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Sequencing Technologies (sequencing + technology)
Kinds of Sequencing Technologies Selected AbstractsA statistical method for scanning the genome for regions with rare disease allelesGENETIC EPIDEMIOLOGY, Issue 5 2010Chad GarnerArticle first published online: 21 JUN 2010 Abstract Studying the role of rare alleles in common disease has been prevented by the impractical task of determining the DNA sequence of large numbers of individuals. Next-generation DNA sequencing technologies are being developed that will make it possible for genetic studies of common disease to study the full frequency spectrum of genetic variation, including rare alleles. This report describes a method for scanning the genome for disease susceptibility regions that show an increased number of rare alleles among a sample of disease cases versus an ethnically matched sample of controls. The method was based on a hidden Markov model and the statistical support for a disease susceptibility region characterized by rare alleles was measured by a likelihood ratio statistic. Due to the lack of empirical data, the method was evaluated through simulation. The performance of the method was tested under the null and alternative hypotheses under a range of sequence generating and hidden Markov models parameters. The results showed that the statistical method performs well at identifying true disease susceptibility regions and that performance was primarily affected by the amount of variation in the neutral sequence and the number of rare disease alleles found in the disease susceptibility region. Genet. Epidemiol. 34: 386,395, 2010. © 2010 Wiley-Liss, Inc. [source] Genome-wide SNP detection in the great tit Parus major using high throughput sequencingMOLECULAR ECOLOGY, Issue 2010NIKKIE E. M. VAN BERS Abstract Identifying genes that underlie ecological traits will open exiting possibilities to study gene,environment interactions in shaping phenotypes and in measuring natural selection on genes. Evolutionary ecology has been pursuing these objectives for decades, but they come into reach now that next generation sequencing technologies have dramatically lowered the costs to obtain the genomic sequence information that is currently lacking for most ecologically important species. Here we describe how we generated over 2 billion basepairs of novel sequence information for an ecological model species, the great tit Parus major. We used over 16 million short sequence reads for the de novo assembly of a reference sequence consisting of 550 000 contigs, covering 2.5% of the genome of the great tit. This reference sequence was used as the scaffold for mapping of the sequence reads, which allowed for the detection of over 20 000 novel single nucleotide polymorphisms. Contigs harbouring 4272 of the single nucleotide polymorphisms could be mapped to a unique location on the recently sequenced zebra finch genome. Of all the great tit contigs, significantly more were mapped to the microchromosomes than to the intermediate and the macrochromosomes of the zebra finch, indicating a higher overall level of sequence conservation on the microchromosomes than on the other types of chromosomes. The large number of great tit contigs that can be aligned to the zebra finch genome shows that this genome provides a valuable framework for large scale genetics, e.g. QTL mapping or whole genome association studies, in passerines. [source] Sequencing breakthroughs for genomic ecology and evolutionary biologyMOLECULAR ECOLOGY RESOURCES, Issue 1 2008MATTHEW E. HUDSON Abstract Techniques involving whole-genome sequencing and whole-population sequencing (metagenomics) are beginning to revolutionize the study of ecology and evolution. This revolution is furthest advanced in the Bacteria and Archaea, and more sequence data are required for genomic ecology to be fully applied to the majority of eukaryotes. Recently developed next-generation sequencing technologies provide practical, massively parallel sequencing at lower cost and without the requirement for large, automated facilities, making genome and transcriptome sequencing and resequencing possible for more projects and more species. These sequencing methods include the 454 implementation of pyrosequencing, Solexa/Illumina reversible terminator technologies, polony sequencing and AB SOLiD. All of these methods use nanotechnology to generate hundreds of thousands of small sequence reads at one time. These technologies have the potential to bring the genomics revolution to whole populations, and to organisms such as endangered species or species of ecological and evolutionary interest. A future is now foreseeable where ecologists may resequence entire genomes from wild populations and perform population genetic studies at a genome, rather than gene, level. The new technologies for high throughput sequencing, their limitations and their applicability to evolutionary and environmental