Table 2.

Overview on the application and limitations of the molecular methods commonly applied in routine diagnostic genetic testing, and future omic technologies which might be implemented in daily routine.

	SNVsa (scale)	CNVsa(scale)	Methylation testing	Balanced SVsa	Main applications/advantages	Limitations	Examples (references)
Single locus tests
Allele-specific PCR assays (e.g. ASO, ARMS)	Single bp	No	Possible	No	Low-cost screening for single SNVs	Only single and preselected SNVs are addressed in one tube	Rarely applied in growth diagnostics, rather used to identify a known variant in (affected) family members
Sanger sequencing	Up to 800 bp	No	Possible	No	Sequencing of small genes (e.g. SHOX)	High costs/bp, time-consuming	SHOX
Methylation tests
MLPA	Possible	Up to 46 targets	Yes	No	CNV detection of specific genes, (multilocus) methylation analysis, low costs, fast	Restricted number of target loci	SHOX, Beckwith–Wiedemann syndrome (67)
Pyrosequencing	40–60 bp	No	Yes	No	SNV and methylation of a small genomic stretch	Only small genomic regions can be addressed in one assay	Beckwith–Wiedemann syndrome (68)
Cytogenomics
Karyotyping	No	>5 Mb	No	>5 Mb	Detection of large structural variants and aneuploidies (e.g. Turner or Klinefelter syndrome)	Low resolution, time-consuming, sample preparation, subjective assessment	Turner syndrome, Klinefelter syndrome
FISH	No	100–200 kb	No	Possible	Second-line test to confirm suspected CNVs	Time-consuming, duplications might be difficult to be detected	Prader–Willi syndrome
Microarray (SNP, CGH)	No	>50 kbd	Possible (SNP array)	No	Detection of (sub)microscopic aberrations (e.g. syndromic patients)	Spatial rearrangements are not detected	Silver–Russell syndrome as second-line test (69)
Optical mapping	No	>500 bp	Research	Yes	High-resolution, spatial rearrangements are detectable	Sample preparation	(70)
NGS assays
NGS panel (might be leaned on https://panelapp.genomicsengland.co.uk/panels)	1.5–3 Mbb	Possiblec	ImprintSeq	No	Suitable for heterogeneous disorders with specific clinical features, high coverage (mosaicism detectable), incidental findings rare	Only targeted loci are covered, untargeted variants escape detection	(40, 71)
Clinical exome	~4000 genes	Possiblec	Research	No	All known disease-associated genes are addressed, suitable for disorders with unspecific clinical features	Increased probability for VUS and incidental findings. Fixed panel, new disease-associated genes not identifiable. Non-coding regions are not covered.	Suitable for prenatal testing to avoid incidental findings.
WES	1.1% of the total genome	Possiblec	Research	No	All protein-coding regions are covered. Identification of new disease-causing genes possible. Suitable for disorders with unspecific phenotypes	Detection of VUS and incidental findings probable. Non-coding regions are not covered. Analysis and interpretation of large datasets required.	Heterogenous and unspecific phenotypes (72)
WGS	Whole genome	Possiblec	Research	Possiblec	Whole genome is analyzed, including non-coding regions. Suitable for disorders with unspecific phenotypes.	Detection of VUS and incidental findings highly probable. Processing, interpretation and storage of datasets required.	Heterogenous and unspecific phenotypes (17)
Future omic technologies
Third-generation sequencing/long read sequencing (PacBio, Nanopore)	Whole genome	Possible	Research	Yes	Identification of all types of SVs, repeats, mosaic detection, determination of physical breakpoints.	General use in diagnostics in evaluation.	Application in progress
Transcriptomics	na	na	na	na	Identification of functional variants. Complementary tool for WES and WGS.	RNAs which are not expressed in the analyzed tissue are missed. Integration with data from other omic assays required	Application in progress

ARMS, amplification refractory mutation system; ASO, allele-specific oligonucleotide; na, not applicable; Possible, not commonly used in diagnostic context; SNVs^a: 91%: substitutions, indel: 6%, deletion: 2%, insertion: 1% (according to Human variant class distribution – Ensembl 106); CNVs^a: >50 bp; SVs^a: inversions, translocations; ^bRecommended size to balance benefits with costs; ^cIn case a bioinformatic analysis pipeline for CNV detection is implemented and validated; ^dThe resolution of a microarray, rather than by the number of probes, is given by their spacing, i.e. the distance between the genomic position of a probe and the position of the next one.