Significance of functional disease-causal/susceptible variants identified by whole-genome analyses for the understanding of human diseases.

PubMed

Hitomi, Yuki; Tokunaga, Katsushi

2017-01-01

Human genome variation may cause differences in traits and disease risks. Disease-causal/susceptible genes and variants for both common and rare diseases can be detected by comprehensive whole-genome analyses, such as whole-genome sequencing (WGS), using next-generation sequencing (NGS) technology and genome-wide association studies (GWAS). Here, in addition to the application of an NGS as a whole-genome analysis method, we summarize approaches for the identification of functional disease-causal/susceptible variants from abundant genetic variants in the human genome and methods for evaluating their functional effects in human diseases, using an NGS and in silico and in vitro functional analyses. We also discuss the clinical applications of the functional disease causal/susceptible variants to personalized medicine.

[Advances in genome editing technologies for treating muscular dystrophy.

PubMed

Makita, Yukimasa; Hozumi, Hiroyuki; Hotta, Akitsu

Recent advances in genome editing technologies have opened the possibility for treating genetic diseases, such as Duchenne muscular dystrophy(DMD), by correcting the causing gene mutations in dystrophin gene. In fact, there are several reports that demonstrated the restoration of the mutated dystrophin gene in DMD patient-derived iPS cell or functional recovery of forelimb grip strength in DMD model mice. For future clinical applications, there are several aspects that need to be taken into consideration:efficient delivery of the genome editing components, risk of off-target mutagenesis and immunogenicity against genome editing enzyme. In this review, we summarize the current status and future prospective of the research in applying genome editing technologies to DMD.

Novel Functional Genomics Approaches: A Promising Future in the Combat Against Plant Viruses.

PubMed

Fondong, Vincent N; Nagalakshmi, Ugrappa; Dinesh-Kumar, Savithramma P

2016-10-01

Advances in functional genomics and genome editing approaches have provided new opportunities and potential to accelerate plant virus control efforts through modification of host and viral genomes in a precise and predictable manner. Here, we discuss application of RNA-based technologies, including artificial micro RNA, transacting small interfering RNA, and Cas9 (clustered regularly interspaced short palindromic repeat-associated protein 9), which are currently being successfully deployed in generating virus-resistant plants. We further discuss the reverse genetics approach, targeting induced local lesions in genomes (TILLING) and its variant, known as EcoTILLING, that are used in the identification of plant virus recessive resistance gene alleles. In addition to describing specific applications of these technologies in plant virus control, this review discusses their advantages and limitations.

The contribution of the DNA microarray technology to gene expression profiling in Leishmania spp.: a retrospective.

PubMed

Alonso, Ana; Larraga, Vicente; Alcolea, Pedro J

2018-05-07

The first genome project of any living organism excluding viruses, the gammaproteobacteria Haemophilus influenzae, was completed in 1995. Until the last decade, genome sequencing was very tedious because genome survey sequences (GSS) and/or expressed sequence tags (ESTs) belonging to plasmid, cosmid and artificial chromosome genome libraries had to be sequenced and assembled in silico. Nowadays, no genome is completely assembled actually, because gaps and unassembled contigs are always remaining. However, most represent the whole genome of the organism of origin from a practical point of view. The first genome sequencing projects of trypanosomatid parasites were completed in 2005 following those strategies, and belong to Leishmania major, Trypanosoma cruzi and T. brucei. The functional genomics era rapidly developed on the basis of the microarray technology and has been evolving. In the case of the genus Leishmania, substantial biological information about differentiation in the digenetic life cycle of the parasite has been obtained. Later on, next generation sequencing has revolutionized genome sequencing and functional genomics, leading to more sensitive, accurate results by using much less resources. This new technology is more advantageous, but does not invalidate microarray results. In fact, promising vaccine candidates and drug targets have been found on the basis of microarray-based screening and preliminary proof-of-concept tests. Copyright © 2018. Published by Elsevier B.V.

Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research.

PubMed

Musunuru, Kiran; Bernstein, Daniel; Cole, F Sessions; Khokha, Mustafa K; Lee, Frank S; Lin, Shin; McDonald, Thomas V; Moskowitz, Ivan P; Quertermous, Thomas; Sankaran, Vijay G; Schwartz, David A; Silverman, Edwin K; Zhou, Xiaobo; Hasan, Ahmed A K; Luo, Xiao-Zhong James

2018-04-01

The National Institutes of Health have made substantial investments in genomic studies and technologies to identify DNA sequence variants associated with human disease phenotypes. The National Heart, Lung, and Blood Institute has been at the forefront of these commitments to ascertain genetic variation associated with heart, lung, blood, and sleep diseases and related clinical traits. Genome-wide association studies, exome- and genome-sequencing studies, and exome-genotyping studies of the National Heart, Lung, and Blood Institute-funded epidemiological and clinical case-control studies are identifying large numbers of genetic variants associated with heart, lung, blood, and sleep phenotypes. However, investigators face challenges in identification of genomic variants that are functionally disruptive among the myriad of computationally implicated variants. Studies to define mechanisms of genetic disruption encoded by computationally identified genomic variants require reproducible, adaptable, and inexpensive methods to screen candidate variant and gene function. High-throughput strategies will permit a tiered variant discovery and genetic mechanism approach that begins with rapid functional screening of a large number of computationally implicated variants and genes for discovery of those that merit mechanistic investigation. As such, improved variant-to-gene and gene-to-function screens-and adequate support for such studies-are critical to accelerating the translation of genomic findings. In this White Paper, we outline the variety of novel technologies, assays, and model systems that are making such screens faster, cheaper, and more accurate, referencing published work and ongoing work supported by the National Heart, Lung, and Blood Institute's R21/R33 Functional Assays to Screen Genomic Hits program. We discuss priorities that can accelerate the impressive but incomplete progress represented by big data genomic research. © 2018 American Heart Association, Inc.

Partnering for functional genomics research conference: Abstracts of poster presentations

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-06-01

This reports contains abstracts of poster presentations presented at the Functional Genomics Research Conference held April 16--17, 1998 in Oak Ridge, Tennessee. Attention is focused on the following areas: mouse mutagenesis and genomics; phenotype screening; gene expression analysis; DNA analysis technology development; bioinformatics; comparative analyses of mouse, human, and yeast sequences; and pilot projects to evaluate methodologies.

Solutions for data integration in functional genomics: a critical assessment and case study.

PubMed

Smedley, Damian; Swertz, Morris A; Wolstencroft, Katy; Proctor, Glenn; Zouberakis, Michael; Bard, Jonathan; Hancock, John M; Schofield, Paul

2008-11-01

The torrent of data emerging from the application of new technologies to functional genomics and systems biology can no longer be contained within the traditional modes of data sharing and publication with the consequence that data is being deposited in, distributed across and disseminated through an increasing number of databases. The resulting fragmentation poses serious problems for the model organism community which increasingly rely on data mining and computational approaches that require gathering of data from a range of sources. In the light of these problems, the European Commission has funded a coordination action, CASIMIR (coordination and sustainability of international mouse informatics resources), with a remit to assess the technical and social aspects of database interoperability that currently prevent the full realization of the potential of data integration in mouse functional genomics. In this article, we assess the current problems with interoperability, with particular reference to mouse functional genomics, and critically review the technologies that can be deployed to overcome them. We describe a typical use-case where an investigator wishes to gather data on variation, genomic context and metabolic pathway involvement for genes discovered in a genome-wide screen. We go on to develop an automated approach involving an in silico experimental workflow tool, Taverna, using web services, BioMart and MOLGENIS technologies for data retrieval. Finally, we focus on the current impediments to adopting such an approach in a wider context, and strategies to overcome them.

Genome Microscale Heterogeneity among Wild Potatoes Revealed by Diversity Arrays Technology Marker Sequences.

PubMed

Traini, Alessandra; Iorizzo, Massimo; Mann, Harpartap; Bradeen, James M; Carputo, Domenico; Frusciante, Luigi; Chiusano, Maria Luisa

2013-01-01

Tuber-bearing potato species possess several genes that can be exploited to improve the genetic background of the cultivated potato Solanum tuberosum. Among them, S. bulbocastanum and S. commersonii are well known for their strong resistance to environmental stresses. However, scant information is available for these species in terms of genome organization, gene function, and regulatory networks. Consequently, genomic tools to assist breeding are meager, and efficient exploitation of these species has been limited so far. In this paper, we employed the reference genome sequences from cultivated potato and tomato and a collection of sequences of 1,423 potato Diversity Arrays Technology (DArT) markers that show polymorphic representation across the genomes of S. bulbocastanum and/or S. commersonii genotypes. Our results highlighted microscale genome sequence heterogeneity that may play a significant role in functional and structural divergence between related species. Our analytical approach provides knowledge of genome structural and sequence variability that could not be detected by transcriptome and proteome approaches.

Genome Engineering and Agriculture: Opportunities and Challenges.

PubMed

Baltes, Nicholas J; Gil-Humanes, Javier; Voytas, Daniel F

2017-01-01

In recent years, plant biotechnology has witnessed unprecedented technological change. Advances in high-throughput sequencing technologies have provided insight into the location and structure of functional elements within plant DNA. At the same time, improvements in genome engineering tools have enabled unprecedented control over genetic material. These technologies, combined with a growing understanding of plant systems biology, will irrevocably alter the way we create new crop varieties. As the first wave of genome-edited products emerge, we are just getting a glimpse of the immense opportunities the technology provides. We are also seeing its challenges and limitations. It is clear that genome editing will play an increased role in crop improvement and will help us to achieve food security in the coming decades; however, certain challenges and limitations must be overcome to realize the technology's full potential. © 2017 Elsevier Inc. All rights reserved.

Enabling functional genomics with genome engineering

PubMed Central

Hilton, Isaac B.; Gersbach, Charles A.

2015-01-01

Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances. PMID:26430154

The most common technologies and tools for functional genome analysis.

PubMed

Gasperskaja, Evelina; Kučinskas, Vaidutis

2017-01-01

Since the sequence of the human genome is complete, the main issue is how to understand the information written in the DNA sequence. Despite numerous genome-wide studies that have already been performed, the challenge to determine the function of genes, gene products, and also their interaction is still open. As changes in the human genome are highly likely to cause pathological conditions, functional analysis is vitally important for human health. For many years there have been a variety of technologies and tools used in functional genome analysis. However, only in the past decade there has been rapid revolutionizing progress and improvement in high-throughput methods, which are ranging from traditional real-time polymerase chain reaction to more complex systems, such as next-generation sequencing or mass spectrometry. Furthermore, not only laboratory investigation, but also accurate bioinformatic analysis is required for reliable scientific results. These methods give an opportunity for accurate and comprehensive functional analysis that involves various fields of studies: genomics, epigenomics, proteomics, and interactomics. This is essential for filling the gaps in the knowledge about dynamic biological processes at both cellular and organismal level. However, each method has both advantages and limitations that should be taken into account before choosing the right method for particular research in order to ensure successful study. For this reason, the present review paper aims to describe the most frequent and widely-used methods for the comprehensive functional analysis.

Emerging applications of genome-editing technology to examine functionality of GWAS-associated variants for complex traits.

PubMed

Smith, Andrew J P; Deloukas, Panos; Munroe, Patricia B

2018-04-13

Over the last decade, genome-wide association studies (GWAS) have propelled the discovery of thousands of loci associated with complex diseases. The focus is now turning towards the function of these association signals, determining the causal variant(s) amongst those in strong linkage disequilibrium, and identifying their underlying mechanisms, such as long-range gene regulation. Genome-editing techniques utilising zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats with Cas9 nuclease (CRISPR-Cas9), are becoming the tools of choice to establish functionality for these variants, due to the ability to assess effects of single variants in vivo. This review will discuss examples of how these technologies have begun to aid functional analysis of GWAS loci for complex traits such as cardiovascular disease, type 2 diabetes, cancer, obesity and autoimmune disease. We focus on analysis of variants occurring within non-coding genomic regions, as these comprise the majority of GWAS variants, providing the greatest challenges to determining functionality, and compare editing strategies that provide different levels of evidence for variant functionality. The review describes molecular insights into some of these potentially causal variants, and how these may relate to the pathology of the trait, and look towards future directions for these technologies in post-GWAS analysis, such as base-editing.

The W22 genome: a foundation for maize functional genomics and transposon biology

USDA-ARS?s Scientific Manuscript database

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using small-read sequencing technologies. We show that significant structural heterogeneity exists in ...

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Recent Advances in Microbial Single Cell Genomics Technology and Applications

NASA Astrophysics Data System (ADS)

Stepanauskas, R.

2016-02-01

Single cell genomics is increasingly utilized as a powerful tool to decipher the metabolic potential, evolutionary histories and in situ interactions of environmental microorganisms. This transformative technology recovers extensive information from cultivation-unbiased samples of individual, unicellular organisms. Thus, it does not require data binning into arbitrary phylogenetic or functional groups and therefore is highly compatible with agent-based modeling approaches. I will present several technological advances in this field, which significantly improve genomic data recovery from individual cells and provide direct linkages between cell's genomic and phenotypic properties. I will also demonstrate how these new technical capabilities help understanding the metabolic potential and viral infections of the "microbial dark matter" inhabiting aquatic and subsurface environments.

Human genetics and genomics a decade after the release of the draft sequence of the human genome.

PubMed

Naidoo, Nasheen; Pawitan, Yudi; Soong, Richie; Cooper, David N; Ku, Chee-Seng

2011-10-01

Substantial progress has been made in human genetics and genomics research over the past ten years since the publication of the draft sequence of the human genome in 2001. Findings emanating directly from the Human Genome Project, together with those from follow-on studies, have had an enormous impact on our understanding of the architecture and function of the human genome. Major developments have been made in cataloguing genetic variation, the International HapMap Project, and with respect to advances in genotyping technologies. These developments are vital for the emergence of genome-wide association studies in the investigation of complex diseases and traits. In parallel, the advent of high-throughput sequencing technologies has ushered in the 'personal genome sequencing' era for both normal and cancer genomes, and made possible large-scale genome sequencing studies such as the 1000 Genomes Project and the International Cancer Genome Consortium. The high-throughput sequencing and sequence-capture technologies are also providing new opportunities to study Mendelian disorders through exome sequencing and whole-genome sequencing. This paper reviews these major developments in human genetics and genomics over the past decade.

Human genetics and genomics a decade after the release of the draft sequence of the human genome

PubMed Central

2011-01-01

Substantial progress has been made in human genetics and genomics research over the past ten years since the publication of the draft sequence of the human genome in 2001. Findings emanating directly from the Human Genome Project, together with those from follow-on studies, have had an enormous impact on our understanding of the architecture and function of the human genome. Major developments have been made in cataloguing genetic variation, the International HapMap Project, and with respect to advances in genotyping technologies. These developments are vital for the emergence of genome-wide association studies in the investigation of complex diseases and traits. In parallel, the advent of high-throughput sequencing technologies has ushered in the 'personal genome sequencing' era for both normal and cancer genomes, and made possible large-scale genome sequencing studies such as the 1000 Genomes Project and the International Cancer Genome Consortium. The high-throughput sequencing and sequence-capture technologies are also providing new opportunities to study Mendelian disorders through exome sequencing and whole-genome sequencing. This paper reviews these major developments in human genetics and genomics over the past decade. PMID:22155605

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2016-01-01

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

Application of genome editing technologies to the study and treatment of hematological disease.

PubMed

Pellagatti, Andrea; Dolatshad, Hamid; Yip, Bon Ham; Valletta, Simona; Boultwood, Jacqueline

2016-01-01

Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enabling functional genomics with genome engineering.

PubMed

Hilton, Isaac B; Gersbach, Charles A

2015-10-01

Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances. © 2015 Hilton and Gersbach; Published by Cold Spring Harbor Laboratory Press.

Genome Editing for the Study of Cardiovascular Diseases.

PubMed

Chadwick, Alexandra C; Musunuru, Kiran

2017-03-01

The opportunities afforded through the recent advent of genome-editing technologies have allowed investigators to more easily study a number of diseases. The advantages and limitations of the most prominent genome-editing technologies are described in this review, along with potential applications specifically focused on cardiovascular diseases. The recent genome-editing tools using programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have rapidly been adapted to manipulate genes in a variety of cellular and animal models. A number of recent cardiovascular disease-related publications report cases in which specific mutations are introduced into disease models for functional characterization and for testing of therapeutic strategies. Recent advances in genome-editing technologies offer new approaches to understand and treat diseases. Here, we discuss genome editing strategies to easily characterize naturally occurring mutations and offer strategies with potential clinical relevance.

Conifer genomics and adaptation: at the crossroads of genetic diversity and genome function.

PubMed

Prunier, Julien; Verta, Jukka-Pekka; MacKay, John J

2016-01-01

Conifers have been understudied at the genomic level despite their worldwide ecological and economic importance but the situation is rapidly changing with the development of next generation sequencing (NGS) technologies. With NGS, genomics research has simultaneously gained in speed, magnitude and scope. In just a few years, genomes of 20-24 gigabases have been sequenced for several conifers, with several others expected in the near future. Biological insights have resulted from recent sequencing initiatives as well as genetic mapping, gene expression profiling and gene discovery research over nearly two decades. We review the knowledge arising from conifer genomics research emphasizing genome evolution and the genomic basis of adaptation, and outline emerging questions and knowledge gaps. We discuss future directions in three areas with potential inputs from NGS technologies: the evolutionary impacts of adaptation in conifers based on the adaptation-by-speciation model; the contributions of genetic variability of gene expression in adaptation; and the development of a broader understanding of genetic diversity and its impacts on genome function. These research directions promise to sustain research aimed at addressing the emerging challenges of adaptation that face conifer trees. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update

PubMed Central

Hu, Yanhui; Comjean, Aram; Roesel, Charles; Vinayagam, Arunachalam; Flockhart, Ian; Zirin, Jonathan; Perkins, Lizabeth; Perrimon, Norbert; Mohr, Stephanie E.

2017-01-01

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches. PMID:27924039

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2014-08-15

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

A New Approach to Dissect Nuclear Organization: TALE-Mediated Genome Visualization (TGV).

PubMed

Miyanari, Yusuke

2016-01-01

Spatiotemporal organization of chromatin within the nucleus has so far remained elusive. Live visualization of nuclear remodeling could be a promising approach to understand its functional relevance in genome functions and mechanisms regulating genome architecture. Recent technological advances in live imaging of chromosomes begun to explore the biological roles of the movement of the chromatin within the nucleus. Here I describe a new technique, called TALE-mediated genome visualization (TGV), which allows us to visualize endogenous repetitive sequence including centromeric, pericentromeric, and telomeric repeats in living cells.

Precision medicine for cancer with next-generation functional diagnostics.

PubMed

Friedman, Adam A; Letai, Anthony; Fisher, David E; Flaherty, Keith T

2015-12-01

Precision medicine is about matching the right drugs to the right patients. Although this approach is technology agnostic, in cancer there is a tendency to make precision medicine synonymous with genomics. However, genome-based cancer therapeutic matching is limited by incomplete biological understanding of the relationship between phenotype and cancer genotype. This limitation can be addressed by functional testing of live patient tumour cells exposed to potential therapies. Recently, several 'next-generation' functional diagnostic technologies have been reported, including novel methods for tumour manipulation, molecularly precise assays of tumour responses and device-based in situ approaches; these address the limitations of the older generation of chemosensitivity tests. The promise of these new technologies suggests a future diagnostic strategy that integrates functional testing with next-generation sequencing and immunoprofiling to precisely match combination therapies to individual cancer patients.

Technological advances and genomics in metazoan parasites.

PubMed

Knox, D P

2004-02-01

Molecular biology has provided the means to identify parasite proteins, to define their function, patterns of expression and the means to produce them in quantity for subsequent functional analyses. Whole genome and expressed sequence tag programmes, and the parallel development of powerful bioinformatics tools, allow the execution of genome-wide between stage or species comparisons and meaningful gene-expression profiling. The latter can be undertaken with several new technologies such as DNA microarray and serial analysis of gene expression. Proteome analysis has come to the fore in recent years providing a crucial link between the gene and its protein product. RNA interference and ballistic gene transfer are exciting developments which can provide the means to precisely define the function of individual genes and, of importance in devising novel parasite control strategies, the effect that gene knockdown will have on parasite survival.

UCSC Xena | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

UCSC Xena securely analyzes and visualizes your private functional genomics data set in the context of public and shared genomic/phenotypic data sets such as TCGA, ICGC, TARGET, GTEx, and GA4GH (TOIL).

Human centromere genomics: now it's personal.

PubMed

Hayden, Karen E

2012-07-01

Advances in human genomics have accelerated studies in evolution, disease, and cellular regulation. However, centromere sequences, defining the chromosomal interface with spindle microtubules, remain largely absent from ongoing genomic studies and disconnected from functional, genome-wide analyses. This disparity results from the challenge of predicting the linear order of multi-megabase-sized regions that are composed almost entirely of near-identical satellite DNA. Acknowledging these challenges, the field of human centromere genomics possesses the potential to rapidly advance given the availability of individual, or personalized, genome projects matched with the promise of long-read sequencing technologies. Here I review the current genomic model of human centromeres in consideration of those studies involving functional datasets that examine the role of sequence in centromere identity.

Assaying gene function by growth competition experiment.

PubMed

Merritt, Joshua; Edwards, Jeremy S

2004-07-01

High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies

PubMed Central

Li, Xueyan; Fan, Dingding; Zhang, Wei; Liu, Guichun; Zhang, Lu; Zhao, Li; Fang, Xiaodong; Chen, Lei; Dong, Yang; Chen, Yuan; Ding, Yun; Zhao, Ruoping; Feng, Mingji; Zhu, Yabing; Feng, Yue; Jiang, Xuanting; Zhu, Deying; Xiang, Hui; Feng, Xikan; Li, Shuaicheng; Wang, Jun; Zhang, Guojie; Kronforst, Marcus R.; Wang, Wen

2015-01-01

Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high-quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 kb, 3.4 Mb) and Papilio machaon (contig and scaffold N50: 81 kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations, and adult and larval colour patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony and frizzled. Our results provide valuable genomic and technological resources for butterflies and unlock their potential as a genetic model system. PMID:26354079

Decoding the genome beyond sequencing: the new phase of genomic research.

PubMed

Heng, Henry H Q; Liu, Guo; Stevens, Joshua B; Bremer, Steven W; Ye, Karen J; Abdallah, Batoul Y; Horne, Steven D; Ye, Christine J

2011-10-01

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing. Copyright © 2011 Elsevier Inc. All rights reserved.

Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology.

PubMed

Khan, Muhammad Hafeez Ullah; Khan, Shahid U; Muhammad, Ali; Hu, Limin; Yang, Yang; Fan, Chuchuan

2018-06-01

Clustered regularly interspaced palindromic repeats associated protein Cas9 (CRISPR-Cas9), originally an adaptive immunity system of prokaryotes, is revolutionizing genome editing technologies with minimal off-targets in the present era. The CRISPR/Cas9 is now highly emergent, advanced, and highly specific tool for genome engineering. The technology is widely used to animal and plant genomes to achieve desirable results. The present review will encompass how CRISPR-Cas9 is revealing its beneficial role in characterizing plant genetic functions, genomic rearrangement, how it advances the site-specific mutagenesis, and epigenetics modification in plants to improve the yield of field crops with minimal side-effects. The possible pitfalls of using and designing CRISPR-Cas9 for plant genome editing are also discussed for its more appropriate applications in plant biology. Therefore, CRISPR/Cas9 system has multiple benefits that mostly scientists select for genome editing in several biological systems. © 2017 Wiley Periodicals, Inc.

Genome Editing for the Study of Cardiovascular Diseases

PubMed Central

Chadwick, Alexandra C.

2018-01-01

Purpose of Review The opportunities afforded through the recent advent of genome-editing technologies have allowed investigators to more easily study a number of diseases. The advantages and limitations of the most prominent genome-editing technologies are described in this review, along with potential applications specifically focused on cardiovascular diseases. Recent Findings The recent genome-editing tools using programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have rapidly been adapted to manipulate genes in a variety of cellular and animal models. A number of recent cardiovascular disease-related publications report cases in which specific mutations are introduced into disease models for functional characterization and for testing of therapeutic strategies. Summary Recent advances in genome-editing technologies offer new approaches to understand and treat diseases. Here, we discuss genome editing strategies to easily characterize naturally occurring mutations and offer strategies with potential clinical relevance. PMID:28220462

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Gene calling and bacterial genome annotation with BG7.

PubMed

Tobes, Raquel; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Kovach, Evdokim; Alekhin, Alexey; Pareja, Eduardo

2015-01-01

New massive sequencing technologies are providing many bacterial genome sequences from diverse taxa but a refined annotation of these genomes is crucial for obtaining scientific findings and new knowledge. Thus, bacterial genome annotation has emerged as a key point to investigate in bacteria. Any efficient tool designed specifically to annotate bacterial genomes sequenced with massively parallel technologies has to consider the specific features of bacterial genomes (absence of introns and scarcity of nonprotein-coding sequence) and of next-generation sequencing (NGS) technologies (presence of errors and not perfectly assembled genomes). These features make it convenient to focus on coding regions and, hence, on protein sequences that are the elements directly related with biological functions. In this chapter we describe how to annotate bacterial genomes with BG7, an open-source tool based on a protein-centered gene calling/annotation paradigm. BG7 is specifically designed for the annotation of bacterial genomes sequenced with NGS. This tool is sequence error tolerant maintaining their capabilities for the annotation of highly fragmented genomes or for annotating mixed sequences coming from several genomes (as those obtained through metagenomics samples). BG7 has been designed with scalability as a requirement, with a computing infrastructure completely based on cloud computing (Amazon Web Services).

The 4D nucleome project.

PubMed

Dekker, Job; Belmont, Andrew S; Guttman, Mitchell; Leshyk, Victor O; Lis, John T; Lomvardas, Stavros; Mirny, Leonid A; O'Shea, Clodagh C; Park, Peter J; Ren, Bing; Politz, Joan C Ritland; Shendure, Jay; Zhong, Sheng

2017-09-13

The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.

CRISPR Mediated Genome Engineering and its Application in Industry.

PubMed

Kaboli, Saeed; Babazada, Hasan

2018-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

PubMed

Wragg, J; Müller, F

2016-01-01

Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic embryological tools and genome-wide assays. In this review we summarize recent advances in the characterization of epigenetic regulation, transcription control, and gene promoter function during zygotic genome activation and how they fit with old models for the mechanisms of the maternal to zygotic transition. This review will focus on the zebrafish embryo but draw comparisons with other vertebrate model systems and refer to invertebrate models where informative. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene correction in patient-specific iPSCs for therapy development and disease modeling

PubMed Central

Jang, Yoon-Young

2018-01-01

The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field. PMID:27256364

BG7: A New Approach for Bacterial Genome Annotation Designed for Next Generation Sequencing Data

PubMed Central

Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Pareja, Eduardo; Tobes, Raquel

2012-01-01

BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version – which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future. PMID:23185310

Molecular inversion probe assay.

PubMed

Absalan, Farnaz; Ronaghi, Mostafa

2007-01-01

We have described molecular inversion probe technologies for large-scale genetic analyses. This technique provides a comprehensive and powerful tool for the analysis of genetic variation and enables affordable, large-scale studies that will help uncover the genetic basis of complex disease and explain the individual variation in response to therapeutics. Major applications of the molecular inversion probes (MIP) technologies include targeted genotyping from focused regions to whole-genome studies, and allele quantification of genomic rearrangements. The MIP technology (used in the HapMap project) provides an efficient, scalable, and affordable way to score polymorphisms in case/control populations for genetic studies. The MIP technology provides the highest commercially available multiplexing levels and assay conversion rates for targeted genotyping. This enables more informative, genome-wide studies with either the functional (direct detection) approach or the indirect detection approach.

A Perspective on the Future of High-Throughput RNAi Screening: Will CRISPR Cut Out the Competition or Can RNAi Help Guide the Way?

PubMed

Taylor, Jessica; Woodcock, Simon

2015-09-01

For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA-Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery. © 2015 Society for Laboratory Automation and Screening.

Gene Editing and Crop Improvement Using CRISPR-Cas9 System

PubMed Central

Arora, Leena; Narula, Alka

2017-01-01

Advancements in Genome editing technologies have revolutionized the fields of functional genomics and crop improvement. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)-Cas9 is a multipurpose technology for genetic engineering that relies on the complementarity of the guideRNA (gRNA) to a specific sequence and the Cas9 endonuclease activity. It has broadened the agricultural research area, bringing in new opportunities to develop novel plant varieties with deletion of detrimental traits or addition of significant characters. This RNA guided genome editing technology is turning out to be a groundbreaking innovation in distinct branches of plant biology. CRISPR technology is constantly advancing including options for various genetic manipulations like generating knockouts; making precise modifications, multiplex genome engineering, and activation and repression of target genes. The review highlights the progression throughout the CRISPR legacy. We have studied the rapid evolution of CRISPR/Cas9 tools with myriad functionalities, capabilities, and specialized applications. Among varied diligences, plant nutritional improvement, enhancement of plant disease resistance and production of drought tolerant plants are reviewed. The review also includes some information on traditional delivery methods of Cas9-gRNA complexes into plant cells and incorporates the advent of CRISPR ribonucleoproteins (RNPs) that came up as a solution to various limitations that prevailed with plasmid-based CRISPR system. PMID:29167680

Gene Editing and Crop Improvement Using CRISPR-Cas9 System.

PubMed

Arora, Leena; Narula, Alka

2017-01-01

Advancements in Genome editing technologies have revolutionized the fields of functional genomics and crop improvement. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)-Cas9 is a multipurpose technology for genetic engineering that relies on the complementarity of the guideRNA (gRNA) to a specific sequence and the Cas9 endonuclease activity. It has broadened the agricultural research area, bringing in new opportunities to develop novel plant varieties with deletion of detrimental traits or addition of significant characters. This RNA guided genome editing technology is turning out to be a groundbreaking innovation in distinct branches of plant biology. CRISPR technology is constantly advancing including options for various genetic manipulations like generating knockouts; making precise modifications, multiplex genome engineering, and activation and repression of target genes. The review highlights the progression throughout the CRISPR legacy. We have studied the rapid evolution of CRISPR/Cas9 tools with myriad functionalities, capabilities, and specialized applications. Among varied diligences, plant nutritional improvement, enhancement of plant disease resistance and production of drought tolerant plants are reviewed. The review also includes some information on traditional delivery methods of Cas9-gRNA complexes into plant cells and incorporates the advent of CRISPR ribonucleoproteins (RNPs) that came up as a solution to various limitations that prevailed with plasmid-based CRISPR system.

Non-coding RNAs in virology: an RNA genomics approach.

PubMed

Isaac, Christopher; Patel, Trushar R; Zovoilis, Athanasios

2018-04-01

Advances in sequencing technologies and bioinformatic analysis techniques have greatly improved our understanding of various classes of RNAs and their functions. Despite not coding for proteins, non-coding RNAs (ncRNAs) are emerging as essential biomolecules fundamental for cellular functions and cell survival. Interestingly, ncRNAs produced by viruses not only control the expression of viral genes, but also influence host cell regulation and circumvent host innate immune response. Correspondingly, ncRNAs produced by the host genome can play a key role in host-virus interactions. In this article, we will first discuss a number of types of viral and mammalian ncRNAs associated with viral infections. Subsequently, we also describe the new possibilities and opportunities that RNA genomics and next-generation sequencing technologies provide for studying ncRNAs in virology.

