Generations of sequencing technologies

Advancements in the field of DNA sequencing are changing the scientific horizon and promising an era of personalized medicine for elevated human health. Although platforms are improving at the rate of Moore's Law, thereby reducing the sequencing costs by a factor of two or three each year, we find ourselves at a point in history where individual genomes are starting to appear but where the cost is still too high for routine sequencing of whole genomes. These needs will be met by miniaturized and parallelized platforms that allow a lower sample and template consumption thereby increasing speed and reducing costs. Current massively parallel, state-of-the-art systems are providing significantly improved throughput over Sanger systems and future single-molecule approaches will continue the exponential improvements in the field.     

Introduction to plant genomics]

The success in complete sequencing of "small" genomes and development of new technologies which sharply accelerate processes of cloning and sequencing made real an intensive development of plant genomics and complete sequencing of DNA of some species. It is assumed that the success in plant genomics will result in revolutionary changes in biotechnology and plant breeding. However, the enormous size of genomes (tens of billions bp), their extraordinary enrichment in repetitive sequences, and allopolyploidy (the presence in a nucleus of several related but not identical genomes) force us to think that only few "basic" will undergo complete sequencing, whereas the genome investigations in other species will follow principles of comparative genomics. By the present time, complete sequencing of the Arabidopsis genome (125 Mbp) is completed and that of the rice genome (about 430 Mbp) is close to its end. Studying other plant genomes, including those economically valuable, already began on the basis of these investigations. Peculiarities of plant genomes make extraordinarily important the knowledge on plant chromosomes which, in its turn, requires expansion of investigations in this direction and development of new chromosome technologies, including the DNA-sparing methods of high-resolution banding.     

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.     

Efficient capture of unique sequences from eukaryotic genomes

Cot-based cloning and sequencing (CBCS), a synthesis of Cot analysis, DNA cloning and high-throughput sequencing, promises to accelerate the study of eukaryotic genomes. In particular, CBCS will (1) permit efficient gene discovery in species with substantial quantities of repetitive DNA, (2) allow the sequence complexity (i.e. all the unique sequence information) of large genomes to be elucidated at a fraction of the cost of shotgun sequencing, and (3) enhance genome sequencing efforts by facilitating capture of low-copy sequences not secured by EST sequencing. CBCS should accelerate comparative genomics research, especially in large genomes such as those of many crops.     

Annotating individual human genomes

Advances in DNA sequencing technologies have made it possible to rapidly, accurately and affordably sequence entire individual human genomes. As impressive as this ability seems, however, it will not likely amount to much if one cannot extract meaningful information from individual sequence data. Annotating variations within individual genomes and providing information about their biological or phenotypic impact will thus be crucially important in moving individual sequencing projects forward, especially in the context of the clinical use of sequence information. In this paper we consider the various ways in which one might annotate individual sequence variations and point out limitations in the available methods for doing so. It is arguable that, in the foreseeable future, DNA sequencing of individual genomes will become routine for clinical, research, forensic, and personal purposes. We therefore also consider directions and areas for further research in annotating genomic variants.     

Advances in sequencing technology

Faster sequencing methods will undoubtedly lead to faster single nucleotide polymorphism (SNP) discovery. The Sanger method has served as the cornerstone for genome sequence production since 1977, close to almost 30 years of tremendous utility [Sanger, F., Nicklen, S., Coulson, A.R, DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A. 74 (1977) 5463-5467]. With the completion of the human genome sequence [Venter, J.C. et al., The sequence of the human genome, Science 291 (2001) 1304-1351; Lander, E.S. et al., Initial sequencing and analysis of the human genome, Nature 409 (2001) 860-921], there is now a focus on developing new sequencing methodologies that will enable "personal genomics", or the routine study of our individual genomes. Technologies that will lead us to this lofty goal are those that can provide improvements in three areas: read length, throughput, and cost. As progress is made in this field, large sections of genomes and then whole genomes of individuals will become increasingly more facile to sequence. SNP discovery efforts will be enhanced lock-step with these improvements. Here, the breadth of new sequencing approaches will be summarized including their status and prospects for enabling personal genomics.     

