Genetic tests differ in what they detect, what they miss, turnaround time, and cost. Choosing the right test starts with understanding what each methodology can and cannot do. This page covers the major test categories — single-gene Sanger, multi-gene NGS panels, whole-exome and whole-genome sequencing, repeat-expansion testing, mitochondrial DNA testing, copy-number variant detection, methylation testing, karyotype, and long-read sequencing.

1. Targeted Single-Gene Sequencing (Sanger or NGS)

What It Detects

• Single-nucleotide variants (SNVs) and small insertions/deletions in one specific gene.

What It Misses

• Large deletions / duplications (unless paired with del/dup analysis).
• Repeat expansions.
• Deep intronic variants.
• Structural variants.
• Variants in other genes that share the phenotype.

Turnaround & Cost

• Typically 2–4 weeks.
• Low cost (US $200–600 commonly).

When to Use

• The clinical syndrome and gene are unambiguous: CADASIL → NOTCH3; Wilson disease → ATP7B; familial AD with strong autosomal dominant pedigree → PSEN1; LHON → the three primary mtDNA mutations.
• Predictive / pre-symptomatic testing for a known familial variant.
• When the differential is so narrow that a panel adds cost without benefit.

Tip

• Pair with del/dup analysis (MLPA or sequencing-based CNV calling) when single-exon or whole-gene deletions are known to cause the disease (e.g., DMD, PMP22, SMN1).

2. Multi-Gene NGS Panels

What It Detects

• SNVs + small indels across a curated gene list — tens to hundreds of genes.
• Most modern commercial panels also include exon-level CNV calling.

What It Misses

• Repeat expansions (largely — some specialized panels add this).
• Deep intronic variants.
• Structural variants outside coding regions.
• Somatic mosaic variants below ~10% allele frequency.
• Variants in genes not on the panel — panel content varies between vendors.

Turnaround & Cost

• 3–6 weeks.
• Moderate cost (US $1,000–3,000); often insurance-covered for symptomatic patients.

When to Use

• The differential includes more than one plausible gene within a clinical category: epilepsy, leukodystrophy, hereditary spastic paraplegia, CMT, LGMD, hereditary ataxia, dystonia, parkinsonism, FTD, ALS, hereditary neuropathy.
• Default choice for most adult neurogenetic workups.

Tips

• Choose a panel with broad and current gene content; many vendors update panels annually.
• Verify the panel includes the gene you most suspect — content varies between vendors.
• For pediatric / ultra-rare suspicion, trio WES may be a better single test than a large panel.

3. Whole-Exome Sequencing (WES)

What It Detects

• SNVs + small indels across all ~20,000 protein-coding genes (~1.5% of the genome).
• Many laboratories now also call CNVs from exome data with reasonable accuracy.

What It Misses

• Non-coding regulatory variants.
• Deep intronic variants (unless transcriptome sequencing is added).
• Repeat expansions (standard analysis).
• Structural variants.
• Mitochondrial DNA variants (some labs add mtDNA reflex; verify with the lab).

Turnaround & Cost

• 4–8 weeks.
• Higher cost (US $3,000–6,000); insurance coverage variable — often requires documented prior negative testing.

When to Use

• Atypical or undifferentiated phenotype.
• Multi-system involvement.
• Suspected ultra-rare disease.
• Prior negative gene panel.

Trio WES — Critical Concept

Trio exome (proband + both parents) substantially increases diagnostic yield:

• Confirms de novo variants (parents wild-type, proband variant new).
• Resolves compound heterozygosity (variants in trans on parental alleles).
• Clarifies inheritance patterns.
• Yield is approximately 5–10% higher than singleton WES in pediatric undiagnosed disease.

4. Whole-Genome Sequencing (WGS)

What It Detects

• SNVs + indels across coding AND non-coding regions.
• Structural variants.
• Copy-number variants (more reliably than exome).
• Mitochondrial variants.
• Increasingly, repeat expansions (with bioinformatics tools like ExpansionHunter).

What It Misses

• Large repeat expansions still require specialized assays for definitive sizing.
• Methylation.
• Somatic mosaicism below threshold.

Turnaround & Cost

• 4–12 weeks.
• Highest cost (US $4,000–10,000+); rarely insurance-covered as first-line.

When to Use

• Prior negative WES with high clinical suspicion of genetic disease.
• Suspected structural variant or deep intronic variant.
• Research-grade comprehensive analysis.
• Increasingly used as first-line in pediatric neurology in specialized health systems (Genomics England, NICUSeq).

5. Repeat Expansion Testing

Critical point: standard NGS panels and WES MISS repeat expansions. Order repeat-expansion testing separately for any disorder where expansion is the mechanism.

What It Detects

• Pathologic repeat expansions in specific loci: HTT CAG (Huntington), FXN GAA (Friedreich ataxia), SCA repeats (SCA1, 2, 3, 6, 7, 8, 10, 12, 17, DRPLA), DMPK CTG (DM1), CNBP CCTG (DM2), C9orf72 GGGGCC (ALS/FTD), AR CAG (Kennedy disease), FMR1 CGG (Fragile X / FXTAS).

Methodologies

• Standard PCR + capillary electrophoresis: normal and small expansions.
• Repeat-primed PCR (RP-PCR): detects large expansions when standard PCR fails. Common for C9orf72.
• Southern blot: sizing very large expansions; turnaround 4–8 weeks; increasingly replaced.
• Long-read sequencing (PacBio HiFi, Oxford Nanopore): now the method of choice for some loci (FXN, RFC1) — sequence-level resolution + interruption detection.

Cost

• Low to moderate per locus; specialized panels available bundling multiple loci (e.g., dominant ataxia panel covering SCAs).

