3.2 Molecular Diagnostics (Sanger, NGS Panels, WES, WGS, MLPA)
Key Takeaways
- Sanger sequencing remains a gold-standard orthogonal method for known single-variant confirmation and small amplicon questions, but it is inefficient as a first-line genome-wide diagnostic.
- NGS gene panels offer deep coverage of curated genes matched to phenotype; they still have coverage gaps, limited structural-variant detection, and panel-content obsolescence risk.
- WES targets mostly coding sequence; WGS sequences broadly across the genome and improves some structural and noncoding detection—neither guarantees perfect sensitivity for all variant classes.
- Secondary findings on exome/genome are a pretest counseling topic (ACMG secondary findings gene list concept); detail on return-of-results processes continues in later interpretation chapters.
- MLPA and related dosage assays detect exon-level deletions/duplications that sequencing-only pipelines may miss when CNV calling is absent or limited.
Why molecular method literacy is a counseling skill
CGC exam stems rarely ask you to run a sequencer. They ask whether the ordered test can answer the clinical question, what it might miss, and what incidental information could return. Molecular methods differ in target space (one variant, a gene list, the exome, the genome), depth, CNV/structural sensitivity, and incidental finding potential. Choosing poorly wastes time, insurance authorization, and family trust.
Sanger sequencing
Sanger sequencing (chain-termination sequencing) reads a defined PCR amplicon at high accuracy for single-nucleotide variants and small insertions/deletions within that amplicon.
| Strength | Limitation |
|---|---|
| Excellent for confirming a known familial variant | Poor throughput for large gene lists |
| Orthogonal validation of NGS calls when labs require it | Does not interrogate unamplified regions |
| Useful for known hotspot / single-exon questions | Allele dropout if primer binding site has a variant |
| Familiar for cascade testing of a reported variant | Will not detect large deletions spanning primer sites without dosage methods |
Clinical pattern: Use Sanger (or an equivalent targeted assay) when the familial pathogenic variant is already defined and you need relatives tested efficiently. Do not start an undiagnosed complex phenotype workup with “Sanger the whole genome”—that is not how Sanger works.
NGS gene panels
Next-generation sequencing (NGS) panels enrich or capture a curated set of genes related to a phenotype (cardiomyopathy panel, epilepsy panel, RASopathy panel, etc.), then sequence at typically high depth.
Advantages
- Phenotype-matched content reduces some noise versus untargeted testing.
- Deep coverage improves sensitivity for mosaic calls in some settings (still assay-dependent).
- Often cheaper and faster than exome/genome with clearer coverage metrics for included genes.
Limitations boards love
| Limitation | Counseling language |
|---|---|
| Panel content | Genes not on the panel are not tested—even if later evidence implicates them |
| Coverage gaps | Homopolymers, high-GC regions, pseudogenes, and repetitive segments may have low/no coverage |
| Structural variants | Many panels have limited or no reliable detection of large rearrangements unless CNV analysis is included and validated |
| Deep intronic / regulatory variants | Usually outside panel design |
| Updates | A “negative” 2018 panel may miss genes added to 2026 panels—reanalysis or broader testing may be warranted |
Exam scenario: Phenotype strongly suggests a gene known to have frequent exon deletions; a sequencing-only panel without del/dup analysis is incomplete—add MLPA, targeted deletion analysis, or a platform with validated CNV calling.
Whole exome sequencing (WES)
WES targets the exome—roughly the ~1–2% of the genome that encodes proteins—plus some flanking bases, depending on kit design.
| What WES does well | What WES struggles with |
|---|---|
| Many Mendelian coding SNVs/indels | Noncoding regulatory variants outside capture |
| Broader differential than a narrow panel | Some exon-level CNVs (better if CNV pipeline included) |
| Discovery when phenotype is heterogeneous | Pseudogenes / homologous regions; mitochondrial genome often separate |
| Trio exome (proband + parents) improves de novo/inheritance filters | Incomplete coverage of some exons despite “exome” label |
Coverage honesty: “Exome” does not mean every coding base is adequately covered. Labs issue coverage metrics and gene-level caveats; a critical exon with poor coverage may need Sanger fill-in.