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.     

The maize genome as a model for efficient sequence analysis of large plant genomes

The genomes of flowering plants vary in size from about 0.1 to over 100 gigabase pairs (Gbp), mostly because of polyploidy and variation in the abundance of repetitive elements in intergenic regions. High-quality sequences of the relatively small genomes of Arabidopsis (0.14 Gbp) and rice (0.4 Gbp) have now been largely completed. The sequencing of plant genomes that have a more representative size (the mean for flowering plant genomes is 5.6 Gbp) has been seen as a daunting task, partly because of their size and partly because of the numerous highly conserved repeats. Nevertheless, creative strategies and powerful new tools have been generated recently in the plant genetics community, so that sequencing large plant genomes is now a realistic possibility. Maize (2.4-2.7 Gbp) will be the first gigabase-size plant genome to be sequenced using these novel approaches. Pilot studies on maize indicate that the new gene-enrichment, gene-finishing and gene-orientation technologies are efficient, robust and comprehensive. These strategies will succeed in sequencing the gene-space of large genome plants, and in locating all of these genes and adjacent sequences on the genetic and physical maps.     

Simultaneous genome sequencing of symbionts and their hosts

Second-generation sequencing has made possible the sequencing of genomes of interest for even small research groups. However, obtaining separate clean cultures and clonal or inbred samples of metazoan hosts and their bacterial symbionts is often difficult. We present a computational pipeline for separating metazoan and bacterial DNA in silico rather than at the bench. The method relies on the generation of deep coverage of all the genomes in a mixed sample using Illumina short-read sequencing technology, and using aggregate properties of the different genomes to identify read sets belonging to each. This inexpensive and rapid approach has been used to sequence several nematode genomes and their bacterial endosymbionts in the last year in our laboratory and can also be used to visualize and identify unexpected contaminants (or possible symbionts) in genomic DNA samples. We hope that this method will enable researchers studying symbiotic systems to move from gene-centric to genome-centric approaches.     

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next-generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively-parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large-scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome-level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.      

Leafing through the genomes of our major crop plants: strategies for capturing unique information

Crop plants not only have economic significance, but also comprise important botanical models for evolution and development. This is reflected by the recent increase in the percentage of publicly available sequence data that are derived from angiosperms. Further genome sequencing of the major crop plants will offer new learning opportunities, but their large, repetitive, and often polyploid genomes present challenges. Reduced-representation approaches - such as EST sequencing, methyl filtration and Cot-based cloning and sequencing - provide increased efficiency in extracting key information from crop genomes without full-genome sequencing. Combining these methods with phylogenetically stratified sampling to allow comparative genomic approaches has the potential to further accelerate progress in angiosperm genomics.     

Effective Extraction and Assembly Methods for Simultaneously Obtaining Plastid and Mitochondrial Genomes

Background

In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously.

Results

First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately.

Conclusion

A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.     

Introduction into Plant Genomics

The success in complete sequencing of small genomes and development of new technologies that markedly speed up the cloning and sequencing processes open the way to intense development of plant genomics and complete sequencing of DNA of some species. It is assumed that success in plant genomics will result in revolutionary changes in biotechnology and plant breeding. However, the enormous size of genomes (tens of billions of base pairs), their extraordinary abundance of repetitive sequences, and allopolyploidy (the presence in a nucleus of several related but not identical genomes) force us to think that only few basic plant species will undergo complete sequencing, whereas genome investigations in other species will follow the principles of comparative genomics. By the present time, sequencing of the Arabidopsis genome (125 Mbp) is completed and that of the rice genome (about 430 Mbp) is close to its end. Studying the genomes of other plants, including economically valuable ones, already began on the basis of these works. The peculiarities of plant genomes make extraordinarily important our detailed knowledge on plant chromosomes which, in its turn, calls for expansion of research in this direction and development of new chromosome technologies, including the DNA-sparing methods of high-resolution banding.     

