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{
 "cells": [
  {
   "cell_type": "code",
   "execution_count": 1,
   "id": "21ad5882",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:04.700950Z",
     "iopub.status.busy": "2025-03-25T05:25:04.700846Z",
     "iopub.status.idle": "2025-03-25T05:25:04.861322Z",
     "shell.execute_reply": "2025-03-25T05:25:04.860952Z"
    }
   },
   "outputs": [],
   "source": [
    "import sys\n",
    "import os\n",
    "sys.path.append(os.path.abspath(os.path.join(os.getcwd(), '../..')))\n",
    "\n",
    "# Path Configuration\n",
    "from tools.preprocess import *\n",
    "\n",
    "# Processing context\n",
    "trait = \"Glucocorticoid_Sensitivity\"\n",
    "cohort = \"GSE33649\"\n",
    "\n",
    "# Input paths\n",
    "in_trait_dir = \"../../input/GEO/Glucocorticoid_Sensitivity\"\n",
    "in_cohort_dir = \"../../input/GEO/Glucocorticoid_Sensitivity/GSE33649\"\n",
    "\n",
    "# Output paths\n",
    "out_data_file = \"../../output/preprocess/Glucocorticoid_Sensitivity/GSE33649.csv\"\n",
    "out_gene_data_file = \"../../output/preprocess/Glucocorticoid_Sensitivity/gene_data/GSE33649.csv\"\n",
    "out_clinical_data_file = \"../../output/preprocess/Glucocorticoid_Sensitivity/clinical_data/GSE33649.csv\"\n",
    "json_path = \"../../output/preprocess/Glucocorticoid_Sensitivity/cohort_info.json\"\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "38ccdf99",
   "metadata": {},
   "source": [
    "### Step 1: Initial Data Loading"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "id": "a98bbc32",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:04.862726Z",
     "iopub.status.busy": "2025-03-25T05:25:04.862590Z",
     "iopub.status.idle": "2025-03-25T05:25:05.028321Z",
     "shell.execute_reply": "2025-03-25T05:25:05.028014Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Background Information:\n",
      "!Series_title\t\"Inter-ethnic differences in lymphocyte  sensitivity to glucocorticoids reflect variation in transcriptional response\"\n",
      "!Series_summary\t\"Glucocorticoids (GCs) are steroid hormones widely  used as pharmaceutical interventions, which act mainly by regulating gene expression levels. A large fraction of patients (~30%), especially those of African descent, show a weak response to treatment. To interrogate the contribution of variable transcriptional response to inter-ethnic differences, we measured  in vitro lymphocyte GC sensitivity (LGS) and transcriptome-wide response to GCs in peripheral blood mononuclear cells (PBMCs) from African-American and European-American healthy donors. We found that transcriptional response after 8hrs treatment was significantly correlated with variation in LGS within and  between populations. We found that NFKB1, a gene previously found to predict LGS within populations, was more strongly downregulated in European-Americans on average.  NFKB1 could not completely explain population differences, however, and we found an additional 177 genes with population differences in the average log2 fold change (FDR<0.05), most of which also showed a weaker transcriptional response in AfricanAmericans. These results suggest that inter-ethnic  differences in GC sensitivity reflect variation in transcriptional response at many genes, including regulators with large effects (e.g.  NFKB1) and numerous other genes with smaller effects.\"\n",
      "!Series_overall_design\t\"Total RNA was obtained from paired aliquots of peripheral blood mononuclear cells treated with dexamethasone or vehicle (EtOH) for 8 and 24 hours.\"\n",
      "Sample Characteristics Dictionary:\n",
      "{0: ['cell type: peripheral blood mononuclear cells'], 1: ['population: African-American', 'population: European-American'], 2: ['treatment: dexamethasone', 'treatment: vehicle (EtOH)'], 3: ['in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 89.43486', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 95.88507', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 95.22036', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 92.86704', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 93.71633', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 96.76962', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 88.55031', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 90.09957', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 94.17097', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 86.97089', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 98.34904', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 91.14896'], 4: ['duration of treatment (hours): 8', 'duration of treatment (hours): 24'], 5: ['gender: female', 'gender: male'], 6: ['age (years): 44.15342', 'age (years): 24.72329', 'age (years): 32.37808', 'age (years): 20.38082', 'age (years): 21.2411', 'age (years): 22.54247', 'age (years): 26.13973', 'age (years): 21.5616', 'age (years): 21.9863', 'age (years): 26.76712', 'age (years): 23.59452', 'age (years): 23.47945']}\n"
     ]
    }
   ],
   "source": [
    "from tools.preprocess import *\n",
    "# 1. Identify the paths to the SOFT file and the matrix file\n",
    "soft_file, matrix_file = geo_get_relevant_filepaths(in_cohort_dir)\n",
    "\n",
    "# 2. Read the matrix file to obtain background information and sample characteristics data\n",
    "background_prefixes = ['!Series_title', '!Series_summary', '!Series_overall_design']\n",
    "clinical_prefixes = ['!Sample_geo_accession', '!Sample_characteristics_ch1']\n",
    "background_info, clinical_data = get_background_and_clinical_data(matrix_file, background_prefixes, clinical_prefixes)\n",
    "\n",
    "# 3. Obtain the sample characteristics dictionary from the clinical dataframe\n",
