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.     

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.     

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.     

Tick genomics: the Ixodes genome project and beyond

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.     

Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry in genomics research

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies.     

Sequencing technologies and genome sequencing

The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.     

Schistosoma mansoni genome project: an update

A schistosome genome project was initiated by the World Health Organization in 1994 with the notion that the best prospects for identifying new targets for drugs, vaccines, and diagnostic development lie in schistosome gene discovery, development of chromosome maps, whole genome sequencing and genome analysis. Schistosoma mansoni has a haploid genome of 270 Mb contained on 8 pairs of chromosomes. It is estimated that the S. mansoni genome contains between 15000 and 25000 genes. There are approximately 16689 ESTs obtained from diverse libraries representing different developmental stages of S. mansoni, deposited in the NCBI EST database. More than half of the deposited sequences correspond to genes of unknown function. Approximately 40-50% of the sequences form unique clusters, suggesting that approximately 20-25% of the total schistosome genes have been discovered. Efforts to develop low resolution chromosome maps are in progress. There is a genome sequencing program underway that will provide 3X sequence coverage of the S. mansoni genome that will result in approximately 95% gene discovery. The genomics era has provided the resources to usher in the era of functional genomics that will involve microarrays to focus on specific metabolic pathways, proteomics to identify relevant proteins and protein-protein interactions to understand critical parasite pathways. Functional genomics is expected to accelerate the development of control and treatment strategies for schistosomiasis.     

The promise and reality of personal genomics

The publication of the highest-quality and best-annotated personal genome yet tells us much about sequencing technology, something about genetic ancestry, but still little of medical relevance.Which country has published the largest per-capita number of personal genomes? The United States, the United Kingdom? Actually, it is Korea. A recent article in Nature by Kim et al. [1] presents the genome sequence of a Korean male, AK1 - the seventh published sequence of an individual human genome and the second from Korea. The rapid progress in personal genome sequencing is possible because so-called ''next-generation'' sequencing technology has decreased costs by orders of magnitude and increased throughput. But those advantages come at a price: short, error-prone reads derived from single molecules that have to be stitched back together to make a best-guess at the starting sequence. We are still at the stage of working out how to apply the available technologies to coax out biological information: the goal of a US$1,000 genome providing life-changing personal medical insights is still some way off.     

Molecular confirmation of the genomic constitution of <Emphasis Type="Italic">Douglasdeweya</Emphasis> (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification

A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae.     

Connexin genes in the mouse and human genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; S?hl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

Insights from human/mouse genome comparisons

Large-scale public genomic sequencing efforts have provided a wealth of vertebrate sequence data poised to provide insights into mammalian biology. These include deep genomic sequence coverage of human, mouse, rat, zebrafish, and two pufferfish (Fugu rubripes and Tetraodon nigroviridis) (Aparicio et al. 2002; Lander et al. 2001; Venter et al. 2001; Waterston et al. 2002). In addition, a high-priority has been placed on determining the genomic sequence of chimpanzee, dog, cow, frog, and chicken (Boguski 2002). While only recently available, whole genome sequence data have provided the unique opportunity to globally compare complete genome contents. Furthermore, the shared evolutionary ancestry of vertebrate species has allowed the development of comparative genomic approaches to identify ancient conserved sequences with functionality. Accordingly, this review focuses on the initial comparison of available mammalian genomes and describes various insights derived from such analysis.     

Biological information: making it accessible and integrated (and trying to make sense of it)

The availability of the genome sequences of human and mouse, human sequence variation data and other large genetic data sets will lead to a revolution in understanding of the human machine and the treatment of its diseases. The success of the international genome sequencing consortiums shows what can be achieved by well coordinated large scale public domain projects and the benefits of data access to all. It is already clear that the availability of this sequence is having a huge impact on research worldwide. Complete genome sequences provide a framework to pull all biological data together such that each piece has the potential to say something about biology as a whole. Biology is too complex for any organisation to have a monopoly of ideas or data, so the collection, analysis and access to this data can be contributed to by research institutes around the world. However, although it is possible for all this data to be accessible to all through the internet, the more organisations provide data or analysis separately, the harder it becomes for anyone to collect and integrate the results. To address these problems of intergration of data, open standards for biological data exchange, such as the 'Distributed Annotation System' (DAS) are being developed and bioinformatics (Dowell et al., 2001) as a whole is now being strongly driven by the open source software (OSS) model for collaborative software development (Hubbard and Birney, 1999). The leading provider of human genome annotation, the Ensembl project (http://www.ensembl.org), is entirely an OSS project and has been widely adopted by academic and commerical organisations alike (Hubbard et al., 2002). Accurate automatic annotation of features such as genes in vertebrate genomes currently relies on supporting evidence in the form of homologies to mRNAs, ESTs or protein. However, it appears that sufficient high quality experimentally curated annotation now exists to be used as a substrate for machine learning algorithms to create effective models of biological signal sequences (Down and Hubbard, 2002). Is there hope for ab initio prediction methods after all?     

