Increasingly, nearly all of molecular diagnostics applications requiring assessment of DNA (or RNA) are being supplanted by so-called “next-generation sequencing” (NGS) technologies, so named because the Sanger sequencing mentioned earlier is now considered “first generation.” The availability of NGS technology has the potential to alter molecular diagnostics radically, by the sheer volume of sequencing data (more than 1 gigabase pairs or 1,000,000,000 base pairs of DNA per day!) at relatively low costs. The entire human genome has a little more than 3 gigabases, so true “whole genome sequencing” can be performed several times in a matter of days. In contrast with Sanger sequencing, NGS technologies use platforms in which sequencing of multiple fragments of the human genome (DNA or cDNA) can occur in parallel (“massively parallel sequencing”), significantly enhancing its speed (
Fig. 7.41 ). Fluorescently labeled nucleotides are incorporated that are complementary to the template DNA strands, which are immobilized on a solid phase, with one nucleotide added per template per cycle. An image is captured at the end of each cycle, and the cycles are repeated until a “read” of sufficient length is generated to map each read back to the human genome using sophisticated bioinformatics. In addition to differences of time and scale, another key difference between NGS and traditional Sanger sequencing is the ability of the former to detect variant alleles that occur at low frequency in samples that contain a heterogeneous mixture of cells. With Sanger sequencing, the lower limit of detection for variant allele frequency is 20% or more, thus requiring either homogeneous samples (such as germline DNA) or some form of enrichment, such as microdissection of tumor cells from a biopsy that also contains normal tissue. The ability of NGS to “read” the same region of the genome multiple times (from 100to 1000-fold redundancy) allows detection of allele frequencies as low as 1%, making NGS particularly useful when searching for mutations in tissue biopsies that contain more nonneoplastic stroma than the tumor cells.
Clinical and research applications of NGS (which can be performed on either germline or somatic DNA, or both) include targeted sequencing (typically a panel of a few to several hundred genes); whole exome sequencing (WES), which interrogates all coding regions of the human genome; and whole genome sequencing (WGS), which includes the remaining 99% of the human genome, including regions that express noncoding RNAs. In addition to DNA, NGS can also be used for measuring the transcriptome (RNA-Seq) and genomewide binding sites for transcription factors or histones (chromatin immunoprecipitation and sequencing, or ChIP-Seq).
Direct diagnosis of mutations is possible only if the gene responsible for a genetic disorder is known and its sequence has been identified. In several diseases that have a genetic basis, including some common disorders, direct genetic diagnosis is not possible, either because the causal gene has not been identified or because the disease is multifactorial (polygenic) and no single gene is involved. In such cases, two types of analyses can be performed for unbiased identification of disease-associated gene(s): linkage analysis and GWASs. In both instances, surrogate markers in the genome, also known as marker loci, must be used to localize the chromosomal regions of interest, based on their linkage to one or more putative disease-causing genes. The marker loci used are naturally occurring variations in DNA sequences known as polymorphisms, typically SNPs, as described previously.