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

PubMed Central

2015-01-01

Conspectus The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein–DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology. PMID:24877793

Schistosoma comparative genomics: integrating genome structure, parasite biology and anthelmintic discovery

PubMed Central

Swain, Martin T.; Larkin, Denis M.; Caffrey, Conor R.; Davies, Stephen J.; Loukas, Alex; Skelly, Patrick J.; Hoffmann, Karl F.

2011-01-01

Schistosoma genomes provide a comprehensive resource for identifying the molecular processes that shape parasite evolution and for discovering novel chemotherapeutic or immunoprophylactic targets. Here, we demonstrate how intra- and intergenus comparative genomics can be used to drive these investigations forward, illustrate the advantages and limitations of these approaches and review how post genomic technologies offer complementary strategies for genome characterisation. While sequencing and functional characterisation of other schistosome/platyhelminth genomes continues to expedite anthelmintic discovery, we contend that future priorities should equally focus on improving assembly quality, and chromosomal assignment, of existing schistosome/platyhelminth genomes. PMID:22024648

New bioinformatic tool for quick identification of functionally relevant endogenous retroviral inserts in human genome.

PubMed

Garazha, Andrew; Ivanova, Alena; Suntsova, Maria; Malakhova, Galina; Roumiantsev, Sergey; Zhavoronkov, Alex; Buzdin, Anton

2015-01-01

Endogenous retroviruses (ERVs) and LTR retrotransposons (LRs) occupy ∼8% of human genome. Deep sequencing technologies provide clues to understanding of functional relevance of individual ERVs/LRs by enabling direct identification of transcription factor binding sites (TFBS) and other landmarks of functional genomic elements. Here, we performed the genome-wide identification of human ERVs/LRs containing TFBS according to the ENCODE project. We created the first interactive ERV/LRs database that groups the individual inserts according to their familial nomenclature, number of mapped TFBS and divergence from their consensus sequence. Information on any particular element can be easily extracted by the user. We also created a genome browser tool, which enables quick mapping of any ERV/LR insert according to genomic coordinates, known human genes and TFBS. These tools can be used to easily explore functionally relevant individual ERV/LRs, and for studying their impact on the regulation of human genes. Overall, we identified ∼110,000 ERV/LR genomic elements having TFBS. We propose a hypothesis of "domestication" of ERV/LR TFBS by the genome milieu including subsequent stages of initial epigenetic repression, partial functional release, and further mutation-driven reshaping of TFBS in tight coevolution with the enclosing genomic loci.

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics

PubMed Central

Ben-Amar, Anis; Daldoul, Samia; Reustle, Götz M.; Krczal, Gabriele; Mliki, Ahmed

2016-01-01

In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies. Integration of different omics platforms along with gene annotation and computational analysis may elucidate a complete view in a system biology level. Extensive investigations on reverse genetics methodologies were deployed for assigning biological function to a specific gene or gene product. We provide here an updated overview of these high throughout strategies highlighting recent advances in the knowledge of functional genomics in plants. PMID:28217003

Information management systems for pharmacogenomics.

PubMed

Thallinger, Gerhard G; Trajanoski, Slave; Stocker, Gernot; Trajanoski, Zlatko

2002-09-01

The value of high-throughput genomic research is dramatically enhanced by association with key patient data. These data are generally available but of disparate quality and not typically directly associated. A system that could bring these disparate data sources into a common resource connected with functional genomic data would be tremendously advantageous. However, the integration of clinical and accurate interpretation of the generated functional genomic data requires the development of information management systems capable of effectively capturing the data as well as tools to make that data accessible to the laboratory scientist or to the clinician. In this review these challenges and current information technology solutions associated with the management, storage and analysis of high-throughput data are highlighted. It is suggested that the development of a pharmacogenomic data management system which integrates public and proprietary databases, clinical datasets, and data mining tools embedded in a high-performance computing environment should include the following components: parallel processing systems, storage technologies, network technologies, databases and database management systems (DBMS), and application services.

Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

PubMed

Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

2015-02-01

Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Perspectives: Gene Expression in Fisheries Management

USGS Publications Warehouse

Nielsen, Jennifer L.; Pavey, Scott A.

2010-01-01

Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.

Accurate evaluation and analysis of functional genomics data and methods

PubMed Central

Greene, Casey S.; Troyanskaya, Olga G.

2016-01-01

The development of technology capable of inexpensively performing large-scale measurements of biological systems has generated a wealth of data. Integrative analysis of these data holds the promise of uncovering gene function, regulation, and, in the longer run, understanding complex disease. However, their analysis has proved very challenging, as it is difficult to quickly and effectively assess the relevance and accuracy of these data for individual biological questions. Here, we identify biases that present challenges for the assessment of functional genomics data and methods. We then discuss evaluation methods that, taken together, begin to address these issues. We also argue that the funding of systematic data-driven experiments and of high-quality curation efforts will further improve evaluation metrics so that they more-accurately assess functional genomics data and methods. Such metrics will allow researchers in the field of functional genomics to continue to answer important biological questions in a data-driven manner. PMID:22268703

Genomics Portals: integrative web-platform for mining genomics data.

PubMed

Shinde, Kaustubh; Phatak, Mukta; Johannes, Freudenberg M; Chen, Jing; Li, Qian; Vineet, Joshi K; Hu, Zhen; Ghosh, Krishnendu; Meller, Jaroslaw; Medvedovic, Mario

2010-01-13

A large amount of experimental data generated by modern high-throughput technologies is available through various public repositories. Our knowledge about molecular interaction networks, functional biological pathways and transcriptional regulatory modules is rapidly expanding, and is being organized in lists of functionally related genes. Jointly, these two sources of information hold a tremendous potential for gaining new insights into functioning of living systems. Genomics Portals platform integrates access to an extensive knowledge base and a large database of human, mouse, and rat genomics data with basic analytical visualization tools. It provides the context for analyzing and interpreting new experimental data and the tool for effective mining of a large number of publicly available genomics datasets stored in the back-end databases. The uniqueness of this platform lies in the volume and the diversity of genomics data that can be accessed and analyzed (gene expression, ChIP-chip, ChIP-seq, epigenomics, computationally predicted binding sites, etc), and the integration with an extensive knowledge base that can be used in such analysis. The integrated access to primary genomics data, functional knowledge and analytical tools makes Genomics Portals platform a unique tool for interpreting results of new genomics experiments and for mining the vast amount of data stored in the Genomics Portals backend databases. Genomics Portals can be accessed and used freely at http://GenomicsPortals.org.

Genomics Portals: integrative web-platform for mining genomics data

PubMed Central

2010-01-01

Background A large amount of experimental data generated by modern high-throughput technologies is available through various public repositories. Our knowledge about molecular interaction networks, functional biological pathways and transcriptional regulatory modules is rapidly expanding, and is being organized in lists of functionally related genes. Jointly, these two sources of information hold a tremendous potential for gaining new insights into functioning of living systems. Results Genomics Portals platform integrates access to an extensive knowledge base and a large database of human, mouse, and rat genomics data with basic analytical visualization tools. It provides the context for analyzing and interpreting new experimental data and the tool for effective mining of a large number of publicly available genomics datasets stored in the back-end databases. The uniqueness of this platform lies in the volume and the diversity of genomics data that can be accessed and analyzed (gene expression, ChIP-chip, ChIP-seq, epigenomics, computationally predicted binding sites, etc), and the integration with an extensive knowledge base that can be used in such analysis. Conclusion The integrated access to primary genomics data, functional knowledge and analytical tools makes Genomics Portals platform a unique tool for interpreting results of new genomics experiments and for mining the vast amount of data stored in the Genomics Portals backend databases. Genomics Portals can be accessed and used freely at http://GenomicsPortals.org. PMID:20070909

Using biotechnology and genomics to improve biotic and abiotic stress in apple

USDA-ARS?s Scientific Manuscript database

Genomic sequencing, molecular biology, and transformation technologies are providing valuable tools to better understand the complexity of how plants develop, function, and respond to biotic and abiotic stress. These approaches should complement but not replace a solid understanding of whole plant ...

AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

PubMed Central

Song, Giltae; Dickins, Benjamin J. A.; Demeter, Janos; Engel, Stacia; Dunn, Barbara; Cherry, J. Michael

2015-01-01

The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community. PMID:25781462

Phylomedicine: An evolutionary telescope to explore and diagnose the universe of disease mutations

PubMed Central

Kumar, Sudhir; Dudley, Joel T.; Filipski, Alan; Liu, Li

2011-01-01

Modern technologies have made the sequencing of personal genomes routine. They have revealed thousands of nonsynonymous (amino-acid altering) single nucleotide variants (nSNVs) of protein coding DNA per genome. What do these variants foretell about an individual’s predisposition to diseases? The experimental technologies required to carry out such evaluations at a genomic scale are not yet available. Fortunately, the process of natural selection has lent us an almost infinite set of tests in nature. During the long-term evolution, new mutations and existing variations have been evaluated for their biological consequences in countless species, and outcomes were readily revealed by multispecies genome comparisons. We review studies that have investigated evolutionary characteristics and in silico functional diagnoses of nSNVs found in thousands of disease-associated genes. We conclude that the patterns of long-term evolutionary conservation and permissible divergence are essential and instructive modalities for functional assessment of human genetic variations. PMID:21764165

Genome Editing Redefines Precision Medicine in the Cardiovascular Field

PubMed Central

Lahm, Harald; Dreßen, Martina; Lange, Rüdiger; Wu, Sean M.; Krane, Markus

2018-01-01

Genome editing is a powerful tool to study the function of specific genes and proteins important for development or disease. Recent technologies, especially CRISPR/Cas9 which is characterized by convenient handling and high precision, revolutionized the field of genome editing. Such tools have enormous potential for basic science as well as for regenerative medicine. Nevertheless, there are still several hurdles that have to be overcome, but patient-tailored therapies, termed precision medicine, seem to be within reach. In this review, we focus on the achievements and limitations of genome editing in the cardiovascular field. We explore different areas of cardiac research and highlight the most important developments: (1) the potential of genome editing in human pluripotent stem cells in basic research for disease modelling, drug screening, or reprogramming approaches and (2) the potential and remaining challenges of genome editing for regenerative therapies. Finally, we discuss social and ethical implications of these new technologies. PMID:29731778

The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

PubMed Central

SHAO, Ming; XU, Tian-Rui; CHEN, Ce-Shi

2016-01-01

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and biomedicine. PMID:27469250

The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

PubMed

Shao, Ming; Xu, Tian-Rui; Chen, Ce-Shi

2016-07-18

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.

PubMed

Yajima, Misako; Ikuta, Kazufumi; Kanda, Teru

2018-04-03

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

PubMed Central

Ikuta, Kazufumi; Kanda, Teru

2018-01-01

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically. PMID:29614006

Functional genomics of lactic acid bacteria: from food to health

PubMed Central

2014-01-01

Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health. PMID:25186768

Functional genomics of lactic acid bacteria: from food to health.

PubMed

Douillard, François P; de Vos, Willem M

2014-08-29

Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.

Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research

DOE PAGES

Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.; ...

2016-02-18

Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. Here, this article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation ofmore » gene expression, as well as identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.« less

Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.

Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. Here, this article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation ofmore » gene expression, as well as identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.« less

Pathways to Genome-targeted Therapies in Serous Ovarian Cancer.

PubMed

Axelrod, Joshua; Delaney, Joe

2017-07-01

Genome sequencing technologies and corresponding oncology publications have generated enormous publicly available datasets for many cancer types. While this has enabled new treatments, and in some limited cases lifetime management of the disease, the treatment options for serous ovarian cancer remain dismal. This review summarizes recent advances in our understanding of ovarian cancer, with a focus on heterogeneity, functional genomics, and actionable data.

Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

PubMed

Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

2016-02-01

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. © FASEB.

Functional annotation of rare gene aberration drivers of pancreatic cancer | Office of Cancer Genomics

Cancer.gov

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC).

Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python)

PubMed Central

Rutllant, Josep

2016-01-01

Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value. PMID:27200191

Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python).

PubMed

Irizarry, Kristopher J L; Rutllant, Josep

2016-01-01

Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value.

Proteomics in the genome engineering era.

PubMed

Vandemoortele, Giel; Gevaert, Kris; Eyckerman, Sven

2016-01-01

Genome engineering experiments used to be lengthy, inefficient, and often expensive, preventing a widespread adoption of such experiments for the full assessment of endogenous protein functions. With the revolutionary clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, genome engineering became accessible to the broad life sciences community and is now implemented in several research areas. One particular field that can benefit significantly from this evolution is proteomics where a substantial impact on experimental design and general proteome biology can be expected. In this review, we describe the main applications of genome engineering in proteomics, including the use of engineered disease models and endogenous epitope tagging. In addition, we provide an overview on current literature and highlight important considerations when launching genome engineering technologies in proteomics workflows. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points.

PubMed

DeVilbiss, Andrew W; Sanalkumar, Rajendran; Johnson, Kirby D; Keles, Sunduz; Bresnick, Emery H

2014-08-01

Hematopoiesis is an exquisitely regulated process in which stem cells in the developing embryo and the adult generate progenitor cells that give rise to all blood lineages. Master regulatory transcription factors control hematopoiesis by integrating signals from the microenvironment and dynamically establishing and maintaining genetic networks. One of the most rudimentary aspects of cell type-specific transcription factor function, how they occupy a highly restricted cohort of cis-elements in chromatin, remains poorly understood. Transformative technologic advances involving the coupling of next-generation DNA sequencing technology with the chromatin immunoprecipitation assay (ChIP-seq) have enabled genome-wide mapping of factor occupancy patterns. However, formidable problems remain; notably, ChIP-seq analysis yields hundreds to thousands of chromatin sites occupied by a given transcription factor, and only a fraction of the sites appear to be endowed with critical, non-redundant function. It has become en vogue to map transcription factor occupancy patterns genome-wide, while using powerful statistical tools to establish correlations to inform biology and mechanisms. With the advent of revolutionary genome editing technologies, one can now reach beyond correlations to conduct definitive hypothesis testing. This review focuses on key discoveries that have emerged during the path from single loci to genome-wide analyses, specifically in the context of hematopoietic transcriptional mechanisms. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Next-generation mammalian genetics toward organism-level systems biology.

PubMed

Susaki, Etsuo A; Ukai, Hideki; Ueda, Hiroki R

2017-01-01

Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.

Genome editing systems in novel therapies.

PubMed

Jang, Yoon-Young; Cai, Liuhong; Ye, Zhaohui

2016-01-01

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

The Human Genome Initiative of the Department of Energy

DOE R&D Accomplishments Database

1988-01-01

The structural characterization of genes and elucidation of their encoded functions have become a cornerstone of modern health research, biology and biotechnology. A genome program is an organized effort to locate and identify the functions of all the genes of an organism. Beginning with the DOE-sponsored, 1986 human genome workshop at Santa Fe, the value of broadly organized efforts supporting total genome characterization became a subject of intensive study. There is now national recognition that benefits will rapidly accrue from an effective scientific infrastructure for total genome research. In the US genome research is now receiving dedicated funds. Several other nations are implementing genome programs. Supportive infrastructure is being improved through both national and international cooperation. The Human Genome Initiative of the Department of Energy (DOE) is a focused program of Resource and Technology Development, with objectives of speeding and bringing economies to the national human genome effort. This report relates the origins and progress of the Initiative.

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

PubMed Central

Abdurakhmonov, Ibrokhim Y.; Ayubov, Mirzakamol S.; Ubaydullaeva, Khurshida A.; Buriev, Zabardast T.; Shermatov, Shukhrat E.; Ruziboev, Haydarali S.; Shapulatov, Umid M.; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z.; Percy, Richard G.; Devor, Eric J.; Sharma, Govind C.; Sripathi, Venkateswara R.; Kumpatla, Siva P.; van der Krol, Alexander; Kater, Hake D.; Khamidov, Khakimdjan; Salikhov, Shavkat I.; Jenkins, Johnie N.; Abdukarimov, Abdusattor; Pepper, Alan E.

2016-01-01

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization. PMID:26941765

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.).

PubMed

Abdurakhmonov, Ibrokhim Y; Ayubov, Mirzakamol S; Ubaydullaeva, Khurshida A; Buriev, Zabardast T; Shermatov, Shukhrat E; Ruziboev, Haydarali S; Shapulatov, Umid M; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z; Percy, Richard G; Devor, Eric J; Sharma, Govind C; Sripathi, Venkateswara R; Kumpatla, Siva P; van der Krol, Alexander; Kater, Hake D; Khamidov, Khakimdjan; Salikhov, Shavkat I; Jenkins, Johnie N; Abdukarimov, Abdusattor; Pepper, Alan E

2016-01-01

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.

Genome-wide prediction of cis-regulatory regions using supervised deep learning methods.

PubMed

Li, Yifeng; Shi, Wenqiang; Wasserman, Wyeth W

2018-05-31

In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions which precisely control the expression of genes. Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide. Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide (6.8% of the genome, of which 40,000 are supported by bidirectional transcription data), and 26,000 candidate promoters (0.6% of the genome). The predicted annotations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.

Reprogramming neurodegeneration in the big data era.

PubMed

Zhou, Lujia; Verstreken, Patrik

2018-02-01

Recent genome-wide association studies (GWAS) have identified numerous genetic risk variants for late-onset Alzheimer's disease (AD) and Parkinson's disease (PD). However, deciphering the functional consequences of GWAS data is challenging due to a lack of reliable model systems to study the genetic variants that are often of low penetrance and non-coding identities. Pluripotent stem cell (PSC) technologies offer unprecedented opportunities for molecular phenotyping of GWAS variants in human neurons and microglia. Moreover, rapid technological advances in whole-genome RNA-sequencing and epigenome mapping fuel comprehensive and unbiased investigations of molecular alterations in PSC-derived disease models. Here, we review and discuss how integrated studies that utilize PSC technologies and genome-wide approaches may bring new mechanistic insight into the pathogenesis of AD and PD. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptome characterization for genome annotation and functional genomics in Theobroma cacao

USDA-ARS?s Scientific Manuscript database

Evidence from leaf transcriptome sequencing using two technology platforms, in combination with protein homology and trained ab initio predictions, previously enabled us to build 35,000 gene models in T. cacao (www.cacaogenomedb.org). Here we review the contribution of each data type to cacao gene a...

Breeding-assisted genomics.

PubMed

Poland, Jesse

2015-04-01

The revolution of inexpensive sequencing has ushered in an unprecedented age of genomics. The promise of using this technology to accelerate plant breeding is being realized with a vision of genomics-assisted breeding that will lead to rapid genetic gain for expensive and difficult traits. The reality is now that robust phenotypic data is an increasing limiting resource to complement the current wealth of genomic information. While genomics has been hailed as the discipline to fundamentally change the scope of plant breeding, a more symbiotic relationship is likely to emerge. In the context of developing and evaluating large populations needed for functional genomics, none excel in this area more than plant breeders. While genetic studies have long relied on dedicated, well-structured populations, the resources dedicated to these populations in the context of readily available, inexpensive genotyping is making this philosophy less tractable relative to directly focusing functional genomics on material in breeding programs. Through shifting effort for basic genomic studies from dedicated structured populations, to capturing the entire scope of genetic determinants in breeding lines, we can move towards not only furthering our understanding of functional genomics in plants, but also rapidly improving crops for increased food security, availability and nutrition. Copyright © 2015 Elsevier Ltd. All rights reserved.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

PubMed

Lowder, Levi G; Zhang, Dengwei; Baltes, Nicholas J; Paul, Joseph W; Tang, Xu; Zheng, Xuelian; Voytas, Daniel F; Hsieh, Tzung-Fu; Zhang, Yong; Qi, Yiping

2015-10-01

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research. © 2015 American Society of Plant Biologists. All Rights Reserved.

Synthetic Genomics and Synthetic Biology Applications Between Hopes and Concerns

PubMed Central

König, Harald; Frank, Daniel; Heil, Reinhard; Coenen, Christopher

2013-01-01

New organisms and biological systems designed to satisfy human needs are among the aims of synthetic genomics and synthetic biology. Synthetic biology seeks to model and construct biological components, functions and organisms that do not exist in nature or to redesign existing biological systems to perform new functions. Synthetic genomics, on the other hand, encompasses technologies for the generation of chemically-synthesized whole genomes or larger parts of genomes, allowing to simultaneously engineer a myriad of changes to the genetic material of organisms. Engineering complex functions or new organisms in synthetic biology are thus progressively becoming dependent on and converging with synthetic genomics. While applications from both areas have been predicted to offer great benefits by making possible new drugs, renewable chemicals or clean energy, they have also given rise to concerns about new safety, environmental and socio-economic risks – stirring an increasingly polarizing debate. Here we intend to provide an overview on recent progress in biomedical and biotechnological applications of synthetic genomics and synthetic biology as well as on arguments and evidence related to their possible benefits, risks and governance implications. PMID:23997647

The interface of genomic technologies and nursing.

PubMed

Loescher, Lois J; Merkle, Carrie J

2005-01-01

(a) to summarize views of the interface of technology, genomic technology, and nursing; (b) provide an overview of current and emerging genomic technologies; (c) present clinical exemplars of uses of genomic technology in two disease conditions; and (d) list genomic-focused nursing research on genomic technologies. A discussion of genomic technology in the context of nurses' views of technology, the importance of genomic technology for nurses, linking the central dogma of molecular biology to state-of-the-art tests and assays, and nurses' current use of technologies. Human genome discoveries will continue to be an integral part of disease prevention, diagnosis, treatment, and management. These discoveries also have the potential for being integrated into nursing science. Genomic technologies are becoming a driving force in patient management, so that nurses will be unable to provide quality care without knowledge of the types of genomic technologies, the rationale for their use, and the possible sequelae that can result from genetic diagnosis or treatment. Many nurses already are using genomic technologies to conduct genomic-focused nursing research. The biobehavioral nature of much of this research further indicates the important contributions of nurses in genomics.

Genome projects and the functional-genomic era.

PubMed

Sauer, Sascha; Konthur, Zoltán; Lehrach, Hans

2005-12-01

The problems we face today in public health as a result of the -- fortunately -- increasing age of people and the requirements of developing countries create an urgent need for new and innovative approaches in medicine and in agronomics. Genomic and functional genomic approaches have a great potential to at least partially solve these problems in the future. Important progress has been made by procedures to decode genomic information of humans, but also of other key organisms. The basic comprehension of genomic information (and its transfer) should now give us the possibility to pursue the next important step in life science eventually leading to a basic understanding of biological information flow; the elucidation of the function of all genes and correlative products encoded in the genome, as well as the discovery of their interactions in a molecular context and the response to environmental factors. As a result of the sequencing projects, we are now able to ask important questions about sequence variation and can start to comprehensively study the function of expressed genes on different levels such as RNA, protein or the cell in a systematic context including underlying networks. In this article we review and comment on current trends in large-scale systematic biological research. A particular emphasis is put on technology developments that can provide means to accomplish the tasks of future lines of functional genomics.

Efficient genome editing of differentiated renal epithelial cells.

PubMed

Hofherr, Alexis; Busch, Tilman; Huber, Nora; Nold, Andreas; Bohn, Albert; Viau, Amandine; Bienaimé, Frank; Kuehn, E Wolfgang; Arnold, Sebastian J; Köttgen, Michael

2017-02-01

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

Molecular Neuroanatomy: A Generation of Progress

PubMed Central

Pollock, Jonathan D.; Wu, Da-Yu; Satterlee, John

2014-01-01

The neuroscience research landscape has changed dramatically over the past decade. An impressive array of neuroscience tools and technologies have been generated, including brain gene expression atlases, genetically encoded proteins to monitor and manipulate neuronal activity and function, cost effective genome sequencing, new technologies enabling genome manipulation, new imaging methods and new tools for mapping neuronal circuits. However, despite these technological advances, several significant scientific challenges must be overcome in the coming decade to enable a better understanding of brain function and to develop next generation cell type-targeted therapeutics to treat brain disorders. For example, we do not have an inventory of the different types of cells that exist in the brain, nor do we know how to molecularly phenotype them. We also lack robust technologies to map connections between cells. This review will provide an overview of some of the tools and technologies neuroscientists are currently using to move the field of molecular neuroanatomy forward and also discuss emerging technologies that may enable neuroscientists to address these critical scientific challenges over the coming decade. PMID:24388609

CMS: A Web-Based System for Visualization and Analysis of Genome-Wide Methylation Data of Human Cancers

PubMed Central

Huang, Yi-Wen; Roa, Juan C.; Goodfellow, Paul J.; Kizer, E. Lynette; Huang, Tim H. M.; Chen, Yidong

2013-01-01

Background DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters. Methodology/Principal Findings Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework. Conclusions/Significance CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible at: http://cbbiweb.uthscsa.edu/KMethylomes/. PMID:23630576

CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.

PubMed

Gu, Fei; Doderer, Mark S; Huang, Yi-Wen; Roa, Juan C; Goodfellow, Paul J; Kizer, E Lynette; Huang, Tim H M; Chen, Yidong

2013-01-01

DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters. Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework. CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible at: http://cbbiweb.uthscsa.edu/KMethylomes/.

[Advances in CRISPR-Cas-mediated genome editing system in plants].

PubMed

Wang, Chun; Wang, Kejian

2017-10-25

Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

Complete Genome Sequence of the Probiotic Strain Lactobacillus salivarius LPM01

PubMed Central

Codoñer, Francisco M.; Martinez-Blanch, Juan F.; Acevedo-Piérart, Marcelo; Ormeño, M. Loreto; Ramón, Daniel

2016-01-01

Lactobacillus salivarius LPM01 (DSM 22150) is a probiotic strain able to improve health status in immunocompromised people. Here, we report its complete genome sequence deciphered by PacBio single-molecule real-time (SMRT) technology. Analysis of the sequence may provide insights into its functional activity and safety assessment. PMID:27881545

Web Apollo: a web-based genomic annotation editing platform.

PubMed

Lee, Eduardo; Helt, Gregg A; Reese, Justin T; Munoz-Torres, Monica C; Childers, Chris P; Buels, Robert M; Stein, Lincoln; Holmes, Ian H; Elsik, Christine G; Lewis, Suzanna E

2013-08-30

Web Apollo is the first instantaneous, collaborative genomic annotation editor available on the web. One of the natural consequences following from current advances in sequencing technology is that there are more and more researchers sequencing new genomes. These researchers require tools to describe the functional features of their newly sequenced genomes. With Web Apollo researchers can use any of the common browsers (for example, Chrome or Firefox) to jointly analyze and precisely describe the features of a genome in real time, whether they are in the same room or working from opposite sides of the world.

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

PubMed

Xiong, Xin; Chen, Meng; Lim, Wendell A; Zhao, Dehua; Qi, Lei S

2016-08-31

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

Web Apollo: a web-based genomic annotation editing platform

PubMed Central

2013-01-01

Web Apollo is the first instantaneous, collaborative genomic annotation editor available on the web. One of the natural consequences following from current advances in sequencing technology is that there are more and more researchers sequencing new genomes. These researchers require tools to describe the functional features of their newly sequenced genomes. With Web Apollo researchers can use any of the common browsers (for example, Chrome or Firefox) to jointly analyze and precisely describe the features of a genome in real time, whether they are in the same room or working from opposite sides of the world. PMID:24000942

University of Texas MD Anderson Cancer Center: High-Throughput Screening Identifying Driving Mutations in Endometrial Cancer | Office of Cancer Genomics

Cancer.gov

Recent advances in next-generation sequencing technology have enabled the unprecedented characterization of a full spectrum of somatic alterations in cancer genomes. Given the large numbers of somatic mutations typically detected by this approach, a key challenge in the downstream analysis is to distinguish “drivers” that functionally contribute to tumorigenesis from “passengers” that occur as the consequence of genomic instability.

Ascribing Functions to Genes: Journey Towards Genetic Improvement of Rice Via Functional Genomics

PubMed Central

Mustafiz, Ananda; Kumari, Sumita; Karan, Ratna

2016-01-01

Rice, one of the most important cereal crops for mankind, feeds more than half the world population. Rice has been heralded as a model cereal owing to its small genome size, amenability to easy transformation, high synteny to other cereal crops and availability of complete genome sequence. Moreover, sequence wealth in rice is getting more refined and precise due to resequencing efforts. This humungous resource of sequence data has confronted research fraternity with a herculean challenge as well as an excellent opportunity to functionally validate expressed as well as regulatory portions of the genome. This will not only help us in understanding the genetic basis of plant architecture and physiology but would also steer us towards developing improved cultivars. No single technique can achieve such a mammoth task. Functional genomics through its diverse tools viz. loss and gain of function mutants, multifarious omics strategies like transcriptomics, proteomics, metabolomics and phenomics provide us with the necessary handle. A paradigm shift in technological advances in functional genomics strategies has been instrumental in generating considerable amount of information w.r.t functionality of rice genome. We now have several databases and online resources for functionally validated genes but despite that we are far from reaching the desired milestone of functionally characterizing each and every rice gene. There is an urgent need for a common platform, for information already available in rice, and collaborative efforts between researchers in a concerted manner as well as healthy public-private partnership, for genetic improvement of rice crop better able to handle the pressures of climate change and exponentially increasing population. PMID:27252584

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

PubMed

Moll, Karen M; Zhou, Peng; Ramaraj, Thiruvarangan; Fajardo, Diego; Devitt, Nicholas P; Sadowsky, Michael J; Stupar, Robert M; Tiffin, Peter; Miller, Jason R; Young, Nevin D; Silverstein, Kevin A T; Mudge, Joann

2017-08-04

Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

From data to function: functional modeling of poultry genomics data.

PubMed

McCarthy, F M; Lyons, E

2013-09-01

One of the challenges of functional genomics is to create a better understanding of the biological system being studied so that the data produced are leveraged to provide gains for agriculture, human health, and the environment. Functional modeling enables researchers to make sense of these data as it reframes a long list of genes or gene products (mRNA, ncRNA, and proteins) by grouping based upon function, be it individual molecular functions or interactions between these molecules or broader biological processes, including metabolic and signaling pathways. However, poultry researchers have been hampered by a lack of functional annotation data, tools, and training to use these data and tools. Moreover, this lack is becoming more critical as new sequencing technologies enable us to generate data not only for an increasingly diverse range of species but also individual genomes and populations of individuals. We discuss the impact of these new sequencing technologies on poultry research, with a specific focus on what functional modeling resources are available for poultry researchers. We also describe key strategies for researchers who wish to functionally model their own data, providing background information about functional modeling approaches, the data and tools to support these approaches, and the strengths and limitations of each. Specifically, we describe methods for functional analysis using Gene Ontology (GO) functional summaries, functional enrichment analysis, and pathways and network modeling. As annotation efforts begin to provide the fundamental data that underpin poultry functional modeling (such as improved gene identification, standardized gene nomenclature, temporal and spatial expression data and gene product function), tool developers are incorporating these data into new and existing tools that are used for functional modeling, and cyberinfrastructure is being developed to provide the necessary extendibility and scalability for storing and analyzing these data. This process will support the efforts of poultry researchers to make sense of their functional genomics data sets, and we provide here a starting point for researchers who wish to take advantage of these tools.

