Sequencing technologies — the next generation

Abstract

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.