studies, are discussed in this review. [source] Plant genome sequencing: applications for crop improvementPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2010David Edwards Summary DNA sequencing technology is undergoing a revolution with the commercialization of second generation technologies capable of sequencing thousands of millions of nucleotide bases in each run. The data explosion resulting from this technology is likely to continue to increase with the further development of second generation sequencing and the introduction of third generation single-molecule sequencing methods over the coming years. The question is no longer whether we can sequence crop genomes which are often large and complex, but how soon can we sequence them? Even cereal genomes such as wheat and barley which were once considered intractable are coming under the spotlight of the new sequencing technologies and an array of new projects and approaches are being established. The increasing availability of DNA sequence information enables the discovery of genes and molecular markers associated with diverse agronomic traits creating new opportunities for crop improvement. However, the challenge remains to convert this mass of data into knowledge that can be applied in crop breeding programs. [source] The evolution of tropical adaptation: comparing taurine and zebu cattleANIMAL GENETICS, Issue 5 2010E. K. F. Chan Summary Beef cattle breeds consist of three major genetic subdivisions. The taurine group is adapted to temperate environments, and the zebu and Sanga groups are both adapted to tropical environments. With the advent of genotyping and sequencing technologies in agriculture, genome-wide exploration of the genetic basis for the differences in tropical adaptation has only just become possible. In this study, approximately 9000 single nucleotide polymorphism markers were genotyped on 317 animals of a selection of taurine, zebu, and composite breeds to characterize any systematic differences between these groups. We identified 91 intra-breed-class markers; 78 were polymorphic only within the zebu animals, while 13 were polymorphic only in the taurine animals. There were no fixed differences (fixed for alternate alleles between the two breed types) between zebu and taurine animals. We found 14 regions with significantly different allele frequencies between zebu and taurine animals indicative of variable selection pressure or genetic drift. We also found 12 independent regions of differential extended haplotype homozygosity (EHH), indicative of recent selection or rapid fixation of the alternate allele within a short period of time in one of the two breed classes. A preliminary functional genomics analysis of these regions pointed towards signatures of tropical attributes including keratins, heat-shock proteins and heat resistance genes. We anticipate this investigation to be a stepping-stone for future studies to identify genomic regions specific to the two cattle groups, and to subsequently assist in the discrimination between temperate and tropically adapted cattle. [source] Non-coding RNAs: Meet thy mastersBIOESSAYS, Issue 7 2010Fabrício F. Costa Abstract New DNA sequencing technologies have provided novel insights into eukaryotic genomes, epigenomes, and the transcriptome, including the identification of new non-coding RNA (ncRNA) classes such as promoter-associated RNAs and long RNAs. Moreover, it is now clear that up to 90% of eukaryotic genomes are transcribed, generating an extraordinary range of RNAs with no coding capacity. Taken together, these new discoveries are modifying the status quo in genomic science by demonstrating that the eukaryotic gene pool is divided into two distinct categories of transcripts: protein-coding and non-coding. The function of the majority of ncRNAs produced by the transcriptome is largely unknown; however, it is probable that many are associated with epigenetic mechanisms. The purpose of this review is to describe the most recent discoveries in the ncRNA field that implicate these molecules as key players in the epigenome. [source] High-throughput DNA sequencing , concepts and limitationsBIOESSAYS, Issue 6 2010Martin Kircher Abstract Recent advances in DNA sequencing have revolutionized the field of genomics, making it possible for even single research groups to generate large amounts of sequence data very rapidly and at a substantially lower cost. These high-throughput sequencing technologies make deep transcriptome sequencing and transcript quantification, whole genome sequencing and resequencing available to many more researchers and projects. However, while the cost and time have been greatly reduced, the error profiles and limitations of the new platforms differ significantly from those of previous sequencing technologies. The selection of an appropriate sequencing platform for particular types of experiments is an important consideration, and requires a detailed understanding of