Documentation / Family Analysis / De Novo Variants

De Novo Variant Analysis in Trio Sequencing

A de novo variant is present in the proband and absent from both parents at the same genomic site. In a clinical trio, this pattern can provide inheritance evidence that is unavailable from proband-only sequencing.

Folklore identifies technically supported de novo candidates. De novo origin does not establish pathogenicity, and a confidence state does not by itself determine whether PS2 or PM6 should be applied.

Current Production Scope

De novo analysis requires a complete trio: proband, mother, and father. The three samples are evaluated from one normalized multi-sample call set. When separate member gVCFs are supplied, they are joint-genotyped before inheritance analysis.

Why Joint Genotyping Matters

Independently called files can differ because of caller thresholds, normalization, or representation of the same allele. A missing row in a parent-only variant file is not equivalent to a confirmed reference genotype.

Folklore evaluates one multi-sample VCF for the proband, mother, and father. The proband classification and the parental genotype evidence are derived from that shared call set, reducing ambiguity at candidate sites.

Candidate Detection

The detector begins with variants present in the proband. For each site, it reads the maternal and paternal genotypes from the joint VCF and checks whether both parents are reference at that position.

Genotype state alone is not enough. The detector also evaluates the configured parental depth and genotype-quality requirements before assigning a confidence state.

De Novo Confidence States

State	Technical Meaning	Clinical Handling
High confidence	The candidate is present in the proband, both parents have reference genotypes at the site, and the configured genotype-quality and depth requirements are satisfied.	Technically supported de novo origin. Pathogenicity and ACMG criterion use still require clinical review.
Low confidence	The inheritance pattern is compatible with de novo origin, but at least one parental genotype is a no-call or does not meet the configured depth and genotype-quality requirements.	Retained for review with the reason for reduced confidence.
Excluded	The alternate allele is present in at least one parent, so de novo origin is not supported by the trio genotypes.	Not reported as a de novo candidate.
Not applicable	The required trio or inheritance conditions are not available for a valid de novo assessment.	No de novo evidence is assigned.

Explicit Genotypes and No-Calls

The joint call set represents each parent at an evaluated site as reference, heterozygous, homozygous alternate, or no-call. A no-call is not treated as a confirmed reference genotype.

When a parental genotype is a no-call, or a reference call does not meet the configured quality requirements, the candidate cannot receive the high-confidence state. If either parent carries the alternate allele, de novo origin is excluded.

Clinical-Grade Reporting Checks

The reporting layer narrows the technically supported candidate set for clinical review. It does not rerun the detector and does not independently change ACMG criterion semantics.

High-confidence technical support from the trio detector.

Variant-level and proband quality status.

Population-frequency limits used by the reporting profile.

Predicted molecular consequence.

Splice-signal rescue when the configured SpliceAI threshold is met.

PS2 and PM6 Remain Review Decisions

ACMG/AMP uses PS2 and PM6 for de novo evidence under different levels of confirmation. Their application depends on more than the observed trio genotype pattern. The reviewer must consider parentage, phenotype consistency, gene-disease mechanism, penetrance, and the quality of the supporting data.

Folklore records the detector result and its technical basis. A qualified genetics professional decides whether the available evidence satisfies the requirements of a criterion and what strength is justified.

Mosaicism and Other Limits

Low-level parental mosaicism may fall below the detection threshold of the source assay. A variant can therefore appear absent in blood-derived parental data even when mosaicism is present in another tissue or at a lower allele fraction.

Mapping ambiguity, segmental duplication, low-complexity sequence, allelic imbalance, and low coverage can also weaken a de novo inference. The confidence state records the available technical support; it cannot remove these assay-level limits.

What the Reviewer Receives

Each candidate carries its proband and parental genotypes, quality measurements, confidence state, exclusion or downgrade reason, population and consequence context, and reporting status. The original proband ACMG classification remains separately visible.