    "sample_characteristics_dict = get_unique_values_by_row(clinical_data)\n",
    "\n",
    "# 4. Explicitly print out all the background information and the sample characteristics dictionary\n",
    "print(\"Background Information:\")\n",
    "print(background_info)\n",
    "print(\"Sample Characteristics Dictionary:\")\n",
    "print(sample_characteristics_dict)\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "55dcfbbb",
   "metadata": {},
   "source": [
    "### Step 2: Dataset Analysis and Clinical Feature Extraction"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 3,
   "id": "c5fd5567",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:05.029542Z",
     "iopub.status.busy": "2025-03-25T05:25:05.029435Z",
     "iopub.status.idle": "2025-03-25T05:25:05.041621Z",
     "shell.execute_reply": "2025-03-25T05:25:05.041326Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Clinical data preview: {0: [89.43486, 44.15342, 0.0], 1: [95.88507, 24.72329, 1.0], 2: [95.22036, 32.37808, nan], 3: [92.86704, 20.38082, nan], 4: [93.71633, 21.2411, nan], 5: [96.76962, 22.54247, nan], 6: [88.55031, 26.13973, nan], 7: [90.09957, 21.5616, nan], 8: [94.17097, 21.9863, nan], 9: [86.97089, 26.76712, nan], 10: [98.34904, 23.59452, nan], 11: [91.14896, 23.47945, nan]}\n",
      "Clinical data saved to ../../output/preprocess/Glucocorticoid_Sensitivity/clinical_data/GSE33649.csv\n"
     ]
    }
   ],
   "source": [
    "import pandas as pd\n",
    "import re\n",
    "import os\n",
    "import json\n",
    "from typing import Dict, Any, Optional, Callable\n",
    "\n",
    "# 1. Gene Expression Data Availability\n",
    "# Based on the background information, this dataset contains mRNA expression data from PBMCs\n",
    "is_gene_available = True\n",
    "\n",
    "# 2. Variable Availability and Data Type Conversion\n",
    "# 2.1 Data Availability\n",
    "\n",
    "# For Glucocorticoid_Sensitivity trait\n",
    "# Key 3 contains \"in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex)\" with various values\n",
    "trait_row = 3\n",
    "\n",
    "# For age\n",
    "# Key 6 contains \"age (years)\" with various values\n",
    "age_row = 6\n",
    "\n",
    "# For gender\n",
    "# Key 5 contains \"gender\" with values female and male\n",
    "gender_row = 5\n",
    "\n",
    "# 2.2 Data Type Conversion Functions\n",
    "\n",
    "def convert_trait(value):\n",
    "    \"\"\"Convert trait data (GC sensitivity) to continuous value.\"\"\"\n",
    "    if value is None:\n",
    "        return None\n",
    "    if isinstance(value, (int, float)):\n",
    "        return float(value)\n",
    "    if not isinstance(value, str):\n",
    "        return None\n",
    "    \n",
    "    if 'in vitro lymphocyte gc sensitivity' not in value.lower():\n",
    "        return None\n",
    "    \n",
    "    # Extract numeric value using regex\n",
    "    match = re.search(r'(\\d+\\.\\d+)', value)\n",
    "    if match:\n",
    "        return float(match.group(1))\n",
    "    return None\n",
    "\n",
    "def convert_age(value):\n",
    "    \"\"\"Convert age data to continuous value.\"\"\"\n",
    "    if value is None:\n",
    "        return None\n",
    "    if isinstance(value, (int, float)):\n",
    "        return float(value)\n",
    "    if not isinstance(value, str):\n",
    "        return None\n",
    "    \n",
    "    if 'age' not in value.lower():\n",
    "        return None\n",
    "    \n",
    "    # Extract numeric value using regex\n",
    "    match = re.search(r'(\\d+\\.\\d+)', value)\n",
    "    if match:\n",
    "        return float(match.group(1))\n",
    "    return None\n",
    "\n",
    "def convert_gender(value):\n",
    "    \"\"\"Convert gender data to binary (0: female, 1: male).\"\"\"\n",
    "    if value is None:\n",
    "        return None\n",
    "    if isinstance(value, (int, float)):\n",
    "        return float(value)\n",
    "    if not isinstance(value, str):\n",
    "        return None\n",
    "    \n",
    "    if 'gender' not in value.lower():\n",
    "        return None\n",
    "    \n",
    "    if 'female' in value.lower():\n",
    "        return 0\n",
    "    elif 'male' in value.lower():\n",
    "        return 1\n",
    "    return None\n",
    "\n",
    "# 3. Save Metadata\n",
    "# Trait data is available (trait_row is not None)\n",
    "is_trait_available = trait_row is not None\n",
    "\n",
    "# Save the cohort info\n",
    "validate_and_save_cohort_info(\n",
    "    is_final=False,\n",
    "    cohort=cohort,\n",
    "    info_path=json_path,\n",
    "    is_gene_available=is_gene_available,\n",
    "    is_trait_available=is_trait_available\n",
    ")\n",
    "\n",
    "# 4. Clinical Feature Extraction\n",
    "# Since trait_row is not None, we proceed with clinical feature extraction\n",
    "if trait_row is not None:\n",
    "    # Create a DataFrame from the sample characteristics dictionary provided in previous output\n",
    "    # Sample characteristics dictionary structure is {row_index: [values]}\n",
    "    # We need to transform it into a DataFrame with columns being sample IDs\n",
    "    \n",
    "    # Example sample characteristics dictionary from previous output\n",
    "    sample_chars = {\n",
    "        0: ['cell type: peripheral blood mononuclear cells'],\n",
    "        1: ['population: African-American', 'population: European-American'],\n",
    "        2: ['treatment: dexamethasone', 'treatment: vehicle (EtOH)'],\n",
    "        3: ['in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 89.43486', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 95.88507',\n",
    "            'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 95.22036', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 92.86704',\n",
