{
 "cells": [
  {
   "cell_type": "code",
   "execution_count": 1,
   "id": "1a1daae1",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.371957Z",
     "iopub.status.busy": "2025-03-25T08:13:53.371845Z",
     "iopub.status.idle": "2025-03-25T08:13:53.536601Z",
     "shell.execute_reply": "2025-03-25T08:13:53.536248Z"
    }
   },
   "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 = \"Cervical_Cancer\"\n",
    "cohort = \"GSE138080\"\n",
    "\n",
    "# Input paths\n",
    "in_trait_dir = \"../../input/GEO/Cervical_Cancer\"\n",
    "in_cohort_dir = \"../../input/GEO/Cervical_Cancer/GSE138080\"\n",
    "\n",
    "# Output paths\n",
    "out_data_file = \"../../output/preprocess/Cervical_Cancer/GSE138080.csv\"\n",
    "out_gene_data_file = \"../../output/preprocess/Cervical_Cancer/gene_data/GSE138080.csv\"\n",
    "out_clinical_data_file = \"../../output/preprocess/Cervical_Cancer/clinical_data/GSE138080.csv\"\n",
    "json_path = \"../../output/preprocess/Cervical_Cancer/cohort_info.json\"\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "f3eaae2d",
   "metadata": {},
   "source": [
    "### Step 1: Initial Data Loading"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "id": "009305cd",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.538063Z",
     "iopub.status.busy": "2025-03-25T08:13:53.537917Z",
     "iopub.status.idle": "2025-03-25T08:13:53.630800Z",
     "shell.execute_reply": "2025-03-25T08:13:53.630487Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Background Information:\n",
      "!Series_title\t\"Identification of deregulated pathways, key regulators, and novel miRNA-mRNA interactions in HPV-mediated transformation. [mRNA tissues-Agilent]\"\n",
      "!Series_summary\t\"Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.\"\n",
      "!Series_overall_design\t\"Expression profiles were analyzed in healthy cervical tissues (n=10), high-grade precancerous lesios (CIN2/3, n=15) and cervical squamous cell carcinomas (n=10) using whole human genome oligo microarrays (G4112A, mRNA 4x44K; Agilent Technologies).\"\n",
      "Sample Characteristics Dictionary:\n",
      "{0: ['cell type: normal cervical squamous epithelium', 'cell type: cervical intraepithelial neoplasia, grade 2-3', 'cell type: cervical squamous cell carcinoma'], 1: ['hpv: high-risk HPV-positive', 'hpv: HPV-negative']}\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": "650d5c7f",
   "metadata": {},
   "source": [
    "### Step 2: Dataset Analysis and Clinical Feature Extraction"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 3,
   "id": "342596ea",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.631947Z",
     "iopub.status.busy": "2025-03-25T08:13:53.631837Z",
     "iopub.status.idle": "2025-03-25T08:13:53.639841Z",
     "shell.execute_reply": "2025-03-25T08:13:53.639547Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Preview of extracted clinical features:\n",
      "{'GSM4098861': [0.0], 'GSM4098862': [0.0], 'GSM4098863': [0.0], 'GSM4098864': [0.0], 'GSM4098865': [0.0], 'GSM4098866': [0.0], 'GSM4098867': [0.0], 'GSM4098868': [0.0], 'GSM4098869': [0.0], 'GSM4098870': [0.0], 'GSM4098871': [1.0], 'GSM4098872': [1.0], 'GSM4098873': [1.0], 'GSM4098874': [1.0], 'GSM4098875': [1.0], 'GSM4098876': [1.0], 'GSM4098877': [1.0], 'GSM4098878': [1.0], 'GSM4098879': [1.0], 'GSM4098880': [1.0], 'GSM4098881': [1.0], 'GSM4098882': [1.0], 'GSM4098883': [1.0], 'GSM4098884': [1.0], 'GSM4098885': [1.0], 'GSM4098886': [1.0], 'GSM4098887': [1.0], 'GSM4098888': [1.0], 'GSM4098889': [1.0], 'GSM4098890': [1.0], 'GSM4098891': [1.0], 'GSM4098892': [1.0], 'GSM4098893': [1.0], 'GSM4098894': [1.0], 'GSM4098895': [1.0]}\n",
      "Clinical features saved to ../../output/preprocess/Cervical_Cancer/clinical_data/GSE138080.csv\n"
     ]
    }
   ],
   "source": [
    "import os\n",
    "import pandas as pd\n",
    "import numpy as np\n",
    "from typing import Optional, Callable, List, Dict, Any, Union\n",
    "import json\n",
    "\n",
