Sequential sequencing by synthesis and the next-generation sequencing revolution

Abstract

Next-generation sequencing (NGS) involving massively parallel DNA analysis has made an enormous impact on life science, medicine, and biotechnology, through a multitude of applications.The vast majority of the major NGS systems are based on the concept of ‘sequencing by synthesis’ (SBS) with sequential detection of nucleotide incorporation using an engineered DNA polymerase.The basic principles of SBS include attachment of DNA fragments to a solid support, conversion to a single-strand template and the annealing of a primer, the incorporation of complementary nucleotides by a polymerase, and detection of this incorporation.The development of NGS spans several decades of innovations, from early systems using natural nucleotides to later systems for massively parallel sequencing systems using reversible fluorescent nucleotides. There is no question that massively parallel sequencing (MPS) technology, often referred to as NGS, has had a unique and huge impact on life science research [1.Gibbs R.A. The Human Genome Project changed everything.Nat. Rev. Genet. 2020; 21: 575-576Crossref PubMed Scopus (54) Google Scholar,2.McGinn S. Gut I.G. DNA sequencing – spanning the generations.New Biotechnol. 2013; 30: 366-372Crossref PubMed Scopus (0) Google Scholar], with a large number of studies using this technology currently published every day. The number of DNA sequences in public databases has exploded since MPS by synthesis was commercially introduced in 2005 [3.Margulies M. et al.Genome sequencing in microfabricated high-density picolitre reactors.Nature. 2005; 437: 376-380Crossref PubMed Scopus (6037) Google Scholar]. This has also led to a dramatic decrease in cost and efforts to sequence whole genomes. This is illustrated by the estimated cost for the first human genome published in 2001 using electrophoretic technology for base calling, which was estimated to be US$2–3 billion, while today it is possible to sequence a human genome with MPS for less than US$1000 [1.Gibbs R.A. The Human Genome Project changed everything.Nat. Rev. Genet. 2020; 21: 575-576Crossref PubMed Scopus (54) Google Scholar], a cost reduction by a factor of over 1 million. This is remarkable and it is hard to find a similar example in scientific history. This has led to an explosion of scientific data and the entrance of a new era in medicine and biology driven by ‘big data’ and data-driven research. In the field of genetics, the creation of maps covering the genetic diversity in various human populations [4.Gudbjartsson D.F. et al.Sequence variants from whole genome sequencing a large group of Icelanders.Sci. Data. 2015; 2150011Crossref Scopus (49) Google Scholar] has greatly increased our understanding of the relationship of genes and diseases. Similar maps across the ‘tree of life’ [5.Hug L.A. et al.A new view of the tree of life.Nat. Microbiol. 2016; 1: 16048Crossref PubMed Google Scholar] have increased our understanding of the parts list of human building blocks. The discovery of a new human ancestor, the Denisovans [6.Meyer M. et al.A high-coverage genome sequence from an archaic Denisovan individual.Science. 2012; 338: 222-226Crossref PubMed Scopus (1185) Google Scholar], was enabled by NGS, and population studies showed widespread remains of both Neanderthal and Denisovan DNA in our genomes. The technology has also led to clinical practice, both to understand and to treat cancers [7.Mardis E.R. Wilson R.K. Cancer genome sequencing: a review.Hum. Mol. Genet. 2009; 18: R163-R168Crossref PubMed Scopus (162) Google Scholar], but also to allow diagnosis in children with unknown disease-causing mutations, resulting in the design of drug treatments [8.Stranneheim H. et al.Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.Genome Med. 2021; 13: 40Crossref PubMed Scopus (61) Google Scholar,9.Marwaha S. et al.A guide for the diagnosis of rare and undiagnosed disease: beyond the exome.Genome Med. 2022; 14: 23Crossref PubMed Scopus (37) Google Scholar]. In the following, the various concepts enabling the rapid development of NGS are discussed. The objective of SBS is to determine the sequencing of a DNA sample by detecting in a sequential manner the incorporation of nucleotides using an engineered DNA polymerase (Figure 1). An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The key parts are highly similar for all embodiments of SBS and include the following:(i)attachment of the DNA to be sequenced to a solid support, usually combined with amplification of the DNA to enhance the subsequent signal;(ii)generation of single-stranded DNA on the solid support;(iii)primer-dependent incorporation of complementary nucleotides using an engineered polymerase; and(iv)detection of the incorporated nucleotide. Steps (iii) and (iv) are repeated and the sequence is assembled from the signals obtained in step (iv). This principle of SBS has been used for almost all MPS efforts and it has contributed to the vast majority of sequence information generated during the past decade [10.Heather J.M. Chain B. The sequence of sequencers: the history of sequencing DNA.Genomics. 2016; 107: 1-8Crossref PubMed Scopus (634) Google Scholar]. The historical development of sequential SBS has been reviewed by others [10.Heather J.M. Chain B. The sequence of sequencers: the history of sequencing DNA.Genomics. 2016; 107: 1-8Crossref PubMed Scopus (634) Google Scholar], which we briefly summarize here. The concept was first described in 1993 [11.Nyren P. et al.Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay.Anal. Biochem. 1993; 208: 171-175Crossref PubMed Scopus (0) Google Scholar] in the form of a technique later known as pyrosequencing. In this case, nucleotide incorporation was detected by measuring pyrophosphate products of incorporation. In the first publication [11.Nyren P. et al.Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay.Anal. Biochem. 1993; 208: 171-175Crossref PubMed Scopus (0) Google Scholar], all of the key concepts of SBS were introduced, including the amplification of DNA to enhance the subsequent signal and attachment of the DNA to be sequenced to a solid support, the generation of single-stranded DNA on the solid support, the incorporation of nucleotides using an engineered polymerase, and light detection of the incorporated nucleotide. This paper also outlines a vision of MPS: ‘Automated on-line methods with multiple samples in parallel can be envisioned’. In a follow-up article [12.Ronaghi M. et al.Real-time DNA sequencing using detection of pyrophosphate release.Anal. Biochem. 1996; 242: 84-89Crossref PubMed Scopus (848) Google Scholar], the concept was further developed, and a few years later Ronaghi, Uhlén and Nyrén [13.Ronaghi M. et al.A sequencing method based on real-time pyrophosphate.Science. 1998; 281: 365Crossref PubMed Scopus (0) Google Scholar] showed that non-incorporated nucleotides could be removed with a fourth enzyme (apyrase) allowing SBS to be performed without the need to wash away non-incorporated nucleotides. A commercial instrument based on SBS (called Pyrosequencing) was launched in 2000 with all key concepts for SBS with real-time detection and with a throughput of 96 samples in parallel [14.Harrington C.T. et al.Fundamentals of pyrosequencing.Arch. Pathol. Lab. Med. 2013; 137: 1296-1303Crossref PubMed Scopus (92) Google Scholar]. A modified version of this instrument is still available and is used for many applications, including DNA methylation/epigenetics [15.Claus R. et al.A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR.Epigenetics. 2012; 7: 772-780Crossref PubMed Scopus (57) Google Scholar,16.Sadikovic B. et al.Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders.Genet. Med. 2021; 23: 1065-1074Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] and forensics studies [17.Ghemrawi M. et al.Pyrosequencing: current forensic methodology and future applications – a review.Electrophoresis. 2023; 44: 298-312Crossref Scopus (1) Google Scholar]. The first next-generation sequencers (Figure 2) were based on pyrosequencing chemistry and were commercialized by Rothberg and coworkers [3.Margulies M. et al.Genome sequencing in microfabricated high-density picolitre reactors.Nature. 2005; 437: 376-380Crossref PubMed Scopus (6037) Google Scholar] from the company 454 Life Sciences in the USA. They showed that sequencing could be performed in a highly parallel manner, and in the paper they described the successful sequencing of the genome of Mycoplasma genitalium, which is also the first description of whole-genome sequencing using MPS. Many important applications were enabled by this pioneering instrument [18.Rothberg J.M. Leamon J.H. The development and impact of 454 sequencing.Nat. Biotechnol. 2008; 26: 1117-1124Crossref PubMed Scopus (366) Google Scholar], including the analysis of the Neanderthal genome [19.Green R.E. et al.Analysis of one million base pairs of Neanderthal DNA.Nature. 2006; 444: 330-336Crossref PubMed Scopus (517) Google Scholar] in collaboration with Paabo and coworkers at the Max Planck Institute in Leipzig, Germany. Others used this approach to study microbial diversity in the deep sea [20.Sogin M.L. et al.Microbial diversity in the deep sea and the underexplored “rare biosphere”.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12115-12120Crossref PubMed Scopus (2774) Google Scholar], to study the microbial genomics of archaea in soil [21.Leininger S. et al.Archaea predominate among ammonia-oxidizing prokaryotes in soils.Nature. 2006; 442: 806-809Crossref PubMed Scopus (1754) Google Scholar], to study the possible causes of honeybee hive collapse [22.Cox-Foster D.L. et al.A metagenomic survey of microbes in honey bee colony collapse disorder.Science. 