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Search Results: 1 - 10 of 4128 matches for " next-generation sequencing "
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Next generation sequencing for profiling expression of miRNAs: technical progress and applications in drug development  [PDF]
Jie Liu, Steven F. Jennings, Weida Tong, Huixiao Hong
Journal of Biomedical Science and Engineering (JBiSE) , 2011, DOI: 10.4236/jbise.2011.410083
Abstract: miRNAs are non-coding RNAs that play a regulatory role in expression of genes and are associated with diseases. Quantitatively measuring expression levels of miRNAs can help understanding the mechanisms of human diseases and discovering new drug targets. There are three major methods that have been used to measure the expression levels of miRNAs: real-time reverse transcription PCR (qRT-PCR), microarray, and the newly introduced next-generation sequencing (NGS). NGS is not only suitable for profiling of known miRNAs that qRT-PCR and microarray can do too but also able to detect unknown miRNAs that the other two methods are incapable. Profiling of miRNAs by NGS has been progressed rapidly and is a promising field for applications in drug development. This paper will review the technical advancement of NGS for profiling miRNAs, including comparative analyses between different platforms and software packages for analyzing NGS data. Examples and future perspectives of applications of NGS profiling miRNAs in drug development will be discussed.
Genome sequencing and next-generation sequence data analysis: A comprehensive compilation of bioinformatics tools and databases  [PDF]
Jose C. Jimenez-Lopez, Emma W. Gachomo, Sweta Sharma, Simeon O. Kotchoni
American Journal of Molecular Biology (AJMB) , 2013, DOI: 10.4236/ajmb.2013.32016

Genomics has become a ground-breaking field in all areas of the life sciences. The advanced genomics and the development of high-throughput techniques have lately provided insight into whole-genome characterization of a wide range of organisms. In the post-genomic era, new technologies have revealed an outbreak of prerequisite genomic sequences and supporting data to understand genome wide functional regulation of gene expression and metabolic pathways reconstruction. However, the availability of this plethora of genomic data presents a significant challenge for storage, analyses and data management. Analysis of this mega-data requires the development and application of novel bioinformatics tools that must include unified functional annotation, structural search, and comprehensive analysis and identification of new genes in a wide range of species with fully sequenced genomes. In addition, generation of systematically and syntactically unambiguous nomenclature systems for genomic data across species is a crucial task. Such systems are necessary for adequate handling genetic information in the context of comparative functional genomics. In this paper, we provide an overview of major advances in bioinformatics and computational biology in genome sequencing and next-generation sequence data analysis. We focus on their potential applications for efficient collection, storage, and analysis of genetic data/information from a wide range of gene banks. We also discuss the importance of establishing a unified nomenclature system through a functional and structural genomics approach.

Development and Characterization of Microsatellite Markers for Three Pollination Morphs of Cimicifuga simplex (Ranunculaceae)  [PDF]
Tsubasa Toji, Yoshiaki Kameyama, Akira S. Hirao, Takao Itino
American Journal of Plant Sciences (AJPS) , 2018, DOI: 10.4236/ajps.2018.94046

Cimicifuga simplex Wormsk. (Ranunculaceae) is a perennial herb distributed in eastern and northeastern Asia for which at least three different pollination morphs have been reported. It is classified as endangered or near threatened in some Japanese regions, and its rhizome is commercially used as a crude drug. To examine genetic differentiation and gene flow among the three morphs, we developed eight microsatellite markers by using next-generation sequencing and estimated the genetic structure of C. simplex. We tested eight primer pairs on 93 individuals from six populations of C. simplex in Nagano, central Japan, and found that heterozygosity in morphs I and III was low compared to expected heterozygosity. Bayesian clustering performed with the STRUCTURE program clearly distinguished the three morphs of C. simplex, and only a little gene flow was detected among the morphs. These eight microsatellite markers are expected to be useful in conservation genetic studies of this species and for future conservation planning.

Identification of avian W-linked contigs by short-read sequencing
Nancy Chen, Daniel W Bellott, David C Page, Andrew G Clark
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-183
Abstract: Using the Illumina Genome Analyzer, we generated sequence reads from a male domestic chicken (ZZ) and mapped them to the existing female (ZW) genome sequence. This method allowed us to identify segments of the female genome that are underrepresented in the male genome and are therefore likely to be female specific. We developed a Bayesian classifier to automate the calling of W-linked contigs and successfully identified more than 60 novel W-specific sequences.Our classifier can be applied to improve heterogametic whole-genome shotgun assemblies of the W or Y chromosome of any organism. This study greatly improves our knowledge of the W chromosome and will enhance future studies of avian sex determination and sex chromosome evolution.
