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Next-generation Sequencing (next-generation + sequencing)

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Life Sciences60%

Terms modified by Next-generation Sequencing

  • next-generation sequencing technology
    		•

    Selected Abstracts

    Directed Evolution of DNA Polymerases for Next-Generation Sequencing,

    ANGEWANDTE CHEMIE, Issue 34 2010
    Aaron
    Evolution mit mehr Tempo: Mit einem aktivitätsbasierten Phagen-Display wurde die Titelaufgabe gelöst. Dabei wurde eine Mutante der Taq-Polymerase gefunden (siehe Schema), die fluorophormarkierte dA, dT, dC und dG 50- bis 400-fach effizienter in ,vernarbte" Primer einbaut und die zudem unter realen Sequenzierungsbedingungen ein deutlich verbessertes Verhalten zeigt. [source]

    Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology

    MOLECULAR PLANT PATHOLOGY, Issue 4 2009
    IAN P. ADAMS
    SUMMARY A novel, unbiased approach to plant viral disease diagnosis has been developed which requires no a priori knowledge of the host or pathogen. Next-generation sequencing coupled with metagenomic analysis was used to produce large quantities of cDNA sequence in a model system of tomato infected with Pepino mosaic virus. The method was then applied to a sample of Gomphrena globosa infected with an unknown pathogen originally isolated from the flowering plant Liatris spicata. This plant was found to contain a new cucumovirus, for which we suggest the name ,Gayfeather mild mottle virus'. In both cases, the full viral genome was sequenced. This method expedites the entire process of novel virus discovery, identification, viral genome sequencing and, subsequently, the development of more routine assays for new viral pathogens. [source]

    2242: Update on ophthalmic molecular genetics

    ACTA OPHTHALMOLOGICA, Issue 2010
    E DE BAERE
    Purpose To provide an overview of the recent technological advances in human molecular genetics that can be applied in ophthalmic genetics. Methods Since the finalization of the Human Genome Project many novel genomic technologies emerged that led to significant advances in gene identification and genetic testing of hereditary eye disorders: (1) genomewide copy number screening (array CGH); (2) genomewide SNP genotyping; (3) next-generation sequencing. Results (1) Microarray comparative genomic hybridisation or array CGH allows genomewide discovery of submicroscopic deletions and duplications in a single experiment. This technique is applied in routine molecular cytogenetic testing. Using array CGH a causal genomic defect can be found in at least 10% of all cases with mental retardation and/or multiple congenital anomalies. In ophthalmic genetics array CGH is mainly useful in the context of developmental eye disorders, with chorioretinal coloboma and anterior segment dysgenesis as an example. (2) Genomewide chip-based SNP genotyping can be used for homozygosity mapping in inbred and outbred pedigrees. Recent successes in gene identification using this approach are illustrated. (3) Next-generation sequencing or NGS. The application of this technology in gene identification and genetic testing of genetically heterogeneous conditions (with LCA as a paradigm) is discussed. Conclusion The rapid progress of genomic technologies such as array CGH, SNP chip analysis and next-generation sequencing lead to a boost in gene identification and genetic testing of both developmental and retinal eye disease. [source]

    Impact of next generation sequencing: The 2009 Human Genome Variation Society Scientific Meeting

    HUMAN MUTATION, Issue 4 2010
    William S. Oetting
    Abstract The annual scientific meeting of the Human Genome Variation Society (HGVS) was held on the 20th of October, 2009, in Honolulu, Hawaii. The theme of this meeting was the "Impact of Next Generation Sequencing." Presenters spoke on issues ranging from advances in the technology of large-scale genome sequencing to how this information can be analyzed to uncover genetic variants associated with disease. Many of the challenges resulting from the implementation of these new technologies were presented, but possible solutions, or at least paths to the solutions, were also given. With the combined efforts of investigators using next-generation sequencing to help understand the impact of genetic variants on disease, the use of the personal genome in medicine will soon become a reality. Hum Mutat 30:1,4, 2010. © 2010 Wiley-Liss, Inc. [source]

    Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus

    MOLECULAR ECOLOGY, Issue 2010
    LAURA 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]

    Discovering genetic polymorphisms in next-generation sequencing data

    PLANT BIOTECHNOLOGY JOURNAL, Issue 4 2009
    Michael 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. [source]

    2161: Development of a next-generation sequencing platform for retinal dystrophies, with LCA and RP as proof of concept

    ACTA OPHTHALMOLOGICA, Issue 2010
    F COPPIETERS
    Purpose Retinal dystrophies represent an emerging group of hereditary disorders that lead to degeneration of the photoreceptors and/or the retinal pigment epithelium, resulting in irreversible blindness. They are genetically complex, with over 200 disease loci identified so far. Current genetic screening consists of microarray analysis (Asper Ophthalmics) for the most recurrent mutations, and subsequent Sanger sequencing. However, the high cost and low throughput of the latter technology limits testing to only the most recurrent genes. This project aims to develop a high throughput and cost-effective platform for screening of all known disease genes for Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP), using the next-generation sequencing (NGS) technology. Methods A NGS panel will be developed for all 16 and 47 known LCA and RP genes, respectively, including coding and untranslated regions, regulatory regions and microRNA binding sites. The protocol will consist of the following steps: 1) high throughput primerdesign and qPCR, 2) ligation, 3) shearing and 4) sequencing on the Illumina Genome Analyser IIx (GAIIx). This innovative protocol overcomes the need for short amplicons in order to render short-read sequences by the GAIIx. This sequencing instrument was chosen because of its high capacity, low cost per base and the absence of interpretation problems at homopolymeric regions. Analysis of the variants will be performed using in-house developed and commercial software, which ranks all variants according to their pathogenic potential. Conclusion Using the proposed protocol, comprehensive screening for all known disease genes for LCA and RP will be available for the first time. [source]

    2242: Update on ophthalmic molecular genetics

    ACTA OPHTHALMOLOGICA, Issue 2010
    E DE BAERE
    Purpose To provide an overview of the recent technological advances in human molecular genetics that can be applied in ophthalmic genetics. Methods Since the finalization of the Human Genome Project many novel genomic technologies emerged that led to significant advances in gene identification and genetic testing of hereditary eye disorders: (1) genomewide copy number screening (array CGH); (2) genomewide SNP genotyping; (3) next-generation sequencing. Results (1) Microarray comparative genomic hybridisation or array CGH allows genomewide discovery of submicroscopic deletions and duplications in a single experiment. This technique is applied in routine molecular cytogenetic testing. Using array CGH a causal genomic defect can be found in at least 10% of all cases with mental retardation and/or multiple congenital anomalies. In ophthalmic genetics array CGH is mainly useful in the context of developmental eye disorders, with chorioretinal coloboma and anterior segment dysgenesis as an example. (2) Genomewide chip-based SNP genotyping can be used for homozygosity mapping in inbred and outbred pedigrees. Recent successes in gene identification using this approach are illustrated. (3) Next-generation sequencing or NGS. The application of this technology in gene identification and genetic testing of genetically heterogeneous conditions (with LCA as a paradigm) is discussed. Conclusion The rapid progress of genomic technologies such as array CGH, SNP chip analysis and next-generation sequencing lead to a boost in gene identification and genetic testing of both developmental and retinal eye disease. [source]

    Revealing the human mutome

    CLINICAL GENETICS, Issue 4 2010
    JM Chen
    Chen JM, Férec C, Cooper DN. Revealing the human mutome. The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the ,mutome'). With the advent of next-generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole-genome sequencing (WGS) has the potential to identify all disease-causing or disease-associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene-coding sequences for those disease-causing or disease-associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation-screening strategies but also for enhancing the interpretation of findings derived from genome-wide association studies, whole-exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology. [source]