Version Compatibility
Reference examples tested with: Ensembl VEP 111+, SnpEff 5.2+, VarDict 1.8+, pandas 2.2+, pysam 0.22+
Before using code patterns, verify installed versions match. If versions differ:
- Python:
pip show <package> then help(module.function) to check signatures
- 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.
ctDNA Mutation Detection
"Detect mutations in my cfDNA sample" → Identify somatic variants at low allele fractions (0.1-1%) from cell-free DNA using error-suppressed consensus calling and specialized callers.
- CLI:
vardict-java for low-VAF variant calling from cfDNA
Detect somatic mutations in cfDNA at low variant allele fractions.
Input Requirements
| Requirement | Specification |
|---|
| Data type | Targeted panel or WES (NOT sWGS) |
| Depth | >= 1000x for low VAF detection |
| UMIs | Highly recommended for < 1% VAF |
| Input | Preprocessed BAM (UMI consensus if available) |
VAF Detection Limits
| VAF Range | Reliability | Notes |
|---|
| > 1% | Reliable | Standard callers work |
| 0.5-1% | Good with UMIs | Requires error suppression |
| 0.1-0.5% | Challenging | Needs deep UMI consensus |
| < 0.1% | Unreliable | Near noise floor |
VarDict for High Sensitivity (Ensembl VEP 111+)
# VarDict is highly sensitive for low VAF
# Use on UMI-consensus BAM for best results
vardict-java \
-G reference.fa \
-f 0.005 \ # Min VAF 0.5%
-N sample_id \
-b sample.bam \
-c 1 -S 2 -E 3 -g 4 \
regions.bed | \
teststrandbias.R | \
var2vcf_valid.pl \
-N sample_id \
-E \
-f 0.005 \
> sample.vcf
Python Implementation
import subprocess
import pandas as pd
import pysam
def call_variants_vardict(bam_file, reference, bed_file, output_vcf, min_vaf=0.005, min_depth=100):
'''
Call variants with VarDict.
Args:
bam_file: UMI-consensus BAM preferred
reference: Reference FASTA
bed_file: Target regions BED
output_vcf: Output VCF path
min_vaf: Minimum VAF (0.005 = 0.5%)
min_depth: Minimum read depth
'''
sample_id = bam_file.split('/')[-1].replace('.bam', '')
cmd = f'''
vardict-java \
-G {reference} \
-f {min_vaf} \
-N {sample_id} \
-b {bam_file} \
-c 1 -S 2 -E 3 -g 4 \
{bed_file} | \
teststrandbias.R | \
var2vcf_valid.pl \
-N {sample_id} \
-E \
-f {min_vaf} \
> {output_vcf}
'''
subprocess.run(cmd, shell=True, check=True)
return output_vcf
def filter_ctdna_variants(vcf_file, chip_genes=None):
'''
Filter ctDNA variants, removing CHIP.
CHIP genes commonly mutated in elderly:
DNMT3A, TET2, ASXL1, PPM1D, TP53, SF3B1, etc.
'''
if chip_genes is None:
chip_genes = ['DNMT3A', 'TET2', 'ASXL1', 'PPM1D', 'JAK2',
'SF3B1', 'SRSF2', 'TP53', 'CBL', 'BCOR']
import vcfpy
reader = vcfpy.Reader.from_path(vcf_file)
somatic = []
chip = []
for record in reader:
gene = record.INFO.get('GENE', [''])[0]
if gene in chip_genes:
chip.append(record)
else:
somatic.append(record)
print(f'Somatic variants: {len(somatic)}')
print(f'Potential CHIP variants: {len(chip)}')
return somatic, chip
UMI-VarCal for Best Specificity (Ensembl VEP 111+)
def call_with_umi_varcal(bam_file, reference, bed_file, output_vcf, min_vaf=0.005):
'''
UMI-VarCal: Best specificity with UMI data.
'''
subprocess.run([
'umi-varcal',
'--bam', bam_file,
'--ref', reference,
'--bed', bed_file,
'--out', output_vcf,
'--min-vaf', str(min_vaf),
'--min-alt-reads', '3',
'--min-depth', '100'
], check=True)
Variant Annotation (Ensembl VEP 111+)
def annotate_ctdna_variants(vcf_file, output_vcf):
'''Annotate variants with clinically relevant information.'''
# Use VEP or snpEff for annotation
subprocess.run([
'vep',
'--input_file', vcf_file,
'--output_file', output_vcf,
'--format', 'vcf',
'--vcf',
'--cache',
'--canonical',
'--protein',
'--sift', 'b',
'--polyphen', 'b',
'--af_gnomad'
], check=True)
Tracking Known Mutations
Goal: Quantify the variant allele fraction of specific known mutations across serial liquid biopsy samples for minimal residual disease monitoring.
Approach: For each target mutation, pileup reads at the variant position, count reference and alternative alleles, and compute VAF with depth statistics.
def track_specific_mutations(bam_file, mutations, min_depth=100):
'''
Track specific known mutations across samples.
Useful for MRD monitoring.
Args:
bam_file: Aligned BAM
mutations: List of (chrom, pos, ref, alt) tuples
'''
import pysam
bam = pysam.AlignmentFile(bam_file, 'rb')
results = []
for chrom, pos, ref, alt in mutations:
counts = {'ref': 0, 'alt': 0, 'other': 0}
for pileupcolumn in bam.pileup(chrom, pos-1, pos):
if pileupcolumn.pos != pos - 1:
continue
for read in pileupcolumn.pileups:
if read.is_del or read.is_refskip:
continue
base = read.alignment.query_sequence[read.query_position]
if base == ref:
counts['ref'] += 1
elif base == alt:
counts['alt'] += 1
else:
counts['other'] += 1
total = counts['ref'] + counts['alt'] + counts['other']
vaf = counts['alt'] / total if total > 0 else 0
results.append({
'chrom': chrom, 'pos': pos, 'ref': ref, 'alt': alt,
'depth': total, 'alt_count': counts['alt'], 'vaf': vaf
})
bam.close()
return pd.DataFrame(results)
Related Skills
- cfdna-preprocessing - Preprocess with UMI consensus
- tumor-fraction-estimation - Estimate overall tumor burden
- longitudinal-monitoring - Track mutations over time
- variant-calling/variant-calling - General variant calling concepts