Survey of Annual and Seasonal Fungal Communities in Japanese Prunus mume Orchard Soil by Next-Generation Sequencing  [PDF]
Yoshinao Aoki, Keiko Fujita, Hiroyuki Shima, Shunji Suzuki
Advances in Microbiology (AiM) , 2015, DOI: 10.4236/aim.2015.513086
Abstract: Fungi play a vital role in the management of soil environment. Although various fungal communities are found in soil, it is difficult to determine the fungal community structure in soil. In this study, we conducted a comprehensive survey of fungal communities in Japanese Prunus mume orchard soil from 2010 to 2012 growing seasons using next-generation sequencing technology. Fungal DNA was directly extracted from the soil samples and the internal transcribed spacer 1 region was amplified by PCR and sequenced. We identified 34,826 fungal clone sequences from the soil samples. The fungal clones were sorted into 2132 operational taxonomic units and a majority of the discriminated clone sequences were classified as Ascomycota and Basidiomycota. The number of fungal species belonging to Ascomycota showed increases in June in the three growing seasons. That belonging to Glomeromycota showed increases in August in the three growing seasons. As Ascomycota fungi are wood decomposers and saprotrophs, the results suggested that the number of plant pathogenic fungi increased in Japanese P. mume orchard soil in June. These findings show for the first time the annual and seasonal fungal community structures in Japanese P. mume orchard soil, and are expected to provide valuable clues for improvement when planting new P. mume trees in Japanese
Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes
Frank A Feltus, Christopher A Saski, Keithanne Mockaitis, Niina Haiminen, Laxmi Parida, Zachary Smith, James Ford, Margaret E Staton, Stephen P Ficklin, Barbara P Blackmon, Chun-Huai Cheng, Raymond J Schnell, David N Kuhn, Juan-Carlos Motamayor
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-379
Abstract: This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight.Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.For more than a decade, whole-genome sequencing strategies have typically employed one of two strategies: the BAC-by-BAC approach in which BAC clones that represent a minimum tiling path (MTP) are sequenced Sanger-style, as was taken for the rice and maize projects [1,2], or whole-genome shotgun (WGS) sequencing using random Sanger-style sequencing of entire genomic libraries of clones with varying insert size, such as was used to sequence the genomes of black cottonwood, grapevine, and sorghum [3-5]. Traditional de novo sequencing of large, complex eukaryotic genomes is plagued with assembly challenges caused by repetitive DNA and segmental duplications. Misassembly of distal genomic regions is
Next Generation Sequencing: Advances in Characterizing the Methylome
Kristen H. Taylor,Huidong Shi,Charles W. Caldwell
Genes , 2010, DOI: 10.3390/genes1020143
Abstract: Epigenetic modifications play an important role in lymphoid malignancies. This has been evidenced by the large body of work published using microarray technologies to generate methylation profiles for numerous types and subtypes of lymphoma and leukemia. These studies have shown the importance of defining the epigenome so that we can better understand the biology of lymphoma. Recent advances in DNA sequencing technology have transformed the landscape of epigenomic analysis as we now have the ability to characterize the genome-wide distribution of chromatin modifications and DNA methylation using next-generation sequencing. To take full advantage of the throughput of next-generation sequencing, there are many methodologies that have been developed and many more that are currently being developed. Choosing the appropriate methodology is fundamental to the outcome of next-generation sequencing studies. In this review, published technologies and methodologies applicable to studying the methylome are presented. In addition, progress towards defining the methylome in lymphoma is discussed and prospective directions that have been made possible as a result of next-generation sequencing technology. Finally, methodologies are introduced that have not yet been published but that are being explored in the pursuit of defining the lymphoma methylome.
