All chapters
Clinical Genomics Pipeline
advancedClinical WES Full Pathway
Clinical WES from Referral to Report
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Clinical Referral + HPO Phenotyping
Geneticist assigns HPO terms; documents family history; consent obtained
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Sample Collection & DNA QC
Blood/saliva; DIN≥7; ≥1 µg DNA; NanoDrop A260/A280≥1.8
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WES Library & Sequencing
Agilent V8 capture; NovaSeq 6000; target 100x mean depth
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GATK Pipeline + QC
BWA → MarkDup → BQSR → HaplotypeCaller → VQSR; coverage pass/fail
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Annotation + Filtering
VEP + ANNOVAR; gnomAD<0.1%, CADD>15, in OMIM gene; inheritance filter
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Exomiser Phenotype Prioritisation
HPO terms matched to variants; combined phenotype+variant score; top 20 candidates
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ACMG Classification
Variant curator applies all 28 criteria; MDT review; Sanger confirmation if P/LP
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Clinical Report
HGVS nomenclature; ACMG tier; clinical interpretation; management recommendations
Clinical WES Workflow
Clinical Referral + Phenotype (HPO)
DNA Extraction + QC
WES Library Prep
100× Illumina Sequencing
BWA-MEM2 Alignment
GATK BQSR
HaplotypeCaller
VQSR
ANNOVAR + VEP Annotation
Phenotype-Driven Filtering
Tier 1–4 Classification
MDT Review
Clinical Report
Sanger Confirmation
Variant Tiering (AMP/ACMG)
- Tier 1 - High clinical significance: Pathogenic or Likely Pathogenic; directly explains phenotype; report in clinical context
- Tier 2 - Potential clinical significance: VUS in gene with strong phenotype match; hotspot or functionally important region
- Tier 3 - Uncertain significance: VUS without functional evidence; report with caveat
- Tier 4 - Likely benign / Benign: do not report; document in laboratory records
Phenotype-Driven Variant Prioritisation
- HPO terms (Human Phenotype Ontology): standardised phenotype vocabulary; >17,000 terms
- Exomiser: ranks variants by combined phenotype + variant evidence score; uses PhenoDigm algorithm
- PhenIX: HPO-driven candidate gene ranking; integrates OMIM/Orphanet
- LIRICAL: likelihood ratio–based interpretation of clinical exomes
- Prioritisation filters: gnomAD AF <0.001, CADD >15, gene in OMIM with matching phenotype
- Trio analysis: affected child + unaffected parents; identifies de novo and compound hets
- Inheritance patterns: autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), mitochondrial
Secondary Findings (ACMG SF3.2)
- ACMG recommends reporting pathogenic variants in 81 genes regardless of indication
- Categories: hereditary cancer (BRCA1/2, MLH1, APC), cardiomyopathy (MYH7, TNNT2), arrhythmia (SCN5A, KCNQ1), aortopathy (FBN1, TGFBR2), pharmacogenomics
- Patient consent required; can opt out of secondary findings
- Incidental findings vs secondary findings: latter are actively sought in recommended genes
Clinical Report Components
- Patient demographics and indication for testing
- Method: sequencing platform, capture kit, coverage metrics (mean depth, % ≥20×)
- Result summary: variant found / no variant found / uncertain
- Variant details: gene, transcript (NM_), cDNA (c.), protein (p.), zygosity, ACMG classification
- Interpretation: evidence summary, phenotype correlation, inheritance pattern
- Recommendations: family testing, surveillance, treatment implications
- Limitations: coverage gaps, variants not detectable by this method
- Confirmation: Sanger sequencing result if performed