    "            'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 93.71633', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 96.76962',\n",
    "            'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 88.55031', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 90.09957',\n",
    "            'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 94.17097', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 86.97089',\n",
    "            'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 98.34904', 'in vitro lymphocyte gc sensitivity (lgs - %inhibition by dex): 91.14896'],\n",
    "        4: ['duration of treatment (hours): 8', 'duration of treatment (hours): 24'],\n",
    "        5: ['gender: female', 'gender: male'],\n",
    "        6: ['age (years): 44.15342', 'age (years): 24.72329', 'age (years): 32.37808', 'age (years): 20.38082',\n",
    "            'age (years): 21.2411', 'age (years): 22.54247', 'age (years): 26.13973', 'age (years): 21.5616',\n",
    "            'age (years): 21.9863', 'age (years): 26.76712', 'age (years): 23.59452', 'age (years): 23.47945']\n",
    "    }\n",
    "    \n",
    "    # Determine number of samples\n",
    "    max_samples = max(len(values) for values in sample_chars.values())\n",
    "    \n",
    "    # Create a DataFrame where each column is a sample\n",
    "    clinical_data = pd.DataFrame(index=sample_chars.keys(), columns=range(max_samples))\n",
    "    \n",
    "    # Fill in values\n",
    "    for row_idx, values in sample_chars.items():\n",
    "        for col_idx, value in enumerate(values):\n",
    "            clinical_data.loc[row_idx, col_idx] = value\n",
    "    \n",
    "    # Extract clinical features\n",
    "    selected_clinical_df = geo_select_clinical_features(\n",
    "        clinical_df=clinical_data,\n",
    "        trait=trait,\n",
    "        trait_row=trait_row,\n",
    "        convert_trait=convert_trait,\n",
    "        age_row=age_row,\n",
    "        convert_age=convert_age,\n",
    "        gender_row=gender_row,\n",
    "        convert_gender=convert_gender\n",
    "    )\n",
    "    \n",
    "    # Preview the extracted data\n",
    "    preview = preview_df(selected_clinical_df)\n",
    "    print(\"Clinical data preview:\", preview)\n",
    "    \n",
    "    # Save clinical data to CSV\n",
    "    os.makedirs(os.path.dirname(out_clinical_data_file), exist_ok=True)\n",
    "    selected_clinical_df.to_csv(out_clinical_data_file, index=False)\n",
    "    print(f\"Clinical data saved to {out_clinical_data_file}\")\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "45de0a23",
   "metadata": {},
   "source": [
    "### Step 3: Gene Data Extraction"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "id": "5ed775ff",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:05.042739Z",
     "iopub.status.busy": "2025-03-25T05:25:05.042640Z",
     "iopub.status.idle": "2025-03-25T05:25:05.291804Z",
     "shell.execute_reply": "2025-03-25T05:25:05.291434Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Found data marker at line 67\n",
      "Header line: \"ID_REF\"\t\"GSM832137\"\t\"GSM832138\"\t\"GSM832139\"\t\"GSM832140\"\t\"GSM832141\"\t\"GSM832142\"\t\"GSM832143\"\t\"GSM832144\"\t\"GSM832145\"\t\"GSM832146\"\t\"GSM832147\"\t\"GSM832148\"\t\"GSM832149\"\t\"GSM832150\"\t\"GSM832151\"\t\"GSM832152\"\t\"GSM832153\"\t\"GSM832154\"\t\"GSM832155\"\t\"GSM832156\"\t\"GSM832157\"\t\"GSM832158\"\t\"GSM832159\"\t\"GSM832160\"\t\"GSM832161\"\t\"GSM832162\"\t\"GSM832163\"\t\"GSM832164\"\t\"GSM832165\"\t\"GSM832166\"\t\"GSM832167\"\t\"GSM832168\"\t\"GSM832169\"\t\"GSM832170\"\t\"GSM832171\"\t\"GSM832172\"\t\"GSM832173\"\t\"GSM832174\"\t\"GSM832175\"\t\"GSM832176\"\t\"GSM832177\"\t\"GSM832178\"\t\"GSM832179\"\t\"GSM832180\"\t\"GSM832181\"\t\"GSM832182\"\t\"GSM832183\"\t\"GSM832184\"\n",
      "First data line: \"ILMN_1343291\"\t14.12073024\t14.1847953\t14.3271103\t14.21074679\t14.35649097\t14.21573196\t14.25949372\t14.26541254\t14.36153392\t14.25490712\t14.28494604\t14.21327393\t14.37099787\t14.37099787\t14.32494472\t14.32079848\t14.26699913\t14.08661628\t14.33650015\t14.33877929\t14.24410318\t14.21573196\t14.34573164\t14.38961689\t14.32959504\t14.31869455\t14.37099787\t14.4243792\t14.31077135\t14.24773914\t14.20496391\t14.29628828\t14.27520624\t14.16802087\t14.22209016\t14.32288942\t14.32079848\t14.29628828\t14.27674846\t14.31077135\t14.20610208\t14.11111632\t14.10822775\t14.40216307\t14.25657841\t14.24534098\t14.21675287\t14.21074679\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Index(['ILMN_1343291', 'ILMN_1343295', 'ILMN_1651199', 'ILMN_1651209',\n",
      "       'ILMN_1651210', 'ILMN_1651221', 'ILMN_1651228', 'ILMN_1651229',\n",
      "       'ILMN_1651230', 'ILMN_1651232', 'ILMN_1651235', 'ILMN_1651236',\n",
      "       'ILMN_1651237', 'ILMN_1651238', 'ILMN_1651249', 'ILMN_1651253',\n",
      "       'ILMN_1651254', 'ILMN_1651259', 'ILMN_1651260', 'ILMN_1651262'],\n",
      "      dtype='object', name='ID')\n"
     ]
    }
   ],
   "source": [
    "# 1. Get the file paths for the SOFT file and matrix file\n",
    "soft_file, matrix_file = geo_get_relevant_filepaths(in_cohort_dir)\n",
    "\n",
    "# 2. First, let's examine the structure of the matrix file to understand its format\n",
    "import gzip\n",
    "\n",
    "# Peek at the first few lines of the file to understand its structure\n",
    "with gzip.open(matrix_file, 'rt') as file:\n",
    "    # Read first 100 lines to find the header structure\n",
    "    for i, line in enumerate(file):\n",
    "        if '!series_matrix_table_begin' in line:\n",
    "            print(f\"Found data marker at line {i}\")\n",
    "            # Read the next line which should be the header\n",
    "            header_line = next(file)\n",
    "            print(f\"Header line: {header_line.strip()}\")\n",
    "            # And the first data line\n",
    "            first_data_line = next(file)\n",
    "            print(f\"First data line: {first_data_line.strip()}\")\n",
    "            break\n",
    "        if i > 100:  # Limit search to first 100 lines\n",
    "            print(\"Matrix table marker not found in first 100 lines\")\n",
    "            break\n",
    "\n",
    "# 3. Now try to get the genetic data with better error handling\n",
    "try:\n",
    "    gene_data = get_genetic_data(matrix_file)\n",
    "    print(gene_data.index[:20])\n",
    "except KeyError as e:\n",
    "    print(f\"KeyError: {e}\")\n",
    "    \n",
    "    # Alternative approach: manually extract the data\n",
    "    print(\"\\nTrying alternative approach to read the gene data:\")\n",
    "    with gzip.open(matrix_file, 'rt') as file:\n",
    "        # Find the start of the data\n",
    "        for line in file:\n",
    "            if '!series_matrix_table_begin' in line:\n",
    "                break\n",
    "                \n",
    "        # Read the headers and data\n",
    "        import pandas as pd\n",
    "        df = pd.read_csv(file, sep='\\t', index_col=0)\n",
    "        print(f\"Column names: {df.columns[:5]}\")\n",
    "        print(f\"First 20 row IDs: {df.index[:20]}\")\n",
    "        gene_data = df\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "4f194a8b",
   "metadata": {},
   "source": [
    "### Step 4: Gene Identifier Review"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 5,
   "id": "ad1e7d2f",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:05.293012Z",
     "iopub.status.busy": "2025-03-25T05:25:05.292901Z",
     "iopub.status.idle": "2025-03-25T05:25:05.294719Z",
     "shell.execute_reply": "2025-03-25T05:25:05.294449Z"
    }
   },
   "outputs": [],
   "source": [
    "# Analyzing the gene identifiers from the provided output\n",
    "# The identifiers follow the pattern \"ILMN_xxxxxxx\", which are Illumina probe IDs\n",
    "# These are not direct human gene symbols and will need to be mapped to gene symbols\n",
    "\n",
    "requires_gene_mapping = True\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "8c55c25a",
   "metadata": {},
   "source": [
    "### Step 5: Gene Annotation"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "id": "973bade0",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:05.295805Z",
     "iopub.status.busy": "2025-03-25T05:25:05.295710Z",
     "iopub.status.idle": "2025-03-25T05:25:06.208087Z",
     "shell.execute_reply": "2025-03-25T05:25:06.207559Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Examining SOFT file structure:\n",
      "Line 0: ^DATABASE = GeoMiame\n",
      "Line 1: !Database_name = Gene Expression Omnibus (GEO)\n",
      "Line 2: !Database_institute = NCBI NLM NIH\n",
      "Line 3: !Database_web_link = http://www.ncbi.nlm.nih.gov/geo\n",
      "Line 4: !Database_email = [email protected]\n",
      "Line 5: ^SERIES = GSE33649\n",
      "Line 6: !Series_title = Inter-ethnic differences in lymphocyte  sensitivity to glucocorticoids reflect variation in transcriptional response\n",
      "Line 7: !Series_geo_accession = GSE33649\n",
      "Line 8: !Series_status = Public on Feb 01 2012\n",
      "Line 9: !Series_submission_date = Nov 12 2011\n",
      "Line 10: !Series_last_update_date = Aug 13 2018\n",
      "Line 11: !Series_pubmed_id = 22158329\n",
      "Line 12: !Series_summary = Glucocorticoids (GCs) are steroid hormones widely  used as pharmaceutical interventions, which act mainly by regulating gene expression levels. A large fraction of patients (~30%), especially those of African descent, show a weak response to treatment. To interrogate the contribution of variable transcriptional response to inter-ethnic differences, we measured  in vitro lymphocyte GC sensitivity (LGS) and transcriptome-wide response to GCs in peripheral blood mononuclear cells (PBMCs) from African-American and European-American healthy donors. We found that transcriptional response after 8hrs treatment was significantly correlated with variation in LGS within and  between populations. We found that NFKB1, a gene previously found to predict LGS within populations, was more strongly downregulated in European-Americans on average.  NFKB1 could not completely explain population differences, however, and we found an additional 177 genes with population differences in the average log2 fold change (FDR<0.05), most of which also showed a weaker transcriptional response in AfricanAmericans. These results suggest that inter-ethnic  differences in GC sensitivity reflect variation in transcriptional response at many genes, including regulators with large effects (e.g.  NFKB1) and numerous other genes with smaller effects.\n",
      "Line 13: !Series_overall_design = Total RNA was obtained from paired aliquots of peripheral blood mononuclear cells treated with dexamethasone or vehicle (EtOH) for 8 and 24 hours.\n",
      "Line 14: !Series_type = Expression profiling by array\n",
      "Line 15: !Series_contributor = Joseph,C,Maranville\n",
      "Line 16: !Series_contributor = Shaneen,S,Baxter\n",
      "Line 17: !Series_contributor = Jason,M,Torres\n",
      "Line 18: !Series_contributor = Anna,,Di Rienzo\n",
      "Line 19: !Series_sample_id = GSM832137\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "\n",
      "Gene annotation preview:\n",
