Next-Generation Sequencing: Principles for Clinical Application

High-throughput, massively parallel DNA sequencing, more commonly termed “next-generation sequencing,” is an innovation in sequencing that emerged during the past decade. Next-generation sequencing (NGS) is not a single technology, but rather several different technologies that share a common feature of massively parallel sequencing of clonally amplified or single DNA molecules in a flow cell or chip. Inherent to NGS technologies are unique sequencing chemistries that differ from the Sanger dideoxynucleotide chain termination chemistry. NGS can generate, in a single instrument run, hundreds of millions to gigabases of nucleotide sequence data depending upon platform configuration, chemistry, and flow cell or chip capacity. This chapter describes principles of NGS and considerations for its application to clinical molecular tests. Although several NGS technologies have been commercialized, technologies finding greatest adoption into clinical laboratories are emphasized. Current clinical testing applications including multigene panels and exome and genome sequencing for candidate and causal gene identification are discussed. While the examples are primarily based on analyses for inherited disorders, the principles described are applicable to oncology and infectious diseases, with certain modifications mostly specific to the specimen characteristics and sensitivity requirements for these other applications.