Analysis Workflow
Family and Trio Analysis
Family context can change the evidence available in a rare Mendelian case. Folklore analyses a complete proband-mother-father trio to identify de novo candidates, phase compound heterozygous pairs, and assess segregation within the limits of a three-member pedigree.
The three member gVCFs are joint-genotyped into one normalized call set. The proband is classified through the standard ACMG workflow, and the family module records inheritance evidence without replacing the upstream classification.
Clinical Positioning
Family analysis adds evidence in three common clinical scenarios.
Three Scenarios
VUS in proband, de novo confirmed by parents. A variant may remain a VUS after proband classification. Trio-supported de novo evidence identifies it for criterion review; final ACMG handling remains explicit and reviewable.
Two heterozygous variants in a recessive gene, phased in trans. A pair of variants in a recessive disease gene, one inherited from each parent, can form a biallelic loss-of-function finding. Parental phasing distinguishes it from an in-cis pair.
Variant segregates with disease. Segregation strength depends on informative meioses. A complete trio has a theoretical LOD ceiling near 0.3, below the configured Supporting threshold of 0.5. Extended pedigrees are required for PP1 evidence.
Each of these is a different inheritance algorithm with different data requirements. The Family Service runs all three on the same trio data and presents the combined evidence to the geneticist alongside the upstream ACMG classification.
Supported Composition
The current production workflow requires a complete trio: proband, mother, and father.
Complete Trio
Proband, mother, and father represented in the same joint-genotyped call set. This supports parental-origin assessment, relationship QC, de novo detection, and in-trans compound heterozygous phasing.
Capabilities: De novo detection, compound heterozygous phasing, segregation context, and PLINK relationship QC.
Pipeline Architecture
The workflow runs sequentially from trio joint genotyping to proband classification and inheritance analysis. Runtime depends on input size, sequencing mode, and available compute resources.
Stage 1
Joint Genotyping
GLnexus joint-genotypes the proband, mother, and father gVCFs into one normalized multi-sample VCF. The same call set becomes the source for both proband classification and trio inheritance analysis.
Stage 2
Proband Classification
A proband-only VCF is extracted from the normalized joint VCF and processed through the standard Variant Analysis workflow. ACMG classification belongs to the proband.
Stage 3
Trio Evidence Construction and QC
Joint-VCF genotypes are combined with the proband classified DuckDB to build the trio evidence store. PLINK evaluates declared parent-child relationships, possible sample swaps, duplicate samples, and parental relatedness.
Stage 4
Inheritance Analysis and Reporting
De novo detection, compound heterozygous phasing, segregation scoring, inheritance-pattern derivation, and the clinical-grade reporting overlay run on the trio evidence store. Results remain separate from the proband ACMG classification.
Sample Quality Control
Before any inheritance analysis, the service verifies that the family relationships in the metadata match the genetic relationships in the data. Sample-swap, accidental duplication, and unreported consanguinity all corrupt downstream inheritance calls if undetected.
PLINK Identity-by-Descent Analysis
PLINK analyses the joint multi-sample VCF produced by the trio workflow. Biallelic SNPs provide pairwise Identity-by-Descent estimates used to check the declared parent-child relationships and identify unexpected relatedness or duplicate samples.
Sample Swap
When the IBD between proband and a declared parent does not match the expected parent-offspring relationship.
Consanguinity
When the IBD between the two declared parents is elevated above the expected unrelated-individuals baseline.
Duplicate Sample
When two declared family members are genetically identical, typically an upload error rather than a real biological scenario.
Alerts surface in the case summary alongside the inheritance evidence. The geneticist sees QC results before reading variant calls.
De Novo Detection
Identifies variants present in the proband but absent in both parents, with explicit confidence tiers reflecting the strength of supporting evidence.
Explicit Confidence States
Technically supported candidates are reported as high or low confidence. Excluded and not-applicable are control states, while an intermediate tier is reserved for future use. Confidence describes the de novo inference, not pathogenicity.
Chromosome-Presence Gate
When a parent genotype is NULL, the system distinguishes confident inferred homozygous-reference (the chromosome was sequenced and called as reference) from suspicious absence (the chromosome may not have been adequately covered). This is a key clinical safety mechanism preventing false de novo calls from coverage gaps.
Gender-Aware Chromosome Exceptions
Biologically expected NULLs are recognised as such: chromosome Y in a female parent, mitochondrial DNA in a father. These do not trigger confidence downgrades. Gender of each family member is part of the input.
Clean ACMG Classification Preserved
The de novo annotation is added on top of the upstream ACMG classification. The original Pathogenic / Likely Pathogenic / VUS / Likely Benign / Benign assignment from variant analysis is preserved verbatim. Geneticists see both: the classification, and the new family-aware evidence.
Compound Heterozygous Phasing
Identifies pairs of heterozygous coding or splicing variants in the same gene that may form biallelic loss of function. Phasing requires parental origin information and distinguishes in-trans (biallelic) from in-cis (single allele) configurations.
Phasing from Parental Origin
When parental genotypes are available, the service determines whether a pair of heterozygous variants in the same gene came from different parents (in trans, biallelic) or from the same parent (in cis, single-allele). Only in-trans pairs constitute genuine biallelic loss-of-function.
Multi-Partner Detection
A variant may participate in multiple candidate compound heterozygous pairs. The service records all candidate pairs in the gene for geneticist review.
Coding and Splicing Restriction
Compound heterozygous candidacy is restricted to coding and splicing consequences. Synonymous, intronic, and regulatory variants are correctly excluded, they do not constitute biallelic loss of function regardless of zygosity.
Symmetric Annotation
When variants A and B form a compound heterozygous pair, both rows in the trio table are annotated. The geneticist can land on either variant and immediately see the partner.
Segregation Scoring
Per-variant likelihood-ratio LOD scores using the Jarvik framework as specified by ClinGen SVI 2021. Mapped to ClinGen-aligned evidence bands with explicit acknowledgement of trio data limits.
ClinGen SVI 2021 Framework
Per-variant LOD scores are computed using the Jarvik likelihood-ratio framework as specified by the ClinGen Sequence Variant Interpretation Working Group. The methodology is published, peer-reviewed, and explicitly designed for clinical use.
LOD Evidence Bands
Computed LOD values map to indeterminate, supporting, moderate, strong, or very strong bands. Not-applicable is a separate state used when no segregation score is computed.
Trio LOD Ceiling
A complete trio contributes at most two informative meioses and has a theoretical LOD ceiling near 0.3. Because the configured Supporting band begins at 0.5, trio-only segregation normally remains indeterminate. Extended pedigrees are required for PP1 evidence.
Hypothesis-Aware
When the inheritance hypothesis is unknown, the segregation phase explicitly writes not_applicable rather than emitting a spurious LOD = 0 result. This prevents misinterpretation of "no evidence calculated" as "evidence against".
Feasibility Planning
Before the pipeline runs, the service evaluates which inheritance phases are feasible given the family composition and inheritance hypothesis. Feasibility flags and a free-form rationale are persisted alongside the trio analysis record, providing clinical audit trail.
| Flag | Meaning |
|---|---|
| de_novo_feasible | True when both parents are sequenced and an inheritance hypothesis consistent with de novo (autosomal dominant or sporadic) is plausible. False otherwise, for example, a duo without the affected parent. |
| compound_het_feasible | True when at least one parent is sequenced and the inheritance hypothesis is autosomal recessive. Phasing requires parental origin information. |
| segregation_feasible | True when at least two family members with affected status are present. Trio with affected proband and one affected parent qualifies; singleton does not. |
| plan_rationale | Free-form structured explanation of why each phase was planned as feasible or not. Provides clinical audit trail, the geneticist can verify which inheritance evidence was looked for and which was not, and why. |
Inputs and Outputs
Three member gVCFs and pedigree metadata produce a normalized joint VCF, a proband classification, and a separate trio inheritance evidence store.