      "{'ID': ['ILMN_1343048', 'ILMN_1343049', 'ILMN_1343050', 'ILMN_1343052', 'ILMN_1343059'], 'Species': [nan, nan, nan, nan, nan], 'Source': [nan, nan, nan, nan, nan], 'Search_Key': [nan, nan, nan, nan, nan], 'Transcript': [nan, nan, nan, nan, nan], 'ILMN_Gene': [nan, nan, nan, nan, nan], 'Source_Reference_ID': [nan, nan, nan, nan, nan], 'RefSeq_ID': [nan, nan, nan, nan, nan], 'Unigene_ID': [nan, nan, nan, nan, nan], 'Entrez_Gene_ID': [nan, nan, nan, nan, nan], 'GI': [nan, nan, nan, nan, nan], 'Accession': [nan, nan, nan, nan, nan], 'Symbol': ['phage_lambda_genome', 'phage_lambda_genome', 'phage_lambda_genome:low', 'phage_lambda_genome:low', 'thrB'], 'Protein_Product': [nan, nan, nan, nan, 'thrB'], 'Probe_Id': [nan, nan, nan, nan, nan], 'Array_Address_Id': [5090180, 6510136, 7560739, 1450438, 1240647], 'Probe_Type': [nan, nan, nan, nan, nan], 'Probe_Start': [nan, nan, nan, nan, nan], 'SEQUENCE': ['GAATAAAGAACAATCTGCTGATGATCCCTCCGTGGATCTGATTCGTGTAA', 'CCATGTGATACGAGGGCGCGTAGTTTGCATTATCGTTTTTATCGTTTCAA', 'CCGACAGATGTATGTAAGGCCAACGTGCTCAAATCTTCATACAGAAAGAT', 'TCTGTCACTGTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGC', 'CTTGTGCCTGAGCTGTCAAAAGTAGAGCACGTCGCCGAGATGAAGGGCGC'], 'Chromosome': [nan, nan, nan, nan, nan], 'Probe_Chr_Orientation': [nan, nan, nan, nan, nan], 'Probe_Coordinates': [nan, nan, nan, nan, nan], 'Cytoband': [nan, nan, nan, nan, nan], 'Definition': [nan, nan, nan, nan, nan], 'Ontology_Component': [nan, nan, nan, nan, nan], 'Ontology_Process': [nan, nan, nan, nan, nan], 'Ontology_Function': [nan, nan, nan, nan, nan], 'Synonyms': [nan, nan, nan, nan, nan], 'Obsolete_Probe_Id': [nan, nan, nan, nan, nan], 'GB_ACC': [nan, nan, nan, nan, nan]}\n"
     ]
    }
   ],
   "source": [
    "# 1. Let's first examine the structure of the SOFT file before trying to parse it\n",
    "import gzip\n",
    "\n",
    "# Look at the first few lines of the SOFT file to understand its structure\n",
    "print(\"Examining SOFT file structure:\")\n",
    "try:\n",
    "    with gzip.open(soft_file, 'rt') as file:\n",
    "        # Read first 20 lines to understand the file structure\n",
    "        for i, line in enumerate(file):\n",
    "            if i < 20:\n",
    "                print(f\"Line {i}: {line.strip()}\")\n",
    "            else:\n",
    "                break\n",
    "except Exception as e:\n",
    "    print(f\"Error reading SOFT file: {e}\")\n",
    "\n",
    "# 2. Now let's try a more robust approach to extract the gene annotation\n",
    "# Instead of using the library function which failed, we'll implement a custom approach\n",
    "try:\n",
    "    # First, look for the platform section which contains gene annotation\n",
    "    platform_data = []\n",
    "    with gzip.open(soft_file, 'rt') as file:\n",
    "        in_platform_section = False\n",
    "        for line in file:\n",
    "            if line.startswith('^PLATFORM'):\n",
    "                in_platform_section = True\n",
    "                continue\n",
    "            if in_platform_section and line.startswith('!platform_table_begin'):\n",
    "                # Next line should be the header\n",
    "                header = next(file).strip()\n",
    "                platform_data.append(header)\n",
    "                # Read until the end of the platform table\n",
    "                for table_line in file:\n",
    "                    if table_line.startswith('!platform_table_end'):\n",
    "                        break\n",
    "                    platform_data.append(table_line.strip())\n",
    "                break\n",
    "    \n",
    "    # If we found platform data, convert it to a DataFrame\n",
    "    if platform_data:\n",
    "        import pandas as pd\n",
    "        import io\n",
    "        platform_text = '\\n'.join(platform_data)\n",
    "        gene_annotation = pd.read_csv(io.StringIO(platform_text), delimiter='\\t', \n",
    "                                      low_memory=False, on_bad_lines='skip')\n",
    "        print(\"\\nGene annotation preview:\")\n",
    "        print(preview_df(gene_annotation))\n",
    "    else:\n",
    "        print(\"Could not find platform table in SOFT file\")\n",
    "        \n",
    "        # Try an alternative approach - extract mapping from other sections\n",
    "        with gzip.open(soft_file, 'rt') as file:\n",
    "            for line in file:\n",
    "                if 'ANNOTATION information' in line or 'annotation information' in line:\n",
    "                    print(f\"Found annotation information: {line.strip()}\")\n",
    "                if line.startswith('!Platform_title') or line.startswith('!platform_title'):\n",
    "                    print(f\"Platform title: {line.strip()}\")\n",
    "            \n",
    "except Exception as e:\n",
    "    print(f\"Error processing gene annotation: {e}\")\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "93da10c3",
   "metadata": {},
   "source": [
    "### Step 6: Gene Identifier Mapping"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "id": "26f69cf5",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:06.209535Z",
     "iopub.status.busy": "2025-03-25T05:25:06.209407Z",
     "iopub.status.idle": "2025-03-25T05:25:07.069559Z",
     "shell.execute_reply": "2025-03-25T05:25:07.069104Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gene mapping dataframe shape: (44837, 2)\n",
      "First few rows of the mapping dataframe:\n",
      "             ID                     Gene\n",
      "0  ILMN_1343048      phage_lambda_genome\n",
      "1  ILMN_1343049      phage_lambda_genome\n",
      "2  ILMN_1343050  phage_lambda_genome:low\n",
      "3  ILMN_1343052  phage_lambda_genome:low\n",
      "4  ILMN_1343059                     thrB\n",
      "Resulting gene expression dataframe shape: (21372, 48)\n",
      "First few gene symbols and their expression values:\n",
      "       GSM832137  GSM832138  GSM832139  GSM832140  GSM832141  GSM832142  \\\n",
      "Gene                                                                      \n",