the technologies available; including sources of error, error rate, as well as the speed and cost of sequencing. We review the relevant concepts and compare the issues raised by the current high-throughput DNA sequencing technologies. We analyze how future developments may overcome these limitations and what challenges remain. [source] Analysis of ancient human genomesBIOESSAYS, Issue 5 2010000-year-old human from Greenland has been obtained, 20-fold coverage of the genome of a , Using next generation sequencing Abstract High-capacity sequencing technologies have dramatically reduced both the cost and time required to generate complete human genome sequences. Besides expanding our knowledge about existing diversity, the nature of these technologies makes it possible to extend knowledge in yet another dimension: time. Recently, the complete genome sequence of a 4,000-year-old human from the Saqqaq culture of Greenland was determined to 20-fold coverage. These data make it possible to investigate the population affinities of this enigmatic culture and, by identifying several phenotypic traits of this individual, provide a limited glimpse into how these people may have looked. While undoubtedly a milestone in ancient DNA research, the cost to generate an ancient genome, even from such an exceptionally preserved specimen, remains out of reach for most. Nonetheless, recently developed DNA capture methods, already applied to Neanderthal and fossil human mitochondrial DNA, may soon make large-scale genome-wide analysis of ancient human diversity a reality, providing a fresh look at human population history. [source] Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencingBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Tomoko Yoshino Abstract There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost. Biotechnol. Bioeng. 2009;103: 130,137. © 2008 Wiley Periodicals, Inc. [source] Genome-based insights into the evolution of enterococciCLINICAL MICROBIOLOGY AND INFECTION, Issue 6 2010W. Van Schaik Clin Microbiol Infect 2010; 16: 527,532 Abstract It is now 15 years since the first genome of a free-living organism was sequenced. Subsequent to this milestone, a veritable avalanche of genome sequence data has revolutionized many aspects of microbiology. In this review, we discuss recent progress on the genomics of Enterococcus faecalis and Enterococcus faecium, which are the two enterococcal species that cause the large majority of enterococcal infections. We focus on the genome-based analysis of enterococcal diversity and phylogeny. Studies based on comparative genome hybridization have shown that both species exhibit considerable inter-strain genomic diversity, which is mainly linked to the variable presence of phages, plasmids, pathogenicity islands and conjugative elements. We also discuss how the advent of next-generation sequencing technologies allows for a comprehensive characterization of the gene repertoire of multiple isolates, which can be used for extremely robust analyses of diversity and population structure. [source] Genome-enabled development of DNA markers for ecology, evolution and conservationMOLECULAR ECOLOGY, Issue 11 2010ROBERT C. THOMSON Abstract Molecular markers have become a fundamental piece of modern biology's toolkit. In the last decade, new genomic resources from model organisms and advances in DNA sequencing technology have altered the way that these tools are developed, alleviating the marker limitation that researchers previously faced and opening new areas of research for studies of non-model organisms. This availability of markers is directly responsible for advances in several areas of research, including fine-scaled estimation of population structure and demography, the inference of species phylogenies, and the examination of detailed selective pressures in non-model organisms. This review summarizes methods for the development of large numbers of DNA markers in non-model organisms, the challenges encountered when utilizing different methods, and new research applications resulting from these advances. [source] Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locusMOLECULAR ECOLOGY, Issue 2010LAURA FERGUSON Abstract The mimetic wing patterns of Heliconius butterflies are an excellent example of both adaptive radiation and convergent evolution. Alleles at the HmYb and HmSb loci control the presence/absence of hindwing bar and hindwing margin phenotypes respectively between divergent races of Heliconius melpomene, and also between sister species. Here, we used fine-scale linkage mapping to identify and sequence a BAC tilepath across the HmYb/Sb loci. We also generated transcriptome sequence data for two wing pattern forms of H. melpomene that differed in HmYb/Sb alleles using 454 sequencing technology. Custom scripts were used to process the sequence traces and generate transcriptome assemblies. Genomic sequence for the HmYb/Sb candidate region was annotated both using the