Genotyping and Sequencing Technologies

Genotyping and sequencing technologies are the laboratory methods that detect the genetic variation a pharmacogenomic test reports. They range from targeted assays that interrogate a fixed list of known variants to next-generation sequencing that reads stretches of DNA base by base. The choice of platform determines which variants can be found, how rare or structurally complex alleles are handled, and therefore how completely a patient's pharmacogene profile can be characterized.

Definition

Genotyping and sequencing technologies are the analytical methods used to detect inherited DNA variation in pharmacogenes, comprising targeted genotyping assays that test for predefined variants and sequencing methods that read the underlying DNA sequence to identify both known and novel variants.

Scope

This entry covers the main classes of detection technology used in pharmacogenomic laboratories: targeted single-nucleotide-variant genotyping (arrays and allele-specific assays), and sequencing approaches including Sanger and next-generation sequencing. It explains why genotyping panels detect only pre-specified variants while sequencing can discover novel and complex ones, and why complex pharmacogenes such as CYP2D6 challenge every platform. It is a reference description of methods, not laboratory protocol or test-ordering guidance.

Core questions

• What is the difference between targeted genotyping and sequencing, and what does each detect?
• Why do genotyping panels report only a limited set of variants?
• How do sequencing platforms handle novel or rare pharmacogene variants?
• Why are genes such as CYP2D6 technically difficult to type accurately?

Key concepts

• Targeted genotyping versus untargeted sequencing
• Single-nucleotide variant arrays
• Next-generation sequencing
• Sanger sequencing
• Star-allele and diplotype calling
• Structural variation and copy-number in pharmacogenes
• Analytic coverage and the variants a panel misses

Mechanisms

Targeted genotyping assays test a DNA sample for a predetermined set of variant positions, typically common, well-characterized single-nucleotide variants and selected structural changes; they are fast and inexpensive but report only variants they are designed to detect. Sequencing instead reads the nucleotide sequence of target regions, so it can identify both established and previously unobserved variants, including rare alleles that targeted panels omit (Tafazoli et al., 2021; Roden, 2019). Detected variants are mapped to standardized star-allele definitions and combined into a diplotype; the Pharmacogene Variation Consortium maintains the reference allele definitions that make this mapping consistent across laboratories (Gaedigk et al., 2017; Gaedigk et al., 2021). Highly polymorphic and structurally variable genes such as CYP2D6, which can carry deletions, duplications, and hybrid alleles, remain technically demanding for every platform.

Clinical relevance

The platform a laboratory uses shapes what a pharmacogenomic result can claim: a normal targeted-genotyping result means only that the tested variants were absent, not that the gene is free of all variation. Recognizing this distinction is part of appraising a pharmacogenomic report. This entry describes how variation is detected and is not guidance on selecting, ordering, or acting on a specific test.

Evidence & guidelines

Reference allele definitions used to interpret genotyping and sequencing output are curated by the Pharmacogene Variation Consortium, which standardizes star-allele nomenclature across pharmacogenes (Gaedigk et al., 2017; Gaedigk et al., 2021). Reviews of next-generation sequencing in clinical pharmacogenomics describe the comparative coverage and limitations of platforms (Tafazoli et al., 2021). These are methodological references rather than prescriptive standards of care.

History

Pharmacogenomic detection began with single-gene assays and Sanger sequencing, then expanded with high-throughput single-nucleotide-variant arrays that allowed many variants to be typed at once. The arrival of next-generation sequencing made it feasible to read whole pharmacogenes and to discover rare and novel alleles, shifting the field from testing only common known variants toward fuller characterization (Tafazoli et al., 2021; Roden, 2019). In parallel, the consolidation of allele nomenclature under the Pharmacogene Variation Consortium gave the field a common reference for naming what these technologies detect (Gaedigk et al., 2017).

Debates

Targeted genotyping versus sequencing for clinical testing

Targeted panels are cheaper and faster but miss rare and novel variants, while sequencing is more complete but costlier and more complex to interpret; the appropriate balance for routine pharmacogenomic testing is still discussed.

Key figures

• Andrea Gaedigk
• Magnus Ingelman-Sundberg
• Teri E. Klein
• Dan M. Roden

Seminal works

• gaedigk-2017
• tafazoli-2021
• roden-2019

Frequently asked questions

Does a normal genotyping result mean a patient has no relevant variants?

Not necessarily. Targeted genotyping detects only the specific variants it is designed to test for, so a normal result rules out those variants but cannot exclude rare or novel ones that only sequencing would find.

Why is CYP2D6 considered hard to genotype?

CYP2D6 is highly polymorphic and prone to structural changes such as gene deletions, duplications, and hybrid alleles, which are technically difficult to detect and resolve accurately on many platforms.