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

New Funding Opportunity - Illuminating the Druggable Genome | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Institutes of Health Common Fund announces two new Funding Opportunity Announcements with a focus on the Illuminating the Druggable Genome (IDG). These funding opportunities are designed to foster the development of technologies and information management to facilitate the unveiling of the functions of the poorly characterized and/or un-annotated members in four protein classes of the Druggable Genome. The IDG project is predicated on the need to fully explore the underlying biology and role in disease of genes linked to already drugged genes within the Druggable Genome.

Improved maize reference genome with single-molecule technologies.

PubMed

Jiao, Yinping; Peluso, Paul; Shi, Jinghua; Liang, Tiffany; Stitzer, Michelle C; Wang, Bo; Campbell, Michael S; Stein, Joshua C; Wei, Xuehong; Chin, Chen-Shan; Guill, Katherine; Regulski, Michael; Kumari, Sunita; Olson, Andrew; Gent, Jonathan; Schneider, Kevin L; Wolfgruber, Thomas K; May, Michael R; Springer, Nathan M; Antoniou, Eric; McCombie, W Richard; Presting, Gernot G; McMullen, Michael; Ross-Ibarra, Jeffrey; Dawe, R Kelly; Hastie, Alex; Rank, David R; Ware, Doreen

2017-06-22

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Leptospiral Pathogenomics

PubMed Central

Lehmann, Jason S.; Matthias, Michael A.; Vinetz, Joseph M.; Fouts, Derrick E.

2014-01-01

Leptospirosis, caused by pathogenic spirochetes belonging to the genus Leptospira, is a zoonosis with important impacts on human and animal health worldwide. Research on the mechanisms of Leptospira pathogenesis has been hindered due to slow growth of infectious strains, poor transformability, and a paucity of genetic tools. As a result of second generation sequencing technologies, there has been an acceleration of leptospiral genome sequencing efforts in the past decade, which has enabled a concomitant increase in functional genomics analyses of Leptospira pathogenesis. A pathogenomics approach, by coupling of pan-genomic analysis of multiple isolates with sequencing of experimentally attenuated highly pathogenic Leptospira, has resulted in the functional inference of virulence factors. The global Leptospira Genome Project supported by the U.S. National Institute of Allergy and Infectious Diseases to which key scientific contributions have been made from the international leptospirosis research community has provided a new roadmap for comprehensive studies of Leptospira and leptospirosis well into the future. This review describes functional genomics approaches to apply the data generated by the Leptospira Genome Project towards deepening our knowledge of virulence factors of Leptospira using the emerging discipline of pathogenomics. PMID:25437801

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

PubMed

Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

2016-01-01

The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. © The Author(s) 2016. Published by Oxford University Press.

Construction of Pará rubber tree genome and multi-transcriptome database accelerates rubber researches.

PubMed

Makita, Yuko; Kawashima, Mika; Lau, Nyok Sean; Othman, Ahmad Sofiman; Matsui, Minami

2018-01-19

Natural rubber is an economically important material. Currently the Pará rubber tree, Hevea brasiliensis is the main commercial source. Little is known about rubber biosynthesis at the molecular level. Next-generation sequencing (NGS) technologies brought draft genomes of three rubber cultivars and a variety of RNA sequencing (RNA-seq) data. However, no current genome or transcriptome databases (DB) are organized by gene. A gene-oriented database is a valuable support for rubber research. Based on our original draft genome sequence of H. brasiliensis RRIM600, we constructed a rubber tree genome and transcriptome DB. Our DB provides genome information including gene functional annotations and multi-transcriptome data of RNA-seq, full-length cDNAs including PacBio Isoform sequencing (Iso-Seq), ESTs and genome wide transcription start sites (TSSs) derived from CAGE technology. Using our original and publically available RNA-seq data, we calculated co-expressed genes for identifying functionally related gene sets and/or genes regulated by the same transcription factor (TF). Users can access multi-transcriptome data through both a gene-oriented web page and a genome browser. For the gene searching system, we provide keyword search, sequence homology search and gene expression search; users can also select their expression threshold easily. The rubber genome and transcriptome DB provides rubber tree genome sequence and multi-transcriptomics data. This DB is useful for comprehensive understanding of the rubber transcriptome. This will assist both industrial and academic researchers for rubber and economically important close relatives such as R. communis, M. esculenta and J. curcas. The Rubber Transcriptome DB release 2017.03 is accessible at http://matsui-lab.riken.jp/rubber/ .

High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

PubMed

Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

2015-01-27

Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. Copyright © 2015 Zhou et al.

High-Throughput Metagenomic Technologies for Complex Microbial Community Analysis: Open and Closed Formats

PubMed Central

He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

2015-01-01

ABSTRACT   Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. PMID:25626903

High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

DOE PAGES

Zhou, Jizhong; He, Zhili; Yang, Yunfeng; ...

2015-01-27

Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications andmore » focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.« less

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform | Office of Cancer Genomics

Cancer.gov

The vast datasets generated by next generation gene sequencing and expression profiling have transformed biological and translational research. However, technologies to produce large-scale functional genomics datasets, such as high-throughput detection of protein-protein interactions (PPIs), are still in early development. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform featured with both high sensitivity and robustness in a mammalian cell environment remains to be established.

GenomeRNAi: a database for cell-based RNAi phenotypes.

PubMed

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

GenomeRNAi: a database for cell-based RNAi phenotypes

PubMed Central

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at PMID:17135194

Generation of Knock-in Mouse by Genome Editing.

PubMed

Fujii, Wataru

2017-01-01

Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-stranded oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.

University of Texas MD Anderson Cancer Center (UT-MDACC): High-Throughput Screening Identifying Driving Mutations in Endometrial Cancer | Office of Cancer Genomics

Cancer.gov

Recent advances in next-generation sequencing technology have enabled the unprecedented characterization of a full spectrum of somatic alterations in cancer genomes. Given the large numbers of somatic mutations typically detected by this approach, a key challenge in the downstream analysis is to distinguish “drivers” that functionally contribute to tumorigenesis from “passengers” that occur as the consequence of genomic instability.

Complete Genome Sequence of the Probiotic Strain Lactobacillus salivarius LPM01.

PubMed

Chenoll, Empar; Codoñer, Francisco M; Martinez-Blanch, Juan F; Acevedo-Piérart, Marcelo; Ormeño, M Loreto; Ramón, Daniel; Genovés, Salvador

2016-11-23

Lactobacillus salivarius LPM01 (DSM 22150) is a probiotic strain able to improve health status in immunocompromised people. Here, we report its complete genome sequence deciphered by PacBio single-molecule real-time (SMRT) technology. Analysis of the sequence may provide insights into its functional activity and safety assessment. Copyright © 2016 Chenoll et al.

Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation.

PubMed

Piao, Hailan; Froula, Jeff; Du, Changbin; Kim, Tae-Wan; Hawley, Erik R; Bauer, Stefan; Wang, Zhong; Ivanova, Nathalia; Clark, Douglas S; Klenk, Hans-Peter; Hess, Matthias

2014-08-01

Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this "genomic dark matter." Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as "genomic dark matter" and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. © 2014 Wiley Periodicals, Inc.

Saccharomyces cerevisiae: gene annotation and genome variability, state of the art through comparative genomics.

PubMed

Louis, Ed

2011-01-01

In the early days of the yeast genome sequencing project, gene annotation was in its infancy and suffered the problem of many false positive annotations as well as missed genes. The lack of other sequences for comparison also prevented the annotation of conserved, functional sequences that were not coding. We are now in an era of comparative genomics where many closely related as well as more distantly related genomes are available for direct sequence and synteny comparisons allowing for more probable predictions of genes and other functional sequences due to conservation. We also have a plethora of functional genomics data which helps inform gene annotation for previously uncharacterised open reading frames (ORFs)/genes. For Saccharomyces cerevisiae this has resulted in a continuous updating of the gene and functional sequence annotations in the reference genome helping it retain its position as the best characterized eukaryotic organism's genome. A single reference genome for a species does not accurately describe the species and this is quite clear in the case of S. cerevisiae where the reference strain is not ideal for brewing or baking due to missing genes. Recent surveys of numerous isolates, from a variety of sources, using a variety of technologies have revealed a great deal of variation amongst isolates with genome sequence surveys providing information on novel genes, undetectable by other means. We now have a better understanding of the extant variation in S. cerevisiae as a species as well as some idea of how much we are missing from this understanding. As with gene annotation, comparative genomics enhances the discovery and description of genome variation and is providing us with the tools for understanding genome evolution, adaptation and selection, and underlying genetics of complex traits.

The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

PubMed Central

Pramstaller, Peter P.; Hicks, Andrew A.; Rossini, Alessandra

2017-01-01

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future. PMID:29434642

Active Transposition in Genomes

PubMed Central

Huang, Cheng Ran Lisa; Burns, Kathleen H.; Boeke, Jef D.

2013-01-01

Transposons are DNA sequences capable of moving in genomes. Early evidence showed their accumulation in many species and suggested their continued activity in at least isolated organisms. In the past decade, with the development of various genomic technologies, it has become abundantly clear that ongoing activity is the rule rather than the exception. Active transposons of various classes are observed throughout plants and animals, including humans. They continue to create new insertions, have an enormous variety of structural and functional impact on genes and genomes, and play important roles in genome evolution. Transposon activities have been identified and measured by employing various strategies. Here, we summarize evidence of current transposon activity in various plant and animal genomes. PMID:23145912

Applications of CRISPR genome editing technology in drug target identification and validation.

PubMed

Lu, Quinn; Livi, George P; Modha, Sundip; Yusa, Kosuke; Macarrón, Ricardo; Dow, David J

2017-06-01

The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems. Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies. Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.

Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function.

PubMed

Zhang, Yaoyao; Tang, Na; Sadigh, Yashar; Baigent, Susan; Shen, Zhiqiang; Nair, Venugopal; Yao, Yongxiu

2018-05-24

Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek's disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Ecological and evolutionary genomics of marine photosynthetic organisms.

PubMed

Coelho, Susana M; Simon, Nathalie; Ahmed, Sophia; Cock, J Mark; Partensky, Frédéric

2013-02-01

Environmental (ecological) genomics aims to understand the genetic basis of relationships between organisms and their abiotic and biotic environments. It is a rapidly progressing field of research largely due to recent advances in the speed and volume of genomic data being produced by next generation sequencing (NGS) technologies. Building on information generated by NGS-based approaches, functional genomic methodologies are being applied to identify and characterize genes and gene systems of both environmental and evolutionary relevance. Marine photosynthetic organisms (MPOs) were poorly represented amongst the early genomic models, but this situation is changing rapidly. Here we provide an overview of the recent advances in the application of ecological genomic approaches to both prokaryotic and eukaryotic MPOs. We describe how these approaches are being used to explore the biology and ecology of marine cyanobacteria and algae, particularly with regard to their functions in a broad range of marine ecosystems. Specifically, we review the ecological and evolutionary insights gained from whole genome and transcriptome sequencing projects applied to MPOs and illustrate how their genomes are yielding information on the specific features of these organisms. © 2012 Blackwell Publishing Ltd.

Repetitive elements dynamics in cell identity programming, maintenance and disease.

PubMed

Bodega, Beatrice; Orlando, Valerio

2014-12-01

The days of 'junk DNA' seem to be over. The rapid progress of genomics technologies has been unveiling unexpected mechanisms by which repetitive DNA and in particular transposable elements (TEs) have evolved, becoming key issues in understanding genome structure and function. Indeed, rather than 'parasites', recent findings strongly suggest that TEs may have a positive function by contributing to tissue specific transcriptional programs, in particular as enhancer-like elements and/or modules for regulation of higher order chromatin structure. Further, it appears that during development and aging genomes experience several waves of TEs activation, and this contributes to individual genome shaping during lifetime. Interestingly, TEs activity is major target of epigenomic regulation. These findings are shedding new light on the genome-phenotype relationship and set the premises to help to explain complex disease manifestation, as consequence of TEs activity deregulation. Copyright © 2014. Published by Elsevier Ltd.

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

PubMed Central

Engel, Krysta L.; Mackiewicz, Mark; Hardigan, Andrew A.; Myers, Richard M.; Savic, Daniel

2016-01-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. PMID:27224938

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation.

PubMed

Engel, Krysta L; Mackiewicz, Mark; Hardigan, Andrew A; Myers, Richard M; Savic, Daniel

2016-09-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.

PubMed

Plaza Reyes, Alvaro; Lanner, Fredrik

2017-01-01

Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof. © 2017. Published by The Company of Biologists Ltd.

A CRISPR toolbox to study virus–host interactions

PubMed Central

Puschnik, Andreas S.; Majzoub, Karim; Ooi, Yaw Shin; Carette, Jan E.

2018-01-01

Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR–Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR–Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR–Cas make it a powerful tool for probing virus–host interactions and for identifying new antiviral targets. PMID:28420884

The Information Technology Infrastructure for the Translational Genomics Core and the Partners Biobank at Partners Personalized Medicine

PubMed Central

Boutin, Natalie; Holzbach, Ana; Mahanta, Lisa; Aldama, Jackie; Cerretani, Xander; Embree, Kevin; Leon, Irene; Rathi, Neeta; Vickers, Matilde

2016-01-01

The Biobank and Translational Genomics core at Partners Personalized Medicine requires robust software and hardware. This Information Technology (IT) infrastructure enables the storage and transfer of large amounts of data, drives efficiencies in the laboratory, maintains data integrity from the time of consent to the time that genomic data is distributed for research, and enables the management of complex genetic data. Here, we describe the functional components of the research IT infrastructure at Partners Personalized Medicine and how they integrate with existing clinical and research systems, review some of the ways in which this IT infrastructure maintains data integrity and security, and discuss some of the challenges inherent to building and maintaining such infrastructure. PMID:26805892

Draft Genome Sequence, and a Sequence-Defined Genetic Linkage Map of the Legume Crop Species Lupinus angustifolius L

PubMed Central

Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W.; Howieson, John G.; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species. PMID:23734219

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W; Howieson, John G; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

PubMed Central

Polstein, Lauren R.; Perez-Pinera, Pablo; Kocak, D. Dewran; Vockley, Christopher M.; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

2015-01-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. PMID:26025803

The Yeast Deletion Collection: A Decade of Functional Genomics

PubMed Central

Giaever, Guri; Nislow, Corey

2014-01-01

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. PMID:24939991

Functional genomics approaches in parasitic helminths.

PubMed

Hagen, J; Lee, E F; Fairlie, W D; Kalinna, B H

2012-01-01

As research on parasitic helminths is moving into the post-genomic era, an enormous effort is directed towards deciphering gene function and to achieve gene annotation. The sequences that are available in public databases undoubtedly hold information that can be utilized for new interventions and control but the exploitation of these resources has until recently remained difficult. Only now, with the emergence of methods to genetically manipulate and transform parasitic worms will it be possible to gain a comprehensive understanding of the molecular mechanisms involved in nutrition, metabolism, developmental switches/maturation and interaction with the host immune system. This review focuses on functional genomics approaches in parasitic helminths that are currently used, to highlight potential applications of these technologies in the areas of cell biology, systems biology and immunobiology of parasitic helminths. © 2011 Blackwell Publishing Ltd.

Genome network medicine: innovation to overcome huge challenges in cancer therapy.

PubMed

Roukos, Dimitrios H

2014-01-01

The post-ENCODE era shapes now a new biomedical research direction for understanding transcriptional and signaling networks driving gene expression and core cellular processes such as cell fate, survival, and apoptosis. Over the past half century, the Francis Crick 'central dogma' of single n gene/protein-phenotype (trait/disease) has defined biology, human physiology, disease, diagnostics, and drugs discovery. However, the ENCODE project and several other genomic studies using high-throughput sequencing technologies, computational strategies, and imaging techniques to visualize regulatory networks, provide evidence that transcriptional process and gene expression are regulated by highly complex dynamic molecular and signaling networks. This Focus article describes the linear experimentation-based limitations of diagnostics and therapeutics to cure advanced cancer and the need to move on from reductionist to network-based approaches. With evident a wide genomic heterogeneity, the power and challenges of next-generation sequencing (NGS) technologies to identify a patient's personal mutational landscape for tailoring the best target drugs in the individual patient are discussed. However, the available drugs are not capable of targeting aberrant signaling networks and research on functional transcriptional heterogeneity and functional genome organization is poorly understood. Therefore, the future clinical genome network medicine aiming at overcoming multiple problems in the new fields of regulatory DNA mapping, noncoding RNA, enhancer RNAs, and dynamic complexity of transcriptional circuitry are also discussed expecting in new innovation technology and strong appreciation of clinical data and evidence-based medicine. The problematic and potential solutions in the discovery of next-generation, molecular, and signaling circuitry-based biomarkers and drugs are explored. © 2013 Wiley Periodicals, Inc.

Oncogenomics and the development of new cancer therapies.

PubMed

Strausberg, Robert L; Simpson, Andrew J G; Old, Lloyd J; Riggins, Gregory J

2004-05-27

Scientists have sequenced the human genome and identified most of its genes. Now it is time to use these genomic data, and the high-throughput technology developed to generate them, to tackle major health problems such as cancer. To accelerate our understanding of this disease and to produce targeted therapies, further basic mutational and functional genomic information is required. A systematic and coordinated approach, with the results freely available, should speed up progress. This will best be accomplished through an international academic and pharmaceutical oncogenomics initiative.

Epigenetics: the language of the cell?

PubMed

Huang, Biao; Jiang, Cizhong; Zhang, Rongxin

2014-02-01

Epigenetics is one of the most rapidly developing fields of biological research. Breakthroughs in several technologies have enabled the possibility of genome-wide epigenetic research, for example the mapping of human genome-wide DNA methylation. In addition, with the development of various high-throughput and high-resolution sequencing technologies, a large number of functional noncoding RNAs have been identified. Massive studies indicated that these functional ncRNA also play an important role in epigenetics. In this review, we gain inspiration from the recent proposal of the ceRNAs hypothesis. This hypothesis proposes that miRNAs act as a language of communication. Accordingly, we further deduce that all of epigenetics may functionally acquire such a unique language characteristic. In summary, various epigenetic markers may not only participate in regulating cellular processes, but they may also act as the intracellular 'language' of communication and are involved in extensive information exchanges within cell.

The Human Microbiome: Our Second Genome*

PubMed Central

Grice, Elizabeth A.; Segre, Julia A.

2012-01-01

The human genome has been referred to as the blueprint of human biology. In this review we consider an essential but largely ignored overlay to that blueprint, the human microbiome, which is composed of those microbes that live in and on our bodies. The human microbiome is a source of genetic diversity, a modifier of disease, an essential component of immunity, and a functional entity that influences metabolism and modulates drug interactions. Characterization and analysis of the human microbiome have been greatly catalyzed by advances in genomic technologies. We discuss how these technologies have shaped this emerging field of study and advanced our understanding of the human microbiome. We also identify future challenges, many of which are common to human genetic studies, and predict that in the future, analyzing genetic variation and risk of human disease will sometimes necessitate the integration of human and microbial genomic data sets. PMID:22703178

Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

PubMed

Aida, Tomomi; Imahashi, Risa; Tanaka, Kohichi

2014-01-01

Gene-targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time-consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra-rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene-targeted mice by in vivo genome editing. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Cancer systems biology in the genome sequencing era: part 1, dissecting and modeling of tumor clones and their networks.

PubMed

Wang, Edwin; Zou, Jinfeng; Zaman, Naif; Beitel, Lenore K; Trifiro, Mark; Paliouras, Miltiadis

2013-08-01

Recent tumor genome sequencing confirmed that one tumor often consists of multiple cell subpopulations (clones) which bear different, but related, genetic profiles such as mutation and copy number variation profiles. Thus far, one tumor has been viewed as a whole entity in cancer functional studies. With the advances of genome sequencing and computational analysis, we are able to quantify and computationally dissect clones from tumors, and then conduct clone-based analysis. Emerging technologies such as single-cell genome sequencing and RNA-Seq could profile tumor clones. Thus, we should reconsider how to conduct cancer systems biology studies in the genome sequencing era. We will outline new directions for conducting cancer systems biology by considering that genome sequencing technology can be used for dissecting, quantifying and genetically characterizing clones from tumors. Topics discussed in Part 1 of this review include computationally quantifying of tumor subpopulations; clone-based network modeling, cancer hallmark-based networks and their high-order rewiring principles and the principles of cell survival networks of fast-growing clones. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

[Tale nucleases--new tool for genome editing].

PubMed

Glazkova, D V; Shipulin, G A

2014-01-01

The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

Complete Genome Sequence of Lactobacillus rhamnosus Strain BPL5 (CECT 8800), a Probiotic for Treatment of Bacterial Vaginosis.

PubMed

Chenoll, Empar; Codoñer, Francisco M; Martinez-Blanch, Juan F; Ramón, Daniel; Genovés, Salvador; Menabrito, Marco

2016-04-21

ITALIC! Lactobacillus rhamnosusBPL5 (CECT 8800), is a probiotic strain suitable for the treatment of bacterial vaginosis. Here, we report its complete genome sequence deciphered by PacBio single-molecule real-time (SMRT) technology. Analysis of the sequence may provide insight into its functional activity. Copyright © 2016 Chenoll et al.

NCBI GEO: archive for functional genomics data sets--10 years on.

PubMed

Barrett, Tanya; Troup, Dennis B; Wilhite, Stephen E; Ledoux, Pierre; Evangelista, Carlos; Kim, Irene F; Tomashevsky, Maxim; Marshall, Kimberly A; Phillippy, Katherine H; Sherman, Patti M; Muertter, Rolf N; Holko, Michelle; Ayanbule, Oluwabukunmi; Yefanov, Andrey; Soboleva, Alexandra

2011-01-01

A decade ago, the Gene Expression Omnibus (GEO) database was established at the National Center for Biotechnology Information (NCBI). The original objective of GEO was to serve as a public repository for high-throughput gene expression data generated mostly by microarray technology. However, the research community quickly applied microarrays to non-gene-expression studies, including examination of genome copy number variation and genome-wide profiling of DNA-binding proteins. Because the GEO database was designed with a flexible structure, it was possible to quickly adapt the repository to store these data types. More recently, as the microarray community switches to next-generation sequencing technologies, GEO has again adapted to host these data sets. Today, GEO stores over 20,000 microarray- and sequence-based functional genomics studies, and continues to handle the majority of direct high-throughput data submissions from the research community. Multiple mechanisms are provided to help users effectively search, browse, download and visualize the data at the level of individual genes or entire studies. This paper describes recent database enhancements, including new search and data representation tools, as well as a brief review of how the community uses GEO data. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/.

Epigenetics and Epigenomics of Plants.

PubMed

Yadav, Chandra Bhan; Pandey, Garima; Muthamilarasan, Mehanathan; Prasad, Manoj

2018-01-23

The genetic material DNA in association with histone proteins forms the complex structure called chromatin, which is prone to undergo modification through certain epigenetic mechanisms including cytosine DNA methylation, histone modifications, and small RNA-mediated methylation. Alterations in chromatin structure lead to inaccessibility of genomic DNA to various regulatory proteins such as transcription factors, which eventually modulates gene expression. Advancements in high-throughput sequencing technologies have provided the opportunity to study the epigenetic mechanisms at genome-wide levels. Epigenomic studies using high-throughput technologies will widen the understanding of mechanisms as well as functions of regulatory pathways in plant genomes, which will further help in manipulating these pathways using genetic and biochemical approaches. This technology could be a potential research tool for displaying the systematic associations of genetic and epigenetic variations, especially in terms of cytosine methylation onto the genomic region in a specific cell or tissue. A comprehensive study of plant populations to correlate genotype to epigenotype and to phenotype, and also the study of methyl quantitative trait loci (QTL) or epiGWAS, is possible by using high-throughput sequencing methods, which will further accelerate molecular breeding programs for crop improvement. Graphical Abstract.

snpGeneSets: An R Package for Genome-Wide Study Annotation

PubMed Central

Mei, Hao; Li, Lianna; Jiang, Fan; Simino, Jeannette; Griswold, Michael; Mosley, Thomas; Liu, Shijian

2016-01-01

Genome-wide studies (GWS) of SNP associations and differential gene expressions have generated abundant results; next-generation sequencing technology has further boosted the number of variants and genes identified. Effective interpretation requires massive annotation and downstream analysis of these genome-wide results, a computationally challenging task. We developed the snpGeneSets package to simplify annotation and analysis of GWS results. Our package integrates local copies of knowledge bases for SNPs, genes, and gene sets, and implements wrapper functions in the R language to enable transparent access to low-level databases for efficient annotation of large genomic data. The package contains functions that execute three types of annotations: (1) genomic mapping annotation for SNPs and genes and functional annotation for gene sets; (2) bidirectional mapping between SNPs and genes, and genes and gene sets; and (3) calculation of gene effect measures from SNP associations and performance of gene set enrichment analyses to identify functional pathways. We applied snpGeneSets to type 2 diabetes (T2D) results from the NHGRI genome-wide association study (GWAS) catalog, a Finnish GWAS, and a genome-wide expression study (GWES). These studies demonstrate the usefulness of snpGeneSets for annotating and performing enrichment analysis of GWS results. The package is open-source, free, and can be downloaded at: https://www.umc.edu/biostats_software/. PMID:27807048

A New Model Army: Emerging fish models to study the genomics of vertebrate Evo-Devo

PubMed Central

Braasch, Ingo; Peterson, Samuel M.; Desvignes, Thomas; McCluskey, Braedan M.; Batzel, Peter; Postlethwait, John H.

2014-01-01

Many fields of biology – including vertebrate Evo-Devo research – are facing an explosion of genomic and transcriptomic sequence information and a multitude of fish species are now swimming in this ‘genomic tsunami’. Here, we first give an overview of recent developments in sequencing fish genomes and transcriptomes that identify properties of fish genomes requiring particular attention and propose strategies to overcome common challenges in fish genomics. We suggest that the generation of chromosome-level genome assemblies - for which we introduce the term ‘chromonome’ – should be a key component of genomic investigations in fish because they enable large-scale conserved synteny analyses that inform orthology detection, a process critical for connectivity of genomes. Orthology calls in vertebrates, especially in teleost fish, are complicated by divergent evolution of gene repertoires and functions following two rounds of genome duplication in the ancestor of vertebrates and a third round at the base of teleost fish. Second, using examples of spotted gar, basal teleosts, zebrafish-related cyprinids, cavefish, livebearers, icefish, and lobefin fish, we illustrate how next generation sequencing technologies liberate emerging fish systems from genomic ignorance and transform them into a new model army to answer longstanding questions on the genomic and developmental basis of their biodiversity. Finally, we discuss recent progress in the genetic toolbox for the major fish models for functional analysis, zebrafish and medaka, that can be transferred to many other fish species to study in vivo the functional effect of evolutionary genomic change as Evo-Devo research enters the postgenomic era. PMID:25111899

The Ever-Evolving Concept of the Gene: The Use of RNA/Protein Experimental Techniques to Understand Genome Functions

PubMed Central

Cipriano, Andrea; Ballarino, Monica

2018-01-01

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as “junk” DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years. PMID:29560353

UCLA's Molecular Screening Shared Resource: enhancing small molecule discovery with functional genomics and new technology.

PubMed

Damoiseaux, Robert

2014-05-01

The Molecular Screening Shared Resource (MSSR) offers a comprehensive range of leading-edge high throughput screening (HTS) services including drug discovery, chemical and functional genomics, and novel methods for nano and environmental toxicology. The MSSR is an open access environment with investigators from UCLA as well as from the entire globe. Industrial clients are equally welcome as are non-profit entities. The MSSR is a fee-for-service entity and does not retain intellectual property. In conjunction with the Center for Environmental Implications of Nanotechnology, the MSSR is unique in its dedicated and ongoing efforts towards high throughput toxicity testing of nanomaterials. In addition, the MSSR engages in technology development eliminating bottlenecks from the HTS workflow and enabling novel assays and readouts currently not available.

Genomic and metagenomic challenges and opportunities for bioleaching: a mini-review.

PubMed

Cárdenas, Juan Pablo; Quatrini, Raquel; Holmes, David S

2016-09-01

High-throughput genomic technologies are accelerating progress in understanding the diversity of microbial life in many environments. Here we highlight advances in genomics and metagenomics of microorganisms from bioleaching heaps and related acidic mining environments. Bioleaching heaps used for copper recovery provide significant opportunities to study the processes and mechanisms underlying microbial successions and the influence of community composition on ecosystem functioning. Obtaining quantitative and process-level knowledge of these dynamics is pivotal for understanding how microorganisms contribute to the solubilization of copper for industrial recovery. Advances in DNA sequencing technology provide unprecedented opportunities to obtain information about the genomes of bioleaching microorganisms, allowing predictive models of metabolic potential and ecosystem-level interactions to be constructed. These approaches are enabling predictive phenotyping of organisms many of which are recalcitrant to genetic approaches or are unculturable. This mini-review describes current bioleaching genomic and metagenomic projects and addresses the use of genome information to: (i) build metabolic models; (ii) predict microbial interactions; (iii) estimate genetic diversity; and (iv) study microbial evolution. Key challenges and perspectives of bioleaching genomics/metagenomics are addressed. Copyright © 2016 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.

Butterfly genomics eclosing.

PubMed

Beldade, P; McMillan, W O; Papanicolaou, A

2008-02-01

Technological and conceptual advances of the last decade have led to an explosion of genomic data and the emergence of new research avenues. Evolutionary and ecological functional genomics, with its focus on the genes that affect ecological success and adaptation in natural populations, benefits immensely from a phylogenetically widespread sampling of biological patterns and processes. Among those organisms outside established model systems, butterflies offer exceptional opportunities for multidisciplinary research on the processes generating and maintaining variation in ecologically relevant traits. Here we highlight research on wing color pattern variation in two groups of Nymphalid butterflies, the African species Bicyclus anynana (subfamily Satyrinae) and species of the South American genus Heliconius (subfamily Heliconiinae), which are emerging as important systems for studying the nature and origins of functional diversity. Growing genomic resources including genomic and cDNA libraries, dense genetic maps, high-density gene arrays, and genetic transformation techniques are extending current gene mapping and expression profiling analysis and enabling the next generation of research questions linking genes, development, form, and fitness. Efforts to develop such resources in Bicyclus and Heliconius underscore the general challenges facing the larger research community and highlight the need for a community-wide effort to extend ongoing functional genomic research on butterflies.

LINE-1 Elements in Structural Variation and Disease

PubMed Central

Beck, Christine R.; Garcia-Perez, José Luis; Badge, Richard M.; Moran, John V.

2014-01-01

The completion of the human genome reference sequence ushered in a new era for the study and discovery of human transposable elements. It now is undeniable that transposable elements, historically dismissed as junk DNA, have had an instrumental role in sculpting the structure and function of our genomes. In particular, long interspersed element-1 (LINE-1 or L1) and short interspersed elements (SINEs) continue to affect our genome, and their movement can lead to sporadic cases of disease. Here, we briefly review the types of transposable elements present in the human genome and their mechanisms of mobility. We next highlight how advances in DNA sequencing and genomic technologies have enabled the discovery of novel retrotransposons in individual genomes. Finally, we discuss how L1-mediated retrotransposition events impact human genomes. PMID:21801021

[Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

PubMed

Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

2015-11-01

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

ISOL@: an Italian SOLAnaceae genomics resource.

PubMed

Chiusano, Maria Luisa; D'Agostino, Nunzio; Traini, Alessandra; Licciardello, Concetta; Raimondo, Enrico; Aversano, Mario; Frusciante, Luigi; Monti, Luigi

2008-03-26

Present-day '-omics' technologies produce overwhelming amounts of data which include genome sequences, information on gene expression (transcripts and proteins) and on cell metabolic status. These data represent multiple aspects of a biological system and need to be investigated as a whole to shed light on the mechanisms which underpin the system functionality. The gathering and convergence of data generated by high-throughput technologies, the effective integration of different data-sources and the analysis of the information content based on comparative approaches are key methods for meaningful biological interpretations. In the frame of the International Solanaceae Genome Project, we propose here ISOLA, an Italian SOLAnaceae genomics resource. ISOLA (available at http://biosrv.cab.unina.it/isola) represents a trial platform and it is conceived as a multi-level computational environment.ISOLA currently consists of two main levels: the genome and the expression level. The cornerstone of the genome level is represented by the Solanum lycopersicum genome draft sequences generated by the International Tomato Genome Sequencing Consortium. Instead, the basic element of the expression level is the transcriptome information from different Solanaceae species, mainly in the form of species-specific comprehensive collections of Expressed Sequence Tags (ESTs). The cross-talk between the genome and the expression levels is based on data source sharing and on tools that enhance data quality, that extract information content from the levels' under parts and produce value-added biological knowledge. ISOLA is the result of a bioinformatics effort that addresses the challenges of the post-genomics era. It is designed to exploit '-omics' data based on effective integration to acquire biological knowledge and to approach a systems biology view. Beyond providing experimental biologists with a preliminary annotation of the tomato genome, this effort aims to produce a trial computational environment where different aspects and details are maintained as they are relevant for the analysis of the organization, the functionality and the evolution of the Solanaceae family.

[The ENCODE project and functional genomics studies].

PubMed

Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

2014-03-01

Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

The future is now: single-cell genomics of bacteria and archaea

PubMed Central

Blainey, Paul C.

2013-01-01

Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology. PMID:23298390

Genome sequence and analysis of Lactobacillus helveticus

PubMed Central

Cremonesi, Paola; Chessa, Stefania; Castiglioni, Bianca

2013-01-01

The microbiological characterization of lactobacilli is historically well developed, but the genomic analysis is recent. Because of the widespread use of Lactobacillus helveticus in cheese technology, information concerning the heterogeneity in this species is accumulating rapidly. Recently, the genome of five L. helveticus strains was sequenced to completion and compared with other genomically characterized lactobacilli. The genomic analysis of the first sequenced strain, L. helveticus DPC 4571, isolated from cheese and selected for its characteristics of rapid lysis and high proteolytic activity, has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions such as proteolysis, lipolysis, and cell lysis. These genes and their derived enzymes can facilitate the production of cheese and cheese derivatives with potential for use as ingredients in consumer foods. In addition, L. helveticus has the potential to produce peptides with a biological function, such as angiotensin converting enzyme (ACE) inhibitory activity, in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of L. helveticus is the remarkable similarity in gene content with many intestinal lactobacilli. Comparative genomics has allowed the identification of key gene sets that facilitate a variety of lifestyles including adaptation to food matrices or the gastrointestinal tract. As genome sequence and functional genomic information continues to explode, key features of the genomes of L. helveticus strains continue to be discovered, answering many questions but also raising many new ones. PMID:23335916

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

PubMed Central

Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

2012-01-01

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

Decoding genes with coexpression networks and metabolomics - 'majority report by precogs'.

PubMed

Saito, Kazuki; Hirai, Masami Y; Yonekura-Sakakibara, Keiko

2008-01-01

Following the sequencing of whole genomes of model plants, high-throughput decoding of gene function is a major challenge in modern plant biology. In view of remarkable technical advances in transcriptomics and metabolomics, integrated analysis of these 'omics' by data-mining informatics is an excellent tool for prediction and identification of gene function, particularly for genes involved in complicated metabolic pathways. The availability of Arabidopsis public transcriptome datasets containing data of >1000 microarrays reinforces the potential for prediction of gene function by transcriptome coexpression analysis. Here, we review the strategy of combining transcriptome and metabolome as a powerful technology for studying the functional genomics of model plants and also crop and medicinal plants.

Detection of active transposable elements in Arabidopsis thaliana using Oxford Nanopore Sequencing technology.

PubMed

Debladis, Emilie; Llauro, Christel; Carpentier, Marie-Christine; Mirouze, Marie; Panaud, Olivier

2017-07-17

Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.

Genomic Aspects of Research Involving Polyploid Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaohan; Ye, Chuyu; Tschaplinski, Timothy J

2011-01-01

Almost all extant plant species have spontaneously doubled their genomes at least once in their evolutionary histories, resulting in polyploidy which provided a rich genomic resource for evolutionary processes. Moreover, superior polyploid clones have been created during the process of crop domestication. Polyploid plants generated by evolutionary processes and/or crop domestication have been the intentional or serendipitous focus of research dealing with the dynamics and consequences of genome evolution. One of the new trends in genomics research is to create synthetic polyploid plants which provide materials for studying the initial genomic changes/responses immediately after polyploid formation. Polyploid plants are alsomore » used in functional genomics research to study gene expression in a complex genomic background. In this review, we summarize the recent progress in genomics research involving ancient, young, and synthetic polyploid plants, with a focus on genome size evolution, genomics diversity, genomic rearrangement, genetic and epigenetic changes in duplicated genes, gene discovery, and comparative genomics. Implications on plant sciences including evolution, functional genomics, and plant breeding are presented. It is anticipated that polyploids will be a regular subject of genomics research in the foreseeable future as the rapid advances in DNA sequencing technology create unprecedented opportunities for discovering and monitoring genomic and transcriptomic changes in polyploid plants. The fast accumulation of knowledge on polyploid formation, maintenance, and divergence at whole-genome and subgenome levels will not only help plant biologists understand how plants have evolved and diversified, but also assist plant breeders in designing new strategies for crop improvement.« less

Genome-wide comparative analysis of four Indian Drosophila species.

PubMed

Mohanty, Sujata; Khanna, Radhika

2017-12-01

Comparative analysis of multiple genomes of closely or distantly related Drosophila species undoubtedly creates excitement among evolutionary biologists in exploring the genomic changes with an ecology and evolutionary perspective. We present herewith the de novo assembled whole genome sequences of four Drosophila species, D. bipectinata, D. takahashii, D. biarmipes and D. nasuta of Indian origin using Next Generation Sequencing technology on an Illumina platform along with their detailed assembly statistics. The comparative genomics analysis, e.g. gene predictions and annotations, functional and orthogroup analysis of coding sequences and genome wide SNP distribution were performed. The whole genome of Zaprionus indianus of Indian origin published earlier by us and the genome sequences of previously sequenced 12 Drosophila species available in the NCBI database were included in the analysis. The present work is a part of our ongoing genomics project of Indian Drosophila species.

Expansion of genetic testing in the division of functional genomics, research center for bioscience and technology, tottori university from 2000 to 2013.

PubMed

Adachi, Kaori

2014-03-01

At the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, we have been making an effort to establish a genetic testing facility that can provide the same screening procedures conducted worldwide. Direct Sequencing of PCR products is the main method to detect point mutations, small deletions and insertions. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect large deletions or insertions. Expansion of the repeat was analyzed for triplet repeat diseases. Original primers were constructed for 41 diseases when the reported primers failed to amplify the gene. Prediction of functional effects of human nsSNPs (PolyPhen) was used for evaluation of novel mutations. From January 2000 to September 2013, a total of 1,006 DNA samples were subjected to genetic testing in the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University. The hospitals that requested genetic testing were located in 43 prefectures in Japan and in 11 foreign countries. The genetic testing covered 62 diseases, and mutations were detected in 287 out of 1,006 with an average mutation detection rate of 24.7%. There were 77 samples for prenatal diagnosis. The number of samples has rapidly increased since 2010. In 2013, the next-generation sequencers were introduced in our facility and are expected to provide more comprehensive genetic testing in the near future. Nowadays, genetic testing is a popular and powerful tool for diagnosis of many genetic diseases. Our genetic testing should be further expanded in the future.

Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

PubMed Central

Silby, Mark W; Cerdeño-Tárraga, Ana M; Vernikos, Georgios S; Giddens, Stephen R; Jackson, Robert W; Preston, Gail M; Zhang, Xue-Xian; Moon, Christina D; Gehrig, Stefanie M; Godfrey, Scott AC; Knight, Christopher G; Malone, Jacob G; Robinson, Zena; Spiers, Andrew J; Harris, Simon; Challis, Gregory L; Yaxley, Alice M; Harris, David; Seeger, Kathy; Murphy, Lee; Rutter, Simon; Squares, Rob; Quail, Michael A; Saunders, Elizabeth; Mavromatis, Konstantinos; Brettin, Thomas S; Bentley, Stephen D; Hothersall, Joanne; Stephens, Elton; Thomas, Christopher M; Parkhill, Julian; Levy, Stuart B; Rainey, Paul B; Thomson, Nicholas R

2009-01-01

Background Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome. PMID:19432983

CRISPR-Cas9 technology: applications and human disease modelling.

PubMed

Torres-Ruiz, Raul; Rodriguez-Perales, Sandra

2017-01-01

Genome engineering is a powerful tool for a wide range of applications in biomedical research and medicine. The development of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has revolutionized the field of gene editing, thus facilitating efficient genome editing through the creation of targeted double-strand breaks of almost any organism and cell type. In addition, CRISPR-Cas9 technology has been used successfully for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The implementation of the CRISPR-Cas9 system has increased the number of available technological alternatives for studying gene function, thus enabling generation of CRISPR-based disease models. Although many mechanistic questions remain to be answered and several challenges have yet to be addressed, the use of CRISPR-Cas9-based genome engineering technologies will increase our knowledge of disease processes and their treatment in the near future. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications.

PubMed

Chang, Chia-Yu; Ting, Hsiao-Chien; Su, Hong-Lin; Jeng, Jing-Ren

2018-01-01

In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource

PubMed Central

Seaver, Samuel M. D.; Gerdes, Svetlana; Frelin, Océane; Lerma-Ortiz, Claudia; Bradbury, Louis M. T.; Zallot, Rémi; Hasnain, Ghulam; Niehaus, Thomas D.; El Yacoubi, Basma; Pasternak, Shiran; Olson, Robert; Pusch, Gordon; Overbeek, Ross; Stevens, Rick; de Crécy-Lagard, Valérie; Ware, Doreen; Hanson, Andrew D.; Henry, Christopher S.

2014-01-01

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today’s annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for cross-kingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of model-based assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed. PMID:24927599

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource.

PubMed

Seaver, Samuel M D; Gerdes, Svetlana; Frelin, Océane; Lerma-Ortiz, Claudia; Bradbury, Louis M T; Zallot, Rémi; Hasnain, Ghulam; Niehaus, Thomas D; El Yacoubi, Basma; Pasternak, Shiran; Olson, Robert; Pusch, Gordon; Overbeek, Ross; Stevens, Rick; de Crécy-Lagard, Valérie; Ware, Doreen; Hanson, Andrew D; Henry, Christopher S

2014-07-01

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for cross-kingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of model-based assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed.

Strategies to explore functional genomics data sets in NCBI's GEO database.

PubMed

Wilhite, Stephen E; Barrett, Tanya

2012-01-01

The Gene Expression Omnibus (GEO) database is a major repository that stores high-throughput functional genomics data sets that are generated using both microarray-based and sequence-based technologies. Data sets are submitted to GEO primarily by researchers who are publishing their results in journals that require original data to be made freely available for review and analysis. In addition to serving as a public archive for these data, GEO has a suite of tools that allow users to identify, analyze, and visualize data relevant to their specific interests. These tools include sample comparison applications, gene expression profile charts, data set clusters, genome browser tracks, and a powerful search engine that enables users to construct complex queries.

Strategies to Explore Functional Genomics Data Sets in NCBI’s GEO Database

PubMed Central

Wilhite, Stephen E.; Barrett, Tanya

2012-01-01

The Gene Expression Omnibus (GEO) database is a major repository that stores high-throughput functional genomics data sets that are generated using both microarray-based and sequence-based technologies. Data sets are submitted to GEO primarily by researchers who are publishing their results in journals that require original data to be made freely available for review and analysis. In addition to serving as a public archive for these data, GEO has a suite of tools that allow users to identify, analyze and visualize data relevant to their specific interests. These tools include sample comparison applications, gene expression profile charts, data set clusters, genome browser tracks, and a powerful search engine that enables users to construct complex queries. PMID:22130872

CRISPR/Cas9: From Genome Engineering to Cancer Drug Discovery

PubMed Central

Luo, Ji

2016-01-01

Advances in translational research are often driven by new technologies. The advent of microarrays, next-generation sequencing, proteomics and RNA interference (RNAi) have led to breakthroughs in our understanding of the mechanisms of cancer and the discovery of new cancer drug targets. The discovery of the bacterial clustered regularly interspaced palindromic repeat (CRISPR) system and its subsequent adaptation as a tool for mammalian genome engineering has opened up new avenues for functional genomics studies. This review will focus on the utility of CRISPR in the context of cancer drug target discovery. PMID:28603775

The Comprehensive Microbial Resource.

PubMed

Peterson, J D; Umayam, L A; Dickinson, T; Hickey, E K; White, O

2001-01-01

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

PubMed

Brown, Nathan M; Mueller, Ryan S; Shepardson, Jonathan W; Landry, Zachary C; Morré, Jeffrey T; Maier, Claudia S; Hardy, F Joan; Dreher, Theo W

2016-06-13

Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

Genome sequence analysis of a flocculant-producing bacterium, Paenibacillus shenyangensis.

PubMed

Fu, Lili; Jiang, Binhui; Liu, Jinliang; Zhao, Xin; Liu, Qian; Hu, Xiaomin

2016-03-01

To explore the metabolic process of Paenibacillus shenyangensis that is an efficient bioflocculant-producing bacterium. The biosynthesis mechanism of bioflocculation was used to enrich the genome of Paenibacillus shenyangensis and provide a basis for molecular genetics and functional genomics analyses. According to the analysis of de novo assembly, a total of 5,501,467 bp clean reads were generated, and were assembled into 92 contigs. 4800 unigenes were predicted of which 4393 were annotated showing a specific gene function in the NCBI-Nr database. 3423 genes were found in the database of cluster of orthologous groups. Among the 168 Kyoto Encyclopedia of Genes and Genomes database, cell growth and metabolism were the main biological processes, and a potential metabolic pathway was predicted from glucose to exopolysaccharide within the starch and sucrose metabolism pathway. By using the high-throughput sequencing technology, we provide a genome analysis of Paenibacillus shenyangensis that predicts the main metabolic processes and a potential pathway of exopolysaccharide biosynthesis.

Harnessing CRISPR-Cas systems for bacterial genome editing.

PubMed

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advances in targeted genome editing.

PubMed

Perez-Pinera, Pablo; Ousterout, David G; Gersbach, Charles A

2012-08-01

New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

[CRISPR/Cas system for genome editing in pluripotent stem cells].

PubMed

Vasil'eva, E A; Melino, D; Barlev, N A

2015-01-01

Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

PubMed

Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

2015-08-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs.

PubMed

Swain, Martin T; Tsai, Isheng J; Assefa, Samual A; Newbold, Chris; Berriman, Matthew; Otto, Thomas D

2012-06-07

Genome projects now produce draft assemblies within weeks owing to advanced high-throughput sequencing technologies. For milestone projects such as Escherichia coli or Homo sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects, and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT) that is used to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence and exploit reference genomes (if available) in order to improve scaffolding and generating annotations. The protocol is most accessible for bacterial and small eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes ∼24 h: it doubles the average contig size and annotates over 4,300 gene models.

Three-Dimensional Genome Organization and Function in Drosophila

PubMed Central

Schwartz, Yuri B.; Cavalli, Giacomo

2017-01-01

Understanding how the metazoan genome is used during development and cell differentiation is one of the major challenges in the postgenomic era. Early studies in Drosophila suggested that three-dimensional (3D) chromosome organization plays important regulatory roles in this process and recent technological advances started to reveal connections at the molecular level. Here we will consider general features of the architectural organization of the Drosophila genome, providing historical perspective and insights from recent work. We will compare the linear and spatial segmentation of the fly genome and focus on the two key regulators of genome architecture: insulator components and Polycomb group proteins. With its unique set of genetic tools and a compact, well annotated genome, Drosophila is poised to remain a model system of choice for rapid progress in understanding principles of genome organization and to serve as a proving ground for development of 3D genome-engineering techniques. PMID:28049701

Genome Consortium for Active Teaching: Meeting the Goals of BIO2010

PubMed Central

Ledbetter, Mary Lee S.; Hoopes, Laura L.M.; Eckdahl, Todd T.; Heyer, Laurie J.; Rosenwald, Anne; Fowlks, Edison; Tonidandel, Scott; Bucholtz, Brooke; Gottfried, Gail

2007-01-01

The Genome Consortium for Active Teaching (GCAT) facilitates the use of modern genomics methods in undergraduate education. Initially focused on microarray technology, but with an eye toward diversification, GCAT is a community working to improve the education of tomorrow's life science professionals. GCAT participants have access to affordable microarrays, microarray scanners, free software for data analysis, and faculty workshops. Microarrays provided by GCAT have been used by 141 faculty on 134 campuses, including 21 faculty that serve large numbers of underrepresented minority students. An estimated 9480 undergraduates a year will have access to microarrays by 2009 as a direct result of GCAT faculty workshops. Gains for students include significantly improved comprehension of topics in functional genomics and increased interest in research. Faculty reported improved access to new technology and gains in understanding thanks to their involvement with GCAT. GCAT's network of supportive colleagues encourages faculty to explore genomics through student research and to learn a new and complex method with their undergraduates. GCAT is meeting important goals of BIO2010 by making research methods accessible to undergraduates, training faculty in genomics and bioinformatics, integrating mathematics into the biology curriculum, and increasing participation by underrepresented minority students. PMID:17548873

Genome Consortium for Active Teaching: meeting the goals of BIO2010.

PubMed

Campbell, A Malcolm; Ledbetter, Mary Lee S; Hoopes, Laura L M; Eckdahl, Todd T; Heyer, Laurie J; Rosenwald, Anne; Fowlks, Edison; Tonidandel, Scott; Bucholtz, Brooke; Gottfried, Gail

2007-01-01

The Genome Consortium for Active Teaching (GCAT) facilitates the use of modern genomics methods in undergraduate education. Initially focused on microarray technology, but with an eye toward diversification, GCAT is a community working to improve the education of tomorrow's life science professionals. GCAT participants have access to affordable microarrays, microarray scanners, free software for data analysis, and faculty workshops. Microarrays provided by GCAT have been used by 141 faculty on 134 campuses, including 21 faculty that serve large numbers of underrepresented minority students. An estimated 9480 undergraduates a year will have access to microarrays by 2009 as a direct result of GCAT faculty workshops. Gains for students include significantly improved comprehension of topics in functional genomics and increased interest in research. Faculty reported improved access to new technology and gains in understanding thanks to their involvement with GCAT. GCAT's network of supportive colleagues encourages faculty to explore genomics through student research and to learn a new and complex method with their undergraduates. GCAT is meeting important goals of BIO2010 by making research methods accessible to undergraduates, training faculty in genomics and bioinformatics, integrating mathematics into the biology curriculum, and increasing participation by underrepresented minority students.

MBGD update 2015: microbial genome database for flexible ortholog analysis utilizing a diverse set of genomic data.

PubMed

Uchiyama, Ikuo; Mihara, Motohiro; Nishide, Hiroyo; Chiba, Hirokazu

2015-01-01

The microbial genome database for comparative analysis (MBGD) (available at http://mbgd.genome.ad.jp/) is a comprehensive ortholog database for flexible comparative analysis of microbial genomes, where the users are allowed to create an ortholog table among any specified set of organisms. Because of the rapid increase in microbial genome data owing to the next-generation sequencing technology, it becomes increasingly challenging to maintain high-quality orthology relationships while allowing the users to incorporate the latest genomic data available into an analysis. Because many of the recently accumulating genomic data are draft genome sequences for which some complete genome sequences of the same or closely related species are available, MBGD now stores draft genome data and allows the users to incorporate them into a user-specific ortholog database using the MyMBGD functionality. In this function, draft genome data are incorporated into an existing ortholog table created only from the complete genome data in an incremental manner to prevent low-quality draft data from affecting clustering results. In addition, to provide high-quality orthology relationships, the standard ortholog table containing all the representative genomes, which is first created by the rapid classification program DomClust, is now refined using DomRefine, a recently developed program for improving domain-level clustering using multiple sequence alignment information. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

NCBI GEO: archive for functional genomics data sets—10 years on

PubMed Central

Barrett, Tanya; Troup, Dennis B.; Wilhite, Stephen E.; Ledoux, Pierre; Evangelista, Carlos; Kim, Irene F.; Tomashevsky, Maxim; Marshall, Kimberly A.; Phillippy, Katherine H.; Sherman, Patti M.; Muertter, Rolf N.; Holko, Michelle; Ayanbule, Oluwabukunmi; Yefanov, Andrey; Soboleva, Alexandra

2011-01-01

A decade ago, the Gene Expression Omnibus (GEO) database was established at the National Center for Biotechnology Information (NCBI). The original objective of GEO was to serve as a public repository for high-throughput gene expression data generated mostly by microarray technology. However, the research community quickly applied microarrays to non-gene-expression studies, including examination of genome copy number variation and genome-wide profiling of DNA-binding proteins. Because the GEO database was designed with a flexible structure, it was possible to quickly adapt the repository to store these data types. More recently, as the microarray community switches to next-generation sequencing technologies, GEO has again adapted to host these data sets. Today, GEO stores over 20 000 microarray- and sequence-based functional genomics studies, and continues to handle the majority of direct high-throughput data submissions from the research community. Multiple mechanisms are provided to help users effectively search, browse, download and visualize the data at the level of individual genes or entire studies. This paper describes recent database enhancements, including new search and data representation tools, as well as a brief review of how the community uses GEO data. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/. PMID:21097893

The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST)

PubMed Central

Overbeek, Ross; Olson, Robert; Pusch, Gordon D.; Olsen, Gary J.; Davis, James J.; Disz, Terry; Edwards, Robert A.; Gerdes, Svetlana; Parrello, Bruce; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Xia, Fangfang; Stevens, Rick

2014-01-01

In 2004, the SEED (http://pubseed.theseed.org/) was created to provide consistent and accurate genome annotations across thousands of genomes and as a platform for discovering and developing de novo annotations. The SEED is a constantly updated integration of genomic data with a genome database, web front end, API and server scripts. It is used by many scientists for predicting gene functions and discovering new pathways. In addition to being a powerful database for bioinformatics research, the SEED also houses subsystems (collections of functionally related protein families) and their derived FIGfams (protein families), which represent the core of the RAST annotation engine (http://rast.nmpdr.org/). When a new genome is submitted to RAST, genes are called and their annotations are made by comparison to the FIGfam collection. If the genome is made public, it is then housed within the SEED and its proteins populate the FIGfam collection. This annotation cycle has proven to be a robust and scalable solution to the problem of annotating the exponentially increasing number of genomes. To date, >12 000 users worldwide have annotated >60 000 distinct genomes using RAST. Here we describe the interconnectedness of the SEED database and RAST, the RAST annotation pipeline and updates to both resources. PMID:24293654

The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST).

PubMed

Overbeek, Ross; Olson, Robert; Pusch, Gordon D; Olsen, Gary J; Davis, James J; Disz, Terry; Edwards, Robert A; Gerdes, Svetlana; Parrello, Bruce; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R; Xia, Fangfang; Stevens, Rick

2014-01-01

In 2004, the SEED (http://pubseed.theseed.org/) was created to provide consistent and accurate genome annotations across thousands of genomes and as a platform for discovering and developing de novo annotations. The SEED is a constantly updated integration of genomic data with a genome database, web front end, API and server scripts. It is used by many scientists for predicting gene functions and discovering new pathways. In addition to being a powerful database for bioinformatics research, the SEED also houses subsystems (collections of functionally related protein families) and their derived FIGfams (protein families), which represent the core of the RAST annotation engine (http://rast.nmpdr.org/). When a new genome is submitted to RAST, genes are called and their annotations are made by comparison to the FIGfam collection. If the genome is made public, it is then housed within the SEED and its proteins populate the FIGfam collection. This annotation cycle has proven to be a robust and scalable solution to the problem of annotating the exponentially increasing number of genomes. To date, >12 000 users worldwide have annotated >60 000 distinct genomes using RAST. Here we describe the interconnectedness of the SEED database and RAST, the RAST annotation pipeline and updates to both resources.

RNA Interference Based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly

USDA-ARS?s Scientific Manuscript database

Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) could offer potential for insect pest management. Insects feeding exclusively on plant sap depend on osmotic pressure...

RNAi for functional genomics in plants.

PubMed

McGinnis, Karen M

2010-03-01

RNAi refers to several different types of gene silencing mediated by small, dsRNA molecules. Over the course of 20 years, the scientific understanding of RNAi has developed from the initial observation of unexpected expression patterns to a sophisticated understanding of a multi-faceted, evolutionarily conserved network of mechanisms that regulate gene expression in many organisms. It has also been developed as a genetic tool that can be exploited in a wide range of species. Because transgene-induced RNAi has been effective at silencing one or more genes in a wide range of plants, this technology also bears potential as a powerful functional genomics tool across the plant kingdom. Transgene-induced RNAi has indeed been shown to be an effective mechanism for silencing many genes in many organisms, but the results from multiple projects which attempted to exploit RNAi on a genome-wide scale suggest that there is a great deal of variation in the silencing efficacy between transgenic events, silencing targets and silencing-induced phenotype. The results from these projects indicate several important variables that should be considered in experimental design prior to the initiation of functional genomics efforts based on RNAi silencing. In recent years, alternative strategies have been developed for targeted gene silencing, and a combination of approaches may also enhance the use of targeted gene silencing for functional genomics.

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

PubMed Central

Zhang, Xiao-Hui; Tee, Louis Y; Wang, Xiao-Gang; Huang, Qun-Shan; Yang, Shi-Hua

2015-01-01

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. PMID:26575098

Factors affecting reproducibility between genome-scale siRNA-based screens

PubMed Central

Barrows, Nicholas J.; Le Sommer, Caroline; Garcia-Blanco, Mariano A.; Pearson, James L.

2011-01-01

RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage. In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies. PMID:20625183

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

PubMed

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

PubMed Central

Grzesik, Peter; Voorhies, Alexander A.; Alperovich, Nina; MacMath, Derek; Najera, Claudia D.; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N.; Montague, Michael G.; Friedman, Robert M.; Desai, Prashant J.

2017-01-01

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats. PMID:28928148

Bioinformatics and genomic analysis of transposable elements in eukaryotic genomes.

PubMed

Janicki, Mateusz; Rooke, Rebecca; Yang, Guojun

2011-08-01

A major portion of most eukaryotic genomes are transposable elements (TEs). During evolution, TEs have introduced profound changes to genome size, structure, and function. As integral parts of genomes, the dynamic presence of TEs will continue to be a major force in reshaping genomes. Early computational analyses of TEs in genome sequences focused on filtering out "junk" sequences to facilitate gene annotation. When the high abundance and diversity of TEs in eukaryotic genomes were recognized, these early efforts transformed into the systematic genome-wide categorization and classification of TEs. The availability of genomic sequence data reversed the classical genetic approaches to discovering new TE families and superfamilies. Curated TE databases and their accurate annotation of genome sequences in turn facilitated the studies on TEs in a number of frontiers including: (1) TE-mediated changes of genome size and structure, (2) the influence of TEs on genome and gene functions, (3) TE regulation by host, (4) the evolution of TEs and their population dynamics, and (5) genomic scale studies of TE activity. Bioinformatics and genomic approaches have become an integral part of large-scale studies on TEs to extract information with pure in silico analyses or to assist wet lab experimental studies. The current revolution in genome sequencing technology facilitates further progress in the existing frontiers of research and emergence of new initiatives. The rapid generation of large-sequence datasets at record low costs on a routine basis is challenging the computing industry on storage capacity and manipulation speed and the bioinformatics community for improvement in algorithms and their implementations.

Genome Editing to Study Ca2+ Homeostasis in Zebrafish Cone Photoreceptors.

PubMed

Brockerhoff, Susan E

2017-01-01

Photoreceptors are specialized sensory neurons with unique biological features. Phototransduction is well understood due in part to the exclusive expression and function of the molecular components of this cascade. Many other processes are less well understood, but also extremely important for understanding photoreceptor function and for treating disease. One example is the role of Ca 2+ in the cell body and overall compartmentalization and regulation of Ca 2+ within the cell. The recent development of CRISPR/Cas9 genome editing techniques has made it possible to rapidly and cheaply alter specific genes. This will help to define the biological function of elusive processes that have been more challenging to study. CRISPR/Cas9 has been optimized in many systems including zebrafish, which already has some distinct advantages for studying photoreceptor biology and function. These new genome editing technologies and the continued use of the zebrafish model system will help advance our understanding of important understudied aspects of photoreceptor biology.

Integration of Structural Dynamics and Molecular Evolution via Protein Interaction Networks: A New Era in Genomic Medicine

PubMed Central

Kumar, Avishek; Butler, Brandon M.; Kumar, Sudhir; Ozkan, S. Banu

2016-01-01

Summary Sequencing technologies are revealing many new non-synonymous single nucleotide variants (nsSNVs) in each personal exome. To assess their functional impacts, comparative genomics is frequently employed to predict if they are benign or not. However, evolutionary analysis alone is insufficient, because it misdiagnoses many disease-associated nsSNVs, such as those at positions involved in protein interfaces, and because evolutionary predictions do not provide mechanistic insights into functional change or loss. Structural analyses can aid in overcoming both of these problems by incorporating conformational dynamics and allostery in nSNV diagnosis. Finally, protein-protein interaction networks using systems-level methodologies shed light onto disease etiology and pathogenesis. Bridging these network approaches with structurally resolved protein interactions and dynamics will advance genomic medicine. PMID:26684487

CRISPR mediated somatic cell genome engineering in the chicken.

PubMed

Véron, Nadège; Qu, Zhengdong; Kipen, Phoebe A S; Hirst, Claire E; Marcelle, Christophe

2015-11-01

Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model. Copyright © 2015 Elsevier Inc. All rights reserved.

Network-assisted crop systems genetics: network inference and integrative analysis.

