Trio Variant Analysis Methodology
Folklore analyses a complete proband-mother-father trio. The member gVCFs are joint-genotyped into one normalized call set, the proband is classified through the standard ACMG workflow, and family-aware evidence is computed from the same joint genotypes.
The family module records inheritance evidence alongside the proband classification. It does not replace clinical review, and the clinical-grade reporting overlay does not independently change PS2 criterion semantics.
Contents
Pipeline overview
Seven-stage production flow from complete-trio validation and joint genotyping to proband classification, relationship QC, inheritance analysis, and reviewable evidence delivery.
Validate the complete trio
The workflow requires exactly one proband, one mother, and one father with compatible gVCF inputs and recorded pedigree metadata.
Joint-genotype the three gVCFs
GLnexus creates one normalized multi-sample VCF. Caller-specific preset selection and contig normalization are resolved before downstream processing.
Extract and classify the proband
A proband-only variant VCF is extracted from the joint call set and sent through Variant Analysis. ACMG classification and variant annotations are produced for the proband only.
Build the trio evidence store
Every joint-VCF site is written to the trio DuckDB with proband, father, and mother genotypes. Proband annotation and ACMG fields are joined from the classified proband database.
Run relationship quality control
PLINK computes pairwise Identity-by-Descent estimates to evaluate declared parent-child relationships, parental relatedness, and possible duplicate samples.
Compute inheritance evidence
De novo confidence, compound heterozygous phase, segregation scores, and categorical inheritance patterns are derived from the trio genotypes.
Apply the reporting overlay
Quality, population-frequency, consequence, and splice-signal filters identify the clinically focused reporting subset. Detector outputs and ACMG criterion semantics remain separately auditable.
Family compositions
The current production workflow accepts one composition: a complete proband-mother-father trio.
| Composition | Requirement | Analysis scope |
|---|---|---|
| Complete trio | Proband + mother + father | Joint genotyping, proband classification, PLINK relationship QC, de novo assessment, compound heterozygous phasing, and trio segregation context |
Duo, sibling-only, extended-pedigree, and multiple-proband workflows are not part of the current production trio pipeline.
Sample quality control (PLINK IBD)
Before inheritance analysis begins, Folklore verifies that declared family relationships are consistent with the genomic data. Sample swap detection is a critical clinical safety check: a mislabelled sample produces invalid inheritance evidence regardless of algorithmic correctness downstream.
Method
PLINK 1.9 computes pairwise Identity-by-Descent (IBD) proportions across all declared family members using biallelic single-nucleotide variants with minor allele frequency at or above 0.05 and missingness at or below 5 percent. Expected IBD for parent-child pairs is approximately 0.50; expected IBD for unrelated parents is below 0.10. Deviations beyond defined thresholds raise alerts that are surfaced to the clinical reviewer.
Alert types
Sample swap
Declared parent-child pair with PI_HAT below 0.40. Pipeline fails; clinical reviewer must validate sample labelling before re-running analysis.
Duplicate sample
Declared parent-child pair with PI_HAT above 0.70, indicating possible sample duplication or extreme consanguinity. Pipeline proceeds; warning surfaced.
Consanguinity
Declared unrelated parents with PI_HAT above 0.125 (third-degree relatives or closer). Pipeline proceeds; warning surfaced for clinical interpretation.
De novo variant detection
De novo variants arise in the germline of a parent or in the post-zygotic period of the proband and are absent from both parental genomes. Their identification relies on confident parental hom_ref calls at the variant position. Folklore applies a tiered confidence assessment that surfaces the strength of de novo inference rather than producing a binary call.
ACMG criteria triggered
ACMG/AMP 2015 (Richards et al., PMID: 25741868) defines PS2 (Strong) for confirmed de novo variants with verified maternity and paternity, and PM6 (Moderate) for assumed de novo variants without confirmation. The ClinGen Sequence Variant Interpretation Working Group specification (Biesecker & Harrison 2018, PMID: 29543229) refined these criteria, distinguishing confidence levels and providing application guidance.
Applicability
De novo detection requires a complete proband-mother-father trio. Both parental genotypes must be available from the same joint-genotyped call set before de novo confidence can be assessed.