Isolation of a D-genome specific repeated DNA sequence fromAegilops squarrosa

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa. Nuclear DNA content varies over a range of 10,000 fold among all organisms (Nagl et al., 1983). Among angiosperms, at least a 65-fold range in genome size occurs in diploid species (Sparrow, Price and Underbrink, 1972; Bennett, Smith and Heslop-Harrison, 1982). This DNA variation has been reported within families, genera, and species (Rothfels et al., 1966; Rees and Jones, 1967; Miksche, 1968; Price, Chambers and Bachmann, 1981). Much of the interspecific variation in genome size among angiosperms appears to be due to amplification and/or deletion of DNA within chromosomes. The variation in genome size does not appear to result in changes in the number of coding genes (Nagl et al., 1983). While the number of coding genes, with the exception of rDNA in specific examples, appears to remain constant, the remaining non-coding regions are quite flexible. This non-coding DNA encompasses over 99% of the plant genome and consists of sequences that exist as multiple copies throughout the genome and are identified as repeated DNA sequences (Flavell et al., 1974). Flavell et al. (1974) have reported that increasing genome size in higher plants is associated with increasing repetitive DNA amounts. Subsequent reports have substantiated this correlation (Bachmann and Price, 1977; Narayan, 1982). In various cereals, heterochromatin, which has been demonstrated to be correlated with the location of specific repeated DNA sequences, has been positively correlated with genome size (Bennett, Gustafson and Smith, 1977; Rayburn et al., 1985). Furuta, Nishikawa and Makino (1975) found significant DNA content variation among different accessions ofAegilops squarrosa L. This species contains the D genome, a pivotal genome in several polyploid species and also found in hexaploid wheat (AABBDD). The importance of this genome to the study of bread wheat genomes makes the mechanism(s) of this genomic plasticity of particular interest. In order to determine which sequences are varying, one must first have a way to identify specific types of chromatin and/or DNA. Specific types of chromosome banding such as C- and N-banding have been used to identity types of chromatin in previous studies. C-banding of the D genome results in very lightly staining bands whose pattern is somewhat indistinct. N-banding alternatively has been shown to be useful in identifying certain chromosomes of hexaploid wheat but is limited by the lack of major bands in the D genome (Endo and Gill, 1984). Specific DNA sequences have been isolated fromTriticum aestivum cultivar “Chinese Spring” (hexaploid wheat). However, these sequences are representatives of the A and/or B genomes of hexaploid wheat and are not found in significant quantities in the D genome (Hutchinson and Lonsdale, 1982). Various other repeated DNA sequences have been successfully isolated from rye (Bedbrook et al., 1980) and identified on rye chromosomes (Appels et al., 1981; Jones and Flavell, 1982). Certain of these sequences are found in wheat genomes, but the sequences are representative of only a minor fraction of the D genome (Bedbrook et al., 1980; Rayburn and Gill, 1985). The purpose of this report is to describe three distinct repeated DNA sequences isolated fromA. squarrosa (D genome). Two clones appear to be distributed throughout the total genome, and the third clone is restricted to specific sites along the chromosomes. This latter clone will prove useful in cytologically defining the D genome chromosomes. These sequences appear representative of two types of repeated DNA genome organization: 1) sequences distributed throughout the genome and 2) specific arrays of repeated sequences. The availability of such repeated DNA sequence clones along with the known intraspecific DNA content variation inA. squarrosa will allow the study of genomic plasticity of this species.     

Intra‐individual polymorphism in chloroplasts from NGS data: where does it come from and how to handle it?

Next‐generation sequencing allows access to a large quantity of genomic data. In plants, several studies used whole chloroplast genome sequences for inferring phylogeography or phylogeny. Even though the chloroplast is a haploid organelle, NGS plastome data identified a nonnegligible number of intra‐individual polymorphic SNPs. Such observations could have several causes such as sequencing errors, the presence of heteroplasmy or transfer of chloroplast sequences in the nuclear and mitochondrial genomes. The occurrence of allelic diversity has practical important impacts on the identification of diversity, the analysis of the chloroplast data and beyond that, significant evolutionary questions. In this study, we show that the observed intra‐individual polymorphism of chloroplast sequence data is probably the result of plastid DNA transferred into the mitochondrial and/or the nuclear genomes. We further assess nine different bioinformatics pipelines’ error rates for SNP and genotypes calling using SNPs identified in Sanger sequencing. Specific pipelines are adequate to deal with this issue, optimizing both specificity and sensitivity. Our results will allow a proper use of whole chloroplast NGS sequence and will allow a better handling of NGS chloroplast sequence diversity.     

Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.     

Molecular Mapping of Restriction-Site Associated DNA Markers in Allotetraploid Upland Cotton

Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits.