While sequencing and alignment produce the data, tertiary analysis provides the meaning. It is the final, most critical stage of the NGS pipeline where genetic variants are annotated, filtered, and interpreted to deliver actionable medical insights.
In the context of Next-Generation Sequencing (NGS), tertiary analysis is the process of translating a raw list of genetic variations (typically in a VCF file) into a clinical diagnosis or research conclusion.
If secondary analysis is about "what is there," tertiary analysis is about "what does it mean for the patient?" It involves comparing variants against massive genomic databases, applying professional guidelines (ACMG/AMP), and filtering out the background noise of common genetic variation.
Base calling & FASTQ generation (The Sequencer)
Alignment & Variant Calling (BAM/VCF)
Interpretation & Reporting (The Diagnostic)
Professional tertiary analysis genomics workflows consist of four interconnected stages that transform VCF data into medical certainty.
Layering variant data with external knowledge: ClinVar classifications, gnomAD population frequencies, and gene impact scores.
Reducing ~4 million variants to a handful of candidates by excluding benign, common, and non-coding variations that don't match the phenotype.
Systematically scoring variants against ACMG (Germline) or AMP (Somatic) guidelines to determine pathogenic potential.
The final step of clinical reporting, where findings are synthesized with the patient's history to provide a definitive medical diagnostic.
While secondary analysis has been largely commoditized and automated, tertiary analysis remains the primary time-sink in the clinical genomics workflow.
Sequencers can run hundreds of genomes a week, but many labs can only interpret 10-20 cases a week due to manual analysis hurdles.
Accreditation bodies (CAP/CLIA) require full audit trails and deterministic results, making "black box" automated tools risky without professional oversight.
"The goal of advanced variant interpretation pipelines is to move from evidence collection to evidence application."
"Is the variant present?"
"Is the variant clinically relevant?"
While NGS tertiary analysis is often treated as a separate manual step, the most efficient laboratories treat secondary and tertiary analysis as a single continuous pipeline.
Golden Helix bridges this gap by offering Sentieon for industry-leading secondary analysis and VarSeq for world-class tertiary analysis in a unified ecosystem. This prevents "data silos" and ensures that clinical interpretation always has access to the underlying quality metrics of the sequencing run.
Learn more about the full NGS workflowGolden Helix's VarSeq is specifically engineered to handle the complexity of clinical interpretation, providing a scalable, deterministic environment for labs of all sizes.
Standardize your variant interpretation with guided workflows for ACMG and AMP guidelines. Surface evidence automatically and reduce interpretation time by over 80%.
Clinical Interpretation →Automate the "hands-off" portion of your tertiary analysis, from VCF import and annotation to automated filtering and draft report generation.
Automation Details →"The ability to handle genomes at scale while maintaining clinical rigor is why leading diagnostic centers choose VarSeq."
The process typically involves: 1) Annotation with genomic databases, 2) Filtering based on population frequency and quality, 3) Scoring variants according to ACMG/AMP guidelines, and 4) Synthesizing findings into a clinical report.
Unlike secondary analysis, which is primarily computational, tertiary analysis requires deep medical and genetic knowledge. It involves weighing conflicting evidence from clinical literature and databases to make a definitive pathogenicity classification.
Germline analysis (inherited disease) follows ACMG guidelines, focusing on pathogenicity. Somatic analysis (cancer) follows AMP/ASCO/CAP guidelines, focusing on therapeutic relevance, actionability, and prognostic significance.
A clinical knowledgebase (like VSWarehouse) allows a laboratory to store and reuse variant assessments. If a lab sees the same variant in multiple patients, they can leverage previous interpretations to ensure consistency and speed up analysis.
Deep dives into variant interpretation, clinical guidelines, and interpretation workflow optimization.
Join thousands of clinical professionals using VarSeq to automate variant interpretation and deliver reliable diagnostic insights.