Contribution of genomics to bacterial pathogenesis

Genomics is changing the landscape of modern biology. The impact is far-reaching because it provides both the most economical means of acquiring large amounts of information and because it has forced the creation of new technologies to exploit this information. Five of the six genomes published in the year from August 1998 to August 1999 were human pathogens, all of which are highly host-adapted. Four of these are obligate intracellular pathogens and the study of these genomes is providing novel insights into the intricacies of pathogen-host interactions and co-evolution. These genomes are also significant because they mark the beginning of an important trend in the sequencing of closely related genomes, including the sequencing of more than one strain from a single pathogenic species. As comparative genomics truly comes of age, the ability to compare the genomes of pathogenic and non-pathogenic organisms will hopefully provide insight into what makes certain bacterial strains and species pathogens.     

Plant genomics in Africa: present and prospects

Plants are the world’s most consumed goods. They are of high economic value and bring many health benefits. In most countries in Africa, the supply and quality of food will rise to meet the growing population’s increasing demand. Genomics and other biotechnology tools offer the opportunity to improve subsistence crops and medicinal herbs in the continent. Significant advances have been made in plant genomics, which have enhanced our knowledge of the molecular processes underlying both plant quality and yield. The sequencing of complex genomes of African plant species, facilitated by the continuously evolving next-generation sequencing technologies and advanced bioinformatics approaches, has provided new opportunities for crop improvement. This review summarizes the achievements of genome sequencing projects of endemic African plants in the last two decades. We also present perspectives and challenges for future plant genomic studies that will accelerate important plant breeding programs for African communities. These challenges include a lack of basic facilities, a lack of sequencing and bioinformatics facilities, and a lack of skills to design genomics studies. However, it is imperative to state that African countries have become key players in the plant genome revolution and genome derived-biotechnology. Therefore, African governments should invest in public plant genomics research and applications, establish bioinformatics platforms and training programs, and stimulate university and industry partnerships to fully deploy plant genomics, particularly in the fields of agriculture and medicine.     

DNA sequencing: bench to bedside and beyond

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment.     

Potenzial und Herausforderungen der personalisierten Genomik und des 1000-Genom-Projekts

The ability to sequence entire individual human genomes has heralded a new era in human genetics. Such advances in sequencing technologies make it possible to address new questions such as the generation of a comprehensive map of common and rare genetic variants in humans. The 1000 Genome Project will analyze 2500 genomes and is expected to greatly expand our knowledge about genomic variation, both on single nucleotide polymorphisms and genomic structural variants in a number of human ethnic populations. Furthermore, the possibility to use these new sequencing technologies for such large scale projects will be evaluated. Finally, new bioinformatics solutions will be developed to efficiently store and process such large volumes of data for the scientific community. This catalogue of common and rare variations will facilitate the development of better methods for phenotype-genotype associations and help uncover the molecular bases for a variety of diseases in the near future.     

Modern methods of genome sequencing and their application for deciphering genomes of microorganisms

Development of new methods for genome sequencing allows rapid and relatively inexpensive determination of the large volumes of nucleotide sequences and opens new possibilities to conduct fundamental and applied research in biology, medicine, and biotechnology. The traditional and the newest methods of genome sequencing of microorganisms including the Sanger sequencing method, parallel pyrosequencing (454 Life Sciences/Roche), Solexa/Illumina technologies, and ligation-based sequencing (SOLiD System) are discussed in the current review. The main areas and prospects of applying various sequencing methods to achieve diverse scientific goals are presented. The possibilities of applying methods of parallel pyrosequencing for deciphering genomes of microorganisms are reviewed, using as an example the genome projects completed at the Bioengineering Center, Russian Academy of Sciences.     

Microbial genomes

Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It is hard for the traditional bacteriologist to grasp the scale and pace of the venture. Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced. Indeed, we will often have more than one sequence from a species or genus--for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus. However, genome sequencing risks becoming expensive molecular stamp-collecting without the tools to mine the data and fuel hypothesis-driven laboratory-based research. Bioinformatics, twinned with the new experimental approaches forming functional genomics', provides some of the needed tools. Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings. Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.