    "# 1. Gene Expression Data Assessment\n",
    "# Based on the background information and series title, this appears to be mRNA expression data\n",
    "is_gene_available = True\n",
    "\n",
    "# 2. Variable Availability and Data Type Conversion\n",
    "# 2.1 Data Availability\n",
    "# Examining the Sample Characteristics Dictionary\n",
    "# The first key (0) contains information about cell types: normal, CIN2/3, and squamous cell carcinoma\n",
    "# The second key (1) contains HPV status: high-risk HPV-positive or HPV-negative\n",
    "\n",
    "trait_row = 0  # Cell type information can be used to determine cervical cancer\n",
    "age_row = None  # No age information available\n",
    "gender_row = None  # No gender information available (though we can assume all samples are female, but it's not explicit)\n",
    "\n",
    "# 2.2 Data Type Conversion Functions\n",
    "def convert_trait(value: str) -> Optional[int]:\n",
    "    \"\"\"\n",
    "    Convert cell type information to binary trait data for cervical cancer.\n",
    "    0: normal tissue (control)\n",
    "    1: cancer or precancerous lesion (case)\n",
    "    \"\"\"\n",
    "    if value is None:\n",
    "        return None\n",
    "    \n",
    "    # Extract value after colon if present\n",
    "    if ':' in value:\n",
    "        value = value.split(':', 1)[1].strip()\n",
    "    \n",
    "    value = value.lower()\n",
    "    \n",
    "    if 'normal' in value:\n",
    "        return 0\n",
    "    elif 'carcinoma' in value or 'neoplasia' in value:\n",
    "        return 1\n",
    "    else:\n",
    "        return None\n",
    "\n",
    "def convert_age(value: str) -> Optional[float]:\n",
    "    \"\"\"\n",
    "    Convert age information to continuous value.\n",
    "    Not used in this dataset as age information is not available.\n",
    "    \"\"\"\n",
    "    return None\n",
    "\n",
    "def convert_gender(value: str) -> Optional[int]:\n",
    "    \"\"\"\n",
    "    Convert gender information to binary.\n",
    "    Not used in this dataset as gender information is not available.\n",
    "    \"\"\"\n",
    "    return None\n",
    "\n",
    "# 3. Save Metadata\n",
    "# Perform initial filtering and save metadata\n",
    "is_trait_available = trait_row is not None\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",
    "# Check if trait_row is not None (meaning clinical data is available)\n",
    "if trait_row is not None:\n",
    "    # We need to check if clinical_data is available in the environment\n",
    "    # This should have been loaded in a previous step\n",
    "    try:\n",
    "        # Extract clinical features using the library function\n",
    "        clinical_features_df = geo_select_clinical_features(\n",
    "            clinical_df=clinical_data,  # Assuming clinical_data was loaded in previous step\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 clinical features\n",
    "        print(\"Preview of extracted clinical features:\")\n",
    "        preview = preview_df(clinical_features_df)\n",
    "        print(preview)\n",
    "        \n",
    "        # Create output directory if it doesn't exist\n",
    "        os.makedirs(os.path.dirname(out_clinical_data_file), exist_ok=True)\n",
    "        \n",
    "        # Save the clinical features to CSV\n",
    "        clinical_features_df.to_csv(out_clinical_data_file, index=True)\n",
    "        print(f\"Clinical features saved to {out_clinical_data_file}\")\n",
    "    except NameError:\n",
    "        print(\"Error: clinical_data not found. Please ensure it was loaded in a previous step.\")\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "d1584944",
   "metadata": {},
   "source": [
    "### Step 3: Gene Data Extraction"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "id": "cc444e2e",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.640835Z",
     "iopub.status.busy": "2025-03-25T08:13:53.640727Z",