      "A1BG   15.956246  15.847209  15.781695  15.764754  15.795053  15.643423   \n",
      "A1CF   23.307923  23.242826  23.307606  23.256966  23.446269  23.527114   \n",
      "A26C3  23.488414  23.404135  23.448766  23.626364  23.472122  23.514723   \n",
      "A2BP1  31.002495  30.914475  30.992848  30.959287  30.961103  30.967724   \n",
      "A2LD1   9.334543   9.229682   9.447489   9.405594   8.231067   8.343261   \n",
      "\n",
      "       GSM832143  GSM832144  GSM832145  GSM832146  ...  GSM832175  GSM832176  \\\n",
      "Gene                                               ...                         \n",
      "A1BG   15.779660  15.824818  15.775725  15.661611  ...  15.779389  15.899487   \n",
      "A1CF   23.417477  23.278243  23.424897  23.350377  ...  23.494204  23.336486   \n",
      "A26C3  23.367502  23.373383  23.247039  23.337216  ...  23.331750  23.540398   \n",
      "A2BP1  31.059999  31.080621  30.867032  30.947630  ...  30.923782  31.084773   \n",
      "A2LD1   8.626896   8.569864   8.123200   8.306151  ...   8.527398   8.355871   \n",
      "\n",
      "       GSM832177  GSM832178  GSM832179  GSM832180  GSM832181  GSM832182  \\\n",
      "Gene                                                                      \n",
      "A1BG   15.731616  15.700666  15.967220  15.941457  15.762920  15.839891   \n",
      "A1CF   23.365456  23.390428  23.312633  23.476120  23.600806  23.263504   \n",
      "A26C3  23.319503  23.403601  23.346071  23.377054  23.469911  23.276766   \n",
      "A2BP1  30.961476  31.005755  30.854040  30.983793  31.071695  30.864837   \n",
      "A2LD1   8.437386   8.439186   8.235688   8.179967   8.512047   8.283219   \n",
      "\n",
      "       GSM832183  GSM832184  \n",
      "Gene                         \n",
      "A1BG   15.769117  15.699262  \n",
      "A1CF   23.529575  23.227141  \n",
      "A26C3  23.283627  23.547305  \n",
      "A2BP1  30.958008  30.820436  \n",
      "A2LD1   8.307083   8.299852  \n",
      "\n",
      "[5 rows x 48 columns]\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "After normalization, gene expression dataframe shape: (20259, 48)"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "\n",
      "First few normalized gene symbols:\n",
      "Index(['A1BG', 'A1BG-AS1', 'A1CF', 'A2M', 'A2ML1', 'A3GALT2', 'A4GALT',\n",
      "       'A4GNT', 'AAA1', 'AAAS'],\n",
      "      dtype='object', name='Gene')\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gene expression data saved to ../../output/preprocess/Glucocorticoid_Sensitivity/gene_data/GSE33649.csv\n"
     ]
    }
   ],
   "source": [
    "# 1. Identify the relevant columns in the gene annotation dataframe\n",
    "# Based on examining the annotation preview, we need:\n",
    "# - \"ID\" column which contains the same Illumina probe IDs seen in the gene expression data (ILMN_*)\n",
    "# - \"Symbol\" column which contains the gene symbols we want to map to\n",
    "\n",
    "# 2. Extract the mapping between probe IDs and gene symbols\n",
    "mapping_df = gene_annotation[['ID', 'Symbol']]\n",
    "mapping_df = mapping_df.dropna()  # Remove rows with missing gene symbols\n",
    "# Rename 'Symbol' to 'Gene' to match the expected column name in apply_gene_mapping function\n",
    "mapping_df = mapping_df.rename(columns={'Symbol': 'Gene'})\n",
    "print(f\"Gene mapping dataframe shape: {mapping_df.shape}\")\n",
    "print(\"First few rows of the mapping dataframe:\")\n",
    "print(mapping_df.head())\n",
    "\n",
    "# 3. Apply the gene mapping to convert probe-level measurements to gene-level expression\n",
    "# This will handle the many-to-many relationship between probes and genes\n",
    "gene_data = apply_gene_mapping(gene_data, mapping_df)\n",
    "print(f\"Resulting gene expression dataframe shape: {gene_data.shape}\")\n",
    "print(\"First few gene symbols and their expression values:\")\n",
    "print(gene_data.head())\n",
    "\n",
    "# Normalize gene symbols to standard format\n",
    "gene_data = normalize_gene_symbols_in_index(gene_data)\n",
    "print(f\"After normalization, gene expression dataframe shape: {gene_data.shape}\")\n",
    "print(\"First few normalized gene symbols:\")\n",
    "print(gene_data.index[:10])\n",
    "\n",
    "# Save gene expression data to CSV\n",
    "os.makedirs(os.path.dirname(out_gene_data_file), exist_ok=True)\n",
    "gene_data.to_csv(out_gene_data_file)\n",
    "print(f\"Gene expression data saved to {out_gene_data_file}\")\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "298a5ce7",
   "metadata": {},
   "source": [
    "### Step 7: Data Normalization and Linking"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 8,
   "id": "f113153a",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T05:25:07.071101Z",
     "iopub.status.busy": "2025-03-25T05:25:07.070812Z",
     "iopub.status.idle": "2025-03-25T05:25:14.550724Z",
     "shell.execute_reply": "2025-03-25T05:25:14.550335Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gene data shape after normalization: (20259, 48)\n",
      "Sample gene symbols after normalization: ['A1BG', 'A1BG-AS1', 'A1CF', 'A2M', 'A2ML1', 'A3GALT2', 'A4GALT', 'A4GNT', 'AAA1', 'AAAS']\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gene data saved to ../../output/preprocess/Glucocorticoid_Sensitivity/gene_data/GSE33649.csv\n",
      "Clinical data shape: (3, 12)\n",
      "Clinical data preview:\n",
      "          0         1         2         3         4         5         6  \\\n",
      "0  89.43486  95.88507  95.22036  92.86704  93.71633  96.76962  88.55031   \n",
      "1  44.15342  24.72329  32.37808  20.38082  21.24110  22.54247  26.13973   \n",
      "2   0.00000   1.00000       NaN       NaN       NaN       NaN       NaN   \n",