MAKER pipeline and manually using transcriptome sequence reads. In total, 28 genes were identified in the HmYb/Sb candidate region, six of which have alternative splice forms. None of these are orthologues of genes previously identified as being expressed in butterfly wing pattern development, implying previously undescribed molecular mechanisms of pattern determination on Heliconius wings. The use of next-generation sequencing has therefore facilitated DNA annotation of a poorly characterized genome, and generated hypotheses regarding the identity of wing pattern at the HmYb/Sb loci. [source] Massive parallel MHC genotyping: titanium that shinesMOLECULAR ECOLOGY, Issue 9 2009K. MATHIAS WEGNER The power of population genetic analyses is often limited by sample size resulting from constraints in financial resources and time to genotype large numbers of individuals. This particularly applies to nonmodel species where detailed genomic knowledge is lacking. Next-generation sequencing technology using primers ,tagged' with an individual barcode of a few nucleotides offers the opportunity to genotype hundreds of individuals at several loci in parallel (Binladen et al. 2007; Meyer et al. 2008). The large number of sequence reads can also be used to identify artefacts by frequency distribution thresholds intrinsically determined for each run and data set. In Babik et al. (2009), next-generation deep sequencing was used to genotype several major histocompatibility complex (MHC) class IIB loci of the European bank vole (Fig. 1). Their approach can be useful for many researchers working with complex multiallelic templates and large sample sizes. Figure 1. Hypothetical example of parallel genotyping of two individuals using individually bar-coded primers. Polymerase chain reactions (PCRs) are performed separately for each individual using a forward primer with a unique Tag -sequence of four nucleotides. After sequencing of pooled PCR products, sequences can be sorted by their forward primer Tag (Tag -sorting error rate was estimated < 0.1%). Rare sequences most likely represent artefacts and due to the large amount of sequences obtained (up to 106) the artefact threshold can be determined intrinsically for each data set and was estimated to be around 3% in the case of bank vole MHC class IIB genes (Babik et al. 2009). Photos by Gabriela Bydlon. [source] 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversityNEW PHYTOLOGIST, Issue 2 2009M. Buée Summary ,,Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). ,,Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. ,,Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. ,,This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems. [source] Plant genome sequencing: applications for crop improvementPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2010David Edwards Summary DNA sequencing technology is undergoing a revolution with the commercialization of second generation technologies capable of sequencing thousands of millions of nucleotide bases in each run. The data explosion resulting from this technology is likely to continue to increase with the further development of second generation sequencing and the introduction of third generation single-molecule sequencing methods over the coming years. The question is no longer whether we can sequence crop genomes which are often large and complex, but how soon can we sequence them? Even cereal genomes such as wheat and barley which were once considered intractable are coming under the spotlight of the new sequencing technologies and an array of new projects and approaches are being established. The increasing availability of DNA sequence information enables the discovery of genes and molecular markers associated with diverse agronomic traits creating new opportunities for crop improvement. However, the challenge remains to convert this mass of data into knowledge that can be applied in crop breeding programs. [source] Discovering genetic polymorphisms in next-generation sequencing dataPLANT BIOTECHNOLOGY JOURNAL, Issue 4 2009Michael Imelfort Summary The ongoing revolution in DNA sequencing technology now enables the reading of thousands of millions of nucleotide bases in a single instrument run. However, this data quantity is often compromised by poor confidence in the read quality. The identification of genetic polymorphisms from this data is therefore problematic and, combined with the vast quantity of data, poses a major bioinformatics challenge. However, once these difficulties have been addressed, next-generation sequencing will offer a means to identify and characterize the wealth of genetic polymorphisms underlying the vast phenotypic variation in biological systems. We describe the recent advances in next-generation sequencing technology, together with preliminary approaches that can be applied for single nucleotide polymorphism discovery in plant species. 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