Tiered confidence assessment
The detector emits high- and low-confidence candidates, plus excluded and not-applicable control states. Confidence reflects technical support for de novo origin, not pathogenicity. The clinical-grade layer is a reporting refinement and does not independently alter PS2 semantics.
High confidence
Both parents meet the configured genotype and coverage requirements with reference genotypes at the candidate position. The result is a technically supported de novo candidate for clinical and ACMG review.
Low confidence
At least one parent has sub-threshold coverage, genotype quality below recommended levels, or chromosome representation that cannot be confirmed. Variant is surfaced for manual reviewer interpretation; PS2 is not auto-applied.
Excluded
Variant present in at least one parent (parent genotype not hom_ref). De novo origin is explicitly disconfirmed.
Chromosome-presence gate
The joint multi-sample VCF provides an explicit genotype state for each trio member at every retained site. Folklore distinguishes confirmed reference genotypes, low-quality calls, no-calls, and biologically expected chromosome exceptions before assigning de novo confidence.
chrY for female parents
Female parents biologically lack chrY. Absence of chrY variants in maternal data is expected, not a coverage gap. The chromosome-presence gate accommodates this exception so chrY proband variants are not unnecessarily downgraded.
chrM for paternal contribution
Mitochondrial DNA is maternally inherited; paternal mitochondria are degraded during fertilisation. Absence of chrM variants in paternal data is expected. The gate excludes paternal chrM coverage from confidence calculations to reflect this biology.
Exclusions
The following situations are excluded from de novo interpretation or trigger downgrades to lower confidence tiers:
Variants present in at least one parent are explicitly excluded; de novo origin is disconfirmed.
Mosaic de novo variants below detection threshold in parental samples may be incorrectly classified as de novo. Clinical interpretation should consider mosaicism when phenotype suggests it.
Regions with known low-quality parental genotyping (low complexity, segmental duplications) may produce sub-threshold coverage and downgrade confidence.
Variants on parent-only positions (present in a parent but not in the proband) are not de novo candidates by definition and are excluded from de novo classification.
Compound heterozygosity
Compound heterozygosity is a recessive mechanism in which the proband carries two distinct heterozygous pathogenic variants in the same gene, each inherited from a different parent. Trio inheritance can establish whether the variants are in trans.
Biological rationale
In autosomal recessive disorders, pathogenicity requires biallelic disruption. Two heterozygous variants in trans configuration (one from each parent) result in functional knock-out of the gene, just as a homozygous loss-of-function variant would. In solo proband whole-exome or whole-genome analysis, phase cannot be determined without parental information or long-read sequencing. Trio data resolves this gap.
ACMG criterion triggered
ACMG/AMP 2015 (Richards et al., PMID: 25741868) defines PM3 (Moderate): "Detected in trans with a pathogenic variant for recessive disorders." Compound heterozygous candidates in trans configuration with a confidently classified partner contribute to PM3 application.
Phasing methodology
If the proband is heterozygous for variant A and variant B in the same gene, and variant A is inherited from the mother (mother heterozygous, father reference) while variant B is inherited from the father (mirror configuration), then A and B are in trans configuration. This is a definitive compound heterozygous setup. When parental genotype data is incomplete (sub-threshold coverage, missing chromosome representation), Folklore surfaces the candidate at lower confidence with explicit reason rather than making a binary determination.
Variant consequence filtering
Candidate variants are restricted to clinically relevant consequence types. Intronic variants distant from splice sites, synonymous variants without splice impact, and UTR-adjacent variants are excluded from compound heterozygosity assessment.
Included consequences
missense_variant
stop_gained
stop_lost
frameshift_variant
inframe_insertion
inframe_deletion
splice_donor_variant
splice_acceptor_variant
splice_region_variant
start_lost
initiator_codon_variant
Excluded consequences
intron_variant
downstream_gene_variant
upstream_gene_variant
synonymous_variant
A pragmatic cap of 50 candidate variants per gene protects against combinatorial explosion in large genes such as TTN, MUC16, and SYNE1. Variants are ranked by ACMG severity (P > LP > VUS > LB > B) and confidence score before the cap is applied, so the highest-priority candidates are retained.
Multi-partner candidates
When a variant has multiple potential compound heterozygous partners in the same gene, Folklore selects a single primary partner deterministically and flags the multi-partner state for clinical reviewer awareness. The reviewer sees that additional partner variants exist and can examine each pairing individually. This avoids hidden ambiguity and preserves clinical decision authority.
Cosegregation scoring (LOD)
Cosegregation of a candidate variant with phenotype across family members provides quantitative evidence for pathogenicity. Folklore implements the classical Jarvik & Browning 2016 LOD framework (PMID: 27236918) with phenotype specificity modulation per ClinGen Sequence Variant Interpretation Working Group 2021 recommendations.
LOD formula
Per-variant LOD scores are computed using the logarithm-of-the-odds framework as described in Jarvik & Browning 2016. Each informative meiotic event consistent with the inheritance hypothesis contributes positively; each violation contributes negatively; uninformative meioses contribute zero. The mathematical formulation is described in detail in the cited reference and is not reproduced here.
Supported inheritance hypotheses
Folklore computes LOD scores for the following modes of inheritance. When the inheritance hypothesis is unspecified, segregation scoring is not performed and per-variant strength is reported as not-applicable; this is documented per Option A of the implementation specification.
Five-band evidence strength scale
The continuous LOD score is mapped to one of five evidence strength bands per ClinGen SVI 2021 recommendations. Each band corresponds to an ACMG modifier strength applied to the PP1 (cosegregation) criterion.
| Strength band | LOD range | ACMG points | ACMG strength |
|---|---|---|---|
| indeterminate | [0.0, 0.5) | 0 | -- |
| supporting | [0.5, 2.0) | 1 | PP1_Supporting |
| moderate | [2.0, 3.0) | 2 | PP1_Moderate |
| strong | [3.0, 5.0) | 4 | PP1_Strong |
| very_strong | [5.0, inf) | 8 | PP1_VeryStrong |
Phenotype specificity multiplier
Per ClinGen SVI 2021, the LOD score is dampened by a multiplier reflecting phenotype specificity. Highly specific phenotypes (syndromic, characteristic of a single gene) receive full weight; broad phenotypes (common, multiple genes possible) receive half weight; unspecified phenotypes receive quarter weight as a conservative default.
specific
x1.0
broad
x0.5
unspecified
x0.25
Trio-only mathematical ceiling
A complete trio contributes at most two informative meioses and has a theoretical LOD ceiling near 0.3. The configured Supporting band begins at 0.5, so trio-only results normally remain indeterminate. Moderate, strong, and very strong PP1 evidence require an extended pedigree, outside the current production scope.
Evidence summary for clinical review
After all phases complete, Folklore delivers a structured evidence summary alongside the per-variant inheritance annotations. The clinical reviewer receives the following information for each completed analysis:
Number of de novo candidate variants per confidence tier (high, low, excluded counts).
Number of compound heterozygous candidate pairs evaluated, plus per-gene breakdown of variants with assigned partners and multi-partner cases.
LOD distribution across strength bands (indeterminate, supporting, moderate, strong, very strong counts), maximum LOD observed, and mean LOD across scored variants.
PLINK IBD quality control results when joint VCF was provided: per-pair PI_HAT values, raised alerts (sample swap, duplicate sample, consanguinity), and PLINK version for audit.
Algorithm versions used for this specific analysis run, recorded immutably so the clinical reviewer always knows which exact version produced each result.
Methodology architecture
Two principles shape the production workflow: one normalized joint call set for classification and inheritance, and immutable algorithm-version recording for traceability.
Joint genotyping before inheritance analysis
Folklore joint-genotypes the three member gVCFs with GLnexus. The proband-only classification input and the trio inheritance table derive from the same normalized multi-sample call set. This prevents independent call-set differences from being interpreted as inheritance.
Write-once algorithm versioning
Algorithm version labels for the joint caller, de novo algorithm, segregation algorithm, and sample QC algorithm are recorded when an analysis is created and cannot be modified afterward. A completed analysis therefore identifies the algorithm version used for each result, supporting reconstruction and audit after platform upgrades.
Reference tools and databases
PLINK
Identity-by-Descent relationship quality control
Source: Purcell Lab, Harvard
Distinctive Folklore features
What Folklore adds beyond the public standards. Each feature is publicly documented; the value is in disciplined integration, not in algorithmic novelty hidden from clinical scrutiny.
Shared call set across classification and inheritance
The proband ACMG classification and parental genotype evidence derive from one normalized joint VCF, preserving positional and allelic consistency across the complete trio.
Tiered de novo confidence assessment
De novo confidence records the technical strength of parental genotype evidence. The clinical-grade subset is a reporting overlay; final ACMG criterion handling remains explicit and reviewable.
Chromosome-aware safeguards
The chromosome-presence gate distinguishes confident hom_ref inference from suspicious absence by verifying parental data coverage on the candidate chromosome. Sex-aware exceptions (chrY for female parents, chrM for paternal contribution) prevent false confidence downgrades arising from biologically expected absence rather than coverage gaps.
LOD ceiling disclosure
The complete trio has a theoretical LOD ceiling near 0.3, below the configured Supporting threshold of 0.5. Extended pedigrees are required before PP1 evidence strength can be reached.
Write-once algorithm versioning for regulatory reproducibility
Algorithm version labels are recorded immutably at analysis creation. Subsequent platform upgrades do not retroactively modify completed analyses. A clinical reviewer always knows which exact version produced a given result, supporting both scientific reproducibility and accreditation audit requirements.
Limitations
The following limitations bound the interpretation of Family Analysis results.
The production workflow requires a complete proband-mother-father trio. Duo, sibling-only, and extended-pedigree workflows are not currently supported.
Trio-only segregation has a theoretical LOD ceiling near 0.3, below the configured Supporting threshold of 0.5. PP1 evidence requires an extended pedigree.
Mosaic de novo variants below the detection threshold in a parental sample may appear absent and require clinical review.
Compound heterozygous findings depend on reliable parental genotypes and establish candidate phase, not the clinical significance of both alleles.
The clinical-grade reporting layer narrows technically supported candidates for review but does not independently change PS2 trigger semantics.
Structural variants and copy-number variants are outside the current small-variant trio workflow.
Results must be interpreted with phenotype, family history, and other clinical evidence by a qualified genetics professional.
Methodology scope
The public methodology describes the current production workflow without binding the page to a transient software release number.
Complete proband-mother-father trio input.
GLnexus joint genotyping and normalized joint VCF.
Proband ACMG classification from the joint call set.
PLINK Identity-by-Descent relationship quality control.
De novo and compound heterozygous candidate detection.
Explicit trio-only segregation ceiling.
Separate clinical-grade reporting overlay.
References
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
Genetics in Medicine. 2015;17(5):405-424.
PMID: 25741868Biesecker LG, Harrison SM; ClinGen Sequence Variant Interpretation Working Group. The ACMG/AMP reputable source criteria for the interpretation of sequence variants.
Genetics in Medicine. 2018;20(12):1687-1688.
PMID: 29543229Abou Tayoun AN, Pesaran T, DiStefano MT, Oza A, Rehm HL, Biesecker LG, Harrison SM. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion.
Human Mutation. 2018;39(11):1517-1524.
PMID: 30192042Jarvik GP, Browning BL. Consideration of cosegregation in the pathogenicity classification of genomic variants.
American Journal of Human Genetics. 2016;98(6):1077-1081.
PMID: 27236918Walker LC, Hoya M, Wiggins GAR, Lindy A, Vincent LM, Parsons MT, et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup.
American Journal of Human Genetics. 2023;110(7):1046-1067.
PMID: 37352859Veltman JA, Brunner HG. De novo mutations in human genetic disease.
Nature Reviews Genetics. 2012;13(8):565-575.
PMID: 22781750Kolesnikov A, Goel S, Nattestad M, Yun T, Baid G, Yang H, et al. DeepTrio: variant calling in families using deep learning.
bioRxiv. 2021.
DOI: 10.1101/2021.04.05.438434Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses.
American Journal of Human Genetics. 2007;81(3):559-575.
PMID: 17701901Manichaikul A, Mychaleckyj JC, Rich SS, Daly K, Sale M, Chen WM. Robust relationship inference in genome-wide association studies.
Bioinformatics. 2010;26(22):2867-2873.
PMID: 20926424McCormick EM, Lott MT, Dulik MC, Shen L, Attimonelli M, Vitale O, et al. Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation.
Human Mutation. 2020;41(12):2028-2057.
PMID: 33058415Bring family-aware variant interpretation into your clinical workflow
Folklore's Family Analysis module integrates with your existing Variant Analysis pipeline. Schedule a discussion with our scientific team to assess the fit for your laboratory.