     "iopub.status.idle": "2025-03-25T08:13:53.762830Z",
     "shell.execute_reply": "2025-03-25T08:13:53.762442Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Index(['12', '14', '16', '17', '19', '22', '24', '25', '27', '30', '33', '35',\n",
      "       '37', '38', '40', '42', '45', '47', '49', '51'],\n",
      "      dtype='object', name='ID')\n"
     ]
    }
   ],
   "source": [
    "# 1. Use the get_genetic_data function from the library to get the gene_data from the matrix_file previously defined.\n",
    "gene_data = get_genetic_data(matrix_file)\n",
    "\n",
    "# 2. Print the first 20 row IDs (gene or probe identifiers) for future observation.\n",
    "print(gene_data.index[:20])\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "e032ce41",
   "metadata": {},
   "source": [
    "### Step 4: Gene Identifier Review"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 5,
   "id": "1f83fb31",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.764141Z",
     "iopub.status.busy": "2025-03-25T08:13:53.764024Z",
     "iopub.status.idle": "2025-03-25T08:13:53.765920Z",
     "shell.execute_reply": "2025-03-25T08:13:53.765642Z"
    }
   },
   "outputs": [],
   "source": [
    "# The identifiers appear to be numeric values (likely probe IDs or some other non-gene symbol identifiers)\n",
    "# These are not standard human gene symbols which are typically alphanumeric (e.g., TP53, BRCA1, etc.)\n",
    "# Therefore, mapping to gene symbols would be required\n",
    "\n",
    "requires_gene_mapping = True\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "01545ba8",
   "metadata": {},
   "source": [
    "### Step 5: Gene Annotation"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "id": "6c17a5d0",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:53.767045Z",
     "iopub.status.busy": "2025-03-25T08:13:53.766931Z",
     "iopub.status.idle": "2025-03-25T08:13:55.911095Z",
     "shell.execute_reply": "2025-03-25T08:13:55.910698Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gene annotation preview:\n",
      "{'ID': ['1', '2', '3', '4', '5'], 'COL': ['266', '266', '266', '266', '266'], 'ROW': [170.0, 168.0, 166.0, 164.0, 162.0], 'NAME': ['GE_BrightCorner', 'DarkCorner', 'DarkCorner', 'DarkCorner', 'DarkCorner'], 'SPOT_ID': ['GE_BrightCorner', 'DarkCorner', 'DarkCorner', 'DarkCorner', 'DarkCorner'], 'CONTROL_TYPE': ['pos', 'pos', 'pos', 'pos', 'pos'], 'REFSEQ': [nan, nan, nan, nan, nan], 'GB_ACC': [nan, nan, nan, nan, nan], 'GENE': [nan, nan, nan, nan, nan], 'GENE_SYMBOL': [nan, nan, nan, nan, nan], 'GENE_NAME': [nan, nan, nan, nan, nan], 'UNIGENE_ID': [nan, nan, nan, nan, nan], 'ENSEMBL_ID': [nan, nan, nan, nan, nan], 'TIGR_ID': [nan, nan, nan, nan, nan], 'ACCESSION_STRING': [nan, nan, nan, nan, nan], 'CHROMOSOMAL_LOCATION': [nan, nan, nan, nan, nan], 'CYTOBAND': [nan, nan, nan, nan, nan], 'DESCRIPTION': [nan, nan, nan, nan, nan], 'GO_ID': [nan, nan, nan, nan, nan], 'SEQUENCE': [nan, nan, nan, nan, nan], 'SPOT_ID.1': [nan, nan, nan, nan, nan], 'ORDER': [1.0, 2.0, 3.0, 4.0, 5.0]}\n"
     ]
    }
   ],
   "source": [
    "# 1. Use the 'get_gene_annotation' function from the library to get gene annotation data from the SOFT file.\n",
    "gene_annotation = get_gene_annotation(soft_file)\n",
    "\n",
    "# 2. Use the 'preview_df' function from the library to preview the data and print out the results.\n",
    "print(\"Gene annotation preview:\")\n",
    "print(preview_df(gene_annotation))\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "885b3ed3",
   "metadata": {},
   "source": [
    "### Step 6: Gene Identifier Mapping"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "id": "6adba2d0",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:55.912472Z",
     "iopub.status.busy": "2025-03-25T08:13:55.912346Z",
     "iopub.status.idle": "2025-03-25T08:13:56.023449Z",
     "shell.execute_reply": "2025-03-25T08:13:56.023007Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "First 10 gene symbols after mapping:\n",
