Summary of genetic techniques used for IEI diagnosis. CNV: copy-number variant; NGS: next-generation sequencing; SVs: structural variants; TGPs: targeted gene panels; VUS: variants of uncertain significance; WES: whole-exome sequencing; WGS: whole-genome sequencing
| Technique . | Function . | Key capabilities . | Limitations . | Diagnostic yield . | Best use . |
|---|---|---|---|---|---|
| Sanger sequencing | Targeted single-gene sequencing | Variant validation; known mutations; family screening for known defects | One gene at a time; cannot detect large deletions/duplications | Validation tool | Confirming specific variants, family cascade testing, and validation of NGS findings |
| TGPs | Pathway-specific multigene analysis | Cost-effective for specific pathways; faster than WES/WGS; good depth of coverage | Limited to preselected genes; lower yield than WES/WGS | 10–25% | Known pathway abnormalities; initial screening |
| CMA | Large CNV detection | Detects large CNVs (≥20–50 kb targeted, ≥100–250 kb nontargeted); identifies aneuploidies; detects microdeletions/microduplications; unbalanced rearrangements | Cannot detect small CNVs (<10–20 kb); no balanced rearrangements; no point mutation detection | +15% when combined with WES | Syndromic presentation; complementary to WES |
| WES | Coding regions | Analyzes ∼20,000 genes simultaneously; detects SNVs and small indels; enables gene discovery; periodic reanalysis possible; high sensitivity for coding variants | No intronic/regulatory variants; limited CNV detection; poor coverage of repetitive regions; VUS interpretation challenges | 25–40% (IEI cases); 30–35% (complex cases); 36–51% (critically ill neonates); up to 50% with CMA | Complex/unknown IEI; negative targeted panel; phenotypically diverse presentation; gene discovery |
| WGS—short-read sequencing | Comprehensive genome-wide analysis | Detects coding, intronic, and regulatory variants; better CNV and SV detection than WES | Expensive; complex data interpretation; struggles with highly repetitive regions; large VUS burden | 43%; superior to WES for undiagnosed cases | WES-negative cases; regulatory variants; complex SVs |
| Long-read sequencing (WGS) | Ultra-long-read genome analysis | Superior repeat expansion detection; resolves highly repetitive regions; better SV detection; identifies variants in previously inaccessible regions | Not yet standard in clinical practice; higher cost; limited clinical validation; requires specialized expertise; lower throughput than short-read sequencing | Better than short-read WGS; identifies novel variants missed by other methods | Short-read negative cases; suspected expansions; complex SVs; research applications |
| RNA-seq | Transcriptome analysis | Validates splice-site variant effects; detects aberrant splicing; identifies allelic imbalance and exon skipping; measures gene expression levels; reveals monoallelic expression | Tissue-specific expression patterns; cannot detect all DNA variants; temporal expression variability | Resolves ∼35% of VUS cases; functional validation tool | Validating variants; explaining splicing defects; complementing DNA sequencing |
| Technique . | Function . | Key capabilities . | Limitations . | Diagnostic yield . | Best use . |
|---|---|---|---|---|---|
| Sanger sequencing | Targeted single-gene sequencing | Variant validation; known mutations; family screening for known defects | One gene at a time; cannot detect large deletions/duplications | Validation tool | Confirming specific variants, family cascade testing, and validation of NGS findings |
| TGPs | Pathway-specific multigene analysis | Cost-effective for specific pathways; faster than WES/WGS; good depth of coverage | Limited to preselected genes; lower yield than WES/WGS | 10–25% | Known pathway abnormalities; initial screening |
| CMA | Large CNV detection | Detects large CNVs (≥20–50 kb targeted, ≥100–250 kb nontargeted); identifies aneuploidies; detects microdeletions/microduplications; unbalanced rearrangements | Cannot detect small CNVs (<10–20 kb); no balanced rearrangements; no point mutation detection | +15% when combined with WES | Syndromic presentation; complementary to WES |
| WES | Coding regions | Analyzes ∼20,000 genes simultaneously; detects SNVs and small indels; enables gene discovery; periodic reanalysis possible; high sensitivity for coding variants | No intronic/regulatory variants; limited CNV detection; poor coverage of repetitive regions; VUS interpretation challenges | 25–40% (IEI cases); 30–35% (complex cases); 36–51% (critically ill neonates); up to 50% with CMA | Complex/unknown IEI; negative targeted panel; phenotypically diverse presentation; gene discovery |
| WGS—short-read sequencing | Comprehensive genome-wide analysis | Detects coding, intronic, and regulatory variants; better CNV and SV detection than WES | Expensive; complex data interpretation; struggles with highly repetitive regions; large VUS burden | 43%; superior to WES for undiagnosed cases | WES-negative cases; regulatory variants; complex SVs |
| Long-read sequencing (WGS) | Ultra-long-read genome analysis | Superior repeat expansion detection; resolves highly repetitive regions; better SV detection; identifies variants in previously inaccessible regions | Not yet standard in clinical practice; higher cost; limited clinical validation; requires specialized expertise; lower throughput than short-read sequencing | Better than short-read WGS; identifies novel variants missed by other methods | Short-read negative cases; suspected expansions; complex SVs; research applications |
| RNA-seq | Transcriptome analysis | Validates splice-site variant effects; detects aberrant splicing; identifies allelic imbalance and exon skipping; measures gene expression levels; reveals monoallelic expression | Tissue-specific expression patterns; cannot detect all DNA variants; temporal expression variability | Resolves ∼35% of VUS cases; functional validation tool | Validating variants; explaining splicing defects; complementing DNA sequencing |