Bio Workflows Biomarker Pipeline

Expression matrix + Metadata
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[1. Data Preparation] -----> StandardScaler, train/test split
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[2. Feature Selection] ----> Boruta or LASSO stability selection
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[3. Model Training] -------> Pipeline with selection inside CV (leakage-safe)
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[4. Model Interpretation] -> SHAP values, feature importance
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[5. Validation] -----------> Hold-out test, bootstrap CI
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Validated biomarker panel + classifier

import pandas as pd
from sklearn.model_selection import StratifiedGroupKFold
from sklearn.preprocessing import StandardScaler

expr = pd.read_csv('expression.csv', index_col=0)
meta = pd.read_csv('metadata.csv', index_col=0)

X = expr.T  # samples x genes
# y must be 0/1: brier_score_loss and calibration_curve raise on string labels, and sklearn orders
# classes alphabetically -- for a case/control column that makes 'control' the positive class, so
# predict_proba[:, 1], the SHAP [:, :, 1] slice, and Brier all silently describe the wrong class.
# AUC is symmetric and will not expose the flip. Encode the disease class as 1 explicitly.
POSITIVE_CLASS = 'disease'
y = (meta.loc[X.index, 'condition'].values == POSITIVE_CLASS).astype(int)
# The critical key: the SUBJECT (patient/donor/site), not the sample. If truly one
# sample per subject, groups = X.index; otherwise it MUST be the subject id.
groups = meta.loc[X.index, 'subject_id'].values

# Group- AND class-aware hold-out: take one StratifiedGroupKFold fold as the test set so no
# subject spans train/test (train_test_split(stratify=y) alone would leak repeated subjects).
sgkf = StratifiedGroupKFold(n_splits=5, shuffle=True, random_state=42)   # 1/5 held out (~0.2)
train_idx, test_idx = next(sgkf.split(X, y, groups))
X_train, X_test = X.iloc[train_idx], X.iloc[test_idx]
y_train, y_test = y[train_idx], y[test_idx]
groups_train = groups[train_idx]

# Fit scaler on training only to prevent data leakage
scaler = StandardScaler()
X_train_scaled = scaler.fit_transform(X_train)
X_test_scaled = scaler.transform(X_test)

import numpy as np
from boruta import BorutaPy
from sklearn.ensemble import RandomForestClassifier
from sklearn.feature_selection import SelectKBest, f_classif

# Pre-filter if >10k features. selected_idx is a positional boolean mask aligned to X_train.columns.
if X_train_scaled.shape[1] > 10000:
    selector = SelectKBest(f_classif, k=5000)
    selector.fit(X_train_scaled, y_train)
    prefilter_idx = np.where(selector.get_support())[0]
    X_train_filt = X_train_scaled[:, prefilter_idx]
else:
    prefilter_idx = None
    X_train_filt = X_train_scaled

# max_depth=5: Shallow trees for stable importances
rf = RandomForestClassifier(n_estimators=100, max_depth=5, n_jobs=-1, random_state=42)
# max_iter=100: Usually sufficient; 200 if many tentative
boruta = BorutaPy(rf, n_estimators='auto', max_iter=100, random_state=42, verbose=0)
boruta.fit(X_train_filt, y_train)

# Map the (possibly pre-filtered) Boruta mask back onto the FULL feature space.
selected_idx = np.zeros(X_train.shape[1], dtype=bool)
selected_idx[prefilter_idx[boruta.support_] if prefilter_idx is not None else boruta.support_] = True
print(f'Selected {selected_idx.sum()} features')

from sklearn.linear_model import LogisticRegressionCV
import numpy as np

# n_bootstrap=100: Quick; use 500 for publication
n_bootstrap = 100
stability_scores = np.zeros(X_train_scaled.shape[1])

for i in range(n_bootstrap):
    idx = np.random.choice(len(y_train), size=len(y_train), replace=True)
    # Cs=10: 10 regularization values to search
    model = LogisticRegressionCV(penalty='l1', solver='saga', Cs=10, cv=3, random_state=i, max_iter=1000)
    model.fit(X_train_scaled[idx], y_train[idx])
    stability_scores += (model.coef_[0] != 0).astype(int)

stability_scores /= n_bootstrap
# stability_threshold=0.6: Standard; 0.8 for strict
selected_idx = stability_scores > 0.6
print(f'Selected {selected_idx.sum()} features (stability >0.6)')

from sklearn.model_selection import StratifiedGroupKFold, cross_val_score
from sklearn.pipeline import Pipeline
from sklearn.preprocessing import StandardScaler
from sklearn.feature_selection import SelectKBest, f_classif
from sklearn.linear_model import LogisticRegression

# Selection lives INSIDE the pipeline -> re-fit per fold, no leakage. Use the unscaled X_train.
pipe = Pipeline([
    ('scaler', StandardScaler()),
    ('select', SelectKBest(f_classif, k=min(50, X_train.shape[1]))),
    ('clf', LogisticRegression(max_iter=5000, class_weight='balanced')),
])
# Group-aware outer CV: pass groups_train so no subject spans a fold boundary.
outer_cv = StratifiedGroupKFold(n_splits=5, shuffle=True, random_state=42)
cv_scores = cross_val_score(pipe, X_train, y_train, groups=groups_train, cv=outer_cv, scoring='roc_auc')
print(f'Leakage-safe CV AUC: {cv_scores.mean():.3f} +/- {cv_scores.std():.3f}')
# If hyperparameters are tuned, wrap a GridSearchCV (inner group CV) as the pipeline's estimator
# and report the OUTER cross_val_score -- flat CV that both tunes and reports is optimistic (Varma & Simon 2006).

import shap
import numpy as np
from sklearn.ensemble import RandomForestClassifier

# Fit the FINAL model on the discovery panel for interpretation and deployment.
sel = X_train.columns[selected_idx]
clf = RandomForestClassifier(n_estimators=300, random_state=42, n_jobs=-1).fit(X_train[sel], y_train)

# Interventional SHAP ('what the model uses') needs a background; set feature_perturbation
# explicitly because the 0.47+ 'auto' default flips the estimand on whether data= is given.
background = shap.utils.sample(X_train[sel], 100)
explainer = shap.TreeExplainer(clf, data=background, feature_perturbation='interventional')
shap_values = explainer(X_test[sel])
# RF returns one output per class in shap 0.47+ (n_samples, n_features, n_classes); keep the positive class.
if shap_values.values.ndim == 3:
    shap_values = shap_values[:, :, 1]
mean_shap = np.abs(shap_values.values).mean(axis=0)