PubMed

Lee, Tak; Kim, Hyojin; Lee, Insuk

2015-04-01

Although next-generation sequencing (NGS) technology has enabled the decoding of many crop species genomes, most of the underlying genetic components for economically important crop traits remain to be determined. Network approaches have proven useful for the study of the reference plant, Arabidopsis thaliana, and the success of network-based crop genetics will also require the availability of a genome-scale functional networks for crop species. In this review, we discuss how to construct functional networks and elucidate the holistic view of a crop system. The crop gene network then can be used for gene prioritization and the analysis of resequencing-based genome-wide association study (GWAS) data, the amount of which will rapidly grow in the field of crop science in the coming years. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-Cell Genomic Analysis in Plants

PubMed Central

Hu, Haifei; Scheben, Armin; Edwards, David

2018-01-01

Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis. PMID:29361790

A decade after the first full human genome sequencing: when will we understand our own genome?

PubMed

Eisenhaber, Frank

2012-10-01

The contrast between the pomp of celebrating the first full human genome sequencing in 2000 and the cautious tone of recollections a decade thereafter could hardly be greater. The promises with regard to medical cures and biotechnology applications have been realized not even nearly to the expectations. Understanding the human genomes means knowing the genes' and proteins' functions and their interconnectedness via biomolecular mechanisms. This articles estimates how long will it take to achieve this goal if we extrapolate from the previous decade (indeed, a century!) and the possible disruptive trends in science, technology and society that may accelerate the pace of progress dramatically.

From genomics to mechanistic insight: a global perspective on molecular deficits induced by environmental agents.

PubMed

Ramos, Kenneth S; Steffen, Marlene C; Falahatpisheh, M H; Nanez, Adrian

2007-06-01

As the postgenomic era continues to unfold, a new wave of scientific investigation is upon us focusing on the application of genomic technologies to study the meanings encrypted on the DNA code and the responses of living organisms to changes in their environment. Recent functional genomics studies in this laboratory have focused on the role of the aryl hydrocarbon receptor, a ubiquitous transcription factor, in genetic programming during renal development. Also of interest is the application of genomics investigations to the study of chronic medical conditions associated with early life exposures to environmental contaminants. Molecular evidence is discussed in this review within the framework of human molecular medicine.

The Comprehensive Microbial Resource

PubMed Central

Peterson, Jeremy D.; Umayam, Lowell A.; Dickinson, Tanja; Hickey, Erin K.; White, Owen

2001-01-01

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes. PMID:11125067

RATT: Rapid Annotation Transfer Tool

PubMed Central

Otto, Thomas D.; Dillon, Gary P.; Degrave, Wim S.; Berriman, Matthew

2011-01-01

Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net. PMID:21306991

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

PubMed Central

Hilson, Pierre; Allemeersch, Joke; Altmann, Thomas; Aubourg, Sébastien; Avon, Alexandra; Beynon, Jim; Bhalerao, Rishikesh P.; Bitton, Frédérique; Caboche, Michel; Cannoot, Bernard; Chardakov, Vasil; Cognet-Holliger, Cécile; Colot, Vincent; Crowe, Mark; Darimont, Caroline; Durinck, Steffen; Eickhoff, Holger; de Longevialle, Andéol Falcon; Farmer, Edward E.; Grant, Murray; Kuiper, Martin T.R.; Lehrach, Hans; Léon, Céline; Leyva, Antonio; Lundeberg, Joakim; Lurin, Claire; Moreau, Yves; Nietfeld, Wilfried; Paz-Ares, Javier; Reymond, Philippe; Rouzé, Pierre; Sandberg, Goran; Segura, Maria Dolores; Serizet, Carine; Tabrett, Alexandra; Taconnat, Ludivine; Thareau, Vincent; Van Hummelen, Paul; Vercruysse, Steven; Vuylsteke, Marnik; Weingartner, Magdalena; Weisbeek, Peter J.; Wirta, Valtteri; Wittink, Floyd R.A.; Zabeau, Marc; Small, Ian

2004-01-01

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics. PMID:15489341

A computational genomics pipeline for prokaryotic sequencing projects.

PubMed

Kislyuk, Andrey O; Katz, Lee S; Agrawal, Sonia; Hagen, Matthew S; Conley, Andrew B; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C; Sammons, Scott A; Govil, Dhwani; Mair, Raydel D; Tatti, Kathleen M; Tondella, Maria L; Harcourt, Brian H; Mayer, Leonard W; Jordan, I King

2010-08-01

New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Using Genome Sequence to Enable the Design of Medicines and Chemical Probes.

PubMed

Angelbello, Alicia J; Chen, Jonathan L; Childs-Disney, Jessica L; Zhang, Peiyuan; Wang, Zi-Fu; Disney, Matthew D

2018-02-28

Rapid progress in genome sequencing technology has put us firmly into a postgenomic era. A key challenge in biomedical research is harnessing genome sequence to fulfill the promise of personalized medicine. This Review describes how genome sequencing has enabled the identification of disease-causing biomolecules and how these data have been converted into chemical probes of function, preclinical lead modalities, and ultimately U.S. Food and Drug Administration (FDA)-approved drugs. In particular, we focus on the use of oligonucleotide-based modalities to target disease-causing RNAs; small molecules that target DNA, RNA, or protein; the rational repurposing of known therapeutic modalities; and the advantages of pharmacogenetics. Lastly, we discuss the remaining challenges and opportunities in the direct utilization of genome sequence to enable design of medicines.

Basics and applications of genome editing technology.

PubMed

Yamamoto, Takashi; Sakamoto, Naoaki

2016-01-01

Genome editing with programmable site-specific nucleases is an emerging technology that enables the manipulation of targeted genes in many organisms and cell lines. Since the development of the CRISPR-Cas9 system in 2012, genome editing has rapidly become an indispensable technology for all life science researchers, applicable in various fields. In this seminar, we will introduce the basics of genome editing and focus on the recent development of genome editing tools and technologies for the modification of various organisms and discuss future directions of the genome editing research field, from basic to medical applications.

Bridging epigenomics and complex disease: the basics.

PubMed

Teperino, Raffaele; Lempradl, Adelheid; Pospisilik, J Andrew

2013-05-01

The DNA sequence largely defines gene expression and phenotype. However, it is becoming increasingly clear that an additional chromatin-based regulatory network imparts both stability and plasticity to genome output, modifying phenotype independently of the genetic blueprint. Indeed, alterations in this "epigenetic" control layer underlie, at least in part, the reason for monozygotic twins being discordant for disease. Functionally, this regulatory layer comprises post-translational modifications of DNA and histones, as well as small and large noncoding RNAs. Together these regulate gene expression by changing chromatin organization and DNA accessibility. Successive technological advances over the past decade have enabled researchers to map the chromatin state with increasing accuracy and comprehensiveness, catapulting genetic research into a genome-wide era. Here, aiming particularly at the genomics/epigenomics newcomer, we review the epigenetic basis that has helped drive the technological shift and how this progress is shaping our understanding of complex disease.

Molecular Basis of Essential Thrombocytosis

DTIC Science & Technology

2008-06-01

Membrane proteins,” below), and 64% were present in the cytoskeleton, endoplasmic reticulum, mitochondria, cytosol, or Golgi apparatus ... perspectives Future advances in proteomic technology that incorporate miniatur- ization,101 coupled with an ability to integrate functional genomics...14. Kralovics R, Passamonti F, Buser AS, et al. A gain-of- function mutation of JAK2 in myeloproliferative disorders. The New England Journal of

The humankind genome: from genetic diversity to the origin of human diseases.

PubMed

Belizário, Jose E

2013-12-01

Genome-wide association studies have failed to establish common variant risk for the majority of common human diseases. The underlying reasons for this failure are explained by recent studies of resequencing and comparison of over 1200 human genomes and 10 000 exomes, together with the delineation of DNA methylation patterns (epigenome) and full characterization of coding and noncoding RNAs (transcriptome) being transcribed. These studies have provided the most comprehensive catalogues of functional elements and genetic variants that are now available for global integrative analysis and experimental validation in prospective cohort studies. With these datasets, researchers will have unparalleled opportunities for the alignment, mining, and testing of hypotheses for the roles of specific genetic variants, including copy number variations, single nucleotide polymorphisms, and indels as the cause of specific phenotypes and diseases. Through the use of next-generation sequencing technologies for genotyping and standardized ontological annotation to systematically analyze the effects of genomic variation on humans and model organism phenotypes, we will be able to find candidate genes and new clues for disease's etiology and treatment. This article describes essential concepts in genetics and genomic technologies as well as the emerging computational framework to comprehensively search websites and platforms available for the analysis and interpretation of genomic data.

Mapping Our Genes: The Genome Projects: How Big, How Fast

DOE R&D Accomplishments Database

1988-04-01

For the past 2 years, scientific and technical journals in biology and medicine have extensively covered a debate about whether and how to determine the function and order of human genes on human chromosomes and when to determine the sequence of molecular building blocks that comprise DNA in those chromosomes. In 1987, these issues rose to become part of the public agenda. The debate involves science, technology, and politics. Congress is responsible for ?writing the rules? of what various federal agencies do and for funding their work. This report surveys the points made so far in the debate, focusing on those that most directly influence the policy options facing the US Congress. Congressional interest focused on how to assess the rationales for conducting human genome projects, how to fund human genome projects (at what level and through which mechanisms), how to coordinate the scientific and technical programs of the several federal agencies and private interests already supporting various genome projects, and how to strike a balance regarding the impact of genome projects on international scientific cooperation and international economic competition in biotechnology. The Office of Technology Assessment (OTA) prepared this report with the assistance of several hundred experts throughout the world.

High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome

PubMed Central

2013-01-01

Background Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. Results Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. Conclusions We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts. PMID:23368932

Genomic and Epigenomic Alterations in Cancer.

PubMed

Chakravarthi, Balabhadrapatruni V S K; Nepal, Saroj; Varambally, Sooryanarayana

2016-07-01

Multiple genetic and epigenetic events characterize tumor progression and define the identity of the tumors. Advances in high-throughput technologies, like gene expression profiling, next-generation sequencing, proteomics, and metabolomics, have enabled detailed molecular characterization of various tumors. The integration and analyses of these high-throughput data have unraveled many novel molecular aberrations and network alterations in tumors. These molecular alterations include multiple cancer-driving mutations, gene fusions, amplification, deletion, and post-translational modifications, among others. Many of these genomic events are being used in cancer diagnosis, whereas others are therapeutically targeted with small-molecule inhibitors. Multiple genes/enzymes that play a role in DNA and histone modifications are also altered in various cancers, changing the epigenomic landscape during cancer initiation and progression. Apart from protein-coding genes, studies are uncovering the critical regulatory roles played by noncoding RNAs and noncoding regions of the genome during cancer progression. Many of these genomic and epigenetic events function in tandem to drive tumor development and metastasis. Concurrent advances in genome-modulating technologies, like gene silencing and genome editing, are providing ability to understand in detail the process of cancer initiation, progression, and signaling as well as opening up avenues for therapeutic targeting. In this review, we discuss some of the recent advances in cancer genomic and epigenomic research. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

HiView: an integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants.

PubMed

Xu, Zheng; Zhang, Guosheng; Duan, Qing; Chai, Shengjie; Zhang, Baqun; Wu, Cong; Jin, Fulai; Yue, Feng; Li, Yun; Hu, Ming

2016-03-11

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with complex traits and diseases. However, most of them are located in the non-protein coding regions, and therefore it is challenging to hypothesize the functions of these non-coding GWAS variants. Recent large efforts such as the ENCODE and Roadmap Epigenomics projects have predicted a large number of regulatory elements. However, the target genes of these regulatory elements remain largely unknown. Chromatin conformation capture based technologies such as Hi-C can directly measure the chromatin interactions and have generated an increasingly comprehensive catalog of the interactome between the distal regulatory elements and their potential target genes. Leveraging such information revealed by Hi-C holds the promise of elucidating the functions of genetic variants in human diseases. In this work, we present HiView, the first integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants. HiView is able to display Hi-C data and statistical evidence for chromatin interactions in genomic regions surrounding any given GWAS variant, enabling straightforward visualization and interpretation. We believe that as the first GWAS variants-centered Hi-C genome browser, HiView is a useful tool guiding post-GWAS functional genomics studies. HiView is freely accessible at: http://www.unc.edu/~yunmli/HiView .

De novo characterization of Lentinula edodes C(91-3) transcriptome by deep Solexa sequencing.

PubMed

Zhong, Mintao; Liu, Ben; Wang, Xiaoli; Liu, Lei; Lun, Yongzhi; Li, Xingyun; Ning, Anhong; Cao, Jing; Huang, Min

2013-02-01

Lentinula edodes, has been utilized as food, as well as, in popular medicine, moreover, its extract isolated from its mycelium and fruiting body have shown several therapeutic properties. Yet little is understood about its genes involved in these properties, and the absence of L.edodes genomes has been a barrier to the development of functional genomics research. However, high throughput sequencing technologies are now being widely applied to non-model species. To facilitate research on L.edodes, we leveraged Solexa sequencing technology in de novo assembly of L.edodes C(91-3) transcriptome. In a single run, we produced more than 57 million sequencing reads. These reads were assembled into 28,923 unigene sequences (mean size=689bp) including 18,120 unigenes with coding sequence (CDS). Based on similarity search with known proteins, assembled unigene sequences were annotated with gene descriptions, gene ontology (GO) and clusters of orthologous group (COG) terms. Our data provides the first comprehensive sequence resource available for functional genomics studies in L.edodes, and demonstrates the utility of Illumina/Solexa sequencing for de novo transcriptome characterization and gene discovery in a non-model mushroom. Copyright © 2012 Elsevier Inc. All rights reserved.

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Luciferase reporter assay in Drosophila and mammalian tissue culture cells

PubMed Central

Yun, Chi

2014-01-01

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development

PubMed Central

Singer, Ruth; Atar, Shimshi; Atias, Osnat; Oron, Efrat; Segal, Daniel; Hirsch, Joel A.; Tuller, Tamir; Orian, Amir; Chamovitz, Daniel A.

2014-01-01

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health. PMID:25106867

Precision Medicine: Functional Advancements.

PubMed

Caskey, Thomas

2018-01-29

Precision medicine was conceptualized on the strength of genomic sequence analysis. High-throughput functional metrics have enhanced sequence interpretation and clinical precision. These technologies include metabolomics, magnetic resonance imaging, and I rhythm (cardiac monitoring), among others. These technologies are discussed and placed in clinical context for the medical specialties of internal medicine, pediatrics, obstetrics, and gynecology. Publications in these fields support the concept of a higher level of precision in identifying disease risk. Precise disease risk identification has the potential to enable intervention with greater specificity, resulting in disease prevention-an important goal of precision medicine.

The 'dark matter' in the plant genomes: non-coding and unannotated DNA sequences associated with open chromatin.

PubMed

Jiang, Jiming

2015-04-01

Sequencing of complete plant genomes has become increasingly more routine since the advent of the next-generation sequencing technology. Identification and annotation of large amounts of noncoding but functional DNA sequences, including cis-regulatory DNA elements (CREs), have become a new frontier in plant genome research. Genomic regions containing active CREs bound to regulatory proteins are hypersensitive to DNase I digestion and are called DNase I hypersensitive sites (DHSs). Several recent DHS studies in plants illustrate that DHS datasets produced by DNase I digestion followed by next-generation sequencing (DNase-seq) are highly valuable for the identification and characterization of CREs associated with plant development and responses to environmental cues. DHS-based genomic profiling has opened a door to identify and annotate the 'dark matter' in sequenced plant genomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.

PubMed

Ren, Xingjie; Sun, Jin; Housden, Benjamin E; Hu, Yanhui; Roesel, Charles; Lin, Shuailiang; Liu, Lu-Ping; Yang, Zhihao; Mao, Decai; Sun, Lingzhu; Wu, Qujie; Ji, Jun-Yuan; Xi, Jianzhong; Mohr, Stephanie E; Xu, Jiang; Perrimon, Norbert; Ni, Jian-Quan

2013-11-19

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

Integration of structural dynamics and molecular evolution via protein interaction networks: a new era in genomic medicine.

PubMed

Kumar, Avishek; Butler, Brandon M; Kumar, Sudhir; Ozkan, S Banu

2015-12-01

Sequencing technologies are revealing many new non-synonymous single nucleotide variants (nsSNVs) in each personal exome. To assess their functional impacts, comparative genomics is frequently employed to predict if they are benign or not. However, evolutionary analysis alone is insufficient, because it misdiagnoses many disease-associated nsSNVs, such as those at positions involved in protein interfaces, and because evolutionary predictions do not provide mechanistic insights into functional change or loss. Structural analyses can aid in overcoming both of these problems by incorporating conformational dynamics and allostery in nSNV diagnosis. Finally, protein-protein interaction networks using systems-level methodologies shed light onto disease etiology and pathogenesis. Bridging these network approaches with structurally resolved protein interactions and dynamics will advance genomic medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adapting CRISPR/Cas9 for functional genomics screens.

PubMed

Malina, Abba; Katigbak, Alexandra; Cencic, Regina; Maïga, Rayelle Itoua; Robert, Francis; Miura, Hisashi; Pelletier, Jerry

2014-01-01

The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

NASA Astrophysics Data System (ADS)

Sakharkar, Kishore R.; Sakharkar, Meena K.

The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

AGORA : Organellar genome annotation from the amino acid and nucleotide references.

PubMed

Jung, Jaehee; Kim, Jong Im; Jeong, Young-Sik; Yi, Gangman

2018-03-29

Next-generation sequencing (NGS) technologies have led to the accumulation of highthroughput sequence data from various organisms in biology. To apply gene annotation of organellar genomes for various organisms, more optimized tools for functional gene annotation are required. Almost all gene annotation tools are mainly focused on the chloroplast genome of land plants or the mitochondrial genome of animals.We have developed a web application AGORA for the fast, user-friendly, and improved annotations of organellar genomes. AGORA annotates genes based on a BLAST-based homology search and clustering with selected reference sequences from the NCBI database or user-defined uploaded data. AGORA can annotate the functional genes in almost all mitochondrion and plastid genomes of eukaryotes. The gene annotation of a genome with an exon-intron structure within a gene or inverted repeat region is also available. It provides information of start and end positions of each gene, BLAST results compared with the reference sequence, and visualization of gene map by OGDRAW. Users can freely use the software, and the accessible URL is https://bigdata.dongguk.edu/gene_project/AGORA/.The main module of the tool is implemented by the python and php, and the web page is built by the HTML and CSS to support all browsers. gangman@dongguk.edu.

Insights into structural variations and genome rearrangements in prokaryotic genomes.

PubMed

Periwal, Vinita; Scaria, Vinod

2015-01-01

Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Fungal Genomics Program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grigoriev, Igor

The JGI Fungal Genomics Program aims to scale up sequencing and analysis of fungal genomes to explore the diversity of fungi important for energy and the environment, and to promote functional studies on a system level. Combining new sequencing technologies and comparative genomics tools, JGI is now leading the world in fungal genome sequencing and analysis. Over 120 sequenced fungal genomes with analytical tools are available via MycoCosm (www.jgi.doe.gov/fungi), a web-portal for fungal biologists. Our model of interacting with user communities, unique among other sequencing centers, helps organize these communities, improves genome annotation and analysis work, and facilitates new larger-scalemore » genomic projects. This resulted in 20 high-profile papers published in 2011 alone and contributing to the Genomics Encyclopedia of Fungi, which targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts). Our next grand challenges include larger scale exploration of fungal diversity (1000 fungal genomes), developing molecular tools for DOE-relevant model organisms, and analysis of complex systems and metagenomes.« less

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rice, Marlen C.; Norton, Jeanette M.; Valois, Frederica

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism withmore » implications for function in soil environments.« less

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil

DOE PAGES

Rice, Marlen C.; Norton, Jeanette M.; Valois, Frederica; ...

2016-07-28

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism withmore » implications for function in soil environments.« less

The Role of Constitutional Copy Number Variants in Breast Cancer

PubMed Central

Walker, Logan C.; Wiggins, George A.R.; Pearson, John F.

2015-01-01

Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans. PMID:27600231

De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes.

PubMed

Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

2015-01-01

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.

Genome analysis of food-processing stressful-resistant probiotic Bifidobacterium animalis subsp. lactis BF052, and its potential application in fermented soymilk.

PubMed

Charnchai, Pattra; Jantama, Sirima Suvarnakuta; Jantama, Kaemwich

2017-09-15

In this study, Bifidobacterium animalis subsp. lactis BF052 was demonstrated the growth capability in soymilk and could be thus supplemented as a probiotic starter that employed soymilk as one of its food vehicles. The complete genome sequence of BF052 was therefore determined to understand the genetic basis of BF052 as a technological and functional probiotic starter. The whole genome sequence of BF052 consists of a circular genome of 1938 624 bp with a G+C content of 60.50%. This research highlights relevant genes involving in its adaptive responses to industrial and/or environmental stresses and utilization of α-galacto-oligosaccharides in BF052 strain compared with other representative bifidobacterial genomes. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Novel genetic tools for studying food-borne Salmonella.

PubMed

Andrews-Polymenis, Helene L; Santiviago, Carlos A; McClelland, Michael

2009-04-01

Nontyphoidal Salmonellae are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates. Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM), pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Complete genome sequences permit the construction of pools of targeted in-frame deletions that have improved STM by minimizing the number of clones and the polarity of each mutant. Together, genome sequences and the continuing development of new tools for functional genomics will drive a revolution in the understanding of Salmonellae in many different niches that are critical for food safety.

Genomics, transcriptomics and proteomics: enabling insights into social evolution and disease challenges for managed and wild bees.

PubMed

Trapp, Judith; McAfee, Alison; Foster, Leonard J

2017-02-01

Globally, there are over 20 000 bee species (Hymenoptera: Apoidea: Anthophila) with a host of biologically fascinating characteristics. Although they have long been studied as models for social evolution, recent challenges to bee health (mainly diseases and pesticides) have gathered the attention of both public and research communities. Genome sequences of twelve bee species are now complete or under progress, facilitating the application of additional 'omic technologies. Here, we review recent developments in honey bee and native bee research in the genomic era. We discuss the progress in genome sequencing and functional annotation, followed by the enabled comparative genomics, proteomics and transcriptomics applications regarding social evolution and health. Finally, we end with comments on future challenges in the postgenomic era. © 2016 John Wiley & Sons Ltd.

Ten steps to get started in Genome Assembly and Annotation

PubMed Central

Dominguez Del Angel, Victoria; Hjerde, Erik; Sterck, Lieven; Capella-Gutierrez, Salvadors; Notredame, Cederic; Vinnere Pettersson, Olga; Amselem, Joelle; Bouri, Laurent; Bocs, Stephanie; Klopp, Christophe; Gibrat, Jean-Francois; Vlasova, Anna; Leskosek, Brane L.; Soler, Lucile; Binzer-Panchal, Mahesh; Lantz, Henrik

2018-01-01

As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project. Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR). PMID:29568489

The genome revolution and its role in understanding complex diseases.

PubMed

Hofker, Marten H; Fu, Jingyuan; Wijmenga, Cisca

2014-10-01

The completion of the human genome sequence in 2003 clearly marked the beginning of a new era for biomedical research. It spurred technological progress that was unprecedented in the life sciences, including the development of high-throughput technologies to detect genetic variation and gene expression. The study of genetics has become "big data science". One of the current goals of genetic research is to use genomic information to further our understanding of common complex diseases. An essential first step made towards this goal was by the identification of thousands of single nucleotide polymorphisms showing robust association with hundreds of different traits and diseases. As insight into common genetic variation has expanded enormously and the technology to identify more rare variation has become available, we can utilize these advances to gain a better understanding of disease etiology. This will lead to developments in personalized medicine and P4 healthcare. Here, we review some of the historical events and perspectives before and after the completion of the human genome sequence. We also describe the success of large-scale genetic association studies and how these are expected to yield more insight into complex disorders. We show how we can now combine gene-oriented research and systems-based approaches to develop more complex models to help explain the etiology of common diseases. This article is part of a Special Issue entitled: From Genome to Function. Copyright © 2014 Elsevier B.V. All rights reserved.

A computational genomics pipeline for prokaryotic sequencing projects

PubMed Central

Kislyuk, Andrey O.; Katz, Lee S.; Agrawal, Sonia; Hagen, Matthew S.; Conley, Andrew B.; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C.; Sammons, Scott A.; Govil, Dhwani; Mair, Raydel D.; Tatti, Kathleen M.; Tondella, Maria L.; Harcourt, Brian H.; Mayer, Leonard W.; Jordan, I. King

2010-01-01

Motivation: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. Results: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. Availability and implementation: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems. Contact: king.jordan@biology.gatech.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20519285

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes.

PubMed

Vorapreeda, Tayvich; Thammarongtham, Chinae; Laoteng, Kobkul

2016-07-01

Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach. The identification of putative lipase genes from microbial genomes and metagenomic libraries using homology-based mining is discussed, with an emphasis on sequence analysis of conserved motifs and enzyme topology. Molecular modelling of three-dimensional structure on the basis of sequence similarity is shown to be a potential approach for exploring the structural and functional relationships of candidate lipase enzymes. The perspectives on a discriminative framework of cutting-edge tools and technologies, including bioinformatics, computational biology, functional genomics and functional proteomics, intended to facilitate rapid progress in understanding lipolysis mechanism and to discover novel lipid-degrading enzymes of microorganisms are discussed.

Synthetic biology: Novel approaches for microbiology.

PubMed

Padilla-Vaca, Felipe; Anaya-Velázquez, Fernando; Franco, Bernardo

2015-06-01

In the past twenty years, molecular genetics has created powerful tools for genetic manipulation of living organisms. Whole genome sequencing has provided necessary information to assess knowledge on gene function and protein networks. In addition, new tools permit to modify organisms to perform desired tasks. Gene function analysis is speed up by novel approaches that couple both high throughput data generation and mining. Synthetic biology is an emerging field that uses tools for generating novel gene networks, whole genome synthesis and engineering. New applications in biotechnological, pharmaceutical and biomedical research are envisioned for synthetic biology. In recent years these new strategies have opened up the possibilities to study gene and genome editing, creation of novel tools for functional studies in virus, parasites and pathogenic bacteria. There is also the possibility to re-design organisms to generate vaccine subunits or produce new pharmaceuticals to combat multi-drug resistant pathogens. In this review we provide our opinion on the applicability of synthetic biology strategies for functional studies of pathogenic organisms and some applications such as genome editing and gene network studies to further comprehend virulence factors and determinants in pathogenic organisms. We also discuss what we consider important ethical issues for this field of molecular biology, especially for potential misuse of the new technologies. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Unlocking Triticeae genomics to sustainably feed the future

PubMed Central

Mochida, Keiichi; Shinozaki, Kazuo

2013-01-01

The tribe Triticeae includes the major crops wheat and barley. Within the last few years, the whole genomes of four Triticeae species—barley, wheat, Tausch’s goatgrass (Aegilops tauschii) and wild einkorn wheat (Triticum urartu)—have been sequenced. The availability of these genomic resources for Triticeae plants and innovative analytical applications using next-generation sequencing technologies are helping to revitalize our approaches in genetic work and to accelerate improvement of the Triticeae crops. Comparative genomics and integration of genomic resources from Triticeae plants and the model grass Brachypodium distachyon are aiding the discovery of new genes and functional analyses of genes in Triticeae crops. Innovative approaches and tools such as analysis of next-generation populations, evolutionary genomics and systems approaches with mathematical modeling are new strategies that will help us discover alleles for adaptive traits to future agronomic environments. In this review, we provide an update on genomic tools for use with Triticeae plants and Brachypodium and describe emerging approaches toward crop improvements in Triticeae. PMID:24204022

Regulatory variation: an emerging vantage point for cancer biology.

PubMed

Li, Luolan; Lorzadeh, Alireza; Hirst, Martin

2014-01-01

Transcriptional regulation involves complex and interdependent interactions of noncoding and coding regions of the genome with proteins that interact and modify them. Genetic variation/mutation in coding and noncoding regions of the genome can drive aberrant transcription and disease. In spite of accounting for nearly 98% of the genome comparatively little is known about the contribution of noncoding DNA elements to disease. Genome-wide association studies of complex human diseases including cancer have revealed enrichment for variants in the noncoding genome. A striking finding of recent cancer genome re-sequencing efforts has been the previously underappreciated frequency of mutations in epigenetic modifiers across a wide range of cancer types. Taken together these results point to the importance of dysregulation in transcriptional regulatory control in genesis of cancer. Powered by recent technological advancements in functional genomic profiling, exploration of normal and transformed regulatory networks will provide novel insight into the initiation and progression of cancer and open new windows to future prognostic and diagnostic tools. © 2013 Wiley Periodicals, Inc.

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

PubMed

Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

2018-05-14

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

The Importance of Normalization on Large and Heterogeneous Microarray Datasets

EPA Science Inventory

DNA microarray technology is a powerful functional genomics tool increasingly used for investigating global gene expression in environmental studies. Microarrays can also be used in identifying biological networks, as they give insight on the complex gene-to-gene interactions, ne...

Customizing the Connectivity Map Approach for Functional Evaluation in Toxicogenomics Studies (SOT)

EPA Science Inventory

Evaluating effects on the transcriptome can provide insight on putative chemical-specific mechanisms of action (MOAs). With whole genome transcriptomics technologies becoming more amenable to high-throughput screening, libraries of chemicals can be evaluated in vitro to produce l...

Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion.

PubMed

Vaschetto, Luis María

2018-04-01

In a recent past, transposable elements (TEs) were referred to as selfish genetic components only capable of copying themselves with the aim of increasing the odds of being inherited. Nonetheless, TEs have been initially proposed as positive control elements acting in synergy with the host. Nowadays, it is well known that TE movement into host genome comprises an important evolutionary mechanism capable of increasing the adaptive fitness. As insights into TE functioning are increasing day to day, the manipulation of transposition has raised an interesting possibility of setting the host functions, although the lack of appropriate genome engineering tools has unpaved it. Fortunately, the emergence of genome editing technologies based on programmable nucleases, and especially the arrival of a multipurpose RNA-guided Cas9 endonuclease system, has made it possible to reconsider this challenge. For such purpose, a particular type of transposons referred to as miniature inverted-repeat transposable elements (MITEs) has shown a series of interesting characteristics for designing functional drivers. Here, recent insights into MITE elements and versatile RNA-guided CRISPR/Cas9 genome engineering system are given to understand how to deploy the potential of TEs for control of the host transcriptional activity.

We have AN Increasing Need to Model Ourselves

NASA Astrophysics Data System (ADS)

Farmer, J. Doyne

Pierre Teilhard de Chardin referred to the fusion of biological life, human culture, and technology as the noosphere. Technological improvement is causing the noosphere to evolve rapidly, driving the enormous increase in human population over the last 10,000 years and the transformation (and devastation) of the biosphere. The rapid proliferation of the internet is changing human culture, including everything from the way we find mates to the way democracy functions, or fails to function. The emergence of the BINC (Bio, Info, Nano, Cogno) technologies promises to further accelerate this change. We are acquiring an ever-increasing ability to engineer devices at a molecular level, to control the genome, and to create new forms of life and intelligence...

New Developments in CRISPR Technology: Improvements in Specificity and Efficiency.

PubMed

Safari, Fatemeh; Farajnia, Safar; Ghasemi, Younes; Zarghami, Nosratollah

2017-01-01

RNA-guided endonuclease as a versatile genome editing technology opened new windows in various fields of biology. The simplicity of this revolutionary technique provides a promising future for its application in a broad range of approaches from functional annotation of genes to diseases, to genetic manipulation and gene therapy. Besides the site-specific activity of Cas9 endonuclease, the unintended cleavage known as off-target effect is still a major challenge for this genome editing technique. Various strategies have been developed to resolve this bottleneck including development of new softwares for designing optimized guide RNA (gRNA), engineering Cas9 enzyme, improvement in off-target detection assays, etc. Results: This review dedicated to discuss on methods that have been used for optimizing Cas9, specificity with the aim of improving this technology for therapeutic applications. In addition, the applications and novel breakthroughs in the field of CRISPR technology will be described. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

PubMed

Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

2018-01-01

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect

PubMed Central

Xu, Hanfu; O'Brochta, David A.

2015-01-01

Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed. PMID:26108630

Comparative Genomics in Drosophila.

PubMed

Oti, Martin; Pane, Attilio; Sammeth, Michael

2018-01-01

Since the pioneering studies of Thomas Hunt Morgan and coworkers at the dawn of the twentieth century, Drosophila melanogaster and its sister species have tremendously contributed to unveil the rules underlying animal genetics, development, behavior, evolution, and human disease. Recent advances in DNA sequencing technologies launched Drosophila into the post-genomic era and paved the way for unprecedented comparative genomics investigations. The complete sequencing and systematic comparison of the genomes from 12 Drosophila species represents a milestone achievement in modern biology, which allowed a plethora of different studies ranging from the annotation of known and novel genomic features to the evolution of chromosomes and, ultimately, of entire genomes. Despite the efforts of countless laboratories worldwide, the vast amount of data that were produced over the past 15 years is far from being fully explored.In this chapter, we will review some of the bioinformatic approaches that were developed to interrogate the genomes of the 12 Drosophila species. Setting off from alignments of the entire genomic sequences, the degree of conservation can be separately evaluated for every region of the genome, providing already first hints about elements that are under purifying selection and therefore likely functional. Furthermore, the careful analysis of repeated sequences sheds light on the evolutionary dynamics of transposons, an enigmatic and fascinating class of mobile elements housed in the genomes of animals and plants. Comparative genomics also aids in the computational identification of the transcriptionally active part of the genome, first and foremost of protein-coding loci, but also of transcribed nevertheless apparently noncoding regions, which were once considered "junk" DNA. Eventually, the synergy between functional and comparative genomics also facilitates in silico and in vivo studies on cis-acting regulatory elements, like transcription factor binding sites, that due to the high degree of sequence variability usually impose increased challenges for bioinformatics approaches.

Opening plenary speaker: Human genomics, precision medicine, and advancing human health.

PubMed

Green, Eric D

2016-08-01

Starting with the launch of the Human Genome Project in 1990, the past quarter-century has brought spectacular achievements in genomics that dramatically empower the study of human biology and disease. The human genomics enterprise is now in the midst of an important transition, as the growing foundation of genomic knowledge is being used by researchers and clinicians to tackle increasingly complex problems in biomedicine. Of particular prominence is the use of revolutionary new DNA sequencing technologies for generating prodigious amounts of DNA sequence data to elucidate the complexities of genome structure, function, and evolution, as well as to unravel the genomic bases of rare and common diseases. Together, these developments are ushering in the era of genomic medicine. Augmenting the advances in human genomics have been innovations in technologies for measuring environmental and lifestyle information, electronic health records, and data science; together, these provide opportunities of unprecedented scale and scope for investigating the underpinnings of health and disease. To capitalize on these opportunities, U.S. President Barack Obama recently announced a major new research endeavor - the U.S. Precision Medicine Initiative. This bold effort will be framed around several key aims, which include accelerating the use of genomically informed approaches to cancer care, making important policy and regulatory changes, and establishing a large research cohort of >1 million volunteers to facilitate precision medicine research. The latter will include making the partnership with all participants a centerpiece feature in the cohort's design and development. The Precision Medicine Initiative represents a broad-based research program that will allow new approaches for individualized medical care to be rigorously tested, so as to establish a new evidence base for advancing clinical practice and, eventually, human health.

Toward Genomics-Based Breeding in C3 Cool-Season Perennial Grasses.

PubMed

Talukder, Shyamal K; Saha, Malay C

2017-01-01

Most important food and feed crops in the world belong to the C3 grass family. The future of food security is highly reliant on achieving genetic gains of those grasses. Conventional breeding methods have already reached a plateau for improving major crops. Genomics tools and resources have opened an avenue to explore genome-wide variability and make use of the variation for enhancing genetic gains in breeding programs. Major C3 annual cereal breeding programs are well equipped with genomic tools; however, genomic research of C3 cool-season perennial grasses is lagging behind. In this review, we discuss the currently available genomics tools and approaches useful for C3 cool-season perennial grass breeding. Along with a general review, we emphasize the discussion focusing on forage grasses that were considered orphan and have little or no genetic information available. Transcriptome sequencing and genotype-by-sequencing technology for genome-wide marker detection using next-generation sequencing (NGS) are very promising as genomics tools. Most C3 cool-season perennial grass members have no prior genetic information; thus NGS technology will enhance collinear study with other C3 model grasses like Brachypodium and rice. Transcriptomics data can be used for identification of functional genes and molecular markers, i.e., polymorphism markers and simple sequence repeats (SSRs). Genome-wide association study with NGS-based markers will facilitate marker identification for marker-assisted selection. With limited genetic information, genomic selection holds great promise to breeders for attaining maximum genetic gain of the cool-season C3 perennial grasses. Application of all these tools can ensure better genetic gains, reduce length of selection cycles, and facilitate cultivar development to meet the future demand for food and fodder.

Discovery of common sequences absent in the human reference genome using pooled samples from next generation sequencing.

PubMed

Liu, Yu; Koyutürk, Mehmet; Maxwell, Sean; Xiang, Min; Veigl, Martina; Cooper, Richard S; Tayo, Bamidele O; Li, Li; LaFramboise, Thomas; Wang, Zhenghe; Zhu, Xiaofeng; Chance, Mark R

2014-08-16

Sequences up to several megabases in length have been found to be present in individual genomes but absent in the human reference genome. These sequences may be common in populations, and their absence in the reference genome may indicate rare variants in the genomes of individuals who served as donors for the human genome project. As the reference genome is used in probe design for microarray technology and mapping short reads in next generation sequencing (NGS), this missing sequence could be a source of bias in functional genomic studies and variant analysis. One End Anchor (OEA) and/or orphan reads from paired-end sequencing have been used to identify novel sequences that are absent in reference genome. However, there is no study to investigate the distribution, evolution and functionality of those sequences in human populations. To systematically identify and study the missing common sequences (micSeqs), we extended the previous method by pooling OEA reads from large number of individuals and applying strict filtering methods to remove false sequences. The pipeline was applied to data from phase 1 of the 1000 Genomes Project. We identified 309 micSeqs that are present in at least 1% of the human population, but absent in the reference genome. We confirmed 76% of these 309 micSeqs by comparison to other primate genomes, individual human genomes, and gene expression data. Furthermore, we randomly selected fifteen micSeqs and confirmed their presence using PCR validation in 38 additional individuals. Functional analysis using published RNA-seq and ChIP-seq data showed that eleven micSeqs are highly expressed in human brain and three micSeqs contain transcription factor (TF) binding regions, suggesting they are functional elements. In addition, the identified micSeqs are absent in non-primates and show dynamic acquisition during primate evolution culminating with most micSeqs being present in Africans, suggesting some micSeqs may be important sources of human diversity. 76% of micSeqs were confirmed by a comparative genomics approach. Fourteen micSeqs are expressed in human brain or contain TF binding regions. Some micSeqs are primate-specific, conserved and may play a role in the evolution of primates.

Genomic and transcriptomic approaches to study immunology in cyprinids: What is next?

PubMed

Petit, Jules; David, Lior; Dirks, Ron; Wiegertjes, Geert F

2017-10-01

Accelerated by the introduction of Next-Generation Sequencing (NGS), a number of genomes of cyprinid fish species have been drafted, leading to a highly valuable collective resource of comparative genome information on cyprinids (Cyprinidae). In addition, NGS-based transcriptome analyses of different developmental stages, organs, or cell types, increasingly contribute to the understanding of complex physiological processes, including immune responses. Cyprinids are a highly interesting family because they comprise one of the most-diversified families of teleosts and because of their variation in ploidy level, with diploid, triploid, tetraploid, hexaploid and sometimes even octoploid species. The wealth of data obtained from NGS technologies provides both challenges and opportunities for immunological research, which will be discussed here. Correct interpretation of ploidy effects on immune responses requires knowledge of the degree of functional divergence between duplicated genes, which can differ even between closely-related cyprinid fish species. We summarize NGS-based progress in analysing immune responses and discuss the importance of respecting the presence of (multiple) duplicated gene sequences when performing transcriptome analyses for detailed understanding of complex physiological processes. Progressively, advances in NGS technology are providing workable methods to further elucidate the implications of gene duplication events and functional divergence of duplicates genes and proteins involved in immune responses in cyprinids. We conclude with discussing how future applications of NGS technologies and analysis methods could enhance immunological research and understanding. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution.

PubMed

Gilles, Anna F; Averof, Michalis

2014-01-01

Developmental biology, as all experimental science, is empowered by technological advances. The availability of genetic tools in some species - designated as model organisms - has driven their use as major platforms for understanding development, physiology and behavior. Extending these tools to a wider range of species determines whether (and how) we can experimentally approach developmental diversity and evolution. During the last two decades, comparative developmental biology (evo-devo) was marked by the introduction of gene knockdown and deep sequencing technologies that are applicable to a wide range of species. These approaches allowed us to test the developmental role of specific genes in diverse species, to study biological processes that are not accessible in established models and, in some cases, to conduct genome-wide screens that overcome the limitations of the candidate gene approach. The recent discovery of CRISPR/Cas as a means of precise alterations into the genome promises to revolutionize developmental genetics. In this review we describe the development of gene editing tools, from zinc-finger nucleases to TALENs and CRISPR, and examine their application in gene targeting, their limitations and the opportunities they present for evo-devo. We outline their use in gene knock-out and knock-in approaches, and in manipulating gene functions by directing molecular effectors to specific sites in the genome. The ease-of-use and efficiency of CRISPR in diverse species provide an opportunity to close the technology gap that exists between established model organisms and emerging genetically-tractable species.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas

The number of genomes from uncultivated microbes will soon surpass the number of isolate genomes in public databases (Hugenholtz, Skarshewski, & Parks, 2016). Technological advancements in high-throughput sequencing and assembly, including single-cell genomics and the computational extraction of genomes from metagenomes (GFMs), are largely responsible. Here we propose community standards for reporting the Minimum Information about a Single-Cell Genome (MIxS-SCG) and Minimum Information about Genomes extracted From Metagenomes (MIxS-GFM) specific for Bacteria and Archaea. The standards have been developed in the context of the International Genomics Standards Consortium (GSC) community (Field et al., 2014) and can be viewed as amore » supplement to other GSC checklists including the Minimum Information about a Genome Sequence (MIGS), Minimum information about a Metagenomic Sequence(s) (MIMS) (Field et al., 2008) and Minimum Information about a Marker Gene Sequence (MIMARKS) (P. Yilmaz et al., 2011). Community-wide acceptance of MIxS-SCG and MIxS-GFM for Bacteria and Archaea will enable broad comparative analyses of genomes from the majority of taxa that remain uncultivated, improving our understanding of microbial function, ecology, and evolution.« less

RNA regulatory networks in animals and plants: a long noncoding RNA perspective.

PubMed

Bai, Youhuang; Dai, Xiaozhuan; Harrison, Andrew P; Chen, Ming

2015-03-01

A recent highlight of genomics research has been the discovery of many families of transcripts which have function but do not code for proteins. An important group is long noncoding RNAs (lncRNAs), which are typically longer than 200 nt, and whose members originate from thousands of loci across genomes. We review progress in understanding the biogenesis and regulatory mechanisms of lncRNAs. We describe diverse computational and high throughput technologies for identifying and studying lncRNAs. We discuss the current knowledge of functional elements embedded in lncRNAs as well as insights into the lncRNA-based regulatory network in animals. We also describe genome-wide studies of large amount of lncRNAs in plants, as well as knowledge of selected plant lncRNAs with a focus on biotic/abiotic stress-responsive lncRNAs. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

ArrayExpress update--trends in database growth and links to data analysis tools.

PubMed

Rustici, Gabriella; Kolesnikov, Nikolay; Brandizi, Marco; Burdett, Tony; Dylag, Miroslaw; Emam, Ibrahim; Farne, Anna; Hastings, Emma; Ison, Jon; Keays, Maria; Kurbatova, Natalja; Malone, James; Mani, Roby; Mupo, Annalisa; Pedro Pereira, Rui; Pilicheva, Ekaterina; Rung, Johan; Sharma, Anjan; Tang, Y Amy; Ternent, Tobias; Tikhonov, Andrew; Welter, Danielle; Williams, Eleanor; Brazma, Alvis; Parkinson, Helen; Sarkans, Ugis

2013-01-01

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is one of three international functional genomics public data repositories, alongside the Gene Expression Omnibus at NCBI and the DDBJ Omics Archive, supporting peer-reviewed publications. It accepts data generated by sequencing or array-based technologies and currently contains data from almost a million assays, from over 30 000 experiments. The proportion of sequencing-based submissions has grown significantly over the last 2 years and has reached, in 2012, 15% of all new data. All data are available from ArrayExpress in MAGE-TAB format, which allows robust linking to data analysis and visualization tools, including Bioconductor and GenomeSpace. Additionally, R objects, for microarray data, and binary alignment format files, for sequencing data, have been generated for a significant proportion of ArrayExpress data.

Breeding nursery tissue collection for possible genomic analysis

USDA-ARS?s Scientific Manuscript database

Phenotyping is considered a major bottleneck in breeding programs. With new genomic technologies, high throughput genotype schemes are constantly being developed. However, every genomic technology requires phenotypic data to inform prediction models generated from the technology. Forage breeders con...

DArT Markers Effectively Target Gene Space in the Rye Genome

PubMed Central

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye (Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes. PMID:27833625

DArT Markers Effectively Target Gene Space in the Rye Genome.

PubMed

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye ( Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes.

GENETICALLY MODIFIED FOODS: TECHNOLOGICAL BREAKTHROUGH OR ECOLOGICAL NIGHMARE?

EPA Science Inventory

Fifty years ago, Wastson and Crick described the structure of DNA, setting the stage for the past decade's biotechnology revolution. Scientists have now broken the code of the entire human genome, and delineated the function of multiple genes; similar strides are being taken with...

Proteomic approaches in brain research and neuropharmacology.

PubMed

Vercauteren, Freya G G; Bergeron, John J M; Vandesande, Frans; Arckens, Lut; Quirion, Rémi

2004-10-01

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

PubMed

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E; Irmler, Stefan; Ahrens, Christian H

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus -to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus . Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level

PubMed Central

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R.; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E.; Irmler, Stefan; Ahrens, Christian H.

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level. PMID:29441050

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development.

PubMed

Singer, Ruth; Atar, Shimshi; Atias, Osnat; Oron, Efrat; Segal, Daniel; Hirsch, Joel A; Tuller, Tamir; Orian, Amir; Chamovitz, Daniel A

2014-09-01

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Analysis of Musculoskeletal Systems and Their Diseases. The research for musculoskeletal disease using genome editing technology].

PubMed

Suzuki, Hidetsugu; Asahara, Hiroshi

2015-08-01

Genome editing is a genetic technology by which any DNA sequence is inserted, replaced or deleted. Genome editing has been making rapid progress recently, with the development of new techniques such as ZFN, TALEN and CRISPR/Cas9. Genome editing can be applied to various fields ranging from the production of knock out animals to gene therapy. This section summarizes these new genome editing technologies and its applications.

Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function.

PubMed

Mehrotra, Shweta; Goyal, Vinod

2014-08-01

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150-400 base pairs (bp) in length. Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as "tuning knobs" in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences. Copyright © 2014 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

PubMed

He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

2017-05-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

A Plant-Associated Microbe Genome Initiative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jan E. Leach; Scott Gold; Sue Tolin

2003-03-06

Plant-associated microorganisms are critical to agricultural and food security and are key components in maintaining the balance of our ecosystems. Some of these diverse microbes, which include viruses, bacteria, oomycetes, fungi, and nematodes, cause plant diseases, whereas others prevent diseases or enhance plant growth. Despite their importance, we know little about them on a genomic level. To intervene in disease and understand the basis of biological control or symbiotic relationships, a concerted and coordinated genomic analysis of these microbes is essential. Genome analysis, in this context, refers to the structural and functional analysis of the microbe DNA including the genes,more » the proteins encoded by those genes, as well as noncoding sequences involved in genome dynamics and function. The ultimate emphasis is on understanding genomic functions involved in plant associations. Members of The American Phytopathological Society (APS) developed a prioritized list of plant-associated microbes for genome analysis. With this list as a foundation for discussions, a Workshop on Genomic Analysis of Plant-Associated Microorganisms was held in Washington, D.C., on 9 to 11 April 2002. The workshop was organized by the Public Policy Board of APS, and was funded by the Department of Energy (DOE), the National Science Foundation (NSF), U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), and USDA-National Research Initiatives (USDA-NRI). The workshop included academic, industrial, and governmental experts from the genomics and microbial research communities and observers from the federal funding agencies. After reviewing current and near-term technologies, workshop participants proposed a comprehensive, international initiative to obtain the genomic information needed to understand these important microbes and their interactions with host plants and the environment. Specifically, the recommendations call for a 5-year, $500 million international public effort for genome analysis of plant-associated microbes. The goals are to (i) obtain genome sequence information for several representative groups of microbes; (ii) identify and determine function for the genes/proteins and other genomic elements involved in plant-microbe interactions; (iii) develop and implement standardized bioinformatic tools and a database system that is applicable across all microbes; and (iv) educate and train scientists with skills and knowledge of biological and computational sciences who will apply the information to the protection of our food sources and environment.« less

Inferring transposons activity chronology by TRANScendence - TEs database and de-novo mining tool.

PubMed

Startek, Michał Piotr; Nogły, Jakub; Gromadka, Agnieszka; Grzebelus, Dariusz; Gambin, Anna

2017-10-16

The constant progress in sequencing technology leads to ever increasing amounts of genomic data. In the light of current evidence transposable elements (TEs for short) are becoming useful tools for learning about the evolution of host genome. Therefore the software for genome-wide detection and analysis of TEs is of great interest. Here we describe the computational tool for mining, classifying and storing TEs from newly sequenced genomes. This is an online, web-based, user-friendly service, enabling users to upload their own genomic data, and perform de-novo searches for TEs. The detected TEs are automatically analyzed, compared to reference databases, annotated, clustered into families, and stored in TEs repository. Also, the genome-wide nesting structure of found elements are detected and analyzed by new method for inferring evolutionary history of TEs. We illustrate the functionality of our tool by performing a full-scale analyses of TE landscape in Medicago truncatula genome. TRANScendence is an effective tool for the de-novo annotation and classification of transposable elements in newly-acquired genomes. Its streamlined interface makes it well-suited for evolutionary studies.

WikiGenomes: an open web application for community consumption and curation of gene annotation data in Wikidata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Putman, Tim E.; Lelong, Sebastien; Burgstaller-Muehlbacher, Sebastian

With the advancement of genome-sequencing technologies, new genomes are being sequenced daily. Although these sequences are deposited in publicly available data warehouses, their functional and genomic annotations (beyond genes which are predicted automatically) mostly reside in the text of primary publications. Professional curators are hard at work extracting those annotations from the literature for the most studied organisms and depositing them in structured databases. However, the resources don’t exist to fund the comprehensive curation of the thousands of newly sequenced organisms in this manner. Here, we describe WikiGenomes (wikigenomes.org), a web application that facilitates the consumption and curation of genomicmore » data by the entire scientific community. WikiGenomes is based on Wikidata, an openly editable knowledge graph with the goal of aggregating published knowledge into a free and open database. WikiGenomes empowers the individual genomic researcher to contribute their expertise to the curation effort and integrates the knowledge into Wikidata, enabling it to be accessed by anyone without restriction.« less

WikiGenomes: an open web application for community consumption and curation of gene annotation data in Wikidata

DOE PAGES

Putman, Tim E.; Lelong, Sebastien; Burgstaller-Muehlbacher, Sebastian; ...

2017-03-06

With the advancement of genome-sequencing technologies, new genomes are being sequenced daily. Although these sequences are deposited in publicly available data warehouses, their functional and genomic annotations (beyond genes which are predicted automatically) mostly reside in the text of primary publications. Professional curators are hard at work extracting those annotations from the literature for the most studied organisms and depositing them in structured databases. However, the resources don’t exist to fund the comprehensive curation of the thousands of newly sequenced organisms in this manner. Here, we describe WikiGenomes (wikigenomes.org), a web application that facilitates the consumption and curation of genomicmore » data by the entire scientific community. WikiGenomes is based on Wikidata, an openly editable knowledge graph with the goal of aggregating published knowledge into a free and open database. WikiGenomes empowers the individual genomic researcher to contribute their expertise to the curation effort and integrates the knowledge into Wikidata, enabling it to be accessed by anyone without restriction.« less

Gene editing tools: state-of-the-art and the road ahead for the model and non-model fishes.

PubMed

Barman, Hirak Kumar; Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Ninawe, A S; Vengayil, Doyil T; Asrafuzzaman, Syed; Sundaray, Jitendra K; Jayasankar, Pallipuram

2017-10-01

Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.

CRISPR/Cas9 for cancer research and therapy.

PubMed

Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

2018-04-16

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

An integrative model for in-silico clinical-genomics discovery science.

PubMed

Lussier, Yves A; Sarkar, Indra Nell; Cantor, Michael

2002-01-01

Human Genome discovery research has set the pace for Post-Genomic Discovery Research. While post-genomic fields focused at the molecular level are intensively pursued, little effort is being deployed in the later stages of molecular medicine discovery research, such as clinical-genomics. The objective of this study is to demonstrate the relevance and significance of integrating mainstream clinical informatics decision support systems to current bioinformatics genomic discovery science. This paper is a feasibility study of an original model enabling novel "in-silico" clinical-genomic discovery science and that demonstrates its feasibility. This model is designed to mediate queries among clinical and genomic knowledge bases with relevant bioinformatic analytic tools (e.g. gene clustering). Briefly, trait-disease-gene relationships were successfully illustrated using QMR, OMIM, SNOMED-RT, GeneCluster and TreeView. The analyses were visualized as two-dimensional dendrograms of clinical observations clustered around genes. To our knowledge, this is the first study using knowledge bases of clinical decision support systems for genomic discovery. Although this study is a proof of principle, it provides a framework for the development of clinical decision-support-system driven, high-throughput clinical-genomic technologies which could potentially unveil significant high-level functions of genes.

In vitro manipulation of gene expression in larval Schistosoma: a model for postgenomic approaches in Trematoda

PubMed Central

YOSHINO, TIMOTHY P.; DINGUIRARD, NATHALIE; DE MORAES MOURÃO, MARINA

2013-01-01

SUMMARY With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches. PMID:19961646

iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

PubMed

Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

2015-09-01

High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. © 2014 Society for Laboratory Automation and Screening.

Genomics and molecular breeding in lesser explored pulse crops: current trends and future opportunities.

PubMed

Bohra, Abhishek; Jha, Uday Chand; Kishor, P B Kavi; Pandey, Shailesh; Singh, Narendra P

2014-12-01

Pulses are multipurpose crops for providing income, employment and food security in the underprivileged regions, notably the FAO-defined low-income food-deficit countries. Owing to their intrinsic ability to endure environmental adversities and the least input/management requirements, these crops remain central to subsistence farming. Given their pivotal role in rain-fed agriculture, substantial research has been invested to boost the productivity of these pulse crops. To this end, genomic tools and technologies have appeared as the compelling supplement to the conventional breeding. However, the progress in minor pulse crops including dry beans (Vigna spp.), lupins, lablab, lathyrus and vetches has remained unsatisfactory, hence these crops are often labeled as low profile or lesser researched. Nevertheless, recent scientific and technological breakthroughs particularly the next generation sequencing (NGS) are radically transforming the scenario of genomics and molecular breeding in these minor crops. NGS techniques have allowed de novo assembly of whole genomes in these orphan crops. Moreover, the availability of a reference genome sequence would promote re-sequencing of diverse genotypes to unlock allelic diversity at a genome-wide scale. In parallel, NGS has offered high-resolution genetic maps or more precisely, a robust genetic framework to implement whole-genome strategies for crop improvement. As has already been demonstrated in lupin, sequencing-based genotyping of the representative sample provided access to a number of functionally-relevant markers that could be deployed straight away in crop breeding programs. This article attempts to outline the recent progress made in genomics of these lesser explored pulse crops, and examines the prospects of genomics assisted integrated breeding to enhance and stabilize crop yields. Copyright © 2014 Elsevier Inc. All rights reserved.

Genomics and transcriptomics in drug discovery.

PubMed

Dopazo, Joaquin

2014-02-01

The popularization of genomic high-throughput technologies is causing a revolution in biomedical research and, particularly, is transforming the field of drug discovery. Systems biology offers a framework to understand the extensive human genetic heterogeneity revealed by genomic sequencing in the context of the network of functional, regulatory and physical protein-drug interactions. Thus, approaches to find biomarkers and therapeutic targets will have to take into account the complex system nature of the relationships of the proteins with the disease. Pharmaceutical companies will have to reorient their drug discovery strategies considering the human genetic heterogeneity. Consequently, modeling and computational data analysis will have an increasingly important role in drug discovery. Copyright © 2013 Elsevier Ltd. All rights reserved.

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Application of metagenomics technologies for antimicrobial resistance and food safety research and beyond

USDA-ARS?s Scientific Manuscript database

Current developments in the field of metagenomics in biological sciences have demonstrated the need and potential usefulness of taxonomical and functional analyses of meta-omics data generated by genomics, transcriptomics, proteomics, and metabolomics. This review will provide a general overview of...

The impact of next-generation sequencing on genomics

PubMed Central

Zhang, Jun; Chiodini, Rod; Badr, Ahmed; Zhang, Genfa

2011-01-01

This article reviews basic concepts, general applications, and the potential impact of next-generation sequencing (NGS) technologies on genomics, with particular reference to currently available and possible future platforms and bioinformatics. NGS technologies have demonstrated the capacity to sequence DNA at unprecedented speed, thereby enabling previously unimaginable scientific achievements and novel biological applications. But, the massive data produced by NGS also presents a significant challenge for data storage, analyses, and management solutions. Advanced bioinformatic tools are essential for the successful application of NGS technology. As evidenced throughout this review, NGS technologies will have a striking impact on genomic research and the entire biological field. With its ability to tackle the unsolved challenges unconquered by previous genomic technologies, NGS is likely to unravel the complexity of the human genome in terms of genetic variations, some of which may be confined to susceptible loci for some common human conditions. The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come. PMID:21477781

[Current advances and future prospects of genome editing technology in the field of biomedicine.

PubMed

Sakuma, Tetsushi

Genome editing technology can alter the genomic sequence at will, contributing the creation of cellular and animal models of human diseases including hereditary disorders and cancers, and the generation of the mutation-corrected human induced pluripotent stem cells for ex vivo regenerative medicine. In addition, novel approaches such as drug development using genome-wide CRISPR screening and cancer suppression using epigenome editing technology, which can change the epigenetic modifications in a site-specific manner, have also been conducted. In this article, I summarize the current advances and future prospects of genome editing technology in the field of biomedicine.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases.

PubMed

Shen, Li; Shao, Ningyi; Liu, Xiaochuan; Nestler, Eric

2014-04-15

Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. We have developed ngs.plot - a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases

PubMed Central

2014-01-01

Background Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. Results We have developed ngs.plot – a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. Conclusions We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data. PMID:24735413

De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes

PubMed Central

Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

2015-01-01

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37–100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

INDIGO – INtegrated Data Warehouse of MIcrobial GenOmes with Examples from the Red Sea Extremophiles

PubMed Central

Alam, Intikhab; Antunes, André; Kamau, Allan Anthony; Ba alawi, Wail; Kalkatawi, Manal; Stingl, Ulrich; Bajic, Vladimir B.

2013-01-01

Background The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes. Results We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments. Conclusions We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo. PMID:24324765

INDIGO - INtegrated data warehouse of microbial genomes with examples from the red sea extremophiles.

PubMed

Alam, Intikhab; Antunes, André; Kamau, Allan Anthony; Ba Alawi, Wail; Kalkatawi, Manal; Stingl, Ulrich; Bajic, Vladimir B

2013-01-01

The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes. We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments. We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo.

Resolving the homology—function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

PubMed Central

Klinger, Christen M.; Ramirez-Macias, Inmaculada; Herman, Emily K.; Turkewitz, Aaron P.; Field, Mark C.; Dacks, Joel B.

2016-01-01

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage. PMID:27444378

Identification of mutated driver pathways in cancer using a multi-objective optimization model.

PubMed

Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

2016-05-01

New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

PubMed Central

Li, Ming; Bui, Michelle; Yang, Ting; Bowman, Christian S.; White, Bradley J.; Akbari, Omar S.

2017-01-01

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives. PMID:29138316

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.

PubMed

Li, Ming; Bui, Michelle; Yang, Ting; Bowman, Christian S; White, Bradley J; Akbari, Omar S

2017-12-05

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives. Copyright © 2017 the Author(s). Published by PNAS.

Genome Editing by CRISPR/Cas9: a Game Change in the Genetic Manipulation of Protists

PubMed Central

Lander, Noelia; Chiurillo, Miguel A.; Docampo, Roberto

2016-01-01

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii. PMID:27315329

Trinity: Transcriptome Assembly for Genetic and Functional Analysis of Cancer | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The cancer transcriptome is shaped by genetic changes, variation in gene transcription, mRNA processing, editing and stability, and the cancer microbiome. Deciphering this variation and understanding its implications on tumorigenesis requires sophisticated computational analyses. Most RNA-Seq analyses rely on methods that first map short reads to a reference genome, and then compare them to annotated transcripts or assemble them. However, this strategy can be limited when the cancer genome is substantially different than the reference or for detecting sequences from the cancer microbiome.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral research position is available in the laboratory of Dr. Natasha Caplen in the Genetics Branch, National Cancer Institute, Center for Cancer Research. Dr. Caplen’s Laboratory conducts multidisciplinary and interdisciplinary research that uses RNAi and other DNA or RNA-based functional genomic technologies to interrogate specific aspects of the genetic,

HCMI Organization | Office of Cancer Genomics

Cancer.gov

Consortium The Human Cancer Models Initiative (HCMI) was created and funded by the US National Cancer Institute, Cancer Research UK, the foundation Hubrecht Organoid Technology, and the Wellcome Sanger Institute. Together, these organizations develop policy and make programmatic decisions to contribute to the function of the HCMI. National Cancer Institute

Mapping DNA Methylation with High Throughput Nanopore Sequencing

PubMed Central

Rand, Arthur C.; Jain, Miten; Eizenga, Jordan M.; Musselman-Brown, Audrey; Olsen, Hugh E.; Akeson, Mark

2017-01-01

Chemical modifications to DNA regulate its biological function. We present a framework for mapping methylation to cytosine and adenosine with the Oxford Nanopore Technologies MinION using its ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in E. coli. PMID:28218897

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

PubMed Central

Barrangou, Rodolphe; Birmingham, Amanda; Wiemann, Stefan; Beijersbergen, Roderick L.; Hornung, Veit; Smith, Anja van Brabant

2015-01-01

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications. PMID:25800748

Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system

NASA Astrophysics Data System (ADS)

Luo, Wei; Marks, Daniel L.; Ralston, Tyler S.; Boppart, Stephen A.