2007; 318: 283-287Crossref PubMed Scopus (1369) Google Scholar], and to perform the first single-cell genome sequencing of an unculturable organism [23.Marcy Y. et al.Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11889-11894Crossref PubMed Scopus (471) Google Scholar]. This approach was also used to determine one of the first individual human genome sequences in 2008 [24.Wheeler D.A. et al.The complete genome of an individual by massively parallel DNA sequencing.Nature. 2008; 452: 872-876Crossref PubMed Scopus (1425) Google Scholar]. A further improvement of MPS by synthesis was the development of reversible and fluorescently labeled terminators to address the issue of homopolymers, which were less adequately determined by the Pyrosequencing detection system. The use of fluorescently labeled nucleotides for sequencing dates to the 1980s using conventional electrophoretic sequencing. Reversible terminators were first described by Metzker and colleagues [25.Metzker M.L. et al.Termination of DNA synthesis by novel 3′-modified-deoxyribonucleoside 5′-triphosphates.Nucleic Acids Res. 1994; 22: 4259-4267Crossref Scopus (75) Google Scholar], showing that 3′-modified nucleotides could be used for base-specific termination and photolytic removal of the 3′-protecting group. A few years later, the two UK chemists Balasubramanian and Klenerman filed a patent [26.Balasubramanian, S. and Klenerman, D. Solexa Ltd. Arrayed biomolecules and their use in sequencing, WO 00/06770Google Scholar] describing ‘Arrayed biomolecules and their use in sequencing’ in which they proposed the use of fluorescently labeled nucleotides combined with reversible terminators to allow SBS. Around the same time, the USA-based Quake group was pursuing a similar strategy of fluorescence photobleaching sequencing, which they described in a grant proposal to the National Institutes of Health (NIH) (https://grantome.com/grant/NIH/R29-HG001642-04). Balasubramanian and Klenerman initially aimed to achieve single-molecule detection but later abandoned this strategy and the concept was combined with in vitro-amplified DNA on a solid support [27.Kawashima, E. et al. Method of nucleic acid amplification, WO 98/44151Google Scholar], whereas the Quake group began with the idea of using multiple copies of a DNA template on a surface but then became the first to demonstrate single-molecule SBS [28.Braslavsky I. et al.Sequence information can be obtained from single DNA molecules.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3960-3964Crossref PubMed Scopus (375) Google Scholar]. An instrument called the ‘Genome Analyzer’ for MPS by synthesis was subsequently launched in 2006 by the company Solexa founded by Balasubramanian and Klenerman. The modified SBS strategy used reversible terminators and involved immobilizing the sequencing templates and primers on a solid support, primer extension by the incorporation of a nucleotide with a 3′-blocking group by a polymerase, detection of the color of the fluorophore by the base away the nucleotides to the incorporated and removal of the fluorescent and the 3′-blocking group and the by incorporation of the nucleotide. 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Biochem. 1993; 208: 171-175Crossref PubMed Scopus (0) Google use of reversible M.L. et al.Termination of DNA synthesis by novel 3′-modified-deoxyribonucleoside 5′-triphosphates.Nucleic Acids Res. 1994; 22: 4259-4267Crossref Scopus (75) Google M. et al.A sequencing method based on real-time pyrophosphate.Science. 1998; 281: 365Crossref PubMed Scopus (0) Google of on surface S. and Klenerman, D. Solexa Ltd. Arrayed biomolecules and their use in sequencing, WO 00/06770Google amplification on surface E. et al. Method of nucleic acid amplification, WO 98/44151Google In amplification and of many individual DNA Acids Res. PubMed Scopus Google I. et al.Sequence information can be obtained from single DNA molecules.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3960-3964Crossref PubMed Scopus (375) Google in et in sequencing on polymerase Biochem. 2003; PubMed Scopus (0) Google concept of M. et al.Genome sequencing in microfabricated high-density picolitre reactors.Nature. 2005; 437: 376-380Crossref PubMed Scopus (6037) Google of Neanderthal genome using R.E. et al.Analysis of one million base pairs of Neanderthal DNA.Nature. 2006; 444: 330-336Crossref PubMed Scopus (517) Google using reversible et whole human genome sequencing using reversible 2008; PubMed Scopus Google analysis A. et and by 2008; PubMed Scopus Google Project Project et al.A of human genome from sequencing.Nature. PubMed Scopus Google genomics et of PubMed Scopus Google genome sequencing D.F. et al.Sequence variants from whole genome sequencing a large group of Icelanders.Sci. 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