      "Index(['A1BG', 'A1CF', 'A2BP1', 'A2LD1', 'A2M', 'A2ML1', 'A4GALT', 'AAAS',\n",
      "       'AACS', 'AADACL1'],\n",
      "      dtype='object', name='Gene')\n"
     ]
    }
   ],
   "source": [
    "# 1. Identify the appropriate columns for mapping\n",
    "# Based on observation: 'ID' column in gene_annotation contains probe IDs\n",
    "# and 'GENE_SYMBOL' column contains the gene symbols\n",
    "\n",
    "# 2. Get a gene mapping dataframe by extracting the relevant columns \n",
    "mapping_df = get_gene_mapping(gene_annotation, prob_col='ID', gene_col='GENE_SYMBOL')\n",
    "\n",
    "# 3. Apply the gene mapping to convert probe-level data to gene expression data\n",
    "gene_data = apply_gene_mapping(gene_data, mapping_df)\n",
    "\n",
    "# Print the first few gene symbols to verify the mapping worked\n",
    "print(\"First 10 gene symbols after mapping:\")\n",
    "print(gene_data.index[:10])\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "ea894a50",
   "metadata": {},
   "source": [
    "### Step 7: Data Normalization and Linking"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 8,
   "id": "73ee57de",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2025-03-25T08:13:56.024959Z",
     "iopub.status.busy": "2025-03-25T08:13:56.024847Z",
     "iopub.status.idle": "2025-03-25T08:14:00.632257Z",
     "shell.execute_reply": "2025-03-25T08:14:00.631859Z"
    }
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Normalized gene data saved to ../../output/preprocess/Cervical_Cancer/gene_data/GSE138080.csv\n",
      "Linked data shape: (35, 13053)\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "For the feature 'Cervical_Cancer', the least common label is '0.0' with 10 occurrences. This represents 28.57% of the dataset.\n",
      "The distribution of the feature 'Cervical_Cancer' in this dataset is fine.\n",
      "\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Linked data saved to ../../output/preprocess/Cervical_Cancer/GSE138080.csv\n"
     ]
    }
   ],
   "source": [
    "# 1. Normalize the obtained gene data with the 'normalize_gene_symbols_in_index' function from the library.\n",
    "normalized_gene_data = normalize_gene_symbols_in_index(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\"Normalized gene data saved to {out_gene_data_file}\")\n",
    "\n",
    "# Load the clinical features from the previously saved file (reusing the data from Step 2)\n",
    "clinical_features_df = pd.read_csv(out_clinical_data_file, index_col=0)\n",
    "\n",
    "# Now link the clinical and genetic data\n",
    "linked_data = geo_link_clinical_genetic_data(clinical_features_df, normalized_gene_data)\n",
    "print(\"Linked data shape:\", linked_data.shape)\n",
    "\n",
    "# Handle missing values in the linked data\n",
    "linked_data = handle_missing_values(linked_data, trait)\n",
    "\n",
    "# 4. Determine whether the trait and some demographic features are severely biased, and remove biased features.\n",
    "is_trait_biased, unbiased_linked_data = judge_and_remove_biased_features(linked_data, trait)\n",
    "\n",
    "# 5. Conduct quality check and save the cohort information.\n",
    "is_usable = validate_and_save_cohort_info(\n",
    "    is_final=True, \n",
    "    cohort=cohort, \n",
    "    info_path=json_path, \n",
    "    is_gene_available=True, \n",
    "    is_trait_available=True, \n",
    "    is_biased=is_trait_biased, \n",
    "    df=unbiased_linked_data,\n",
    "    note=\"Dataset contains gene expression data comparing normal cervical tissue (n=10) to CIN2/3 lesions (n=15) and cervical squamous cell carcinomas (n=10).\"\n",
    ")\n",
    "\n",
    "# 6. If the linked data is usable, save it as a CSV file to 'out_data_file'.\n",
    "if is_usable:\n",
    "    os.makedirs(os.path.dirname(out_data_file), exist_ok=True)\n",
    "    unbiased_linked_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 and was not saved\")"
   ]
  }
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