2006-03-01

Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-µm axial and 15-µm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics.

An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.

PubMed

Hay, Elizabeth Anne; Khalaf, Abdulla Razak; Marini, Pietro; Brown, Andrew; Heath, Karyn; Sheppard, Darrin; MacKenzie, Alasdair

2017-08-01

We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of "off target" effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genomics and integrated systems biology in Plasmodium falciparum: a path to malaria control and eradication.

PubMed

Le Roch, K G; Chung, D-W D; Ponts, N

2012-01-01

The first draft of the human malaria parasite's genome was released in 2002. Since then, the malaria scientific community has witnessed a steady embrace of new and powerful functional genomic studies. Over the years, these approaches have slowly revolutionized malaria research and enabled the comprehensive, unbiased investigation of various aspects of the parasite's biology. These genome-wide analyses delivered a refined annotation of the parasite's genome, delivered a better knowledge of its RNA, proteins and metabolite derivatives, and fostered the discovery of new vaccine and drug targets. Despite the positive impacts of these genomic studies, most research and investment still focus on protein targets, drugs and vaccine candidates that were known before the publication of the parasite genome sequence. However, recent access to next-generation sequencing technologies, along with an increased number of genome-wide applications, is expanding the impact of the parasite genome on biomedical research, contributing to a paradigm shift in research activities that may possibly lead to new optimized diagnosis and treatments. This review provides an update of Plasmodium falciparum genome sequences and an overview of the rapid development of genomics and system biology applications that have an immense potential of creating powerful tools for a successful malaria eradication campaign. © 2011 Blackwell Publishing Ltd.

Whole Genome Sequencing of Greater Amberjack (Seriola dumerili) for SNP Identification on Aligned Scaffolds and Genome Structural Variation Analysis Using Parallel Resequencing

PubMed Central

Aokic, Jun-ya; Kawase, Junya; Hamada, Kazuhisa; Fujimoto, Hiroshi; Yamamoto, Ikki; Usuki, Hironori

2018-01-01

Greater amberjack (Seriola dumerili) is distributed in tropical and temperate waters worldwide and is an important aquaculture fish. We carried out de novo sequencing of the greater amberjack genome to construct a reference genome sequence to identify single nucleotide polymorphisms (SNPs) for breeding amberjack by marker-assisted or gene-assisted selection as well as to identify functional genes for biological traits. We obtained 200 times coverage and constructed a high-quality genome assembly using next generation sequencing technology. The assembled sequences were aligned onto a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map by sequence homology. A total of 215 of the longest amberjack sequences, with a total length of 622.8 Mbp (92% of the total length of the genome scaffolds), were lined up on the yellowtail RH map. We resequenced the whole genomes of 20 greater amberjacks and mapped the resulting sequences onto the reference genome sequence. About 186,000 nonredundant SNPs were successfully ordered on the reference genome. Further, we found differences in the genome structural variations between two greater amberjack populations using BreakDancer. We also analyzed the greater amberjack transcriptome and mapped the annotated sequences onto the reference genome sequence. PMID:29785397

Metabolic Network Modeling of Microbial Communities

PubMed Central

Biggs, Matthew B.; Medlock, Gregory L.; Kolling, Glynis L.

2015-01-01

Genome-scale metabolic network reconstructions and constraint-based analysis are powerful methods that have the potential to make functional predictions about microbial communities. Current use of genome-scale metabolic networks to characterize the metabolic functions of microbial communities includes species compartmentalization, separating species-level and community-level objectives, dynamic analysis, the “enzyme-soup” approach, multi-scale modeling, and others. There are many challenges inherent to the field, including a need for tools that accurately assign high-level omics signals to individual community members, new automated reconstruction methods that rival manual curation, and novel algorithms for integrating omics data and engineering communities. As technologies and modeling frameworks improve, we expect that there will be proportional advances in the fields of ecology, health science, and microbial community engineering. PMID:26109480

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

PubMed Central

Swanton, Charles; Szallasi, Zoltan; Brenton, James D; Downward, Julian

2008-01-01

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP) as well as endocrine therapies such as tamoxifen. Given the limited power of microarray signatures to predict therapeutic response in associative studies of small clinical trial cohorts, the use of functional genomic data combined with expression or sequence analysis of genes and microRNAs implicated in drug response in human tumours may provide a more robust method to guide adjuvant treatment strategies in breast cancer that are transferable across different expression platforms and patient cohorts. PMID:18986507

Global Mapping of Transcription Factor Binding Sites by Sequencing Chromatin Surrogates: a Perspective on Experimental Design, Data Analysis, and Open Problems.

PubMed

Wei, Yingying; Wu, George; Ji, Hongkai

2013-05-01

Mapping genome-wide binding sites of all transcription factors (TFs) in all biological contexts is a critical step toward understanding gene regulation. The state-of-the-art technologies for mapping transcription factor binding sites (TFBSs) couple chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-seq) or tiling array hybridization (ChIP-chip). These technologies have limitations: they are low-throughput with respect to surveying many TFs. Recent advances in genome-wide chromatin profiling, including development of technologies such as DNase-seq, FAIRE-seq and ChIP-seq for histone modifications, make it possible to predict in vivo TFBSs by analyzing chromatin features at computationally determined DNA motif sites. This promising new approach may allow researchers to monitor the genome-wide binding sites of many TFs simultaneously. In this article, we discuss various experimental design and data analysis issues that arise when applying this approach. Through a systematic analysis of the data from the Encyclopedia Of DNA Elements (ENCODE) project, we compare the predictive power of individual and combinations of chromatin marks using supervised and unsupervised learning methods, and evaluate the value of integrating information from public ChIP and gene expression data. We also highlight the challenges and opportunities for developing novel analytical methods, such as resolving the one-motif-multiple-TF ambiguity and distinguishing functional and non-functional TF binding targets from the predicted binding sites. The online version of this article (doi:10.1007/s12561-012-9066-5) contains supplementary material, which is available to authorized users.

Transcriptionally active PCR for antigen identification and vaccine development: in vitro genome-wide screening and in vivo immunogenicity

PubMed Central

Regis, David P.; Dobaño, Carlota; Quiñones-Olson, Paola; Liang, Xiaowu; Graber, Norma L.; Stefaniak, Maureen E.; Campo, Joseph J.; Carucci, Daniel J.; Roth, David A.; He, Huaping; Felgner, Philip L.; Doolan, Denise L.

2009-01-01

We have evaluated a technology called Transcriptionally Active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data. PMID:18164079

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE PAGES

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.; ...

2015-01-14

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification†

PubMed Central

Wu, Liyou; Liu, Xueduan; Schadt, Christopher W.; Zhou, Jizhong

2006-01-01

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. PMID:16820490

Transcriptionally active PCR for antigen identification and vaccine development: in vitro genome-wide screening and in vivo immunogenicity.

PubMed

Regis, David P; Dobaño, Carlota; Quiñones-Olson, Paola; Liang, Xiaowu; Graber, Norma L; Stefaniak, Maureen E; Campo, Joseph J; Carucci, Daniel J; Roth, David A; He, Huaping; Felgner, Philip L; Doolan, Denise L

2008-03-01

We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data.

Understanding our Genetic Inheritance: The U.S. Human Genome Project, The First Five Years FY 1991--1995

DOE R&D Accomplishments Database

1990-04-01

The Human Genome Initiative is a worldwide research effort with the goal of analyzing the structure of human DNA and determining the location of the estimated 100,000 human genes. In parallel with this effort, the DNA of a set of model organisms will be studied to provide the comparative information necessary for understanding the functioning of the human genome. The information generated by the human genome project is expected to be the source book for biomedical science in the 21st century and will by of immense benefit to the field of medicine. It will help us to understand and eventually treat many of the more than 4000 genetic diseases that affect mankind, as well as the many multifactorial diseases in which genetic predisposition plays an important role. A centrally coordinated project focused on specific objectives is believed to be the most efficient and least expensive way of obtaining this information. The basic data produced will be collected in electronic databases that will make the information readily accessible on convenient form to all who need it. This report describes the plans for the U.S. human genome project and updates those originally prepared by the Office of Technology Assessment (OTA) and the National Research Council (NRC) in 1988. In the intervening two years, improvements in technology for almost every aspect of genomics research have taken place. As a result, more specific goals can now be set for the project.

Exploring the possibilities and limitations of a nanomaterials genome.

PubMed

Qian, Chenxi; Siler, Todd; Ozin, Geoffrey A

2015-01-07

What are we going to do with the cornucopia of nanomaterials appearing in the open and patent literature, every day? Imagine the benefits of an intelligent and convenient means of categorizing, organizing, sifting, sorting, connecting, and utilizing this information in scientifically and technologically innovative ways by building a Nanomaterials Genome founded upon an all-purpose Periodic Table of Nanomaterials. In this Concept article, inspired by work on the Human Genome project, which began in 1989 together with motivation from the recent emergence of the Materials Genome project initiated in 2011 and the Nanoinformatics Roadmap 2020 instigated in 2010, we envision the development of a Nanomaterials Genome (NMG) database with the most advanced data-mining tools that leverage inference engines to help connect and interpret patterns of nanomaterials information. It will be equipped with state-of-the-art visualization techniques that rapidly organize and picture, categorize and interrelate the inherited behavior of complex nanomatter from the information programmed in its constituent nanomaterials building blocks. A Nanomaterials Genome Initiative (NMGI) of the type imagined herein has the potential to serve the global nanoscience community with an opportunity to speed up the development continuum of nanomaterials through the innovation process steps of discovery, structure determination and property optimization, functionality elucidation, system design and integration, certification and manufacturing to deployment in technologies that apply these versatile nanomaterials in environmentally responsible ways. The possibilities and limitations of this concept are critically evaluated in this article. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome

USDA-ARS?s Scientific Manuscript database

Modern biological analyses are often assisted by recent technologies making the sequencing of complex genomes both technically possible and feasible. We recently sequenced the tomato genome that, like many eukaryotic genomes, is large and complex. Current sequencing technologies allow the developmen...

Integrating functional genomics to accelerate mechanistic personalized medicine.

PubMed

Tyner, Jeffrey W

2017-03-01

The advent of deep sequencing technologies has resulted in the deciphering of tremendous amounts of genetic information. These data have led to major discoveries, and many anecdotes now exist of individual patients whose clinical outcomes have benefited from novel, genetically guided therapeutic strategies. However, the majority of genetic events in cancer are currently undrugged, leading to a biological gap between understanding of tumor genetic etiology and translation to improved clinical approaches. Functional screening has made tremendous strides in recent years with the development of new experimental approaches to studying ex vivo and in vivo drug sensitivity. Numerous discoveries and anecdotes also exist for translation of functional screening into novel clinical strategies; however, the current clinical application of functional screening remains largely confined to small clinical trials at specific academic centers. The intersection between genomic and functional approaches represents an ideal modality to accelerate our understanding of drug sensitivities as they relate to specific genetic events and further understand the full mechanisms underlying drug sensitivity patterns.

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

PubMed Central

JIN, Li-Fang; LI, Jin-Song

2016-01-01

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

Embedded CMOS basecalling for nanopore DNA sequencing.

PubMed

Chengjie Wang; Junli Zheng; Magierowski, Sebastian; Ghafar-Zadeh, Ebrahim

2016-08-01

DNA sequencing based on nanopore sensors is now entering the marketplace. The ability to interface this technology to established CMOS microelectronics promises significant improvements in functionality and miniaturization. Among the key functions to benefit from this interface will be basecalling, the conversion of raw electronic molecular signatures to nucleotide sequence predictions. This paper presents the design and performance potential of custom CMOS base-callers embedded alongside nanopore sensors. A basecalliing architecture implemented in 32-nm technology is discussed with the ability to process the equivalent of 20 human genomes per day in real-time at a power density of 5 W/cm2 assuming a 3-mer nanopore sensor.

Translational bioinformatics in the cloud: an affordable alternative

PubMed Central

2010-01-01

With the continued exponential expansion of publicly available genomic data and access to low-cost, high-throughput molecular technologies for profiling patient populations, computational technologies and informatics are becoming vital considerations in genomic medicine. Although cloud computing technology is being heralded as a key enabling technology for the future of genomic research, available case studies are limited to applications in the domain of high-throughput sequence data analysis. The goal of this study was to evaluate the computational and economic characteristics of cloud computing in performing a large-scale data integration and analysis representative of research problems in genomic medicine. We find that the cloud-based analysis compares favorably in both performance and cost in comparison to a local computational cluster, suggesting that cloud computing technologies might be a viable resource for facilitating large-scale translational research in genomic medicine. PMID:20691073

Yeast 2.0-connecting the dots in the construction of the world's first functional synthetic eukaryotic genome.

PubMed

Pretorius, I S; Boeke, J D

2018-06-01

Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the biosafety, bioethics and regulatory aspects of their pioneering work. This article presents a shared vision of constructing a synthetic eukaryotic genome in a safe model organism by using novel concepts and advanced technologies. This multidisciplinary and collaborative project is conducted under a sound governance structure that does not only respect the scientific achievements and lessons from the past, but that is also focussed on leading the present and helping to secure a brighter future for all.

Yeast 2.0—connecting the dots in the construction of the world's first functional synthetic eukaryotic genome

PubMed Central

Boeke, J D

2018-01-01

Abstract Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors—driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell—are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders’ concerns about the biosafety, bioethics and regulatory aspects of their pioneering work. This article presents a shared vision of constructing a synthetic eukaryotic genome in a safe model organism by using novel concepts and advanced technologies. This multidisciplinary and collaborative project is conducted under a sound governance structure that does not only respect the scientific achievements and lessons from the past, but that is also focussed on leading the present and helping to secure a brighter future for all. PMID:29648592

The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: a structural biology web resource.

PubMed

Gifford, Lida K; Carter, Lester G; Gabanyi, Margaret J; Berman, Helen M; Adams, Paul D

2012-06-01

The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/ ) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB ( http://sbkb.org ), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a functional sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.

Technological Developments in lncRNA Biology.

PubMed

Jathar, Sonali; Kumar, Vikram; Srivastava, Juhi; Tripathi, Vidisha

2017-01-01

It is estimated that more than 90% of the mammalian genome is transcribed as non-coding RNAs. Recent evidences have established that these non-coding transcripts are not junk or just transcriptional noise, but they do serve important biological purpose. One of the rapidly expanding fields of this class of transcripts is the regulatory lncRNAs, which had been a major challenge in terms of their molecular functions and mechanisms of action. The emergence of high-throughput technologies and the development in various conventional approaches have led to the expansion of the lncRNA world. The combination of multidisciplinary approaches has proven to be essential to unravel the complexity of their regulatory networks and helped establish the importance of their existence. Here, we review the current methodologies available for discovering and investigating functions of long non-coding RNAs (lncRNAs) and focus on the powerful technological advancement available to specifically address their functional importance.

A survey of tools for variant analysis of next-generation genome sequencing data

PubMed Central

Pabinger, Stephan; Dander, Andreas; Fischer, Maria; Snajder, Rene; Sperk, Michael; Efremova, Mirjana; Krabichler, Birgit; Speicher, Michael R.; Zschocke, Johannes

2014-01-01

Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers. PMID:23341494

Supporting community annotation and user collaboration in the integrated microbial genomes (IMG) system.

PubMed

Chen, I-Min A; Markowitz, Victor M; Palaniappan, Krishna; Szeto, Ernest; Chu, Ken; Huang, Jinghua; Ratner, Anna; Pillay, Manoj; Hadjithomas, Michalis; Huntemann, Marcel; Mikhailova, Natalia; Ovchinnikova, Galina; Ivanova, Natalia N; Kyrpides, Nikos C

2016-04-26

The exponential growth of genomic data from next generation technologies renders traditional manual expert curation effort unsustainable. Many genomic systems have included community annotation tools to address the problem. Most of these systems adopted a "Wiki-based" approach to take advantage of existing wiki technologies, but encountered obstacles in issues such as usability, authorship recognition, information reliability and incentive for community participation. Here, we present a different approach, relying on tightly integrated method rather than "Wiki-based" method, to support community annotation and user collaboration in the Integrated Microbial Genomes (IMG) system. The IMG approach allows users to use existing IMG data warehouse and analysis tools to add gene, pathway and biosynthetic cluster annotations, to analyze/reorganize contigs, genes and functions using workspace datasets, and to share private user annotations and workspace datasets with collaborators. We show that the annotation effort using IMG can be part of the research process to overcome the user incentive and authorship recognition problems thus fostering collaboration among domain experts. The usability and reliability issues are addressed by the integration of curated information and analysis tools in IMG, together with DOE Joint Genome Institute (JGI) expert review. By incorporating annotation operations into IMG, we provide an integrated environment for users to perform deeper and extended data analysis and annotation in a single system that can lead to publications and community knowledge sharing as shown in the case studies.

Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery.

PubMed

Kaur, Sukhjiwan; Cogan, Noel O I; Pembleton, Luke W; Shinozuka, Maiko; Savin, Keith W; Materne, Michael; Forster, John W

2011-05-25

Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality. Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism. A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

Zinc-finger nucleases-based genome engineering to generate isogenic human cell lines.

PubMed

Dreyer, Anne-Kathrin; Cathomen, Toni

2012-01-01

Customized zinc-finger nucleases (ZFNs) have developed into a promising technology to precisely alter mammalian genomes for biomedical research, biotechnology, or human gene therapy. In the context of synthetic biology, the targeted integration of a transgene or reporter cassette into a "neutral site" of the human genome, such as the AAVS1 locus, permits the generation of isogenic human cell lines with two major advantages over standard genetic manipulation techniques: minimal integration site-dependent effects on the transgene and, vice versa, no functional perturbation of the host-cell transcriptome. Here we describe in detail how ZFNs can be employed to target integration of a transgene cassette into the AAVS1 locus and how to characterize the targeted cells by PCR-based genotyping.

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

Genetic and epigenetic control of gene expression by CRISPR–Cas systems

PubMed Central

Lo, Albert; Qi, Lei

2017-01-01

The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

Frequently Asked Questions about Genetic and Genomic Science

MedlinePlus

... of the new genetic and genomic techniques and technologies? Proteomics The suffix "-ome" comes from the Greek ... pharmacogenomics is one of the large-scale "omic" technologies, it can examine the entirety of the genome, ...

Using comparative genome analysis to identify problems in annotated microbial genomes.

PubMed

Poptsova, Maria S; Gogarten, J Peter

2010-07-01

Genome annotation is a tedious task that is mostly done by automated methods; however, the accuracy of these approaches has been questioned since the beginning of the sequencing era. Genome annotation is a multilevel process, and errors can emerge at different stages: during sequencing, as a result of gene-calling procedures, and in the process of assigning gene functions. Missed or wrongly annotated genes differentially impact different types of analyses. Here we discuss and demonstrate how the methods of comparative genome analysis can refine annotations by locating missing orthologues. We also discuss possible reasons for errors and show that the second-generation annotation systems, which combine multiple gene-calling programs with similarity-based methods, perform much better than the first annotation tools. Since old errors may propagate to the newly sequenced genomes, we emphasize that the problem of continuously updating popular public databases is an urgent and unresolved one. Due to the progress in genome-sequencing technologies, automated annotation techniques will remain the main approach in the future. Researchers need to be aware of the existing errors in the annotation of even well-studied genomes, such as Escherichia coli, and consider additional quality control for their results.

The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data

PubMed Central

Clarke, Laura; Fairley, Susan; Zheng-Bradley, Xiangqun; Streeter, Ian; Perry, Emily; Lowy, Ernesto; Tassé, Anne-Marie; Flicek, Paul

2017-01-01

The International Genome Sample Resource (IGSR; http://www.internationalgenome.org) expands in data type and population diversity the resources from the 1000 Genomes Project. IGSR represents the largest open collection of human variation data and provides easy access to these resources. IGSR was established in 2015 to maintain and extend the 1000 Genomes Project data, which has been widely used as a reference set of human variation and by researchers developing analysis methods. IGSR has mapped all of the 1000 Genomes sequence to the newest human reference (GRCh38), and will release updated variant calls to ensure maximal usefulness of the existing data. IGSR is collecting new structural variation data on the 1000 Genomes samples from long read sequencing and other technologies, and will collect relevant functional data into a single comprehensive resource. IGSR is extending coverage with new populations sequenced by collaborating groups. Here, we present the new data and analysis that IGSR has made available. We have also introduced a new data portal that increases discoverability of our data—previously only browseable through our FTP site—by focusing on particular samples, populations or data sets of interest. PMID:27638885

Smoking Gun or Circumstantial Evidence? Comparison of Statistical Learning Methods using Functional Annotations for Prioritizing Risk Variants.

PubMed

Gagliano, Sarah A; Ravji, Reena; Barnes, Michael R; Weale, Michael E; Knight, Jo

2015-08-24

Although technology has triumphed in facilitating routine genome sequencing, new challenges have been created for the data-analyst. Genome-scale surveys of human variation generate volumes of data that far exceed capabilities for laboratory characterization. By incorporating functional annotations as predictors, statistical learning has been widely investigated for prioritizing genetic variants likely to be associated with complex disease. We compared three published prioritization procedures, which use different statistical learning algorithms and different predictors with regard to the quantity, type and coding. We also explored different combinations of algorithm and annotation set. As an application, we tested which methodology performed best for prioritizing variants using data from a large schizophrenia meta-analysis by the Psychiatric Genomics Consortium. Results suggest that all methods have considerable (and similar) predictive accuracies (AUCs 0.64-0.71) in test set data, but there is more variability in the application to the schizophrenia GWAS. In conclusion, a variety of algorithms and annotations seem to have a similar potential to effectively enrich true risk variants in genome-scale datasets, however none offer more than incremental improvement in prediction. We discuss how methods might be evolved for risk variant prediction to address the impending bottleneck of the new generation of genome re-sequencing studies.

Clinical Applications of Genome Editing to HIV Cure.

PubMed

Wang, Cathy X; Cannon, Paula M

2016-12-01

Despite significant advances in HIV drug treatment regimens, which grant near-normal life expectancies to infected individuals who have good virological control, HIV infection itself remains incurable. In recent years, novel gene- and cell-based therapies have gained increasing attention due to their potential to provide a functional or even sterilizing cure for HIV infection with a one-shot treatment. A functional cure would keep the infection in check and prevent progression to AIDS, while a sterilizing cure would eradicate all HIV viruses from the patient. Genome editing is the most precise form of gene therapy, able to achieve permanent genetic disruption, modification, or insertion at a predesignated genetic locus. The most well-studied candidate for anti-HIV genome editing is CCR5, an essential coreceptor for the majority of HIV strains, and the lack of which confers HIV resistance in naturally occurring homozygous individuals. Genetic disruption of CCR5 to treat HIV has undergone clinical testing, with seven completed or ongoing trials in T cells and hematopoietic stem and progenitor cells, and has shown promising safety and potential efficacy profiles. Here we summarize clinical findings of CCR5 editing for HIV therapy, as well as other genome editing-based approaches under pre-clinical development. The anticipated development of more sophisticated genome editing technologies should continue to benefit HIV cure efforts.

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

PubMed Central

Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

2012-01-01

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

Noncoding RNAs in DNA Repair and Genome Integrity

PubMed Central

Wan, Guohui; Liu, Yunhua; Han, Cecil; Zhang, Xinna

2014-01-01

Abstract Significance: The well-studied sequences in the human genome are those of protein-coding genes, which account for only 1%–2% of the total genome. However, with the advent of high-throughput transcriptome sequencing technology, we now know that about 90% of our genome is extensively transcribed and that the vast majority of them are transcribed into noncoding RNAs (ncRNAs). It is of great interest and importance to decipher the functions of these ncRNAs in humans. Recent Advances: In the last decade, it has become apparent that ncRNAs play a crucial role in regulating gene expression in normal development, in stress responses to internal and environmental stimuli, and in human diseases. Critical Issues: In addition to those constitutively expressed structural RNA, such as ribosomal and transfer RNAs, regulatory ncRNAs can be classified as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs). However, little is known about the biological features and functional roles of these ncRNAs in DNA repair and genome instability, although a number of miRNAs and lncRNAs are regulated in the DNA damage response. Future Directions: A major goal of modern biology is to identify and characterize the full profile of ncRNAs with regard to normal physiological functions and roles in human disorders. Clinically relevant ncRNAs will also be evaluated and targeted in therapeutic applications. Antioxid. Redox Signal. 20, 655–677. PMID:23879367

Reference quality assembly of the 3.5 Gb genome of Capsicum annuum form a single linked-read library

USDA-ARS?s Scientific Manuscript database

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Cap...

ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data

PubMed Central

2010-01-01

Background Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome. Results We have developed ChIPpeakAnno as a Bioconductor package within the statistical programming environment R to facilitate batch annotation of enriched peaks identified from ChIP-seq, ChIP-chip, cap analysis of gene expression (CAGE) or any experiments resulting in a large number of enriched genomic regions. The binding sites annotated with ChIPpeakAnno can be viewed easily as a table, a pie chart or plotted in histogram form, i.e., the distribution of distances to the nearest genes for each set of peaks. In addition, we have implemented functionalities for determining the significance of overlap between replicates or binding sites among transcription factors within a complex, and for drawing Venn diagrams to visualize the extent of the overlap between replicates. Furthermore, the package includes functionalities to retrieve sequences flanking putative binding sites for PCR amplification, cloning, or motif discovery, and to identify Gene Ontology (GO) terms associated with adjacent genes. Conclusions ChIPpeakAnno enables batch annotation of the binding sites identified from ChIP-seq, ChIP-chip, CAGE or any technology that results in a large number of enriched genomic regions within the statistical programming environment R. Allowing users to pass their own annotation data such as a different Chromatin immunoprecipitation (ChIP) preparation and a dataset from literature, or existing annotation packages, such as GenomicFeatures and BSgenome, provides flexibility. Tight integration to the biomaRt package enables up-to-date annotation retrieval from the BioMart database. PMID:20459804

Jatropha curcas, a biofuel crop: Functional genomics for understanding metabolic pathways and genetic improvement

PubMed Central

Maghuly, Fatemeh; Laimer, Margit

2013-01-01

Jatropha curcas is currently attracting much attention as an oilseed crop for biofuel, as Jatropha can grow under climate and soil conditions that are unsuitable for food production. However, little is known about Jatropha, and there are a number of challenges to be overcome. In fact, Jatropha has not really been domesticated; most of the Jatropha accessions are toxic, which renders the seedcake unsuitable for use as animal feed. The seeds of Jatropha contain high levels of polyunsaturated fatty acids, which negatively impact the biofuel quality. Fruiting of Jatropha is fairly continuous, thus increasing costs of harvesting. Therefore, before starting any improvement program using conventional or molecular breeding techniques, understanding gene function and the genome scale of Jatropha are prerequisites. This review presents currently available and relevant information on the latest technologies (genomics, transcriptomics, proteomics and metabolomics) to decipher important metabolic pathways within Jatropha, such as oil and toxin synthesis. Further, it discusses future directions for biotechnological approaches in Jatropha breeding and improvement. PMID:24092674

NCBI GEO: archive for high-throughput functional genomic data.

PubMed

Barrett, Tanya; Troup, Dennis B; Wilhite, Stephen E; Ledoux, Pierre; Rudnev, Dmitry; Evangelista, Carlos; Kim, Irene F; Soboleva, Alexandra; Tomashevsky, Maxim; Marshall, Kimberly A; Phillippy, Katherine H; Sherman, Patti M; Muertter, Rolf N; Edgar, Ron

2009-01-01

The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest public repository for high-throughput gene expression data. Additionally, GEO hosts other categories of high-throughput functional genomic data, including those that examine genome copy number variations, chromatin structure, methylation status and transcription factor binding. These data are generated by the research community using high-throughput technologies like microarrays and, more recently, next-generation sequencing. The database has a flexible infrastructure that can capture fully annotated raw and processed data, enabling compliance with major community-derived scientific reporting standards such as 'Minimum Information About a Microarray Experiment' (MIAME). In addition to serving as a centralized data storage hub, GEO offers many tools and features that allow users to effectively explore, analyze and download expression data from both gene-centric and experiment-centric perspectives. This article summarizes the GEO repository structure, content and operating procedures, as well as recently introduced data mining features. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/.

GeoChip 3.0: A High Throughput Tool for Analyzing Microbial Community, Composition, Structure, and Functional Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Zhili; Deng, Ye; Nostrand, Joy Van

2010-05-17

Microarray-based genomic technology has been widely used for microbial community analysis, and it is expected that microarray-based genomic technologies will revolutionize the analysis of microbial community structure, function and dynamics. A new generation of functional gene arrays (GeoChip 3.0) has been developed, with 27,812 probes covering 56,990 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance, and organic contaminant degradation. Those probes were derived from 2,744, 140, and 262 species for bacteria, archaea, and fungi, respectively. GeoChip 3.0 has several other distinct features, such as a common oligomore » reference standard (CORS) for data normalization and comparison, a software package for data management and future updating, and the gyrB gene for phylogenetic analysis. Our computational evaluation of probe specificity indicated that all designed probes had a high specificity to their corresponding targets. Also, experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036percent-0.025percent false positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which demonstrated that the structure, composition, and potential activity of soil microbial communities significantly changed with the plant species diversity. All results indicate that GeoChip 3.0 is a high throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. To our knowledge, GeoChip 3.0 is the most comprehensive microarrays currently available for studying microbial communities associated with geobiochemical cycling, global climate change, bioenergy, agricuture, land use, ecosystem management, environmental cleanup and restoration, bioreactor systems, and human health.« less

New Approaches and Technologies to Sequence de novo Plant reference Genomes (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

ScienceCinema

Schmutz, Jeremy

2018-02-01

Jeremy Schmutz of the HudsonAlpha Institute for Biotechnology on New approaches and technologies to sequence de novo plant reference genomes at the 8th Annual Genomics of Energy Environment Meeting on March 27, 2013 in Walnut Creek, CA.

Swine transcriptome characterization by combined Iso-Seq and RNA-seq for annotating the emerging long read-based reference genome

USDA-ARS?s Scientific Manuscript database

PacBio long-read sequencing technology is increasingly popular in genome sequence assembly and transcriptome cataloguing. Recently, a new-generation pig reference genome was assembled based on long reads from this technology. To finely annotate this genome assembly, transcriptomes of nine tissues fr...

New Approaches and Technologies to Sequence de novo Plant reference Genomes (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmutz, Jeremy

2013-03-01

Jeremy Schmutz of the HudsonAlpha Institute for Biotechnology on New approaches and technologies to sequence de novo plant reference genomes at the 8th Annual Genomics of Energy Environment Meeting on March 27, 2013 in Walnut Creek, CA.

