Orchestrates the cell-free DNA / liquid-biopsy pipeline from plasma sequencing to tumor monitoring, forking tumor-naive (screening) vs tumor-informed (MRD), and chaining pre-analytic QC, UMI/duplex error-suppression (fgbio), fragment QC, ichorCNA tumor fraction (sWGS) or VarDict low-VAF calling (panel), CHIP subtraction against matched WBC, optional fragmentomics/methylation, and longitudinal tracking. Use when treating pre-analytics as the irreversible sensitivity ceiling (tube/time-to-plasma/hemolysis), running error-suppression BEFORE calling (single-strand consensus does not remove deamination; only duplex does), reporting a VAF only with input genome-equivalents (TF ~ 2x VAF only for clonal-het-diploid), subtracting CHIP before reporting somatic, or keeping tube/panel/pipeline identical across a longitudinal MRD series. Hands mechanism to the liquid-biopsy component skills; not a re-teach of any single step.
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Orchestrates the cell-free DNA / liquid-biopsy pipeline from plasma sequencing to tumor monitoring, forking tumor-naive (screening) vs tumor-informed (MRD), and chaining pre-analytic QC, UMI/duplex error-suppression (fgbio), fragment QC, ichorCNA tumor fraction (sWGS) or VarDict low-VAF calling (panel), CHIP subtraction against matched WBC, optional fragmentomics/methylation, and longitudinal tracking. Use when treating pre-analytics as the irreversible sensitivity ceiling (tube/time-to-plasma/hemolysis), running error-suppression BEFORE calling (single-strand consensus does not remove deamination; only duplex does), reporting a VAF only with input genome-equivalents (TF ~ 2x VAF only for clonal-het-diploid), subtracting CHIP before reporting somatic, or keeping tube/panel/pipeline identical across a longitudinal MRD series. Hands mechanism to the liquid-biopsy component skills; not a re-teach of any single step.
[{"pre_analytics":"Tube type/time-to-plasma in window; double-spin; low hemolysis; gDNA-contamination fragment check"},{"after_consensus":"Unique-molecule coverage / genome-equivalents recovered (NOT raw depth); duplication plateaued -- read both off the GroupReadsByUmi --family-size-histogram output"},{"after_fragment":"Modal insert ~167bp (150-180); mononucleosome fraction >0.3"},{"after_tf":"TF above the ~3% ichorCNA LoD to trust the value (below = below detection, not low burden)"},{"after_mutation":"VarDict -f 0.005 is a reporting floor, NOT a detection threshold: confirm each call against a per-locus background-error model (PoN or smCounter2) before reporting; CHIP-subtracted against matched WBC"}]
Before using code patterns, verify installed versions match. If versions differ:
Python: pip show <package> then help(module.function) to check signatures
R: packageVersion('<pkg>') then ?function_name to verify parameters
CLI: <tool> --version then <tool> --help to confirm flags
If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.
Liquid Biopsy Analysis Pipeline
"Analyze my liquid biopsy cfDNA data end-to-end" -> Orchestrate UMI-aware preprocessing (fgbio), ctDNA mutation detection (VarDict), tumor fraction estimation (ichorCNA), fragmentomics analysis, and longitudinal monitoring for treatment response.
This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step. Every step below cross-references the component skill that teaches its mechanism.
The governing principle
A liquid-biopsy result is decided at four seams, two of them set before the sequencer ever runs.
Pre-analytics is irreversible and is the sensitivity ceiling โ set at the blood draw, not the pipeline. WBC lysis dumps high-quality germline/CHIP DNA into the denominator; a 0.5% tumor fraction diluted to 0.1% by a processing delay is a false negative with NO bioinformatic recovery. Tube chemistry (Streck ~7d RT vs EDTA <6h), double-spin, and hemolysis are the FIRST gate the whole pipeline is conditional on. Mixing tube types within a longitudinal series is a batch confound.
The error-suppression / UMI scheme is committed at library prep and cannot be upgraded after sequencing. Single-strand UMI consensus floors error ~1e-4 to 1e-5 but does NOT remove deamination (C>T) or oxidation (G>T) โ those lesions are on the template, so every PCR copy inherits them and the family votes unanimously for the artifact. Only DUPLEX (both-strand concordance) reaches <1e-7. Call on the consensus BAM, never the raw BAM.
A VAF is undefined without input genome-equivalents, and TF != VAF. 0.1% on 100 GE is noise; on 30,000 GE it is solid. The LoD is set by input GE and assay design (tumor-informed bespoke integrates 16-50 loci to ppm; tumor-naive fixed panel is error/sampling-limited ~0.1-0.5%; ichorCNA sWGS floors ~3% TF). TF ~ 2x VAF only for a clonal, heterozygous, diploid locus โ copy number breaks the factor of 2.
Matched buffy-coat/WBC is the CHIP-subtraction commitment. ~81.6% of cfDNA variants in controls and ~53.2% in cancer patients are CHIP, not tumor (Razavi 2019). Without matched WBC, a tumor-naive cfDNA call is presumptively CHIP-contaminated; a gene-list filter is a weak fallback. Decide at study design.
Made-once commitments
Commitment
Consequence inherited downstream
Pre-analytics (tube/time-to-plasma/double-spin)
Irreversible TF dilution; the whole pipeline's sensitivity ceiling; consistent across a longitudinal series
Error-suppression scheme (single-strand vs DUPLEX)
defcheck_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
Step 1: cfDNA Preprocessing with UMI Consensus
# For UMI-tagged libraries (targeted panels)# fgbio pipeline# Extract UMIs. Read-structure is library-specific; see liquid-biopsy/cfdna-preprocessing.
fgbio ExtractUmisFromBam \
--input raw.bam \
--output with_umis.bam \
--read-structure 3M2S+T 3M2S+T \
--single-tag RX
# Align. bwa reads FASTQ, not BAM, and stripping to FASTQ drops the RX/UMI tag -- so emit FASTQ# carrying RX (samtools fastq -T RX), align, then re-zip the uBAM tags back on with fgbio ZipperBams# so GroupReadsByUmi still sees RX.
samtools fastq -T RX with_umis.bam | \
bwa mem -C -p -t 8 -Y reference.fa - | \
fgbio ZipperBams --unmapped with_umis.bam --ref reference.fa --output aligned.bam
# Group by UMI. --family-size-histogram is not optional here: rule 3 says a VAF is undefined without# input genome-equivalents, and this histogram is where GE and the duplication plateau are read off.# Raw depth after consensus collapse is not a sensitivity measure -- unique molecules are.
fgbio GroupReadsByUmi \
--input aligned.bam \
--output grouped.bam \
--strategy adjacency \
--edits 1 \
--family-size-histogram qc/family_sizes.txt
# Consensus calling: keep the caller permissive (fgbio #1009), apply strictness at the filter
fgbio CallMolecularConsensusReads \
--input grouped.bam \
--output consensus.bam \
--min-reads 1
# Filter: this is the real quality gate
fgbio FilterConsensusReads \
--input consensus.bam \
--output final.bam \
--ref reference.fa \
--min-reads 2
Step 2: Fragment QC Checkpoint
import pysam
import numpy as np
defverify_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():
# cfDNA fragments are short; cap at 400 bp (fragments beyond are gDNA/noise) so the# modal-size bincount is bounded and not skewed by a few long outliers.if read.is_proper_pair andnot read.is_secondary and0 < read.template_length <= 400:
sizes.append(read.template_length)
bam.close()
sizes = np.array(sizes)
modal_size = np.bincount(sizes).argmax()
mono_frac = np.sum((sizes >= 150) & (sizes <= 180)) / len(sizes)
qc_pass = 150 <= modal_size <= 180and mono_frac > 0.3return {
'modal_size': modal_size,
'mononucleosome_fraction': mono_frac,
'qc_pass': qc_pass,
'message': 'Good cfDNA profile'if qc_pass else'Atypical fragment distribution'
}
Step 3a: Tumor Fraction Estimation (sWGS)
ichorCNA is a command-line script (Rscript scripts/runIchorCNA.R), NOT an importable runIchorCNA() function, and it is preceded by HMMcopy readCounter to bin the BAM. The ~3% tumor-fraction floor is an analytical limit of detection; below it, route to fragmentomics or methylation rather than trusting a low value (see liquid-biopsy/analytical-validation and liquid-biopsy/tumor-fraction-estimation).