Whole Genome Sequencing and a New Bioinformatics Platform Allow for Rapid Gene Identification in D. melanogaster EMS Screens
Michael A. Gonzalez,Derek Van Booven,William Hulme,Rick H. Ulloa,Rafael F. Acosta Lebrigio,Jeannette Osterloh,Mary Logan,Marc Freeman,Stephan Zuchner
Biology , 2012, DOI: 10.3390/biology1030766
Abstract: Forward genetic screens in Drosophila melanogaster using ethyl methanesulfonate (EMS) mutagenesis are a powerful approach for identifying genes that modulate specific biological processes in an in vivo setting. The mapping of genes that contain randomly-induced point mutations has become more efficient in Drosophila thanks to the maturation and availability of many types of genetic tools. However, classic approaches to gene mapping are relatively slow and ultimately require extensive Sanger sequencing of candidate chromosomal loci. With the advent of new high-throughput sequencing techniques, it is increasingly efficient to directly re-sequence the whole genome of model organisms. This approach, in combination with traditional chromosomal mapping, has the potential to greatly simplify and accelerate mutation identification in mutants generated in EMS screens. Here we show that next-generation sequencing (NGS) is an accurate and efficient tool for high-throughput sequencing and mutation discovery in Drosophila melanogaster. As a test case, mutant strains of Drosophila that exhibited long-term survival of severed peripheral axons were identified in a forward EMS mutagenesis. All mutants were recessive and fell into a single lethal complementation group, which suggested that a single gene was responsible for the protective axon degenerative phenotype. Whole genome sequencing of these genomes identified the underlying gene ect4. To improve the process of genome wide mutation identification, we developed Genomes Management Application (GEM.app, https://genomics.med.miami.edu), a graphical online user interface to a custom query framework. Using a custom GEM.app query, we were able to identify that each mutant carried a unique non-sense mutation in the gene ect4 ( dSarm), which was recently shown by Osterloh et al. to be essential for the activation of axonal degeneration. Our results demonstrate the current advantages and limitations of NGS in Drosophila and we introduce GEM.app as a simple yet powerful genomics analysis tool for the Drosophila community. At a current cost of <$1,000 per genome, NGS should thus become a standard gene discovery tool in EMS induced genetic forward screens.
Personalized Targeted Therapy for Lung Cancer
Kehua Wu,Larry House,Wanqing Liu,William C.S. Cho
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms130911471
Abstract: Lung cancer has long been recognized as an extremely heterogeneous disease, since its development is unique in every patient in terms of clinical characterizations, prognosis, response and tolerance to treatment. Personalized medicine refers to the use of markers to predict which patient will most likely benefit from a treatment. In lung cancer, the well-developed epidermal growth factor receptor (EGFR) and the newly emerging EML4-anaplastic lymphoma kinase (ALK) are important therapeutic targets. This review covers the basic mechanism of EGFR and EML4-ALK activation, the predictive biomarkers, the mechanism of resistance, and the current targeted tyrosine kinase inhibitors. The efficacy of EGFR and ALK targeted therapies will be discussed in this review by summarizing the prospective clinical trials, which were performed in biomarker-based selected patients. In addition, the revolutionary sequencing and systems strategies will also be included in this review since these technologies will provide a comprehensive understanding in the molecular characterization of cancer, allow better stratification of patients for the most appropriate targeted therapies, eventually resulting in a more promising personalized treatment. The relatively low incidence of EGFR and ALK in non-Asian patients and the lack of response in mutant patients limit the application of the therapies targeting EGFR or ALK. Nevertheless, it is foreseeable that the sequencing and systems strategies may offer a solution for those patients.
ASAP: an environment for automated preprocessing of sequencing data
Eric S Torstenson, Bingshan Li, Chun Li
BMC Research Notes , 2013, DOI: 10.1186/1756-0500-6-5
Abstract: Advanced Sequence Automated Pipeline (ASAP) was developed to provide a framework for automating the translation of sequencing data into annotated variant calls with the goal of minimizing user involvement without the need for dedicated hardware or administrative rights. ASAP works both on computer clusters and on standalone machines with minimal human involvement and maintains high data integrity, while allowing complete control over the configuration of its component programs. It offers an easy-to-use interface for submitting and tracking jobs as well as resuming failed jobs. It also provides tools for quality checking and for dividing jobs into pieces for maximum throughput.ASAP provides an environment for building an automated pipeline for NGS data preprocessing. This environment is flexible for use and future development. It is freely available at http://biostat.mc.vanderbilt.edu/ASAP webcite.Modern sequencing technologies have greatly improved our capability of acquiring deep sequencing data on a large scale and in a timely fashion. However, the large amount of data presents many new challenges to researchers, including a significant amount of time and effort on preprocessing raw sequencing reads into variant calls that are ready for statistical analyses. This process involves multiple steps and several independent programs. For example, for species with a reference genome available, sequence reads are often initially aligned to the reference genome using a mapping program such as BWA (Li & Durbin [1]). Additionally, reads aligned to insertion-deletion regions may require local realignment to minimize false variant calls, and base quality scores may require recalibration to reflect empirical error rates; these can be achieved with GATK (McKenna et al. [2]). Moreover, variant calls may require filtering for false call removal and annotation for downstream analyses. The various steps require different programs and there may be multiple programs available for some
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