The diagnostic analysis of a gene typically targets the exons (the protein-coding parts of the gene) because about 85% of disease-causing mutations localize in the exons or the directly adjacent sequences.
Sequencing of DNA describes a process that determines the succession (sequence) of its smallest building blocks, the nucleotides (abbreviated as A, C, T and G) which compose the genetic code. The Sanger sequencing method has been the gold standard for decades : Each exon of a gene known to be associated with a given disease first needs to be amplified by the polymerase chain reaction (PCR). Millions of copies are generated using the patient’s gene copies as a template.
In a sequencing PCR (chain termination method), the fluorescence-labelled PCR products are then separated and visualized on a capillary sequencer and subsequently compared with the normal (“wild-type”) sequence. The applicability of this exon-by-exon and gene-by-gene approach is limited in case of disorders that are due to mutations in large and/or many genes (e.g. retinal dystrophies, deafness, developmental delay, epilepsy). Until recently, a genetic diagnosis has therefore been the exception for most patients.
The establishment of NGS technology in a diagnostic setting (which started around 2010) has truly revolutionized the field of medical genetics and – since genetic disorders concern every medical speciality – medicine as a whole. Genetic disorders have now become accessible to comprehensive diagnostic analysis.
NGS of gene panels plays a predominant role, but exome and genome sequencing are constantly gaining importance. In exome sequencing, either the exons of all genes knowingly associated with inherited diseases (Clinical Exome Sequencing, CES) or the exons of all human genes (Whole-Exome Sequencing, WES) are being sequenced. In genome sequencing (Whole-Genome Sequencing, WGS), also the non-coding parts (corresponding to 99% of the genome) are sequenced. Mutations in those parts can, for instance, prevent exons or complete genes from being translated into the corresponding protein.
In short, NGS technologies are based on the following concept: The patient’s DNA, which comprises the genome with its roughly 19,000 genes, is being extracted from a blood sample (or, occasionally, from other tissues). For the analysis of gene panels, the exons of the relevant genes (~6,000 exons in case of retinal dystrophies) are captured from the fragmented DNA through binding to complementary stretches of synthetic nucleic acid (sequence capture) and subsequently sequenced.
The essential difference to the conventional Sanger method (see above) is that DNA amplification and sequencing for many (even hundreds or thousands) genes and exons are carried out simultaneously in a massively paralleled fashion – and not in singular reactions. Through sequence barcodes, sequences can unambiguously be assigned to a patient, which enables “pooling” of samples from many patients. Currently, the Solexa sequencing technology is the most widely used one: Adaptors are ligated to both ends of template DNA fragments and immobilized on a solid phase (flow cell). Based on the starter molecule, clusters of same sequences are being generated (bridge amplification). In a sequencing-by-synthesis-PCR with four chain termination substrates of different fluorescent colours, the nucleotide (which is complementary to the template) incorporated in the cluster in a cycle is being determined (through the emission of light when the fluorescent dye dissociates). To obtain reliable sequences, the target sequences should be covered by a high number of sequence reads (sequence coverage).