = prediction_probabilities_test[:, i]

                # Evaluate performance for the current fold
                fold_report = classification_report(y_test, predictions_test, output_dict=True)
                fold_metrics.append(pd.DataFrame(fold_report).transpose())

                if verbose:
                    print(f"Fold {fold_idx} Classification Report:")
                    print(classification_report(y_test, predictions_test))

            # Aggregate metrics across all folds
            metrics_df = pd.concat(fold_metrics).groupby(level=0).mean()

            # Re-train on full data (X, y) and then apply to entire df
            model.fit(X, y)
            all_predictions = model.predict(df[features])  # Predict on entire df
            df['predictions'] = all_predictions

            # Get prediction probabilities for all rows in df
            prediction_probabilities = model.predict_proba(df[features])
            for i in range(prediction_probabilities.shape[1]):
                df[f'prediction_probability_class_{i}'] = prediction_probabilities[:, i]

            #if verbose:
            #    print("\nFinal Classification Report on Full Dataset:")
            #    print(classification_report(y, all_predictions))

            # Generate metrics DataFrame
            #final_report_dict = classification_report(y, all_predictions, output_dict=True)
            #metrics_df = pd.DataFrame(final_report_dict).transpose()

        else:
            model.fit(X_train, y_train)
            # Predicting the target variable for the test set
            predictions_test = model.predict(X_test)
            combined_df.loc[X_test.index, 'predictions'] = predictions_test

            # Get prediction probabilities for the test set
            prediction_probabilities_test = model.predict_proba(X_test)

            # Find the optimal threshold
            optimal_threshold = find_optimal_threshold(y_test, prediction_probabilities_test[:, 1])
            if verbose:
                print(f'Optimal threshold: {optimal_threshold}')

            # Predicting the target variable for all other rows in the dataframe
            X_all = df[features]
            all_predictions = model.predict(X_all)
            df['predictions'] = all_predictions

            # Get prediction probabilities for all rows in the dataframe
            prediction_probabilities = model.predict_proba(X_all)
            for i in range(prediction_probabilities.shape[1]):
                df[f'prediction_probability_class_{i}'] = prediction_probabilities[:, i]

            if verbose:
                print("\nClassification Report:")
                print(classification_report(y_test, predictions_test))

            report_dict = classification_report(y_test, predictions_test, output_dict=True)
            metrics_df = pd.DataFrame(report_dict).transpose()

        perm_importance = permutation_importance(model, X_train, y_train, n_repeats=n_repeats, random_state=random_state, n_jobs=n_jobs)

        # Create a DataFrame for permutation importances
        permutation_df = pd.DataFrame({
            'feature': [features[i] for i in perm_importance.importances_mean.argsort()],
            'importance_mean': perm_importance.importances_mean[perm_importance.importances_mean.argsort()],
            'importance_std': perm_importance.importances_std[perm_importance.importances_mean.argsort()]
        }).tail(top_features)

        permutation_fig = plot_permutation(permutation_df)
        if verbose:
            permutation_fig.show()

        # Feature importance for models that support it
        if model_type in ['random_forest', 'xgboost', 'gradient_boosting']:
            feature_importances = model.feature_importances_
            feature_importance_df = pd.DataFrame({
                'feature': features,
                'importance': feature_importances
            }).sort_values(by='importance', ascending=False).head(top_features)

            feature_importance_fig = plot_feature_importance(feature_importance_df)
            if verbose:
                feature_importance_fig.show()

        else:
            feature_importance_df = pd.DataFrame()

        df = _calculate_similarity(df, features, location_column, positive_control, negative_control)

        df['prcfo'] = df.index.astype(str)
        df[['plateID', 'rowID', 'columnID', 'fieldID', 'object']] = df['prcfo'].str.split('_', expand=True)
        df['prc'] = df['plateID'] + '_' + df['rowID'] + '_' + df['columnID']

        return [df, permutation_df, feature_importance_df, model, X_train, X_test, y_train, y_test, metrics_df, features], [permutation_fig, feature_importance_fig]

    def shap_analysis(model, X_train, X_test):

        """
        Performs SHAP analysis on the given model and data.

        Args:
        model: The trained model.
        X_train (pandas.DataFrame): Training feature set.
        X_test (pandas.DataFrame): Testing feature set.
        Returns:
        fig: Matplotlib figure object containing the SHAP summary plot.
        """

        explainer = shap.Explainer(model, X_train)
        shap_values = explainer(X_test)
        # Create a new figure
        fig, ax = plt.subplots()
        # Summary plot
        shap.summary_plot(shap_values, X_test, show=False)
        # Save the current figure (the one that SHAP just created)
        fig = plt.gcf()
        plt.close(fig)  # Close the figure to prevent it from displaying immediately
        return fig

    def find_optimal_threshold(y_true, y_pred_proba):
        """
        Find the optimal threshold for binary classification based on the F1-score.

