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It is mandatory to procure user consent prior to running these cookies on your website. Hit enter to search or ESC to close. November July 29th, The early beginning of DNA sequencing The progress from the first isolation of DNA by Friedrich Mietscher in to next generation sequencing high-throughput sequencing was the result of continuous efforts of the science community.
Figure 1: Frederick Sanger. Figure 3: Roche Sequencing System. Enter the third generation of DNA sequencing It is difficult to distinguish the third generation sequencing technologies from the second generation. How does NGS work? Adapter molecules act in the hybridisation of the library fragments to the matrix. Moreover, adapter molecules provide a priming site.
Amplification and sequencing : The library is converted into single stranded molecules. Amplification, subsequently, creates clusters of DNA molecules. Each cluster acts as an individual reaction where sequencing, called run, is performed. Data output and analysis : At the end of the reaction, each NGS run provides a large amount of raw data.
This data can be analysed by using a variety of available software. References Declercq, W. Cell 6 : Behjati, S. Berg, P. PNAS 3 : Declercq, W. Cell, 6 : Fiers, W. Ysebaert, M. Nature J Med Biochem. Check Hayden, E. Technique promises it will produce a human genome in 15 minutes.
Heather, J. The sequence of sequencers: The history of sequencing DNA. Genomics 1 : Holley, R. Genomics 1 3 : Ilyas, M. Pathobiology, 84 6 , Lu, H. Genomics Proteomics Bioinformatics 14 5 The combination of these pioneering discoveries paved the way for the sequencing of DNA.
A timeline illustrating the milestones in major genome assembly achievements. The background colour indicates what type of sequencing was used: Red represents early sequencing methods, yellow is Sanger-based shotgun methods, green is NGS and blue represents third-generation sequencing.
Image credit: Formenti et al. NGS became available at the beginning of the 21 st century. Perhaps the biggest advance that NGS offered was the ability to produce a huge amount of data, alongside its ability to provide a highly efficient, rapid, low-cost approach and accurate to DNA sequencing, beyond the reach of traditional Sanger methods.
The cost of sequencing a single human genome from to Organisations, such as Illumina, Pacific Biosciences, Life Sciences and Oxford Technologies Nanopore are all still working tirelessly to reduce the price even further.
The industry is continuing to expand, with companies now marketing NGS bench top platforms to bring these technologies into as many laboratories as possible. NGS will continue to become increasingly efficient and affordable, revolutionising several fields related to genomics.
At the moment, all NGS approaches require library preparation. Indeed, there are essential genes that are indispensable for the survival of an organism and therefore are considered a foundation of life.
The database of essential genes DEG Table 3 catalogues known essential genomic elements, such as protein-coding genes and noncoding RNAs, within the bacteria, archaea, and eukaryotes that constitute a minimal genome and are useful for annotating newly sequenced genomes [ ].
Phylomes provide the combined analysis of genome-wide collections of phylogenetic trees to aid in the inference of orthological and paralogical relationships and the detection of evolutionary events such as whole-genome duplication polyploidization , gene family expansion and contraction, horizontal gene transfer, recombination, inversion, and incomplete lineage sorting [ , ].
The online PhylomeDB v4 database was created as a phylogenomic repository and is useful for preliminary phylogenetic data analysis of genomes of interest from various phyla as well as for annotating newly derived genomic sequences [ ].
As an example, Fig. The functions of RLTPR are not well characterized, but its distinct functional domains suggest that it may multitask in protein-protein interactions, as recently demonstrated in the development of regulatory T cells in mice [ ]. The analytical approach to find orthologous and paralogous relationships with maximum genomic coverage for the RLTPR gene is both gene-centric and genome-wide in PhylomeDB.
Also of particular interest are the well-conserved genomic mechanisms of innate immunity, such as Apolipoprotein B Editing Catalytic subunit proteins 3 APOBEC3s in mammals that mutate and inactivate viral genomes [ ].
The tree shows the speciation events blue squares and three duplication events red squares at the nodes with the first duplication event arising early in vertebrate evolution before the divergence of fish and mammalian lineages [ ]. The science of mobile genetic elements mobilomics developed long before the advent of genomics and NGS [ ].
The Nobel Prize winner Barbara McClintock first reported the existence of mobile elements as jumping genes in maize in the late s [ ]. Since then, RepeatMasker Table 3 and other tools such as Mobster [ ], Red [ ], and Visual TE [ ] have followed on to help define the mobilome, the totality of mobile genetic elements in a particular genome.
