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  • Review Article
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Advanced sequencing technologies: methods and goals

Nature Reviews Genetics volume 5, pages 335–344 (2004)Cite this article

Key Points

  • Several academic and commercial research groups are working to develop new ultra-low-cost sequencing (ULCS) technologies. These aim to reduce the cost of DNA sequencing by several orders of magnitude.

  • ULCS technology could potentially have an important impact on human health by enabling the sequencing of 'personal genomes' as a component of individualized health care.

  • Microelectrophoretic approaches borrow microfabrication techniques from the semiconductor industry to miniaturize and integrate the amplification, purification and electrophoretic sequencing of DNA.

  • Sequencing by hybridization involves highly parallel genomic resequencing. It is carried out by hybridizing target DNA to high-density microarrays that are designed to query the identity of individual bases.

  • Cyclic-array methods that operate on amplified templates include 'fluorescent in situ sequencing', Pyrosequencing and 'massively parallel signature sequencing'. Cyclic-array methods that aim to directly sequence single molecules are also under development.

  • Methods such as nanopore sequencing offer the prospect of non-cyclic, real-time, single-molecule sequencing.

  • The prospect of ULCS and personal genomes raises various important ethical, legal and social questions.

Abstract

Nearly three decades have passed since the invention of electrophoretic methods for DNA sequencing. The exponential growth in the cost-effectiveness of sequencing has been driven by automation and by numerous creative refinements of Sanger sequencing, rather than through the invention of entirely new methods. Various novel sequencing technologies are being developed, each aspiring to reduce costs to the point at which the genomes of individual humans could be sequenced as part of routine health care. Here, we review these technologies, and discuss the potential impact of such a 'personal genome project' on both the research community and on society.

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Figure 1: Exponential growth in computing and sequencing.
Figure 2: Examples of microelectrophoretic sequencing and nanopore sequencing.
Figure 3: Examples of cyclic-array sequencing and sequencing by hybridization.

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Acknowledgements

The authors thank members of the polony community and C. Hennig for sharing unpublished work, T. Wu and G. Porreca for helpful discussions, and R. Shendure and K. McKernan for critical reading of the manuscript.

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Authors and Affiliations

  1. Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Jay Shendure, Chris Varma & George M. Church

  2. Deptartment of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, 63110, Missouri, USA

    Robi D. Mitra

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DATABASES

LocusLink

CD44

OMIM

malaria

sickle-cell anaemia

FURTHER INFORMATION

International HapMap Project

Personal Genome Project

Revolutionary Genome Sequencing Technologies — The $1000 Genome

Glossary

BERMUDA PRINCIPLES

A commitment that was made in Bermuda (February 1996) by an international assortment of genome-research sponsors to the principles of public sharing and the rapid release of human genome sequence information.

'COMMON' SINGLE NUCLEOTIDE POLYMORPHISMS

(SNPs). Those single-nucleotide substitutions that occur with an allelic frequency of more than 1% in a given population.

HAPLOTYPE MAPPING

A technique that involves the use of combinations of 'common' DNA polymorphisms to find blocks of association with phenotypic traits.

GENETIC HETEROGENEITY

Describes situations in which a similar phenotype can result from various genetic defects.

SYNTHETIC BIOLOGY

A discipline that embraces the emerging ability to design, synthesize and evolve new genomes or biomimetic systems.

PHYLOGENETIC FOOTPRINTING AND SHADOWING

The annotation of functional elements in a genome through bioinformatic comparisons to the genomes of one or more related species.

DIRECTED EVOLUTION

The evolution of a protein (or organism) in the laboratory through rounds of mutation and selection for a particular activity or trait.

FLUORESCENT IN SITU SEQUENCING (FISSEQ).

A cyclical, polymerase-driven sequencing method in which nucleotides are modified with fluorescent labels that can be chemically removed at each step.

PHARMACOGENETIC

The heritable component of variation among individuals with respect to drug response or adverse reaction.

SANGER SEQUENCING

(Chain termination or dideoxy method). A technique that uses an enzymatic procedure to synthesize DNA chains of varying length in four different reactions, stopping the DNA replication at positions that are occupied by one of the four bases, and then determining the resulting fragment lengths to decipher the sequence.

POLONY

A colony of PCR amplicons that is derived from a single molecule of nucleic acid, amplified in situ in an acrylamide gel.

TYPE IIS RESTRICTION ENZYME

A type of restriction endonuclease that is characterized by an asymmetric recognition site and cleavage at a fixed distance outside the recognition site.

RAW READ

The actual nucleotide sequence that is generated by a sequencing instrument. This contrasts with the finished sequence, which is produced by obtaining the consensus sequence of many overlapping raw reads.

FLUORESCENCE RESONANCE ENERGY TRANSFER

(FRET). A phenomenon by which excitation is transferred from a donor fluorescent molecule to an acceptor fluorescent molecule; the interaction is distance-dependent and can be used to probe molecular interactions at distances below the limit of optical resolution.

ZERO-MODE WAVEGUIDE

A nanostructure device with physical properties that markedly limit the effective volume of observation.

DEPHASING

The progressive loss of synchronization between templates within features as a consequence of the failure to achieve 100% extension at each extension cycle.

MULTIPLE-DISPLACEMENT AMPLIFICATION

A technique for achieving whole-genome amplification that uses a strand-displacing polymerase to catalyse the isothermal (that is, at a constant temperature) amplification of DNA.

WHOLE-GENOME AMPLIFICATION

(WGA). The in vitro amplification of a full genome sequence, ideally with even representation of the genome in the amplified product. Techniques for achieving WGA include PCR primed with random or degenerate oligonucleotides, or multiple-displacement amplification.

COMPUTED TOMOGRAPHY

(CT). An imaging technology that uses computer processing of X-ray images to visualize cross-sectional (transverse) slices of internal structures; the advantage of CT over conventional radiography is the ability to eliminate superimposition.

MAGNETIC RESONANCE [IMAGING]

(MRI). A non-invasive technique that is used to generate multi-dimensional proton-density images of internal organs, structures and lesions.

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Shendure, J., Mitra, R., Varma, C. et al. Advanced sequencing technologies: methods and goals. Nat Rev Genet 5, 335–344 (2004). https://doi.org/10.1038/nrg1325

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