Next-Generation Sequencing: Methodology and Application

Abstract

inTrOducTiOn Nucleic acid sequencing is a method for determining the exact order of nucleotides present in a given DNA or RNA molecule. In the past decade, the use of nucleic acid sequencing has increased exponentially as the ability to sequence has become accessible to research and clinical labs all over the world. The first major foray into DNA sequencing was the Human Genome Project, a $3 billion, 13-year-long endeavor, completed in 2003. The Human Genome Project was accomplished with first-generation sequencing, known as Sanger sequencing. Sanger sequencing (the chain-termination method), developed in 1975 by Edward Sanger, was considered the gold standard for nucleic acid sequencing for the subsequent two and a half decades (Sanger et al., 1977). Since completion of the first human genome sequence, demand for cheaper and faster sequencing methods has increased greatly. This demand has driven the development of second-generation sequencing methods, or nextgeneration sequencing (NGS). NGS platforms perform massively parallel sequencing, during which millions of fragments of DNA from a single sample are sequenced in unison. Massively parallel sequencing technology facilitates high-throughput sequencing, which allows an entire genome to be sequenced in less than one day. In the past decade, several NGS platforms have been developed that provide low-cost, high-throughput sequencing. Here we highlight two of the most commonly used platforms in research and clinical labs today: the LifeTechnologies Ion Torrent Personal Genome Machine (PGM) and the Illumina MiSeq. The creation of these and other NGS platforms has made sequencing accessible to more labs, rapidly increasing the amount of research and clinical diagnostics being performed with nucleic acid sequencing.