# For shallow WGS data (0.1-1x coverage); GavinHaLab fork
readCounter --window 1000000 --quality 20 \
--chromosome "chr1,chr2,chr3,chr4,chr5,chr6,chr7,chr8,chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr20,chr21,chr22,chrX" \
sample.bam > sample.wig
Rscript scripts/runIchorCNA.R \
--id sample_id --WIG sample.wig \
--gcWig gc_hg38_1000kb.wig --mapWig map_hg38_1000kb.wig \
--centromere GRCh38.GCA_000001405.2_centromere_acen.txt \
--normalPanel HD_ULP_PoN_hg38_1Mb_normAutosomes_median.rds \ # GavinHaLab/ichorCNA extdata name; PoN build MUST match the gc/map/centromere build (the non-hg38 1Mb PoN is hg19)
--normal "c(0.5,0.6,0.7,0.8,0.9)" --ploidy "c(2,3)" --maxCN 7 \
--estimateNormal TRUE --estimatePloidy TRUE --estimateScPrevalence TRUE \
--outDir ichor_results/
# Tumor fraction = 1 - n in sample_id.params.txt
Clonal hematopoiesis (CHIP) is the dominant false-positive source in plasma: ~81.6% of cfDNA variants in controls and ~53.2% in cancer patients trace to white blood cells (Razavi 2019 Nat Med 25:1928). A gene-list filter is a weak fallback; the definitive control is sequencing matched buffy-coat/WBC DNA and subtracting any variant present there. See liquid-biopsy/ctdna-mutation-detection.
CHIP_GENES = ['DNMT3A', 'TET2', 'ASXL1', 'PPM1D', 'JAK2', 'SF3B1', 'SRSF2', 'TP53']
deffilter_chip(variants_df, wbc_variants=None, chip_genes=CHIP_GENES):
'''Subtract WBC-matched variants when available; else fall back to a CHIP gene list.'''if wbc_variants isnotNone:
# REF must be in the key. Left-aligned indels share (chrom, pos, alt): a 1bp deletion# (REF=AT, ALT=A) and a 2bp deletion (REF=ATT, ALT=A) collide, so a (chrom, pos, alt) key# would silently subtract a real somatic indel as CHIP.
key = ['chrom', 'pos', 'ref', 'alt']
wbc_keys = set(map(tuple, wbc_variants[key].itertuples(index=False, name=None)))
in_wbc = variants_df[key].apply(lambda r: tuple(r) in wbc_keys, axis=1)
return variants_df[~in_wbc], variants_df[in_wbc]
chip = variants_df[variants_df['gene'].isin(chip_genes)]
somatic = variants_df[~variants_df['gene'].isin(chip_genes)]
return somatic, chip
Step 5: Fragmentomics Analysis (Optional)
FinaleToolkit (MIT license, not DELFI software) exposes real hyphenated CLI subcommands and an underscored finaletoolkit.frag Python API; delfi GC-corrects the short/long ratio (raw ratios are dominated by GC and sequencing batch). DELFI is a methodology and a company, not a pip install-able tool.
# GC-corrected genome-wide DELFI profile and end-motif diversity.# delfi positionals: input chrom_sizes reference bins_file; GC correction is on by default.# --no-remove-nocov keeps all bins on non-hg19 references (the default removes two hardcoded hg19 no-coverage regions).
finaletoolkit delfi consensus.bam hg38.chrom.sizes hg38.2bit bins_100kb.bed -g gaps.bed --no-remove-nocov -o sample.delfi.bed
finaletoolkit end-motifs consensus.bam hg38.2bit -o sample.end_motifs.tsv
finaletoolkit mds sample.end_motifs.tsv
from finaletoolkit.frag import delfi # see liquid-biopsy/fragment-analysisdefrun_fragmentomics(bam_path, chrom_sizes, reference, bins_bed, gap_bed):
'''GC-corrected DELFI short/long profile (MDS comes from end_motifs().motif_diversity_score()).
