Sequencing technologies — the next generation

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.

Abstract

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.

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Figure 1: Template immobilization strategies.
Figure 2: Four-colour and one-colour cyclic reversible termination methods.
Figure 3: Next-generation sequencing technologies that use emulsion PCR.
Figure 4: Real-time sequencing.
Figure 5: Targeted capture scheme.

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Acknowledgements

I am extremely grateful to S.-M. Ahn, J. Edwards, J. W. Efcavitch, R. A. Gibbs, T. Harkin, E. Mardis, K. McKernan, D. Muzny, S. Turner and D. Wheeler for providing current performance data for the NGS platforms, and to the National Human Genome Research Institute for their support from grants R01 HG003573, R41 HG003072, R41 HG003265 and R21 HG002443.

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Michael L. Metzker is President and CeO of LaserGen, Inc.

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1000 Genomes Project

Advances in Genome Biology and Technology meeting

The Cancer Genome Atlas

The Exome Project

Human Microbiome Project

National Human Genome Research Institute

National Human Genome Research Institute — Fruitfly Genome Sequencing

Nature Reviews Genetics poster 'Sequencing technologies — the next generation'

Personal Genome Project

Glossary

Automated Sanger sequencing

This process involves a mixture of techniques: bacterial cloning or PCR; template purification; labelling of DNA fragments using the chain termination method with energy transfer, dye-labelled dideoxynucleotides and a DNA polymerase; capillary electrophoresis; and fluorescence detection that provides four-colour plots to reveal the DNA sequence.

Finished grade

A quality measure for a sequenced genome. A finished-grade genome, commonly referred to as a finished genome, is of higher quality than a draft-grade genome, with more base coverage and fewer errors and gaps (for example,the human genome reference contains 2.85 Gb, covers 99% of the genome with 341 gaps, and has an error rate of 1 in every 100,000 bp).

Template

This recombinant DNA molecule is made up of a known region, usually a vector or adaptor sequence to which a universal primer can bind, and the target sequence, which is typically an unknown portion to be sequenced.

Seq-based methods

Assays that use next-generation sequencing technologies. They include methods for determining the sequence content and abundance of mRNAs, non-coding RNAs and small RNAs (collectively called RNA–seq) and methods for measuring genome-wide profiles of immunoprecipitated DNA–protein complexes (ChIP–seq), methylation sites (methyl–seq) and DNase I hypersensitivity sites (DNase–seq).

Polonator

This Review mostly describes technology platforms that are associated with a respective company, but the Polonator G.007 instrument, which is manufactured and distributed by Danaher Motions (a Dover Company), is an open source platform with freely available software and protocols. Users manufacture their own reagents based on published reports or by collaborating with George Church and colleagues or other technology developers.

Fragment templates

A fragment library is prepared by randomly shearing genomic DNA into small sizes of <1kb, and requires less DNA than would be needed for a mate-pair library.

Mate-pair templates

A genomic library is prepared by circularizing sheared DNA that has been selected for a given size, such as 2 kb, therefore bringing the ends that were previously distant from one another into close proximity. Cutting these circles into linear DNA fragments creates mate-pair templates.

Dephasing

This occurs with step-wise addition methods when growing primers move out of synchronicity for any given cycle. Lagging strands (for example, n − 1 from the expected cycle) result from incomplete extension, and leading strands (for example, n + 1) result from the addition of multiple nucleotides or probes in a population of identical templates.

Dark nucleotides or probes

A nucleotide or probe that does not contain a fluorescent label. It can be generated from its cleavage and carry-over from the previous cycle or be hydrolysed in situ from its dye-labelled counterpart in the current cycle.

Total internal reflection fluorescence

A total internal reflection fluorescence imaging device produces an evanescent wave that is, a near-field stationary excitation wave — with an intensity that decreases exponentially away from the surface. This wave propagates across a boundary surface, such as a glass slide, resulting in the excitation of fluorescent molecules near (<200 nm) or at the surface and the subsequent collection of their emission signals by a detector.