Secondary findings preview
Exome and genome sequencing can reveal secondary findings—medically actionable variants in genes unrelated to the primary indication (classic framework: ACMG secondary findings gene list, updated periodically). Pretest counseling should cover:
- That secondary findings may be analyzed and reported depending on lab policy and patient opt-in/opt-out rules.
- That this is distinct from the primary diagnostic search.
- That detailed classification, disclosure logistics, and family cascade planning continue in interpretation and results-delivery chapters.
For Domain 3A, remember the concept and counseling trigger, not every gene on the current ACMG list.
Whole genome sequencing (WGS)
WGS sequences across the genome (with platform-specific gaps) rather than capturing only exons.
| Relative advantage vs WES | Remaining limitations |
|---|---|
| Better access to noncoding and some structural variant signatures | Short-read WGS still struggles with some repeats, highly homologous regions |
| More uniform coverage of many coding regions vs capture bias | Interpretation burden; VUS volume; cost/authorization barriers |
| Improved detection of some CNVs/SVs with appropriate pipelines | Mitochondrial and epigenetic disorders may need dedicated tests |
| Single assay sometimes replaces stepwise panel→exome cascades | “Negative genome” ≠ “not genetic” for all mechanisms (imprinting, repeat expansions, etc.) |
Teaching point: WGS is more comprehensive for many DNA sequence and structural questions than WES, but it is not omniscient. Match mechanism to method: fragile X–type repeat expansions, methylation disorders, and some RNA/protein assays still need specialized tests.
MLPA and dosage methods
MLPA (multiplex ligation-dependent probe amplification) detects copy-number changes at exon or gene resolution for targeted loci. Related approaches include quantitative PCR, chromosomal microarray for larger CNVs, and NGS-based CNV calling.
| Use MLPA (or equivalent) when | Do not assume |
|---|---|
| Gene has frequent exon del/dup architecture (classic teaching examples include dystrophinopathy deletion/duplication patterns, some hereditary cancer genes historically) | Sequencing alone always catches dosage changes |
| A “negative” sequence test leaves del/dup untested | MLPA surveys the whole genome |
| You need orthogonal confirmation of a suspected exon deletion | MLPA replaces CMA for genome-wide pediatric CNV first-line evaluation |
Method comparison table
| Method | Primary target | Best for | Classic miss |
|---|---|---|---|
| Sanger | Single amplicon / known variant | Confirmation; cascade testing | Everything outside amplicons; large dels |
| NGS panel | Curated gene set | Focused phenotype with known gene set | Off-panel genes; some SVs; coverage holes |
| WES | Mostly coding genome | Broad Mendelian differential | Many noncoding/SV classes; coverage gaps |
| WGS | Broad genome sequence | Broader SV/noncoding reach; complex undiagnosed disease | Some repeats/homology; non-sequence mechanisms |
| MLPA | Targeted exon/gene dosage | Del/dup at known loci | Untargeted loci; balanced rearrangements |
Building a molecular testing strategy
- Define the variant class you must detect (SNV, indel, exon del/dup, balanced rearrangement, aneuploidy, repeat expansion).
- Choose the narrowest adequate test that covers that class and the differential—then escalate if negative and suspicion remains.
- Confirm whether the lab’s panel/exome/genome pipeline includes validated CNV analysis.
- Counsel coverage limitations and secondary findings before send-out.
- Plan orthogonal confirmation when management hinges on a single call (especially unexpected secondary findings or atypical variants).
Molecular literacy on the CGC exam is less about chemistry and more about what the report cannot see.
A pathogenic familial BRCA1 variant is already known. A sister wants testing only for that variant. Which approach is most appropriate as first-line?
Which limitation is most characteristic of chromosomal microarray when counseling about molecular sequencing options?
A phenotype is strongly associated with a gene in which exon deletions are a common pathogenic mechanism. Sequence NGS of that gene is negative and the report does not include del/dup analysis. What is the best next methodological consideration?