Landscape of Next-Generation Sequencing Technologies

TLDR: This Review concentrates on the technology behind the third- and fourth-generation sequencing methods: their challenges, current limitations, and tantalizing promise.

Abstract: DNA sequencing is in the throes of an enormous technological shift marked by dramatic throughput increases, a precipitously dropping per-base cost of raw sequence, and an accompanying requirement for substantial investment in large capital equipment in order to utilize the technology. Investigations that were, for most, unreachable luxuries just a few years ago (individual genome sequencing, metagenomics studies, and the sequencing of myriad organisms of interest) are being increasingly enabled, at a rapid pace. This Review concentrates on the technology behind the third- and fourth-generation sequencing methods: their challenges, current limitations, and tantalizing promise. First-generation sequencing encompasses the chain termination method pioneered by Sanger and Coulson1 in 1975 or the chemical method of Maxam and Gilbert in 1976–1977.2 In 1977, Sanger sequenced the first genome, bacteriophage ΦX 174, which is 5375 bases in length.3 These methods and their early history4 have been reviewed in detail previously.5 Four-color fluorescent Sanger sequencing, where each color corresponds to one of the four DNA bases, is the method used by the automated capillary electrophoresis (CE) systems marketed by Applied Biosystems Inc., now integrated into Life Technologies, and by Beckman Coulter Inc. (Table 1).6 The first composite human genome sequence, reported in 2001, was obtained largely using CE, at great cost and with intense human effort over more than a decade.7,8 While the genome reported in 2001 was a work in progress, the availability of an ever-improving “reference” genome is the basis of an ongoing transformation of biological science and remains fundamental to investigations of genotype–phenotype relationships. Considering reports that have appeared (and not appeared) in the literature to date, it could well be that medically meaningful (actionable) insights into complex diseases will require additional types of “personal” genomic data, for instance, tissue-specific mRNA expression profiling and mRNA sequencing, individualized analysis of gene regulatory regions, epigenetic profiling, and high-quality, long-range chromosome mapping to catalog significant deletions, insertions, rearrangements, etc. Correlation of such integrated genomic data sets with comprehensive medical histories for hundreds or thousands of individuals may be what it takes to reach an era of personalized medicine.9–11 Large-scale sequencing centers are now completing the conversion to next-generation sequencers; the Joint Genome Institute (JGI) has retired all of their Sanger sequencing instruments.12 At the other extreme, until small-scale next-generation sequencers can outperform CE on a cost per accurate base called as well as read length, CE systems will likely remain in heavy use for benchtop-scale, targeted sequencing for directed investigations such as quantitative gene expression, biomarker identification, and pathway analysis. Table 1 First- and Second-Generation Sequencing Technologies