Libraries of mutant DNA polymerases

Large numbers of genetically engineered DNA polymerases can be created by either site-directed or random mutagenesis, which leads to one or more amino acid substitutions, insertions and/or deletions in the polymerase. The goal of this approach is to incorporate modified nucleotides more efficiently during the sequencing reaction.

Consensus reads

These are only useful for single-molecule techniques and are produced by sequencing the same template molecule more than once. The data are then aligned to produce a 'consensus read', reducing stochastic errors that may occur in a given sequence read.

One-base-encoded probe

An oligonucleotide sequence in which one interrogation base is associated with a particular dye (for example,A in the first position corresponds to a green dye). An example of a one-base degenerate probe set is '1-probes', which indicates that the first nucleotide is the interrogation base. The remaining bases consist of either degenerate (four possible bases) or universal bases.

Two-base-encoded probe

An oligonucleotide sequence in which two interrogation bases are associated with a particular dye (for example, AA, CC, GG and TT are coded with a blue dye). '1,2-probes' indicates that the first and second nucleotides are the interrogation bases. The remaining bases consist of either degenerate or universal bases.

Adjacent valid colour

A nucleotide substitution will have two colour calls, one from the 5′ position and one from the 3′ position of the dinucleotide sequence. When compared with a reference genome, base substitution in the target sequence is encoded by two specific, adjacent colours. In Figure 3b, the sequence 'CCT' is encoded as blue-yellow ('CC' = blue; 'CT' = yellow), but substituting the middle 'C' for 'A' would result in two colour changes to green-red. Any other colour sequence can be discarded as an error.

Colour space

With two-base-encoded probes, the fluorescent signal or colour obtained during imaging is associated with four dinucleotide sequences having a 5′- and 3′-base. Colour space is the sequence of overlapping dinucleotides that codes four simultaneous nucleotide sequences. Alignment with a reference genome is the most accurate method for translating colour space into a single nucleotide sequence.

Zero-mode waveguide detectors

This nanostructure device is 100 nm in diameter, which is smaller than the 532 nm and 643 nm laser wavelengths used in the Pacific Biosciences platform. Light cannot propagate through these small waveguides, hence the term zero-mode. These aluminium-clad waveguides are designed to produce an evanescent wave (see the 'total internal reflection fluorescence' glossary term) that substantially reduces the observation volume at the surface of the polymerase reaction down to the zeptolitre range (10−21 l). This provides an advantage for the polymerization reaction, which can be performed at higher dye-labelled nucleotide concentrations.

Fluorescence resonance energy transfer

This is generally a system that consists of two fluorescent dyes, one being a donor dye (a bluer fluorophore) and the other an acceptor dye (a redder fluorophore). When the two dye molecules are brought into close proximity (usually ≤30 nm), the energy from the excited donor dye is transferred to the acceptor dye, increasing its emission intensity signal.

Structural variants

All sequence variants other than single-nucleotide variants, including block substitutions, insertions or deletions, inversions, segmental duplications and copy-number differences.

1000 Genomes Project

A project aimed at discovering rare sequence variants with minor allele frequencies of 1% in normal genomes derived from HapMap samples.

The Exome Project

A project aimed at developing and validating cost-effective, high-throughput technologies for resequencing all of the protein-coding regions of the human genome.

Metagenomics

The study of communities of mixed microbial genomes that reside in animals, plants and environmental niches. Samples are collected and analysed without the need to culture isolated microbes in the laboratory. The Human Microbiome Project aims to characterize a reference set of microbial genomes from different habitats within the human body, including nasal, oral, skin, gastrointestinal and urogenital regions, and to determine how changes in the human microbiome affect health and disease.

The Cancer Genome Atlas

A project aimed at discovering single-nucleotide variants and structural variants that are associated with major cancers, such as brain cancer (glioblastoma multiforme), lung cancer (squamous carcinoma) and ovarian cancer (serous cystadenocarcinoma).

Personal Genome Project

A project aimed at providing open access to human genome sequences from volunteers and to develop tools for interpreting this information and correlating it with related personal medical information.

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Metzker, M. Sequencing technologies — the next generation. Nat Rev Genet 11, 31–46 (2010). https://doi.org/10.1038/nrg2626

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