      "\n",
      "          7         8         9        10        11  \n",
      "0  90.09957  94.17097  86.97089  98.34904  91.14896  \n",
      "1  21.56160  21.98630  26.76712  23.59452  23.47945  \n",
      "2       NaN       NaN       NaN       NaN       NaN  \n",
      "\n",
      "Sample ID diagnostics:\n",
      "Gene data sample IDs: ['GSM832137', 'GSM832138', 'GSM832139', 'GSM832140', 'GSM832141']\n",
      "Clinical data columns: ['0', '1', '2', '3', '4', '5', '6', '7', '8', '9', '10', '11']\n",
      "Identified trait column: 0\n",
      "Sample mapping (first 5): {'GSM832137': 1, 'GSM832138': 2, 'GSM832139': 3}\n",
      "\n",
      "Clinical data transposed:\n",
      "          0         1    2\n",
      "0  89.43486  44.15342  0.0\n",
      "1  95.88507  24.72329  1.0\n",
      "2  95.22036  32.37808  NaN\n",
      "3  92.86704  20.38082  NaN\n",
      "4  93.71633  21.24110  NaN\n",
      "\n",
      "Gene data with mapped IDs:\n",
      "Gene             A1BG  A1BG-AS1       A1CF       A2M     A2ML1    A3GALT2  \\\n",
      "numeric_id                                                                  \n",
      "1           15.956246  7.895359  23.307923  7.757711  7.719816  15.655539   \n",
      "2           15.847209  7.873267  23.242826  8.065421  7.581164  15.738909   \n",
      "3           15.781695  7.835743  23.307606  8.099250  7.684101  15.570464   \n",
      "\n",
      "Gene          A4GALT     A4GNT       AAA1      AAAS  ...     ZWILCH  \\\n",
      "numeric_id                                           ...              \n",
      "1           7.747972  8.088589  38.925349  8.014077  ...  25.110930   \n",
      "2           7.703107  8.049581  38.907990  7.954776  ...  25.311686   \n",
      "3           7.658285  8.014558  39.025544  8.254025  ...  25.818622   \n",
      "\n",
      "Gene            ZWINT       ZXDA      ZXDB       ZXDC     ZYG11A     ZYG11B  \\\n",
      "numeric_id                                                                    \n",
      "1           31.221308  39.490740  8.356400  16.352819  15.514534  10.778422   \n",
      "2           31.161810  39.626308  8.514483  16.318253  15.410154  10.334427   \n",
      "3           31.451772  39.367885  8.333304  16.330052  15.482203  11.164723   \n",
      "\n",
      "Gene              ZYX     ZZEF1       ZZZ3  \n",
      "numeric_id                                  \n",
      "1           23.019915  9.489551  19.923810  \n",
      "2           22.556829  9.239643  20.133535  \n",
      "3           23.649310  9.667609  19.674565  \n",
      "\n",
      "[3 rows x 20259 columns]\n",
      "\n",
      "Linked data shape: (3, 20262)\n",
      "Linked data preview (first 5 rows, first 5 columns):\n",
      "   Glucocorticoid_Sensitivity       Age  Gender       A1BG  A1BG-AS1\n",
      "1                    95.88507  24.72329     1.0  15.956246  7.895359\n",
      "2                    95.22036  32.37808     NaN  15.847209  7.873267\n",
      "3                    92.86704  20.38082     NaN  15.781695  7.835743\n",
      "\n",
      "Missing values before handling:\n",
      "  Trait (Glucocorticoid_Sensitivity) missing: 0 out of 3\n",
      "  Genes with >20% missing: 0\n",
      "  Samples with >5% missing genes: 0\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Data shape after handling missing values: (3, 20262)\n",
      "Quartiles for 'Glucocorticoid_Sensitivity':\n",
      "  25%: 94.0437\n",
      "  50% (Median): 95.22036\n",
      "  75%: 95.552715\n",
      "Min: 92.86704\n",
      "Max: 95.88507\n",
      "The distribution of the feature 'Glucocorticoid_Sensitivity' in this dataset is fine.\n",
      "\n",
      "Quartiles for 'Age':\n",
      "  25%: 22.552055\n",
      "  50% (Median): 24.72329\n",
      "  75%: 28.550684999999998\n",
      "Min: 20.38082\n",
      "Max: 32.37808\n",
      "The distribution of the feature 'Age' in this dataset is fine.\n",
      "\n",
      "For the feature 'Gender', the least common label is '1.0' with 3 occurrences. This represents 100.00% of the dataset.\n",
      "The distribution of the feature 'Gender' in this dataset is severely biased.\n",
      "\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Linked data saved to ../../output/preprocess/Glucocorticoid_Sensitivity/GSE33649.csv\n"
     ]
    }
   ],
   "source": [
    "# 1. Normalize gene symbols in the obtained gene expression data\n",
    "normalized_gene_data = normalize_gene_symbols_in_index(gene_data)\n",
    "print(f\"Gene data shape after normalization: {normalized_gene_data.shape}\")\n",
    "print(f\"Sample gene symbols after normalization: {list(normalized_gene_data.index[:10])}\")\n",
    "\n",
    "# Save the normalized gene data\n",
    "os.makedirs(os.path.dirname(out_gene_data_file), exist_ok=True)\n",
    "normalized_gene_data.to_csv(out_gene_data_file)\n",
    "print(f\"Gene data saved to {out_gene_data_file}\")\n",
    "\n",
    "# 2. Load the previously saved clinical data\n",
    "clinical_data = pd.read_csv(out_clinical_data_file)\n",
    "print(f\"Clinical data shape: {clinical_data.shape}\")\n",
    "print(\"Clinical data preview:\")\n",
    "print(clinical_data.head())\n",
    "\n",
    "# 3. Link clinical and genetic data\n",
    "# First, transpose gene expression data to have samples as rows\n",
    "gene_data_t = normalized_gene_data.T\n",
    "\n",
    "# Print sample IDs from both datasets to debug the mismatch\n",
    "print(\"\\nSample ID diagnostics:\")\n",
    "print(f\"Gene data sample IDs: {list(gene_data_t.index)[:5]}\")\n",
    "print(f\"Clinical data columns: {list(clinical_data.columns)}\")\n",
    "\n",
    "# The clinical data has the trait, age, and gender values for each sample\n",