A list and description of some of the families, types, and classes of transposons and retrotransposons in prokaryotes and eukaryotes can be found in the following reviews [ , — ].
A recent survey of repeats and mobile elements that affect genomic stability has elucidated how some bacteria can control the mobilome through postsegregation killing systems [ — , ].
Indeed, many of the TE ancient relics have undergone exaptation and developed new functions, such as transcript repeat elements, within regulatory gene networks to generate lineage-specific adaptation [ , ]. The importance of widespread HGT in creating genomic diversity in microbes has been highlighted by the many comparative genomic studies using metagenome data [ ]. Comparative genomic analysis of different strains of E.
Comparative genomics of photosynthetic prokaryotes revealed that they have evolved as complex mosaics via multiple HGT events [ ]. Similarly, photosynthetic gene clusters and gene clusters that encode various toxins, resistance genes, metabolic genes, and components of secretion systems appear to be the products of HGT [ , — ]. Before the new millennium, transposons and repeat elements were largely viewed as junk and as parasites that created unnecessary burden on the genome.
Comparative genomics and online databases dedicated to transposons and repeat elements such as SINES, LINES, and ERVs, however, began to change this picture in the s, and it soon became evident that these elements were the drivers of evolutionary innovation. Many integrated transposons mutate with time to interact with the host transcriptional machinery and therefore provide a useful substrate for evolution of novel regulatory elements [ , , — ]. Moreover, some of the ancient integrated retrotransposons appear to have been involved in advantageous segmental genomic duplications such as in the major histocompatibility complex region [ — ], and others have dispersed regulatory controls to provide coordinated regulation across the genome [ , ].
Agrigenomics or agricultural genomics can be defined as the research and development activities that translate NGS and genomics technology into a better understanding of plant biology and advancing crop improvements. Since the publication in of the first plant genome, Arabidopsis thaliana , 54 new plant genomes were published by [ ] followed by at least another 6 plant genomes including the hexaploid bread wheat genome [ ].
The accumulation of knowledge on the human genome and its genetic and molecular processes humanomics has amplified considerably since the first draft assembly was published in [ ]. Ten years after the publication of the first human draft sequence, six more human genome sequences were completed with a much greater coverage and accuracy, enabling more informative comparisons to be made between them [ 7 , 8 , 79 ]. Since then, Ethiopian and Egyptian genomes were compared to reconstruct their population history out of Africa [ ], genomes from 10 populations of African, East Asian, and European ancestries were sequenced to elucidate novel patterns and signatures of genetic differentiation [ ], and whole-exome sequences from genomes of a ClinSeq cohort were compared to discover new loss-of-function mutations [ ].
Today, there are many human genome projects, and WGS of the human genome for personalized medicine personomics is already a reality for 2, Icelanders [ 9 ] and for some others [ , ] of the 7.
Veeramah and Hammer [ ] recently reviewed the usefulness of NGS to sequence ancient DNA samples for phylogenetic and evolutionary studies and for the reconstruction of human population history. Some of these NGS studies have helped to refine the demographic histories of human evolution. These studies include those of the ancient DNA of extinct hominins Neanderthals, Denisovans and ancient modern humans such as 7,year-old Mesolithic hunter-gathers in northwestern Spain, Neolithic and post-Neolithic 5, to 4,year-old hunter-gathers and farmers in Scandinavia, a 4,year-old Paleo-Eskimo from southern Greenland, and a 24, and 17,year-old South-Central Siberian [ ].
NGS of ancient nonhuman genomes such as those of pathogens, parasites, and domesticated animals and plants also can provide new information about human history in regard to life styles, health, and the spread of agriculture [ ].
NGS has allowed a detailed analysis of single nucleotide variants SNVs , structural variants SV , and methylations in coding and noncoding regions and to assess their role in human disease [ 9 , 14 , 15 , 19 , 22 , 25 , 29 , 30 , , — , , , , ]. More than 97 million validated SNPs dbSNP have been discovered from human genome sequencing projects and many of the variants have been linked to a range of medical and phenotypic conditions and catalogued at dbGAP Table 3 , the database of genotype and phenotype [ ].