6. Mitochondrial DNA Testing

What It Detects

• Mitochondrial DNA (mtDNA) point mutations.
• Small deletions.
• Large-scale deletions / duplications.
• Quantification of heteroplasmy (the percentage of mutant vs. wild-type mtDNA per cell).

Methodologies

• Targeted mtDNA point mutation testing: common mutations — m.3243A>G (MELAS), m.8344A>G (MERRF), the three LHON mutations.
• Whole mtDNA sequencing by NGS: detects all point mutations and small deletions; quantifies heteroplasmy down to ~5–10%.
• Long-range PCR / Southern blot: large mtDNA deletions (Kearns-Sayre, CPEO).

Sample — Critical

• Blood is convenient but heteroplasmy may be lower in blood than in affected tissue (muscle, liver).
• For suspected mitochondrial disease with negative blood: send muscle biopsy mtDNA and/or buccal swab / urine sediment (urothelial cells often retain higher heteroplasmy).
• Send both whole mtDNA sequencing AND a nuclear mitochondrial NGS panel — >75% of pediatric mitochondrial disease and a substantial fraction of adult-onset disease is caused by nuclear gene mutations, not mtDNA.

7. Copy-Number Variant (CNV) / Dosage Testing

What It Detects

• Deletions and duplications too large for standard sequencing to detect reliably (typically > single exon, often > 1 kb).

Methodologies

• MLPA (multiplex ligation-dependent probe amplification): gene-specific dosage analysis. Gold standard for DMD deletions/duplications, SMN1 exon 7 deletion, PMP22 duplication (CMT1A), NF1 deletions.
• Chromosomal microarray (CMA / aCGH / SNP array): genome-wide CNV detection; first-line for unexplained developmental delay, intellectual disability, autism, multiple congenital anomalies.
• Exome / genome NGS with CNV calling: increasingly accurate; many labs report exon-level CNVs from sequencing data.

When to Use

• Any disease where large deletions / duplications are a known mechanism: DMD/BMD, SMA, CMT1A (PMP22 dup), NF1, NF2.
• Developmental delay / intellectual disability workup (microarray as first-line).

8. Methylation Testing

What It Detects

• Abnormal DNA methylation at imprinted loci or in repeat expansions where hypermethylation drives the phenotype (e.g., fragile X full mutation).

Methodologies

• Methylation-specific PCR.
• Methylation-sensitive MLPA.
• Bisulfite sequencing.

When to Use

• Fragile X syndrome / FXTAS workup (FMR1 expansion ± methylation).
• Imprinting disorders (Prader-Willi, Angelman) when suspected.
• Rare epilepsies with imprinting defects.

9. Karyotype & Chromosomal Analysis

What It Detects

• Large chromosomal abnormalities — trisomies, translocations, large deletions/duplications (> ~5–10 Mb), inversions visible by light microscopy.

Methodology

• G-banding karyotype.
• FISH (fluorescence in situ hybridization) for targeted loci.

When to Use

• Dysmorphism + neurologic phenotype.
• Multiple congenital anomalies.
• Suspected Down syndrome / trisomy with seizures.
• Largely replaced by chromosomal microarray for routine diagnostic workup — CMA detects much smaller CNVs.

10. Long-Read Sequencing (Emerging)

What It Detects

• Same as short-read NGS PLUS:
  • Accurate sizing of large repeat expansions.
  • Structural variants.
  • Phasing of variants across long distances.
  • Methylation calling on the same molecule.

Methodologies

• PacBio HiFi sequencing: high accuracy, longer reads.
• Oxford Nanopore: portable, real-time, very long reads.

When to Use

• Now first-line for some repeat-expansion loci: FXN GAA (Friedreich ataxia), RFC1 AAGGG (CANVAS).
• Structural variants in undiagnosed cases.
• Comprehensive single-test diagnostic workup in specialized centers.
• Expect broader adoption 2025–2027 as costs continue to fall.

Comparison Table — When to Use Which

Pitfalls and Pearls

• NGS misses repeat expansions — always order separately when an expansion mechanism is plausible.
• NGS panels vary between vendors — confirm the gene you suspect is on the panel before ordering.
• WES misses mtDNA in many labs — verify whether mtDNA is included or reflex.
• Trio WES > singleton WES when parents are available.
• WGS detects more than WES but is rarely insurance-covered as first-line — emerging.
• Mitochondrial blood testing can be negative with positive muscle / urine — heteroplasmy is tissue-specific.
• MLPA is the gold standard for DMD del/dup, SMN1 exon 7 deletion, PMP22 dup, NF1 large deletions.
• Chromosomal microarray has largely replaced karyotype for developmental delay / intellectual disability workup.
• Long-read sequencing is now first-line for FXN, RFC1, and some other complex loci.
• Sponsored testing programs often cover panel costs — check before ordering at full price.

References

1. Rexach J, Lee H, Martinez-Agosto JA, et al. Clinical application of next-generation sequencing to the practice of neurology. Lancet Neurol. 2019;18(5):492-503.
2. Klein CJ, Foroud TM. Neurology individualized medicine: when to use next-generation sequencing panels. Mayo Clin Proc. 2017;92(2):292-305.
3. Miller DT, Adam MP, Aradhya S, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010;86(5):749-764.
4. Schon KR, Ratnaike T, van den Ameele J, et al. Mitochondrial diseases: a diagnostic revolution. Trends Genet. 2020;36(9):702-717.
5. Tafazoli A, Khalili M, Ranjbar V, et al. Long-read sequencing in neurogenetic disorders: a review. Mov Disord Clin Pract. 2024;11(3):236-251.