        Args:
        y_true (array-like): True binary labels.
        y_pred_proba (array-like): Predicted probabilities for the positive class.

        Returns:
        float: The optimal threshold.
        """
        precision, recall, thresholds = precision_recall_curve(y_true, y_pred_proba)
        f1_scores = 2 * (precision * recall) / (precision + recall)
        optimal_idx = np.argmax(f1_scores)
        optimal_threshold = thresholds[optimal_idx]
        return optimal_threshold

    def _calculate_similarity(df, features, col_to_compare, val1, val2):
        """
        Calculate similarity scores of each well to the positive and negative controls using various metrics.

        Args:
        df (pandas.DataFrame): DataFrame containing the data.
        features (list): List of feature columns to use for similarity calculation.
        col_to_compare (str): Column name to use for comparing groups.
        val1, val2 (str): Values in col_to_compare to create subsets for comparison.

        Returns:
        pandas.DataFrame: DataFrame with similarity scores.
        """
        # Separate positive and negative control wells
        if isinstance(val1, str):
            pos_control = df[df[col_to_compare] == val1][features].mean()
        elif isinstance(val1, list):
            pos_control = df[df[col_to_compare].isin(val1)][features].mean()
        if isinstance(val2, str):
            neg_control = df[df[col_to_compare] == val2][features].mean()
        elif isinstance(val2, list):
            neg_control = df[df[col_to_compare].isin(val2)][features].mean()

        # Standardize features for Mahalanobis distance
        scaler = StandardScaler()
        scaled_features = scaler.fit_transform(df[features])

        # Regularize the covariance matrix to avoid singularity
        cov_matrix = np.cov(scaled_features, rowvar=False)
        inv_cov_matrix = None
        try:
            inv_cov_matrix = np.linalg.inv(cov_matrix)
        except np.linalg.LinAlgError:
            # Add a small value to the diagonal elements for regularization
            epsilon = 1e-5
            inv_cov_matrix = np.linalg.inv(cov_matrix + np.eye(cov_matrix.shape[0]) * epsilon)

        # Calculate similarity scores
        def safe_similarity(func, row, control, *args, **kwargs):
            try:
                return func(row, control, *args, **kwargs)
            except Exception:
                return np.nan

        # Calculate similarity scores
        try:
            df['similarity_to_pos_euclidean'] = df[features].apply(lambda row: safe_similarity(euclidean, row, pos_control), axis=1)
            df['similarity_to_neg_euclidean'] = df[features].apply(lambda row: safe_similarity(euclidean, row, neg_control), axis=1)
            df['similarity_to_pos_cosine'] = df[features].apply(lambda row: safe_similarity(cosine, row, pos_control), axis=1)
            df['similarity_to_neg_cosine'] = df[features].apply(lambda row: safe_similarity(cosine, row, neg_control), axis=1)
            df['similarity_to_pos_mahalanobis'] = df[features].apply(lambda row: safe_similarity(mahalanobis, row, pos_control, inv_cov_matrix), axis=1)
            df['similarity_to_neg_mahalanobis'] = df[features].apply(lambda row: safe_similarity(mahalanobis, row, neg_control, inv_cov_matrix), axis=1)
            df['similarity_to_pos_manhattan'] = df[features].apply(lambda row: safe_similarity(cityblock, row, pos_control), axis=1)
            df['similarity_to_neg_manhattan'] = df[features].apply(lambda row: safe_similarity(cityblock, row, neg_control), axis=1)
            df['similarity_to_pos_minkowski'] = df[features].apply(lambda row: safe_similarity(minkowski, row, pos_control, p=3), axis=1)
            df['similarity_to_neg_minkowski'] = df[features].apply(lambda row: safe_similarity(minkowski, row, neg_control, p=3), axis=1)
            df['similarity_to_pos_chebyshev'] = df[features].apply(lambda row: safe_similarity(chebyshev, row, pos_control), axis=1)
            df['similarity_to_neg_chebyshev'] = df[features].apply(lambda row: safe_similarity(chebyshev, row, neg_control), axis=1)
            df['similarity_to_pos_braycurtis'] = df[features].apply(lambda row: safe_similarity(braycurtis, row, pos_control), axis=1)
            df['similarity_to_neg_braycurtis'] = df[features].apply(lambda row: safe_similarity(braycurtis, row, neg_control), axis=1)
        except Exception as e:
            print(f"Error calculating similarity scores: {e}")
        return df

    def interperate_vision_model(settings={}):

        from .io import _read_and_merge_data, _results_to_csv
        from .settings import set_interperate_vision_model_defaults
        from .utils import save_settings

        settings = set_interperate_vision_model_defaults(settings)
        save_settings(settings, name='interperate_vision_model', show=True)

        # Function to create radar plot for individual and combined values
        def create_extended_radar_plot(values, labels, title):
            values = list(values) + [values[0]]  # Close the loop for radar chart
            angles = [n / float(len(labels)) * 2 * pi for n in range(len(labels))]
            angles += angles[:1]

            fig, ax = plt.subplots(figsize=(8, 8), subplot_kw=dict(polar=True))
            ax.plot(angles, values, linewidth=2, linestyle='solid')
            ax.fill(angles, values, alpha=0.25)

            ax.set_xticks(angles[:-1])
            ax.set_xticklabels(labels, fontsize=10, rotation=45, ha='right')
            plt.title(title, pad=20)
            plt.show()

        def extract_compartment_channel(feature_name):
            # Identify compartment as the first part before an underscore
            compartment = feature_name.split('_')[0]

            if compartment == 'cells':
                compartment = 'cell'

            # Identify channels based on substring presence
            channels = []
            if 'channel_0' in feature_name:
                channels.append('channel_0')
            if 'channel_1' in feature_name:
                channels.append('channel_1')
            if 'channel_2' in feature_name:
                channels.append('channel_2')
            if 'channel_3' in feature_name:
                channels.append('channel_3')

            # If multiple channels are found, join them with a '+'
            if channels:
                channel = ' + '.join(channels)
            else:
                channel = 'morphology'  # Use 'morphology' if no channel identifier is found

            return (compartment, channel)

        def read_and_preprocess_data(settings):

            df, _ = _read_and_merge_data(
                locs=[settings['src']+'/measurements/measurements.db'],
                tables=settings['tables'],
                verbose=True,
                nuclei_limit=settings['nuclei_limit'],
                pathogen_limit=settings['pathogen_limit']
            )

            scores_df = pd.read_csv(settings['scores'])

            # Clean and align columns for merging
            df['object_label'] = df['object_label'].str.replace('o', '')

            if 'rowID' not in scores_df.columns:
                if 'row' in scores_df.columns:
                    scores_df['rowID'] = scores_df['row']
                if 'row_name' in scores_df.columns:
                    scores_df['rowID'] = scores_df['row_name']

            if 'columnID' not in scores_df.columns:
                if 'col' in scores_df.columns:
                    scores_df['columnID'] = scores_df['col']
                if 'column' in scores_df.columns:
                    scores_df['columnID'] = scores_df['column']

            if 'object_label' not in scores_df.columns:
                scores_df['object_label'] = scores_df['object']

            # Remove the 'o' prefix from 'object_label' in df, ensuring it is a string type
            df['object_label'] = df['object_label'].str.replace('o', '').astype(str)

            # Ensure 'object_label' in scores_df is also a string
            scores_df['object_label'] = scores_df['object'].astype(str)

            # Ensure all join columns have the same data type in both DataFrames
            df[['plateID', 'rowID', 'columnID', 'fieldID', 'object_label']] = df[['plateID', 'rowID', 'columnID', 'fieldID', 'object_label']].astype(str)
            scores_df[['plateID', 'rowID', 'columnID', 'fieldID', 'object_label']] = scores_df[['plateID', 'rowID', 'columnID', 'fieldID', 'object_label']].astype(str)

            # Select only the necessary columns from scores_df for merging
            scores_df = scores_df[['plateID', 'rowID', 'columnID', 'fieldID', 'object_label', settings['score_column']]]

            # Now merge DataFrames
            merged_df = pd.merge(df, scores_df, on=['plateID', 'rowID', 'columnID', 'fieldID', 'object_label'], how='inner')

            # Separate numerical features and the score column
            X = merged_df.select_dtypes(include='number').drop(columns=[settings['score_column']])
            y = merged_df[settings['score_column']]

            return X, y, merged_df

        X, y, merged_df = read_and_preprocess_data(settings)

        # Step 1: Feature Importance using Random Forest
        if settings['feature_importance'] or settings['feature_importance']:
            model = RandomForestClassifier(random_state=42, n_jobs=settings['n_jobs'])
            model.fit(X, y)

            if settings['feature_importance']:
                print(f"Feature Importance ...")
                feature_importances = model.feature_importances_
                feature_importance_df = pd.DataFrame({'feature': X.columns, 'importance': feature_importances})
                feature_importance_df = feature_importance_df.sort_values(by='importance', ascending=False)
                top_feature_importance_df = feature_importance_df.head(settings['top_features'])

                # Plot Feature Importance
                plt.figure(figsize=(10, 6))
                plt.barh(top_feature_importance_df['feature'], top_feature_importance_df['importance'])
                plt.xlabel('Importance')
                plt.title(f"Top {settings['top_features']} Features - Feature Importance")
                plt.gca().invert_yaxis()
                plt.show()

            if settings['save']:
                _results_to_csv(feature_importance_df, filename='feature_importance.csv')

        # Step 2: Permutation Importance
        if settings['permutation_importance']:
            print(f"Permutation Importance ...")
            perm_importance = permutation_importance(model, X, y, n_repeats=10, random_state=42, n_jobs=settings['n_jobs'])
            perm_importance_df = pd.DataFrame({'feature': X.columns, 'importance': perm_importance.importances_mean})
            perm_importance_df = perm_importance_df.sort_values(by='importance', ascending=False)
            top_perm_importance_df = perm_importance_df.head(settings['top_features'])

            # Plot Permutation Importance
            plt.figure(figsize=(10, 6))
            plt.barh(top_perm_importance_df['feature'], top_perm_importance_df['importance'])
            plt.xlabel('Importance')
            plt.title(f"Top {settings['top_features']} Features - Permutation Importance")
            plt.gca().invert_yaxis()
            plt.show()

            if settings['save']:
                _results_to_csv(perm_importance_df, filename='permutation_importance.csv')

        # Step 3: SHAP Analysis
        if settings['shap']:
            print(f"SHAP Analysis ...")

            # Select top N features based on Random Forest importance and fit the model on these features only
            top_features = feature_importance_df.head(settings['top_features'])['feature']
            X_top = X[top_features]

            # Refit the model on this subset of features
            model = RandomForestClassifier(random_state=42, n_jobs=settings['n_jobs'])
            model.fit(X_top, y)

            # Sample a smaller subset of rows to speed up SHAP
            if settings['shap_sample']:
                sample = int(len(X_top) / 100)
                X_sample = X_top.sample(min(sample, len(X_top)), random_state=42)
            else:
                X_sample = X_top

            # Initialize SHAP explainer with the same subset of features
            explainer = shap.Explainer(model.predict, X_sample)
            shap_values = explainer(X_sample, max_evals=1500)

            # Plot SHAP summary for the selected sample and top features
            shap.summary_plot(shap_values, X_sample, max_display=settings['top_features'])

            # Convert SHAP values to a DataFrame for easier manipulation
            shap_df = pd.DataFrame(shap_values.values, columns=X_sample.columns)

            # Apply the function to create MultiIndex columns with compartment and channel
            shap_df.columns = pd.MultiIndex.from_tuples(
                [extract_compartment_channel(feat) for feat in shap_df.columns],
                names=['compartment', 'channel']
            )

            # Aggregate SHAP values by compartment and channel
            compartment_mean = shap_df.abs().groupby(level='compartment', axis=1).mean().mean(axis=0)
            channel_mean = shap_df.abs().groupby(level='channel', axis=1).mean().mean(axis=0)

            # Calculate combined importance for each pair of compartments and channels
            combined_compartment = {}
            for i, comp1 in enumerate(compartment_mean.index):
                for comp2 in compartment_mean.index[i+1:]:
                    combined_compartment[f"{comp1} + {comp2}"] = shap_df.loc[:, (comp1, slice(None))].abs().mean().mean() + \
                                                                  shap_df.loc[:, (comp2, slice(None))].abs().mean().mean()

            combined_channel = {}
            for i, chan1 in enumerate(channel_mean.index):
                for chan2 in channel_mean.index[i+1:]:
                    combined_channel[f"{chan1} + {chan2}"] = shap_df.loc[:, (slice(None), chan1)].abs().mean().mean() + \
                                                              shap_df.loc[:, (slice(None), chan2)].abs().mean().mean()

            # Prepare values and labels for radar charts
            all_compartment_importance = list(compartment_mean.values) + list(combined_compartment.values())
            all_compartment_labels = list(compartment_mean.index) + list(combined_compartment.keys())

            all_channel_importance = list(channel_mean.values) + list(combined_channel.values())
            all_channel_labels = list(channel_mean.index) + list(combined_channel.keys())

            # Create radar plots for compartments and channels
            create_extended_radar_plot(all_compartment_importance, all_compartment_labels, "SHAP Importance by Compartment (Individual and Combined)")
            create_extended_radar_plot(all_channel_importance, all_channel_labels, "SHAP Importance by Channel (Individual and Combined)")

        return merged_df