Inputs from the Pipeline
Proband, mother, and father gVCFs from a compatible calling workflow
One normalized joint multi-sample VCF generated by GLnexus
Proband classified_variants.duckdb generated from the extracted proband VCF
Reference genome and contig mapping required for normalization
Inputs from the Geneticist
Complete trio composition: proband, mother, and father
Per-member sample identifier, sex, family role, and affected status
Inheritance hypothesis and phenotype specificity when available
Outputs for the Geneticist
De novo annotation per variant: confidence tier and supporting evidence
Compound heterozygous pairs: phase determination, partner variants, multi-partner flags
Segregation evidence: per-variant LOD score and ClinGen-aligned band
Sample QC summary: IBD analysis results, alerts for sample-swap or consanguinity
Feasibility and rationale: which phases were planned as feasible and why
Evidence summary JSON denormalised on the trio analysis record for fast retrieval
Outputs for Downstream Services
Persistent trio_variants DuckDB consumed by the AI Service for the family analysis report
Per-case data available to cohort-level analytics for population work
Standards and Boundaries
The methods below govern de novo analysis, phasing, segregation, sample quality control, and clinical review.
ACMG/AMP
Variant classification follows ACMG/AMP 2015 with subsequent ClinGen specifications. Performed upstream by the Variant Analysis Service. The Family Service consumes that classification and adds family-aware evidence on top, it does not reclassify.
Reference: Richards et al., Genetics in Medicine, 2015, PMID: 25741868
ClinGen SVI 2021 Segregation
Segregation LOD scoring follows the ClinGen Sequence Variant Interpretation Working Group framework for clinical use. The Jarvik likelihood-ratio methodology is published and validated.
Reference: ClinGen Sequence Variant Interpretation Working Group, 2021
Jarvik Likelihood Framework
Per-variant likelihood-based segregation LOD computation.
Reference: Jarvik and Browning, AJHG, 2016, PMID: 27374771
PLINK 1.9
Sample identity-by-descent analysis for sample-swap, consanguinity, and duplicate-sample detection.
Reference: Chang et al., GigaScience, 2015, PMID: 25722852
Joint-Genotyped Trio Workflow
The member gVCFs are joint-genotyped inside Folklore. The proband classification and trio inheritance table derive from the same normalized call set, preventing cross-sample call-set drift.
Reporting Boundary
Family and Trio Analysis adds inheritance annotations, confidence tiers, evidence bands, and feasibility rationale to the upstream classification. A qualified clinical geneticist decides how those results affect the case.
Data Residency
The service runs within the Folklore platform on EU-based infrastructure compliant with GDPR Article 9 and 1+MG technical requirements. No family genomic data leaves the platform during analysis.
What Sets It Apart
Inheritance logic, feasibility checks, algorithm versioning, and sample quality control define the family workflow.
One call set for classification and inheritance
The proband classification and parental genotype evidence derive from the same normalized joint VCF. Independently called missing rows are not treated as confirmed reference genotypes.
Three inheritance algorithms in one pipeline
De novo detection, compound heterozygous phasing, and segregation scoring run sequentially on the same trio data. The geneticist sees the full inheritance picture in one place.
Trio segregation limit stated explicitly
The theoretical trio-only LOD ceiling is near 0.3, below the configured Supporting threshold of 0.5. Folklore presents this as limited segregation context rather than PP1-strength evidence.
Chromosome-presence gate
Distinguishes confident inferred homozygous-reference from suspicious absence when parent genotype is NULL. A clinical safety mechanism that prevents false de novo calls from coverage gaps.
Gender-aware exceptions
chrY NULL for a female parent and chrM NULL for a father are biologically expected, not coverage gaps. The system recognises this and does not penalise confidence.
Feasibility flags with rationale
Each analysis records which phases were feasible, which ran, and why, allowing the geneticist to review the planned evidence search.
Atomic phase semantics
If a phase fails, later phases do not run. The service records the failure stage and preserves partial state for operator inspection.
Sample QC built in
PLINK IBD analysis runs as part of the standard pipeline when joint VCF is available. Sample-swap, consanguinity, and duplicate-sample alerts surface before clinical interpretation.
See Family Analysis in Practice
Follow de novo detection, compound heterozygous phasing, and segregation scoring for a trio.