Pre-analytical QC → cfDNA Preprocessing → Fragment QC
                          ↓
        ┌─────────────────┴─────────────────┐
        ↓                                   ↓
   sWGS Branch                        Panel Branch
        ↓                                   ↓
   ichorCNA                          VarDict/smCounter2
   (Tumor Fraction)                  (Mutation Detection)
        ↓                                   ↓
        └─────────────────┬─────────────────┘
                          ↓
                 Longitudinal Tracking

def check_preanalytical_quality(sample_metadata):
    '''
    Pre-analytical factors critical for cfDNA quality.

    Requirements:
    - Streck tube: up to 7 days at room temperature
    - EDTA tube: process within 6 hours
    - Avoid hemolysis
    - Store extracted DNA at -80C
    '''
    issues = []

    if sample_metadata['tube_type'] == 'EDTA':
        if sample_metadata['processing_delay_hours'] > 6:
            issues.append('EDTA tube processed > 6 hours - risk of gDNA contamination')

    if sample_metadata['hemolysis_score'] > 1:
        issues.append('Hemolysis detected - expect cellular DNA contamination')

    return issues

# For UMI-tagged libraries (targeted panels)
# fgbio pipeline

# Extract UMIs
fgbio ExtractUmisFromBam \
    --input raw.bam \
    --output with_umis.bam \
    --read-structure 3M2S+T 3M2S+T \
    --single-tag RX

# Align
bwa mem -t 8 -Y reference.fa with_umis.bam | \
    samtools view -bS - > aligned.bam

# Group by UMI
fgbio GroupReadsByUmi \
    --input aligned.bam \
    --output grouped.bam \
    --strategy adjacency \
    --edits 1

# Consensus calling
fgbio CallMolecularConsensusReads \
    --input grouped.bam \
    --output consensus.bam \
    --min-reads 2

# Filter
fgbio FilterConsensusReads \
    --input consensus.bam \
    --output final.bam \
    --ref reference.fa \
    --min-reads 2

import pysam
import numpy as np

def verify_cfdna_quality(bam_path):
    '''
    QC Checkpoint: Verify cfDNA fragment profile.
    Expected: peak at ~167bp (mononucleosome)
    '''
    bam = pysam.AlignmentFile(bam_path, 'rb')
    sizes = []

    for read in bam.fetch():
        if read.is_proper_pair and not read.is_secondary and read.template_length > 0:
            sizes.append(read.template_length)

    bam.close()
    sizes = np.array(sizes)

    modal_size = np.bincount(sizes[:400]).argmax()
    mono_frac = np.sum((sizes >= 150) & (sizes <= 180)) / len(sizes)

    qc_pass = 150 <= modal_size <= 180 and mono_frac > 0.3

    return {
        'modal_size': modal_size,
        'mononucleosome_fraction': mono_frac,
        'qc_pass': qc_pass,
        'message': 'Good cfDNA profile' if qc_pass else 'Atypical fragment distribution'
    }

# For shallow WGS data (0.1-1x coverage)
library(ichorCNA)

runIchorCNA(
    WIG = 'sample.wig',
    gcWig = 'gc_hg38_1mb.wig',
    mapWig = 'map_hg38_1mb.wig',
    normalPanel = 'pon_median.rds',
    centromere = 'centromeres.txt',
    outDir = 'ichor_results/',
    id = 'sample_id',
    normal = c(0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.99),
    ploidy = c(2, 3),
    maxCN = 5
)

# For deep targeted sequencing
# Use UMI-consensus BAM from Step 1

vardict-java \
    -G reference.fa \
    -f 0.005 \
    -N sample_id \
    -b consensus.bam \
    -c 1 -S 2 -E 3 -g 4 \
    panel.bed | \
teststrandbias.R | \
var2vcf_valid.pl \
    -N sample_id \
    -E \
    -f 0.005 \
    > sample.vcf

CHIP_GENES = ['DNMT3A', 'TET2', 'ASXL1', 'PPM1D', 'JAK2', 'SF3B1', 'SRSF2', 'TP53']

def filter_chip(variants_df, chip_genes=CHIP_GENES):
    '''
    Filter out clonal hematopoiesis variants.
    Critical for elderly patients (>5% have CHIP).
    '''
    chip = variants_df[variants_df['gene'].isin(chip_genes)]
    somatic = variants_df[~variants_df['gene'].isin(chip_genes)]

    print(f'Potential CHIP variants: {len(chip)}')
    print(f'Likely somatic: {len(somatic)}')

    return somatic, chip

import finaletoolkit as ft

def run_fragmentomics(bam_path, output_prefix):
    '''
    DELFI-style fragmentation analysis.
    Use FinaleToolkit (MIT license, not DELFI software).
    '''
    fragments = ft.read_fragments(bam_path)

    profile = ft.calculate_fragmentation_profile(
        fragments,
        bin_size=5_000_000,
        short_range=(100, 150),
        long_range=(151, 220)
    )

    profile.to_csv(f'{output_prefix}_frag_profile.csv')
    return profile

import pandas as pd
import numpy as np

def track_longitudinal(samples_df):
    '''
    Track ctDNA over treatment.

    samples_df columns: [sample_id, timepoint, tumor_fraction, mutations...]
    '''
    samples_df = samples_df.sort_values('timepoint')

    baseline = samples_df.iloc[0]['tumor_fraction']
    samples_df['log2_fc'] = np.log2(samples_df['tumor_fraction'] / baseline)

    nadir = samples_df['tumor_fraction'].min()

    response = 'unknown'
    if nadir < 0.001:
        response = 'Complete molecular response'
    elif nadir < baseline * 0.01:
        response = 'Major molecular response (>2 log)'
    elif nadir < baseline * 0.5:
        response = 'Partial molecular response'

    return samples_df, response

def run_liquid_biopsy_pipeline(sample_config):
    '''
    Complete liquid biopsy analysis pipeline.

    sample_config: dict with keys:
        - bam_file: Input BAM
        - data_type: 'swgs' or 'panel'
        - reference: Reference FASTA
        - bed_file: Panel BED (for panel data)
        - output_dir: Output directory
    '''
    results = {}

    # Step 1: Preprocess (if UMI data)
    if sample_config.get('has_umis'):
        preprocessed_bam = preprocess_with_fgbio(sample_config['bam_file'])
    else:
        preprocessed_bam = sample_config['bam_file']

    # Step 2: Fragment QC
    frag_qc = verify_cfdna_quality(preprocessed_bam)
    if not frag_qc['qc_pass']:
        print(f"WARNING: {frag_qc['message']}")
    results['fragment_qc'] = frag_qc

    # Step 3: Analysis based on data type
    if sample_config['data_type'] == 'swgs':
        # Tumor fraction estimation
        results['tumor_fraction'] = run_ichorcna(preprocessed_bam)
    elif sample_config['data_type'] == 'panel':
        # Mutation detection
        variants = call_variants(preprocessed_bam, sample_config['bed_file'])
        somatic, chip = filter_chip(variants)
        results['variants'] = somatic
        results['chip_variants'] = chip

    # Step 4: Optional fragmentomics
    if sample_config.get('run_fragmentomics'):
        results['fragmentomics'] = run_fragmentomics(preprocessed_bam)

    return results