Coming of age: ten years of next-generation sequencing technologies

Key Points There are two major paradigms in next-generation sequencing (NGS) technology: short-read sequencing and long-read sequencing. Short-read sequencing approaches provide lower-cost, higher-accuracy data that are useful for population-level research and clinical variant discovery. By contrast, long-read approaches provide read lengths that are well suited for de novo genome assembly applications and full-length isoform sequencing. NGS technologies have been evolving over the past 10 years, leading to substantial improvements in quality and yield; however, certain approaches have proven to be more effective and adaptable than others. Recent improvements in chemistry, costs, throughput and accessibility are driving the emergence of new, varied technologies to address applications that were not previously possible. These include integrated long-read and short-read sequencing studies, routine clinical DNA sequencing, real-time pathogen DNA monitoring and massive population-level projects. Although massive strides are being made in this technology, several notable limitations remain. The time required to sequence and analyse data limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical considerations can limit the public and private use of genetic data. We can expect increasing democratization and options for NGS in the future. Many new instruments with varied chemistries and applications are being released or being developed. Advances in DNA sequencing technologies have led to vast increases in the diversity of sequencing-based applications and in the amount of data generated. This Review discusses the current state-of-the-art technologies in both short-read and long-read DNA sequencing, their underlying mechanisms, relative strengths and limitations, and emerging applications. Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alter