Journal Article

Sequencing technologies — the next generation

Metzker, Michael L.

2010

Overview

Key Points The major advance offered by next-generation sequencing (NGS) technologies is the ability to produce, in some cases, in excess of one billion short reads per instrument run, which makes them useful for many biological applications. The variety of NGS features makes it likely that multiple platforms will coexist in the marketplace, with some having clear advantages for particular applications over others. The leading NGS platforms use clonally amplified templates, which are not affected by the arbitrary losses of genomic sequences that are inherent in bacterial cloning methods. An important advantage of single-molecule template platforms is that PCR is not required. PCR can create mutations that masquerade as sequence variants and amplification bias that underrepresents AT-rich and GC-rich regions in target sequences. There are four primary NGS chemistry methods: cyclic reversible termination, sequencing by ligation, pyrosequencing and real-time sequencing, which are described in this Review. To call sequence variants in genomes, NGS reads are aligned to a reference sequence using various bioinformatics mapping tools. Whole-genome sequencing using current NGS platforms is still expensive, but targeting regions of interest may provide an interim solution to analysing hundreds, if not thousands, of samples. To date, the sequences of twelve human genomes have been published using a number of NGS platforms, marking the beginning of personalized genomics. NGS costs will continue to drop in the foreseeable future, although the cost reduction should be weighed against the quality of the produced genome sequence. There is an increasing demand for next-generation sequencing technologies that rapidly deliver high volumes of accurate genome information at a low cost. This Review provides a guide to the features of the different platforms, and describes the recent advances in this fast-moving area. Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Agriculture / Animal Genetics and Genomics / Automation / Biological and medical sciences / Biomedical and Life Sciences / Biomedicine / Cancer Research / DNA methylation / DNA polymerase / DNA sequencing / Fundamental and applied biological sciences. Psychology / Gene Function / Genetic Techniques / Genetics of eukaryotes. Biological and molecular evolution / Genome / Genome, Human / Genomes / Genomics - methods / Human Genetics / Humans / Methods / Models, Genetic / Molecular Biology - methods / Nucleotide sequencing / Oligonucleotides - genetics / Polymerase Chain Reaction / review-article / Sequence Analysis, DNA - economics / Sequence Analysis, DNA - instrumentation / Sequence Analysis, DNA - methods / Sequence Analysis, DNA - trends / Technology