Table 4 Key analytic techniques and their uses in diagnosing undiagnosed genetic conditions. Information in table adapted from [1, 72]

Chromosomal microarray analysis

Low-cost detection of chromosomal copy-number variation associated with unbalanced chromosomal structural changes

Gene panel

NGS analysis of one or a small number of genes; selected genes often indicated by clinical features

Detection of sequence and structural variants

Exome sequencing

NGS analysis of the exome. Detection of sequence variants and whole exon deletions, potential to detect structural variants and mosaicism

Short-read whole genome sequencing

NGS analysis of the whole genome, with read lengths of 100-250bp

When compared to exome sequencing, whole genome sequencing has more comprehensive exon coverage, coverage of non-coding regions, and increased sensitivity to detect structural variants

Detection of SNVs, small indels, complex structural variants, non-coding splicing or regulatory genomic variants, variants in the mitochondrial genome, and expansion variants

Long-read whole genome sequencing

NGS analysis of the whole genome, with read lengths of > 10,000bp

When compared with short-read sequencing, long-read sequencing has better detection of nucleotide repeat expansions, distinguishing between regions of high homology

Accurate detection of structural variants and phase variable genes

RNA sequencing

NGS and analysis following conversion of RNA to cDNA

Detection of abnormal expression and splicing, allele-specific expression, RNA abundance and can aid in interpretation of germline variants

Methylation profiling

Methylation-specific microarray or sequencing analysis

Detection of imprinting defects, mutations in epigenetic regulators

Metabolomics

Targeted analysis of small-molecule substrates, intermediates, and metabolites

Detection of altered biochemical functions