In July , dbGAP had links to disease and phenotype studies and 3, data sets. In addition to SNV, small and large SVs that are duplicated, deleted, or rearranged relative to the reference sequences and individuals have been identified in NGS studies and associated with various diseases [ 9 , 30 , ].
NGS has been used to diagnose rare Mendelian diseases and genetically heterogeneous complex disorders, such as X-linked intellectual disability, congenital disorders, cancer genome heterogeneity, and fetal aneuploidy [ 13 , 15 , , , , , , ]. The impact of NGS on the diagnosis of rare genetic diseases is evidenced by the growth of the genes and OMIM database [ 49 , ] that has doubled in data since [ ]. However, it should be noted that NGS does not always reveal causative mutations but instead may provide a list of possible candidates.
NGS in human studies must be used with caution because of the significant levels of false-positive and false-negative rates in sequencing errors and amplification biases.
Soon et al. The Regulome DB based on the accumulation of nongenic functional regulatory regions obtained from ENCODE is a useful resource for the evaluation of polymorphisms of regulatory regions [ ]. Although disease-associated SNPs obtained from GWAS studies may point to gene coding regions, they actually might reside in regulatory sites of downstream genes that are in linkage disequilibrium with the reported SNPs [ ].
Some of these are the interspersed retroelements such as Alu and L1 and endogenous retroviruses ERVs that have evolved before and during primate history to function as regulators of transcription and translation [ 24 , 25 , , , , ].
NGS has especially revolutionized the field of cancer genomes revealing mutations, amplifications, deletions, translocations, and dysregulation of noncoding and coding RNAs to provide a better understanding of the complex genetics and loss of regulation in cancer [ 15 , 25 , 29 , , , ]. For example, paired end sequencing showed that about half of structural rearrangements in breast cancer genomes were fusion transcripts resulting from the rearrangements of segmental tandem duplications involving multiple genes [ ].
NGS also has been applied to circulating tumor cells isolated from the body fluids blood, urine, sputum, saliva, and stools [ 30 , ]. Thus, NGS potentially provides cancer patients with opportunities for personalized diagnosis and optimized therapeutic treatment [ , ]. The integration of NGS data obtained from whole genome, exome, transcriptome, and methylome to build up individual genomic profiles is a growing reality in human health care. Recently, Chen et al. Others have performed similar studies demonstrating that monitoring the longitudinal trends and changes within individuals is an important future protocol for the diagnosis, management, and treatment of disease [ 9 , 81 , ].
The cost benefits of NGS for personomics have still to be assessed with many economic, securities, personal, familial, social, and ethical issues to be considered and resolved. The first-generation sequencing technologies and the pioneering computing and bioinformatics tools produced the initial sequencing data and information within a framework of structural and functional genomics in readiness for the following NGS developments. NGS provides substantially cheaper, friendlier, and more flexible high-throughput sequencing options with a quantum leap towards the generation of much more data on genomics, transcriptomics, and methylomics that translate more productively into proteomics, metabolomics, and systeomics.
A few years ago, the McKinsey Global Institute produced a report predicting that NGS and genomics, including the sequencing of a million human genomes, would become an economically and socially disruptive technology as well as an annual trillion dollar industry by [ ]. The authors assessed that next-generation genomics would affect many high impact areas of molecular biology and bioindustry such as improving genetic engineering tools to custom build organisms, genetically engineer biofuels, modify crops to improve farming practices and food stocks, and develop drugs to treat cancers and other diseases.
Although these technologies promise huge benefits, they also come with social, ethical, and regulatory risks in regard to privacy and security of personal genetic information, the dangerous effects of modified organisms on the environment, the spectre of bioterrorism, eugenics, and concerns about the ownership and commercialization of genomic information.
The application of prenatal genome sequencing for genetic screening already points to the potential of producing genetically modified babies with desired traits.
Much will need to be done to educate and inform regulators and society about the risks and benefits when formulating the regulatory policies about the advances and applications of these next-generation technologies. Future advancements will undoubtedly rely on new technologies and large-scale collaborative efforts from multidisciplinary and international teams to continue generating comprehensive, high-throughput data production and analysis.
The availability of economically friendlier bench-top sequencers and third-generation sequencing tools will allow smaller laboratories and individual scientists to participate in the genomics revolution and contribute new knowledge to the different fields of structural and functional genomics in the life sciences.
The authors of the following chapters in this book present additional examples, more detailed information, and a broader view of the methods and many advances, applications, and challenges of NGS that were either missed or not covered adequately in this opening chapter, particularly in regard to the RNA sequencing and transcriptome methods and data that provide us with a better understanding of functional genomics in microorganisms, plants, animals, and humans.
Te volo, bonam lectionem. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers. Login to your personal dashboard for more detailed statistics on your publications. Edited by Jerzy Kulski. Edited by Marina Silva-Opps. We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals.
Downloaded: Keywords Next-generation sequencing tools platforms applications omics. Introduction Next-generation sequencing NGS refers to the deep, high-throughput, in-parallel DNA sequencing technologies developed a few decades after the Sanger DNA sequencing method first emerged in and then dominated for three decades [ 1 , 2 ].
DNA and RNA library preparations for second-generation sequencing The general workflow for second-generation sequencing is the preparation and amplification of libraries prepared from DNA or RNA samples, clonal formation, sequencing, and analysis [ 55 — 59 ]. NGS platforms The main features and performances of five commonly used second-generation sequencing technologies that have been reviewed in detail by others [ 2 — 4 , 11 , 36 , 54 ] are shown in Table 1. Table 1. Helicos sequencing by the genetic analysis system The Helicos sequencing system was the first commercial implementation of single-molecule fluorescent sequencing [ 66 , 78 ], marketed by the now bankrupt Helicos Biosciences.
NGS by electron microscopy The sequence of long, intact DNA molecules can be visualized and identified by using electron microscopy. Table 2. Program Website 1. Table 3. Useful websites for NGS tools, browsers, portals, providers, and online databases. Genomics A detailed organizational analysis and an understanding of the full landscape of a genome are possible only after de novo whole-genome shotgun sequencing and annotation has been performed [ 11 ].
Methylomics and epigenomics Epigenomics is the study of heritable gene regulation that does not involve the DNA nucleotide coding sequence itself but acts on a genome-wide scale via DNA nucleotide methylation and posttranslational modifications of histones, the interaction between transcription factors and their targets, and nucleosome positioning [ 23 — 30 ].
Proteomics, metabolomics, and systeomics Proteomics is the large-scale study of the structure, function, identification, and characterization of peptides and proteins [ , , ]. Metagenomics and microbiomes Metagenomics, or beyond genomics, is the study of the total genomic content of a microbial community that bridges the three domains of life, Archaea, Bacteria, and Eukaryotes [ , — , — ]. Comparative genomics, phylogenomics, and the phylomes of life Comparative genomics and phylogenomics via NGS and the phylome complete collection of all gene phylogenies in a genome provide powerful applications for classifying and understanding the differences and similarities of all life forms and for unraveling their evolutionary histories [ , , , — ].
Agrigenomics Agrigenomics or agricultural genomics can be defined as the research and development activities that translate NGS and genomics technology into a better understanding of plant biology and advancing crop improvements. Humanomics, personomics, and health The accumulation of knowledge on the human genome and its genetic and molecular processes humanomics has amplified considerably since the first draft assembly was published in [ ]. More Print chapter. How to cite and reference Link to this chapter Copy to clipboard.
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Time h or days. Cost per 10 6 bases. Platform cost USD approx. First generation. Dideoxy terminator. Second generation. Reversible terminators. Proton detection. Third generation. Service provider. Web address. Illumina Ion Torrent. SeqWright Genomic. Illumina Ion Torrent Roche Centrillion Genomic. Broad Institute. Eurofins Genomics. Millennium Science. Oxford Nanopore Technologies. Complete Genomics.
Aligner, assembly, and postassembly tools. TopHat RNA-seq aligner. Soap de novo assembler. Allpaths-LG assembler. Celera assembler. Velvet assembler. SPAdes assembler. BaseSpace Illumina. Torrent Suite Software. RATT: rapid annotation transfer tool. Prokaryote annotation web servers.
Eukaryote annotation web servers. Archives and databases. Complete Genomics data. Roadmap epigenomics. Blueprint epigenomics. ExPASy proteomics. PRIDE proteomics. FAME metabolomics. UCbase 2. Compara GeneTrees. Gene ontology databases and tools. Gene Ontology Consortium. KEGG Pathway database. Genome browsers, projects, and fourth tier providers. Earth Microbiome Project. Terragenome Project. Tara Oceans Project. MetaHit project. Vertebrate Genome 10K. Human Microbiome. Personal Genome Project.
Ensembl browser.
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