Python positional order is (input, chrom_sizes, bins_file, reference_file) - note this differs
from the CLI order (input, chrom_sizes, reference, bins), so pass by keyword to be safe.'''return delfi(bam_path, chrom_sizes=chrom_sizes, bins_file=bins_bed,
reference_file=reference, gap_file=gap_bed)
Step 6: Longitudinal Tracking
import pandas as pd
import numpy as np
deftrack_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
Complete Pipeline Script
defrun_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)
ifnot frag_qc['qc_pass']:
print(f"WARNING: {frag_qc['message']}")
results['fragment_qc'] = frag_qc
# Step 3: Analysis based on data typeif sample_config['data_type'] == 'swgs':
# Tumor fraction estimation
results['tumor_fraction'] = run_ichorcna(preprocessed_bam)
elif sample_config['data_type'] == 'panel':
# Mutation detection. CHIP subtraction is not optional (rule 4): without matched WBC the# calls are presumptively CHIP-contaminated and the gene list is only a weak fallback.
variants = call_variants(preprocessed_bam, sample_config['bed_file'])
wbc = call_variants(sample_config['wbc_bam'], sample_config['bed_file']) if sample_config.get('wbc_bam') elseNone
somatic, chip = filter_chip(variants, wbc_variants=wbc)
results['variants'] = somatic
results['chip_variants'] = chip
# Step 4: Optional fragmentomicsif sample_config.get('run_fragmentomics'):
results['fragmentomics'] = run_fragmentomics(preprocessed_bam)
return results
Choosing the branch (tumor-naive vs tumor-informed)
Pipeline-level selection only; mechanism lives in the component skills.
Situation
Branch
Hand off to
Screening / no known tumor / MCED
tumor-NAIVE (fixed panel, sWGS, or methylation)
liquid-biopsy/methylation-based-detection
MRD/recurrence of a KNOWN tumor
tumor-INFORMED bespoke (16-50 clonal variants from tissue WES)
liquid-biopsy/longitudinal-monitoring
Tumor fraction from sWGS (0.1-1x)
ichorCNA (copy-number-based, floor ~3% TF)
liquid-biopsy/tumor-fraction-estimation
Low-VAF mutations from a deep panel
VarDict / smCounter2 on the UMI-consensus BAM
liquid-biopsy/ctdna-mutation-detection
Below mutation LoD, still need signal
fragmentomics (DELFI/end-motifs) or methylation
liquid-biopsy/fragment-analysis
Stating/trusting a sensitivity claim
LoB/LoD/LoD95/LoQ, per-locus vs panel-integrated
liquid-biopsy/analytical-validation
Tumor-informed reaches ppm by integrating across 16-50 loci (beats the single-locus Poisson floor) but is BLIND by design to new/resistance variants; tumor-naive sees any panel variant but is CHIP-dominated and error-limited.
Common Errors
Symptom
Cause
Fix
False negative on a real low-TF sample
Bad blood draw irreversibly diluted TF
Pre-analytic gate; cannot be rescued downstream
Residual C>T / G>T false positives
Single-strand consensus assumed to remove damage
Duplex, or UDG for deamination; flag the substitution spectrum
Money spent, no sensitivity gain
Sequenced past molecular saturation
Report unique-molecule coverage; invest in plasma volume / conversion efficiency
Blood clones reported as tumor
CHIP not subtracted (esp. TP53/PPM1D, both CHIP and driver)
Matched buffy-coat/WBC sequencing (gene list is a weak fallback)
Apparent burden halved or doubled
VAF reported as TF (or vice versa)
State VAF vs TF; TF ~ 2x VAF only for clonal-het-diploid; prefer CNA-based TF when CN is non-neutral
A "low" TF trusted below the assay floor
~3% ichorCNA LoD ignored
Below the floor = below detection; route to fragmentomics/methylation
Longitudinal "response" that is a batch shift
Tube/panel/pipeline changed between draws
Same tube, same panel, same pipeline across timepoints
References
Razavi P, Li BT, Brown DN, et al (2019) High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants. Nature Medicine 25:1928-1937. DOI 10.1038/s41591-019-0652-7. (CHIP dominance of cfDNA variants.)
Adalsteinsson VA, Ha G, Freeman SS, et al (2017) Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nature Communications 8:1324. DOI 10.1038/s41467-017-00965-y. (ichorCNA; ~3% TF LoD.)
Schmitt MW, Kennedy SR, Salk JJ, et al (2012) Detection of ultra-rare mutations by next-generation sequencing. PNAS 109:14508-14513. DOI 10.1073/pnas.1208715109. (duplex error floor.)
Reinert T, Henriksen TV, Christensen E, et al (2019) Analysis of plasma cell-free DNA by ultradeep sequencing in patients with stages I to III colorectal cancer. JAMA Oncology 5:1124-1131. DOI 10.1001/jamaoncol.2019.0528. (tumor-informed bespoke MRD.)