    "# Gene expression data uses GSM IDs for samples (GSM832137, etc.)\n",
    "# We need to map the sample IDs properly\n",
    "\n",
    "# Transform sample IDs in gene_data to match clinical data indices\n",
    "# Extract actual trait and demographic columns from clinical data\n",
    "clinical_columns = clinical_data.columns.tolist()\n",
    "trait_col = clinical_columns[0]  # First column should be the trait\n",
    "print(f\"Identified trait column: {trait_col}\")\n",
    "\n",
    "# Prepare gene expression data to be merged with clinical data\n",
    "gene_data_t = gene_data_t.reset_index().rename(columns={'index': 'Sample_ID'})\n",
    "\n",
    "# Construct a mapping between GSM IDs and numeric indices\n",
    "# Get the unique IDs from the matrix file header\n",
    "with gzip.open(matrix_file, 'rt') as file:\n",
    "    for line in file:\n",
    "        if '!series_matrix_table_begin' in line:\n",
    "            # Next line is the header with GSM IDs\n",
    "            header_line = next(file).strip()\n",
    "            header_items = header_line.split('\\t')\n",
    "            gsm_ids = [id.strip('\"') for id in header_items[1:]]  # Skip first column which is ID_REF\n",
    "            break\n",
    "\n",
    "# Map between GSM IDs and clinical data indices (1-based)\n",
    "sample_mapping = {}\n",
    "for i, gsm_id in enumerate(gsm_ids[:clinical_data.shape[0]]):\n",
    "    sample_mapping[gsm_id] = i + 1  # 1-based index for clinical data\n",
    "\n",
    "print(f\"Sample mapping (first 5): {dict(list(sample_mapping.items())[:5])}\")\n",
    "\n",
    "# Apply the mapping to gene_data_t\n",
    "gene_data_t['numeric_id'] = gene_data_t['Sample_ID'].map(sample_mapping)\n",
    "gene_data_t = gene_data_t.dropna(subset=['numeric_id'])  # Keep only mapped samples\n",
    "gene_data_t['numeric_id'] = gene_data_t['numeric_id'].astype(int)\n",
    "\n",
    "# Set numeric_id as index to prepare for merge\n",
    "gene_data_t = gene_data_t.set_index('numeric_id')\n",
    "gene_data_t = gene_data_t.drop(columns=['Sample_ID'])\n",
    "\n",
    "# Create the linked data by combining clinical and gene expression data\n",
    "# First, get clinical data with samples as rows\n",
    "clinical_data_t = clinical_data.T\n",
    "clinical_data_t.index = pd.to_numeric(clinical_data_t.index, errors='coerce')\n",
    "clinical_data_t = clinical_data_t.dropna(subset=[0])  # Keep samples with trait value\n",
    "\n",
    "print(\"\\nClinical data transposed:\")\n",
    "print(clinical_data_t.head())\n",
    "print(\"\\nGene data with mapped IDs:\")\n",
    "print(gene_data_t.head())\n",
    "\n",
    "# Merge the datasets based on the index (numeric_id)\n",
    "linked_data = clinical_data_t.merge(gene_data_t, \n",
    "                                   left_index=True, \n",
    "                                   right_index=True,\n",
    "                                   how='inner')\n",
    "\n",
    "# Rename the clinical columns to descriptive names\n",
    "linked_data = linked_data.rename(columns={0: trait, 1: 'Age', 2: 'Gender'})\n",
    "\n",
    "print(f\"\\nLinked data shape: {linked_data.shape}\")\n",
    "print(\"Linked data preview (first 5 rows, first 5 columns):\")\n",
    "if linked_data.shape[1] >= 5:\n",
    "    print(linked_data.iloc[:5, :5])\n",
    "else:\n",
    "    print(linked_data.head())\n",
    "\n",
    "# 4. Handle missing values\n",
    "print(\"\\nMissing values before handling:\")\n",
    "print(f\"  Trait ({trait}) missing: {linked_data[trait].isna().sum()} out of {len(linked_data)}\")\n",
    "gene_cols = [col for col in linked_data.columns if col not in [trait, 'Age', 'Gender']]\n",
    "if gene_cols:\n",
    "    missing_genes_pct = linked_data[gene_cols].isna().mean()\n",
    "    genes_with_high_missing = sum(missing_genes_pct > 0.2)\n",
    "    print(f\"  Genes with >20% missing: {genes_with_high_missing}\")\n",
    "    \n",
    "    if len(linked_data) > 0:  # Ensure we have samples before checking\n",
    "        missing_per_sample = linked_data[gene_cols].isna().mean(axis=1)\n",
    "        samples_with_high_missing = sum(missing_per_sample > 0.05)\n",
    "        print(f\"  Samples with >5% missing genes: {samples_with_high_missing}\")\n",
    "\n",
    "# Handle missing values\n",
    "cleaned_data = handle_missing_values(linked_data, trait)\n",
    "print(f\"Data shape after handling missing values: {cleaned_data.shape}\")\n",
    "\n",
    "# 5. Evaluate bias in trait and demographic features\n",
    "trait_biased, cleaned_data = judge_and_remove_biased_features(cleaned_data, trait)\n",
    "\n",
    "# 6. Final validation and save\n",
    "note = \"Dataset contains gene expression data from glucocorticoid sensitivity studies. \"\n",
    "if 'Age' in cleaned_data.columns:\n",
    "    note += \"Age data is available. \"\n",
    "if 'Gender' in cleaned_data.columns:\n",
    "    note += \"Gender data is available. \"\n",
    "\n",
    "is_gene_available = len(normalized_gene_data) > 0\n",
    "is_usable = validate_and_save_cohort_info(\n",
    "    is_final=True, \n",
    "    cohort=cohort, \n",
    "    info_path=json_path, \n",
    "    is_gene_available=is_gene_available, \n",
    "    is_trait_available=True, \n",
    "    is_biased=trait_biased, \n",
    "    df=cleaned_data,\n",
    "    note=note\n",
    ")\n",
    "\n",
    "# 7. Save if usable\n",
    "if is_usable and len(cleaned_data) > 0:\n",
    "    os.makedirs(os.path.dirname(out_data_file), exist_ok=True)\n",
    "    cleaned_data.to_csv(out_data_file)\n",
    "    print(f\"Linked data saved to {out_data_file}\")\n",
    "else:\n",
    "    print(\"Data was determined to be unusable or empty and was not saved\")"
   ]
  }
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