Key Points
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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.
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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.
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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.
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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.
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There are four primary NGS chemistry methods: cyclic reversible termination, sequencing by ligation, pyrosequencing and real-time sequencing, which are described in this Review.
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To call sequence variants in genomes, NGS reads are aligned to a reference sequence using various bioinformatics mapping tools.
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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.
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To date, the sequences of twelve human genomes have been published using a number of NGS platforms, marking the beginning of personalized genomics.
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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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References
International Human Genome Consortium. Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 (2004).
Metzker, M. L. Emerging technologies in DNA sequencing. Genome Res. 15, 1767–1776 (2005).
Hutchison, C. A. III. DNA sequencing: bench to bedside and beyond. Nucleic Acids Res. 35, 6227–6237 (2007).
Wold, B. & Myers, R. M. Sequence census methods for functional genomics. Nature Methods 5, 19–21 (2008).
Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Rev. Genet. 10, 57–63 (2009). The Review provides a comprehensive overview of recent advances and challenges in techniques that are used in transcriptome profiling methods that use NGS technologies (RNA–seq).
Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotech. 26, 1146–1153 (2008). An excellent review of the current state of nanopore sequencing that highlights recent accomplishments and remaining challenges in the field.
Fan, J.-B., Chee, M. S. & Gunderson, K. L. Highly parallel genomic assays. Nature Rev. Genet. 7, 632–644 (2006).
Pop, M. & Salzberg, S. L. Bioinformatics challenges of new sequencing technology. Trends Genet. 24, 142–149 (2008).
Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. USA 100, 8817–8822 (2003).
Fedurco, M., Romieu, A., Williams, S., Lawrence, I. & Turcatti, G. BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies. Nucleic Acids Res. 34, e22 (2006).
Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005). This paper describes the development of the non-cleavable SBL method and shows its feasibility by sequencing the E. coli genome. The prototype described led to the development of the Polonator instrument.
Kim, J. B. et al. Polony multiplex analysis of gene expression (PMAGE) in mouse hypertrophic cardiomyopathy. Science 316, 1481–1484 (2007).
Leamon, J. H. A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis 24, 3769–3777 (2003).
Harris, T. D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008). Developers from Helicos BioSciences and colleagues describe the development of the first single-molecule sequencing method using reversible terminators and demonstrate the technology by sequencing the M13 genome.
Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009). The authors describe the development of a real-time sequencing method using their ZMW detection system and demonstrate its feasibility by sequencing synthetic templates.
Hardin, S., Gao, X., Briggs, J., Willson, R. & Tu, S.-C. Methods for real-time single molecule sequence determination. US Patent 7,329,492 (2000).
Williams, J. G. K. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis. US Patent 6,255,083 (1998).
Erlich, Y., Mitra, P. P., delaBastide, M., McCombie, W. R. & Hannon, G. J. Alta-Cyclic: a self-optimizing base caller for next-generation sequencing. Nature Methods 5, 679–682 (2008).
Metzker, M. L. et al. Termination of DNA synthesis by novel 3′-modified deoxyribonucleoside triphosphates. Nucleic Acids Res. 22, 4259–4267 (1994).
Canard, B. & Sarfati, R. DNA polymerase fluorescent substrates with reversible 3′-tags. Gene 148, 1–6 (1994).
Ju, J. et al. Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc. Natl. Acad. Sci. USA 103, 19635–19640 (2006).
Guo, J. et al. Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc. Natl Acad. Sci. USA 105, 9145–9150 (2008).
Bentley, D. R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008). Developers from Illumina/Solexa and colleagues report details on their reversible terminator platform and demonstrate the technology by sequencing a flow-sorted X chromosome and the genome from a Yoruban male.
Dohm, J. C., Lottaz, C., Borodina, T. & Himmelbauer, H. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 36, e105 (2008).
Hillier, L. W. et al. Whole-genome sequencing and variant discovery in C. elegans. Nature Methods 5, 183–188 (2008).
Harismendy, O. et al. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol. 10, R32 (2009).
Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).
Frazer, K. A., Murray, S. S., Schork, N. J. & Topol, E. J. Human genetic variation and its contribution to complex traits. Nature Rev. Genet. 10, 241–251 (2009).
Ley, T. J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008).
Sarin, S., Prabhu, S., O'Meara, M. M., Pe'er, I. & Hobert, O. Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nature Methods 5, 865–867 (2008).
Wu, W. et al. Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates. Nucleic Acid Res. 35, 6339–6349 (2007).
Wu, W., Litosh, V. A., Stupi, B. P. & Metzker, M. L. Photocleavable labeled nucleotides and nucleosides and methods for their use in DNA sequencing. US Patent Application 11/567,189 (2009).
Bowers, J. et al. Virtual terminator nucleotides for next-generation DNA sequencing. Nature Methods 6, 593–595 (2009).
Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S. R. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA 100, 3960–3964 (2003).
Tomkinson, A. E., Vijayakumar, S., Pascal, J. M. & Ellenberger, T. DNA ligases: structure, reaction mechanism, and function. Chem. Rev. 106, 687–699 (2006).
Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science 241, 1077–1080 (1988).
Valouev, A. et al. A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Res. 18, 1051–1063 (2008). This paper describes Life/APG's SBL method, which uses cleavable two-base-encoded probes on the SOLiD platform. The authors demonstrate the technology through the application of genome-wide nucleosome mapping in C. elegans .
Shen, Y., Sarin, S., Liu, Y., Hobert, O. & Pe'er, I. Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing. PLoS ONE 3, e4012 (2008).
Ronaghi, M., Uhlén, M. & Nyrén, P. A sequencing method based on real-time pyrophosphate. Science 281, 363–365 (1998).
Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlén, M. & Nyrén, P. Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89 (1996).
Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005). The authors describe the development of the first NGS technology using the pyrosequencing method and demonstrate its feasibility through the sequencing and de novo assembly of the Mycoplasma genitalium genome.
Metzker, M. L. Sequencing in real time. Nature Biotech. 27, 150–151 (2009).
Levene, M. J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).
Trapnell, C. & Salzberg, S. L. How to map billions of short reads onto genomes. Nature Biotech. 27, 455–457 (2009).
Chaisson, M. J., Brinza, D. & Pevzner, P. A. De novo fragment assembly with short mate-paired reads: does the read length matter? Genome Res. 19, 336–346 (2009).
Hofreuter, D. et al. Unique features of a highly pathogenic Campylobacter jejuni strain. Infect. Immun. 74, 4694–4707 (2006).
Holt, K. E. et al. High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nature Genet. 40, 987–993 (2008).
Srivatsan, A. et al. High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies. PLoS Genet. 4, e1000139 (2008).
Suchland, R. J. et al. Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations. Infect. Immun. 76, 5438–5446 (2008).
Nusbaum, C. et al. Sensitive, specific polymorphism discovery in bacteria using massively parallel sequencing. Nature Methods 6, 67–69 (2009).
Moran, N. A., McLaughlin, H. J. & Sorek, R. The dynamics and time scale of ongoing genomic erosion in symbiotic bacteria. Science 323, 379–382 (2009).
Ossowski, S. et al. Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Res. 18, 2024–2033 (2008).
Korbel, J. O. et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318, 420–426 (2007).
Kidd, J. M. et al. Mapping and sequencing of structural variation from eight human genomes. Nature 453, 56–64 (2008).
Warren, R. L., Sutton, G. G., Jones, S. J. M. & Holt, R. A. Assembling millions of short DNA sequences using SSAKE. Bioinformatics 23, 500–501 (2007).
Chaisson, M. J. & Pevzner, P. A. Short read fragment assembly of bacterial genomes. Genome Res. 18, 324–330 (2008).
Hernandez, D., François, P., Farinelli, L., Østerås, M. & Schrenzel, J. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 18, 802–809 (2008).
Butler, J. et al. ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res. 18, 810–820 (2008).
Zerbino, D. R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).
Aury, J.-M. et al. High quality draft sequences for prokaryotic genomes using a mix of new sequencing technologies. BMC Genomics 9, 603 (2008).
Reinhardt, J. A. et al. De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae. Genome Res. 19, 294–305 (2009).
Schloss, J. A. How to get genomes at one ten-thousandth the cost. Nature Biotech. 26, 1113–1115 (2008).
Altshuler, D., Daly, M. J. & Lander, E. S. Genetic mapping in human disease. Science 322, 881–888 (2008).
Wang, L. & Weinshilboum, R. M. Pharmacogenomics: candidate gene identification, functional validation and mechanisms. Hum. Mol. Genet. 17, R174–R179 (2008).
Haaland, W. C. et al. A–β– subtype of ketosis-prone diabetes is not predominantly a monogenic diabetic syndrome. Diabetes Care 32, 873–877 (2009).
Tewhey, R. et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nature Biotech. 27, 1025–1031 (2009).
Singh-Gasson, S. et al. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nature Biotech. 17, 974–978 (1999).
Albert, T. J. et al. Direct selection of human genomic loci by microarray hybridization. Nature Methods 4, 903–905 (2007).
Hodges, E. et al. Genome-wide in situ exon capture for selective resequencing. Nature Genet. 39, 1522–1527 (2007).
Okou, D. T. et al. Microarray-based genomic selection for high-throughput resequencing. Nature Methods 4, 907–909 (2007).
Porreca, G. J. et al. Multiplex amplification of large sets of human exons. Nature Methods 4, 931–936 (2007).
Krishnakumar, S. et al. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc. Natl Acad. Sci. USA 105, 9296–9301 (2008).
Turner, E. H., Lee, C., Ng, S. B., Nickerson, D. A. & Shendure, J. Massively parallel exon capture and library-free resequencing across 16 genomes. Nature Methods 6, 315–316 (2009).
Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nature Biotech. 27, 182–189 (2009).
Olson, M. Enrichment of super-sized resequencing targets from the human genome. Nature Methods 4, 891–892 (2007).
Garber, K. Fixing the front end. Nature Biotech. 26, 1101–1104 (2008).
Petrosino, J. F., Highlander, S., Luna, R. A., Gibbs, R. A. & Versalovic, J. Metagenomic pyrosequencing and microbial identification. Clin. Chem. 55, 856–866 (2009). These authors describe the current state of metagenomics research and highlight the use of the Roche/454 platform for microbial identification through16S ribosomal DNA phylogenetic analysis; other NGS platforms may be better suited for gene discovery efforts (see Table 2).
Lipson, D. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nature Biotech. 27, 652–658 (2009).
Ozsolak, F. et al. Direct RNA sequencing. Nature 461, 814–818 (2009).
Park, P. J. ChIP–seq: advantages and challenges of a maturing technology. Nature Rev. Genet. 10, 669–680 (2009). The article provides a comprehensive review of recent technological advances and challenges in genome-wide profiling of DNA-binding proteins, histone modifications and nucleosomes using NGS technologies (ChIP–seq).
Levy, S. et al. The diploid genome sequence of an individual human. PLoS Biol. 5, e254 (2007).
Wheeler, D. A. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876 (2008).
Iafrate, A. J. et al. Detection of large-scale variation in the human genome. Nature Genet. 36, 949–951 (2004).
Sebat, J. et al. Large-scale copy number polymorphism in the human genome. Science 305, 525–528 (2004).
Tuzun, E. et al. Fine-scale structural variation of the human genome. Nature Genet. 37, 727–732 (2005).
Stranger, B. E. et al. Relative impact of nucleotide and copy number variation on gene expression phenotypes. Science 315, 848–853 (2007).
Wang, J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60–65 (2008).
Kim, J. I. et al. A highly annotated whole-genome sequence of a Korean individual. Nature 460, 1011–1015 (2009).
Ahn, S. M. et al. The first Korean genome sequence and analysis: full genome sequencing for a socio-ethnic group. Genome Res. 19, 1622–1629 (2009).
McKernan, K. J. et al. Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding. Genome Res. 19, 1527–1541 (2009).
Pushkarev, D., Neff, N. F. & Quake, S. R. Single-molecule sequencing of an individual human genome. Nature Biotech. 27, 847–850 (2009).
Mardis, E. R. et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N. Engl. J. Med. 361, 1058–1066 (2009).
Lupski, J. R. et al. Complete genome sequencing identifies recessive alleles in SH3TC2 causing a CMT1 neuropathy. N. Engl. J. Med. (in the press).
Collins, F. S. & Barker, A. D. Mapping the cancer genome. Sci. Am. 296, 50–57 (2007).
Drmanac, R. et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 5 Nov 2009 (doi:10.1126/science.1181498).
Sanderson, K. Personal genomes: standard and pores. Nature 456, 23–25 (2008).
Green, R. E. et al. Analysis of one million base pairs of Neanderthal DNA. Nature 444, 330–336 (2006).
Briggs, A. W. et al. Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325, 318–321 (2009).
Ponting, C. P., Oliver, P. L. & Reik, W. Evolution and functions of long noncoding RNAs. Cell 136, 629–641 (2009).
Carthew, R. W. & Sontheimer, E. J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009).
Barnes, C., Balasubramanian, S., Liu, X., Swerdlow, H. & Milton, J. Labelled nucleotides. US Patent 7,057,026 (2002).
Mitra, R. D., Shendure, J., Olejnik, J., Edyta-Krzymanska-Olejnik & Church, G. M. Fluorescent in situ sequencing on polymerase colonies. Anal. Biochem. 320, 55–65 (2003).
Turcatti, G., Romieu, A., Fedurco, M. & Tairi, A.-P. A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acids Res. 36, e25 (2008).
Yarbrough, L. R., Schlageck, J. G. & Baughman, M. Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. J. Biol. Chem. 254, 12069–12073 (1979).
Kumar, S. et al. Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases. Nucleosides Nucleotides Nucleic Acids 24, 401–408 (2005).
McKernan, K., Blanchard, A., Kotler, L. & Costa, G. Reagents, methods, and libraries for bead-based sequencing. US Patent Application 11/345,979 (2005).
Macevicz, S. C. DNA sequencing by parallel oligonucleotide extensions. US Patent 5,969,119 (1995).
Mir, K. U., Qi, H., Salata, O. & Scozzafava, G. Sequencing by cyclic ligation and cleavage (CycLiC) directly on a microarray captured template. Nucleic Acids Res. 37, e5 (2009).
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.
Glossary
- Automated Sanger sequencing
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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All sequence variants other than single-nucleotide variants, including block substitutions, insertions or deletions, inversions, segmental duplications and copy-number differences.
- 1000 Genomes Project
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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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DOI: https://doi.org/10.1038/nrg2626
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