Advances in Omics and Bioinformatics Tools for Systems Analyses of Plant Functions

PubMed Central

Mochida, Keiichi; Shinozaki, Kazuo

2011-01-01

Omics and bioinformatics are essential to understanding the molecular systems that underlie various plant functions. Recent game-changing sequencing technologies have revitalized sequencing approaches in genomics and have produced opportunities for various emerging analytical applications. Driven by technological advances, several new omics layers such as the interactome, epigenome and hormonome have emerged. Furthermore, in several plant species, the development of omics resources has progressed to address particular biological properties of individual species. Integration of knowledge from omics-based research is an emerging issue as researchers seek to identify significance, gain biological insights and promote translational research. From these perspectives, we provide this review of the emerging aspects of plant systems research based on omics and bioinformatics analyses together with their associated resources and technological advances. PMID:22156726

Microbial Interactions in Plants: Perspectives and Applications of Proteomics.

PubMed

Imam, Jahangir; Shukla, Pratyoosh; Mandal, Nimai Prasad; Variar, Mukund

2017-01-01

The structure and function of proteins involved in plant-microbe interactions is investigated through large-scale proteomics technology in a complex biological sample. Since the whole genome sequences are now available for several plant species and microbes, proteomics study has become easier, accurate and huge amount of data can be generated and analyzed during plant-microbe interactions. Proteomics approaches are highly important and relevant in many studies and showed that only genomics approaches are not sufficient enough as much significant information are lost as the proteins and not the genes coding them are final product that is responsible for the observed phenotype. Novel approaches in proteomics are developing continuously enabling the study of the various aspects in arrangements and configuration of proteins and its functions. Its application is becoming more common and frequently used in plant-microbe interactions with the advancement in new technologies. They are more used for the portrayal of cell and extracellular destructiveness and pathogenicity variables delivered by pathogens. This distinguishes the protein level adjustments in host plants when infected with pathogens and advantageous partners. This review provides a brief overview of different proteomics technology which is currently available followed by their exploitation to study the plant-microbe interaction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Applications of Gene Targeting Technology to Mental Retardation and Developmental Disability Research

ERIC Educational Resources Information Center

Pimenta, Aurea F.; Levitt, Pat

2005-01-01

The human and mouse genome projects elucidated the sequence and position map of innumerous genes expressed in the central nervous system (CNS), advancing our ability to manipulate these sequences and create models to investigate regulation of gene expression and function. In this article, we reviewed gene targeting methodologies with emphasis on…

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

Treesearch

Chaoyang Zhao; Miguel A. Alvarez Gonzales; Therese M. Poland; Omprakash Mittapalli

2015-01-01

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong...

USE OF GENOMIC TECHNOLOGIES AND ISOTONIC DOSE-RESPONSE MODELING IN THE DEVELOPMENT OF A BIOCHEMICAL MARKER OF EFFECT FOR PYRETHROID INSECTICIDE.

EPA Science Inventory

Pyrethroids are pesticides that disrupt nervous system function by prolongation of sodium currents

through voltage-sensitive sodium channels present in nerve membranes. Pyrethroid usage has

increased as use of other pesticides has declined. A sensitive, dose-respons...

mySyntenyPortal: an application package to construct websites for synteny block analysis.

PubMed

Lee, Jongin; Lee, Daehwan; Sim, Mikang; Kwon, Daehong; Kim, Juyeon; Ko, Younhee; Kim, Jaebum

2018-06-05

Advances in sequencing technologies have facilitated large-scale comparative genomics based on whole genome sequencing. Constructing and investigating conserved genomic regions among multiple species (called synteny blocks) are essential in the comparative genomics. However, they require significant amounts of computational resources and time in addition to bioinformatics skills. Many web interfaces have been developed to make such tasks easier. However, these web interfaces cannot be customized for users who want to use their own set of genome sequences or definition of synteny blocks. To resolve this limitation, we present mySyntenyPortal, a stand-alone application package to construct websites for synteny block analyses by using users' own genome data. mySyntenyPortal provides both command line and web-based interfaces to build and manage websites for large-scale comparative genomic analyses. The websites can be also easily published and accessed by other users. To demonstrate the usability of mySyntenyPortal, we present an example study for building websites to compare genomes of three mammalian species (human, mouse, and cow) and show how they can be easily utilized to identify potential genes affected by genome rearrangements. mySyntenyPortal will contribute for extended comparative genomic analyses based on large-scale whole genome sequences by providing unique functionality to support the easy creation of interactive websites for synteny block analyses from user's own genome data.

Tapping the promise of genomics in species with complex, nonmodel genomes.

PubMed

Hirsch, Candice N; Buell, C Robin

2013-01-01

Genomics is enabling a renaissance in all disciplines of plant biology. However, many plant genomes are complex and remain recalcitrant to current genomic technologies. The complexities of these nonmodel plant genomes are attributable to gene and genome duplication, heterozygosity, ploidy, and/or repetitive sequences. Methods are available to simplify the genome and reduce these barriers, including inbreeding and genome reduction, making these species amenable to current sequencing and assembly methods. Some, but not all, of the complexities in nonmodel genomes can be bypassed by sequencing the transcriptome rather than the genome. Additionally, comparative genomics approaches, which leverage phylogenetic relatedness, can aid in the interpretation of complex genomes. Although there are limitations in accessing complex nonmodel plant genomes using current sequencing technologies, genome manipulation and resourceful analyses can allow access to even the most recalcitrant plant genomes.

Multi-scale structural community organisation of the human genome.

PubMed

Boulos, Rasha E; Tremblay, Nicolas; Arneodo, Alain; Borgnat, Pierre; Audit, Benjamin

2017-04-11

Structural interaction frequency matrices between all genome loci are now experimentally achievable thanks to high-throughput chromosome conformation capture technologies. This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the structural motifs characteristic of genome organisation. We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci. Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.

RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes

DOE PAGES

Brettin, Thomas; Davis, James J.; Disz, Terry; ...

2015-02-10

The RAST (Rapid Annotation using Subsystem Technology) annotation engine was built in 2008 to annotate bacterial and archaeal genomes. It works by offering a standard software pipeline for identifying genomic features (i.e., protein-encoding genes and RNA) and annotating their functions. Recently, in order to make RAST a more useful research tool and to keep pace with advancements in bioinformatics, it has become desirable to build a version of RAST that is both customizable and extensible. In this paper, we describe the RAST tool kit (RASTtk), a modular version of RAST that enables researchers to build custom annotation pipelines. RASTtk offersmore » a choice of software for identifying and annotating genomic features as well as the ability to add custom features to an annotation job. RASTtk also accommodates the batch submission of genomes and the ability to customize annotation protocols for batch submissions. This is the first major software restructuring of RAST since its inception.« less

Application of CRISPR/Cas9 system in breeding of new antiviral plant germplasm.

PubMed

Zhang, Dao-wei; Zhang, Chao-fan; Dong, Fang; Huang, Yan-lan; Zhang, Ya; Zhou, Hong

2016-09-01

With the development and improvement of CRISPR/Cas9 system in genomic editing technology, the system has been applied to the prevention and control of animal viral infectious diseases, which has made considerable achievements. It has also been applied to the study of highly efficient gene targeting editing in plant virus genomes. The CRISPR/Cas9-mediated targeted gene modification has not only achieved the genome editing of plant DNA virus, but also showed the genome editing potential of plant RNA virus. In addition, the CRISPR/Cas9 system functions at the gene transcriptional and post-transcriptional level, indicating that the system could regulate the replication of plant viruses through different ways. Compared with other plant viral disease control strategies, this system is more accurate in genome editing, more stable in gene expression regulation, and has broader spectrum of resistance to virus disease. In this review, we summarized the advantages, main problems and development tendency of CRISPR/cas9 system in breeding of new antiviral plant germplasms.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

PubMed

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-02-24

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-01-01

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. PMID:28233845

Hallmarks of cancer: The CRISPR generation.

PubMed

Moses, Colette; Garcia-Bloj, Benjamin; Harvey, Alan R; Blancafort, Pilar

2018-04-01

The hallmarks of cancer were proposed as a logical framework to guide research efforts that aim to understand the molecular mechanisms and derive treatments for this highly complex disease. Recent technological advances, including comprehensive sequencing of different cancer subtypes, have illuminated how genetic and epigenetic alterations are associated with specific hallmarks of cancer. However, as these associations are purely descriptive, one particularly exciting development is the emergence of genome editing technologies, which enable rapid generation of precise genetic and epigenetic modifications to assess the consequences of these perturbations on the cancer phenotype. The most recently developed of these tools, the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), consists of an RNA-guided endonuclease that can be repurposed to edit both genome and epigenome with high specificity, and facilitates the functional interrogation of multiple loci in parallel. This system has the potential to dramatically accelerate progress in cancer research, whether by modelling the genesis and progression of cancer in vitro and in vivo, screening for novel therapeutic targets, conducting functional genomics/epigenomics, or generating targeted cancer therapies. Here, we discuss CRISPR research on each of the ten hallmarks of cancer, outline potential barriers for its clinical implementation and speculate on the advances it may allow in cancer research in the near future. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Family genome browser: visualizing genomes with pedigree information.

PubMed

Juan, Liran; Liu, Yongzhuang; Wang, Yongtian; Teng, Mingxiang; Zang, Tianyi; Wang, Yadong

2015-07-15

Families with inherited diseases are widely used in Mendelian/complex disease studies. Owing to the advances in high-throughput sequencing technologies, family genome sequencing becomes more and more prevalent. Visualizing family genomes can greatly facilitate human genetics studies and personalized medicine. However, due to the complex genetic relationships and high similarities among genomes of consanguineous family members, family genomes are difficult to be visualized in traditional genome visualization framework. How to visualize the family genome variants and their functions with integrated pedigree information remains a critical challenge. We developed the Family Genome Browser (FGB) to provide comprehensive analysis and visualization for family genomes. The FGB can visualize family genomes in both individual level and variant level effectively, through integrating genome data with pedigree information. Family genome analysis, including determination of parental origin of the variants, detection of de novo mutations, identification of potential recombination events and identical-by-decent segments, etc., can be performed flexibly. Diverse annotations for the family genome variants, such as dbSNP memberships, linkage disequilibriums, genes, variant effects, potential phenotypes, etc., are illustrated as well. Moreover, the FGB can automatically search de novo mutations and compound heterozygous variants for a selected individual, and guide investigators to find high-risk genes with flexible navigation options. These features enable users to investigate and understand family genomes intuitively and systematically. The FGB is available at http://mlg.hit.edu.cn/FGB/. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

RPAN: rice pan-genome browser for ∼3000 rice genomes.

PubMed

Sun, Chen; Hu, Zhiqiang; Zheng, Tianqing; Lu, Kuangchen; Zhao, Yue; Wang, Wensheng; Shi, Jianxin; Wang, Chunchao; Lu, Jinyuan; Zhang, Dabing; Li, Zhikang; Wei, Chaochun

2017-01-25

A pan-genome is the union of the gene sets of all the individuals of a clade or a species and it provides a new dimension of genome complexity with the presence/absence variations (PAVs) of genes among these genomes. With the progress of sequencing technologies, pan-genome study is becoming affordable for eukaryotes with large-sized genomes. The Asian cultivated rice, Oryza sativa L., is one of the major food sources for the world and a model organism in plant biology. Recently, the 3000 Rice Genome Project (3K RGP) sequenced more than 3000 rice genomes with a mean sequencing depth of 14.3×, which provided a tremendous resource for rice research. In this paper, we present a genome browser, Rice Pan-genome Browser (RPAN), as a tool to search and visualize the rice pan-genome derived from 3K RGP. RPAN contains a database of the basic information of 3010 rice accessions, including genomic sequences, gene annotations, PAV information and gene expression data of the rice pan-genome. At least 12 000 novel genes absent in the reference genome were included. RPAN also provides multiple search and visualization functions. RPAN can be a rich resource for rice biology and rice breeding. It is available at http://cgm.sjtu.edu.cn/3kricedb/ or http://www.rmbreeding.cn/pan3k. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterizing visible and invisible cell wall mutant phenotypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carpita, Nicholas C.; McCann, Maureen C.

2015-04-06

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basismore » of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.« less

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

PubMed Central

Tycko, Josh; Myer, Vic E.; Hsu, Patrick D.

2016-01-01

Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.

PubMed

Strefford, Jonathan C

2015-04-01

Chronic lymphocytic leukaemia (CLL) remains at the forefront of the genetic analysis of human tumours, principally due its prevalence, protracted natural history and accessibility to suitable material for analysis. With the application of high-throughput genetic technologies, we have an unbridled view of the architecture of the CLL genome, including a comprehensive description of the copy number and mutational landscape of the disease, a detailed picture of clonal evolution during pathogenesis, and the molecular mechanisms that drive genomic instability and therapeutic resistance. This work has nuanced the prognostic importance of established copy number alterations, and identified novel prognostically relevant gene mutations that function within biological pathways that are attractive treatment targets. Herein, an overview of recent genomic discoveries will be reviewed, with associated biological and clinical implications, and a view into how clinical implementation may be facilitated. © 2014 John Wiley & Sons Ltd.

ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

PubMed

Chen, Xingqi; Shen, Ying; Draper, Will; Buenrostro, Jason D; Litzenburger, Ulrike; Cho, Seung Woo; Satpathy, Ansuman T; Carter, Ava C; Ghosh, Rajarshi P; East-Seletsky, Alexandra; Doudna, Jennifer A; Greenleaf, William J; Liphardt, Jan T; Chang, Howard Y

2016-12-01

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

Issues with RNA-seq analysis in non-model organisms: A salmonid example.

PubMed

Sundaram, Arvind; Tengs, Torstein; Grimholt, Unni

2017-10-01

High throughput sequencing (HTS) is useful for many purposes as exemplified by the other topics included in this special issue. The purpose of this paper is to look into the unique challenges of using this technology in non-model organisms where resources such as genomes, functional genome annotations or genome complexity provide obstacles not met in model organisms. To describe these challenges, we narrow our scope to RNA sequencing used to study differential gene expression in response to pathogen challenge. As a demonstration species we chose Atlantic salmon, which has a sequenced genome with poor annotation and an added complexity due to many duplicated genes. We find that our RNA-seq analysis pipeline deciphers between duplicates despite high sequence identity. However, annotation issues provide problems in linking differentially expressed genes to pathways. Also, comparing results between approaches and species are complicated due to lack of standardized annotation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Merlin: Computer-Aided Oligonucleotide Design for Large Scale Genome Engineering with MAGE.

PubMed

Quintin, Michael; Ma, Natalie J; Ahmed, Samir; Bhatia, Swapnil; Lewis, Aaron; Isaacs, Farren J; Densmore, Douglas

2016-06-17

Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off-target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.

Design principles for nuclease-deficient CRISPR-based transcriptional regulators.

PubMed

Jensen, Michael K

2018-06-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies.

CRISPR/Cas9 and genome editing in Drosophila.

PubMed

Bassett, Andrew R; Liu, Ji-Long

2014-01-20

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Whole-genome sequence of Schistosoma haematobium.

PubMed

Young, Neil D; Jex, Aaron R; Li, Bo; Liu, Shiping; Yang, Linfeng; Xiong, Zijun; Li, Yingrui; Cantacessi, Cinzia; Hall, Ross S; Xu, Xun; Chen, Fangyuan; Wu, Xuan; Zerlotini, Adhemar; Oliveira, Guilherme; Hofmann, Andreas; Zhang, Guojie; Fang, Xiaodong; Kang, Yi; Campbell, Bronwyn E; Loukas, Alex; Ranganathan, Shoba; Rollinson, David; Rinaldi, Gabriel; Brindley, Paul J; Yang, Huanming; Wang, Jun; Wang, Jian; Gasser, Robin B

2012-01-15

Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma; schistosomes) and affecting 200 million people worldwide. No vaccines are available, and treatment relies on one drug, praziquantel. Schistosoma haematobium has come into the spotlight as a major cause of urogenital disease, as an agent linked to bladder cancer and as a predisposing factor for HIV/AIDS. The parasite is transmitted to humans from freshwater snails. Worms dwell in blood vessels and release eggs that become embedded in the bladder wall to elicit chronic immune-mediated disease and induce squamous cell carcinoma. Here we sequenced the 385-Mb genome of S. haematobium using Illumina-based technology at 74-fold coverage and compared it to sequences from related parasites. We included genome annotation based on function, gene ontology, networking and pathway mapping. This genome now provides an unprecedented resource for many fundamental research areas and shows great promise for the design of new disease interventions.

Personal genomics and individual identities: motivations and moral imperatives of early users

PubMed Central

McGowan, Michelle L.; Fishman, Jennifer R.; Lambrix, Marcie A.

2010-01-01

Since 2007, consumer genomics companies have marketed personal genome scanning services to assess users’ genetic predispositions to a variety of complex diseases and traits. This study investigates early users’ reasons for utilizing personal genome services, their evaluation of the technology, how they interpret the results, and how they incorporate the results into health-related decision-making. The analysis contextualizes early users’ relationships to the technology, the knowledge generated by it, and how it mediates their relationship to their own health and to biomedicine more broadly. The results reveal that early users approach personal genome scanning with both optimism for genomic research and scepticism about the technology’s current capabilities, which runs contrary to concerns that consumers may be ill equipped to interpret and understand genome scan results. These findings provide important qualitative insight into early users’ conceptualizations of personal genomic risk assessment and illuminate their involvement in configuring this technology in the making. PMID:21076647

Big Data and Genome Editing Technology: A New Paradigm of Cardiovascular Genomics.

PubMed

Krittanawong, Chayakrit; Sun, Tao; Herzog, Eyal

2017-01-01

Opinion Statements: Cardiovascular diseases (CVDs) encompass a range of conditions extending from congenital heart disease to acute coronary syndrome most of which are heterogenous in nature and some of them are multiple genetic loci. However, the pathogenesis of most CVDs remains incompletely understood. The advance in genome-editing technologies, an engineering process of DNA sequences at precise genomic locations, has enabled a new paradigm that human genome can be precisely modified to achieve a therapeutic effect. Genome-editing includes the correction of genetic variants that cause disease, the addition of therapeutic genes to specific sites in the genomic locations, and the removal of deleterious genes or genome sequences. Site-specific genome engineering can be used as nucleases (known as molecular scissors) including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems to provide remarkable opportunities for developing novel therapies in cardiovascular clinical care. Here we discuss genetic polymorphisms and mechanistic insights in CVDs with an emphasis on the impact of genome-editing technologies. The current challenges and future prospects for genomeediting technologies in cardiovascular medicine are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Whole-genome sequencing in bacteriology: state of the art

PubMed Central

Dark, Michael J

2013-01-01

Over the last ten years, genome sequencing capabilities have expanded exponentially. There have been tremendous advances in sequencing technology, DNA sample preparation, genome assembly, and data analysis. This has led to advances in a number of facets of bacterial genomics, including metagenomics, clinical medicine, bacterial archaeology, and bacterial evolution. This review examines the strengths and weaknesses of techniques in bacterial genome sequencing, upcoming technologies, and assembly techniques, as well as highlighting recent studies that highlight new applications for bacterial genomics. PMID:24143115

Molecular Markers and Cotton Genetic Improvement: Current Status and Future Prospects

PubMed Central

Malik, Waqas; Iqbal, Muhammad Zaffar; Ali Khan, Asif; Qayyum, Abdul; Ali Abid, Muhammad; Noor, Etrat; Qadir Ahmad, Muhammad; Hasan Abbasi, Ghulam

2014-01-01

Narrow genetic base and complex allotetraploid genome of cotton (Gossypium hirsutum L.) is stimulating efforts to avail required polymorphism for marker based breeding. The availability of draft genome sequence of G. raimondii and G. arboreum and next generation sequencing (NGS) technologies facilitated the development of high-throughput marker technologies in cotton. The concepts of genetic diversity, QTL mapping, and marker assisted selection (MAS) are evolving into more efficient concepts of linkage disequilibrium, association mapping, and genomic selection, respectively. The objective of the current review is to analyze the pace of evolution in the molecular marker technologies in cotton during the last ten years into the following four areas: (i) comparative analysis of low- and high-throughput marker technologies available in cotton, (ii) genetic diversity in the available wild and improved gene pools of cotton, (iii) identification of the genomic regions within cotton genome underlying economic traits, and (iv) marker based selection methodologies. Moreover, the applications of marker technologies to enhance the breeding efficiency in cotton are also summarized. Aforementioned genomic technologies and the integration of several other omics resources are expected to enhance the cotton productivity and meet the global fiber quantity and quality demands. PMID:25401149

Extensive sequencing of seven human genomes to characterize benchmark reference materials

PubMed Central

Zook, Justin M.; Catoe, David; McDaniel, Jennifer; Vang, Lindsay; Spies, Noah; Sidow, Arend; Weng, Ziming; Liu, Yuling; Mason, Christopher E.; Alexander, Noah; Henaff, Elizabeth; McIntyre, Alexa B.R.; Chandramohan, Dhruva; Chen, Feng; Jaeger, Erich; Moshrefi, Ali; Pham, Khoa; Stedman, William; Liang, Tiffany; Saghbini, Michael; Dzakula, Zeljko; Hastie, Alex; Cao, Han; Deikus, Gintaras; Schadt, Eric; Sebra, Robert; Bashir, Ali; Truty, Rebecca M.; Chang, Christopher C.; Gulbahce, Natali; Zhao, Keyan; Ghosh, Srinka; Hyland, Fiona; Fu, Yutao; Chaisson, Mark; Xiao, Chunlin; Trow, Jonathan; Sherry, Stephen T.; Zaranek, Alexander W.; Ball, Madeleine; Bobe, Jason; Estep, Preston; Church, George M.; Marks, Patrick; Kyriazopoulou-Panagiotopoulou, Sofia; Zheng, Grace X.Y.; Schnall-Levin, Michael; Ordonez, Heather S.; Mudivarti, Patrice A.; Giorda, Kristina; Sheng, Ying; Rypdal, Karoline Bjarnesdatter; Salit, Marc

2016-01-01

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly. PMID:27271295

Nuclease-mediated genome editing: At the front-line of functional genomics technology.

PubMed

Sakuma, Tetsushi; Woltjen, Knut

2014-01-01

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

The impact of post-genomics approaches in neurodegenerative demyelinating diseases: the case of Guillain-Barré syndrome.

PubMed

Villar, Margarita; Mateos-Hernandez, Lourdes; de la Fuente, Jose

2018-03-14

Why an autoimmune disease that is the main cause of the acute neuromuscular paralysis worldwide has not yet a well-characterized cause or an effective treatment? The existence of different clinical variants for the Guillain-Barré syndrome (GBS) coupled with the fact that a high number of pathogens can cause an infection that sometimes, but not always, precedes the development of the syndrome, confers a high degree of uncertainty for both prognosis and treatment. In the post-genomic era, the development of omics technologies for the high-throughput analysis of biological molecules is allowing the characterization of biological systems in a degree of depth unimaginable before. In this context, this work summarize the application of post-genomics technologies to the study of GBS. We performed a structured search of bibliographic databases for peer-reviewed research literature to outline the state of the art with regard the application of post-genomics technologies to the study of GBS. The quality of retrieved papers was assessed using standard tools and thirty-four were included in the review. To date, transcriptomics and proteomics have been the unique post-genomics approaches applied to GBS study. Most of these studies have been performed on cerebrospinal fluid samples and only few studies have been conducted with other samples such as serum, Schwann cells and human peripheral nerve. In the post-genomics era, transcriptomics and proteomics have shown the possibilities that omics technologies can offer for a better understanding of the immunological and pathological mechanisms involved in GBS and the identification of potential biomarkers, but these results have only shown the tip of the iceberg and there is still a long way to exploit the full potential that post-genomics approaches could offer to the study of the GBS. The integration of different omics datasets through a systems biology approach could allow network-based analyses to describe the complexity and functionality of the molecular mechanisms involved in the course of disease facilitating the discovery of novel biomarkers that could be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology, and serve as therapeutic targets for GBS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Research Update: The materials genome initiative: Data sharing and the impact of collaborative ab initio databases

DOE PAGES

Jain, Anubhav; Persson, Kristin A.; Ceder, Gerbrand

2016-03-24

Materials innovations enable new technological capabilities and drive major societal advancements but have historically required long and costly development cycles. The Materials Genome Initiative (MGI) aims to greatly reduce this time and cost. Here, we focus on data reuse in the MGI and, in particular, discuss the impact of three different computational databases based on density functional theory methods to the research community. Finally, we discuss and provide recommendations on technical aspects of data reuse, outline remaining fundamental challenges, and present an outlook on the future of MGI's vision of data sharing.

The functional genomic studies of curcumin.

PubMed

Huminiecki, Lukasz; Horbańczuk, Jarosław; Atanasov, Atanas G

2017-10-01

Curcumin is a natural plant-derived compound that has attracted a lot of attention for its anti-cancer activities. Curcumin can slow proliferation of and induce apoptosis in cancer cell lines, but the precise mechanisms of these effects are not fully understood. However, many lines of evidence suggested that curcumin has a potent impact on gene expression profiles; thus, functional genomics should be the key to understanding how curcumin exerts its anti-cancer activities. Here, we review the published functional genomic studies of curcumin focusing on cancer. Typically, a cancer cell line or a grafted tumor were exposed to curcumin and profiled with microarrays, methylation assays, or RNA-seq. Crucially, these studies are in agreement that curcumin has a powerful effect on gene expression. In the majority of the studies, among differentially expressed genes we found genes involved in cell signaling, apoptosis, and the control of cell cycle. Curcumin can also induce specific methylation changes, and is a powerful regulator of the expression of microRNAs which control oncogenesis. We also reflect on how the broader technological progress in transcriptomics has been reflected on the field of curcumin. We conclude by discussing the areas where more functional genomic studies are highly desirable. Integrated OMICS approaches will clearly be the key to understanding curcumin's anticancer and chemopreventive effects. Such strategies may become a template for elucidating the mode of action of other natural products; many natural products have pleiotropic effects that are well suited for a systems-level analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Illuminating the Black Box of Genome Sequence Assembly: A Free Online Tool to Introduce Students to Bioinformatics

ERIC Educational Resources Information Center

Taylor, D. Leland; Campbell, A. Malcolm; Heyer, Laurie J.

2013-01-01

Next-generation sequencing technologies have greatly reduced the cost of sequencing genomes. With the current sequencing technology, a genome is broken into fragments and sequenced, producing millions of "reads." A computer algorithm pieces these reads together in the genome assembly process. PHAST is a set of online modules…

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

PubMed

Lander, Noelia; Chiurillo, Miguel A; Docampo, Roberto

2016-09-01

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

CRISPR editing in biological and biomedical investigation.

PubMed

Huang, Jiaojiao; Wang, Yanfang; Zhao, Jianguo

2018-05-01

Recently, clustered regularly interspaced short palindromic repeats (CRISPR) based genomic editing technologies have armed researchers with powerful new tools to biological and biomedical investigations. To further improve and expand its functionality, natural, and engineered CRISPR associated nine proteins (Cas9s) have been investigated, various CRISPR delivery strategies have been tested and optimized, and multiple schemes have been developed to ensure precise mammalian genome editing. Benefiting from those in-depth understanding and further development of CRISPR, versatile CRISPR-based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine. In biological research area, CRISPR has been widely adopted in both fundamental and applied research fields, such as accurate base editing, transcriptional regulation, and genome-wide screening. In biomedical research area, CRISPR has also shown its extensive applicability in the establishment of animal models for genetic disorders especially those large animals and non-human primates models, and gene therapy to combat virus infectious diseases, to correct monogenic disorders in vivo or in pluripotent cells. In this prospect article, after highlighting recent developments of CRISPR systems, we outline different applications and current limitations of CRISPR use in biological and biomedical investigation. Finally, we provide a perspective for future development and potential risks of this multifunctional technology. © 2017 Wiley Periodicals, Inc.

Metabolomic strategies to map functions of metabolic pathways

PubMed Central

Mulvihill, Melinda M.

2014-01-01

Genome sequencing efforts have revealed a strikingly large number of unannotated and uncharacterized genes that fall into metabolic enzymes classes, likely indicating that our current knowledge of biochemical pathways in normal physiology, let alone in disease states, remains largely incomplete. This realization presents a daunting challenge for post-genomic-era scientists in deciphering the biochemical and (patho)physiological roles of these enzymes and their metabolites and metabolic networks. This is further complicated by many recent studies showing a rewiring of normal metabolic networks in disease states to give rise to unique pathophysiological functions of enzymes, metabolites, and metabolic pathways. This review focuses on recent discoveries made using metabolic mapping technologies to uncover novel pathways and metabolite-mediated posttranslational modifications and epigenetic alterations and their impact on physiology and disease. PMID:24918200

Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways

PubMed Central

2016-01-01

Covering: 2003 to 2016 The last decade has seen the first major discoveries regarding the genomic basis of plant natural product biosynthetic pathways. Four key computationally driven strategies have been developed to identify such pathways, which make use of physical clustering, co-expression, evolutionary co-occurrence and epigenomic co-regulation of the genes involved in producing a plant natural product. Here, we discuss how these approaches can be used for the discovery of plant biosynthetic pathways encoded by both chromosomally clustered and non-clustered genes. Additionally, we will discuss opportunities to prioritize plant gene clusters for experimental characterization, and end with a forward-looking perspective on how synthetic biology technologies will allow effective functional reconstitution of candidate pathways using a variety of genetic systems. PMID:27321668

Microbial Ecology and Evolution in the Acid Mine Drainage Model System.

PubMed

Huang, Li-Nan; Kuang, Jia-Liang; Shu, Wen-Sheng

2016-07-01

Acid mine drainage (AMD) is a unique ecological niche for acid- and toxic-metals-adapted microorganisms. These low-complexity systems offer a special opportunity for the ecological and evolutionary analyses of natural microbial assemblages. The last decade has witnessed an unprecedented interest in the study of AMD communities using 16S rRNA high-throughput sequencing and community genomic and postgenomic methodologies, significantly advancing our understanding of microbial diversity, community function, and evolution in acidic environments. This review describes new data on AMD microbial ecology and evolution, especially dynamics of microbial diversity, community functions, and population genomes, and further identifies gaps in our current knowledge that future research, with integrated applications of meta-omics technologies, will fill. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using induced pluripotent stem cells to explore genetic and epigenetic variation associated with Alzheimer's disease.

PubMed

Imm, Jennifer; Kerrigan, Talitha L; Jeffries, Aaron; Lunnon, Katie

2017-11-01

It is thought that both genetic and epigenetic variation play a role in Alzheimer's disease initiation and progression. With the advent of somatic cell reprogramming into induced pluripotent stem cells it is now possible to generate patient-derived cells that are able to more accurately model and recapitulate disease. Furthermore, by combining this with recent advances in (epi)genome editing technologies, it is possible to begin to examine the functional consequence of previously nominated genetic variants and infer epigenetic causality from recently identified epigenetic variants. In this review, we explore the role of genetic and epigenetic variation in Alzheimer's disease and how the functional relevance of nominated loci can be investigated using induced pluripotent stem cells and (epi)genome editing techniques.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE PAGES

Schumann, Kathrin; Lin, Steven; Boyer, Eric; ...

2015-07-27

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schumann, Kathrin; Lin, Steven; Boyer, Eric

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Mining biological databases for candidate disease genes

NASA Astrophysics Data System (ADS)

Braun, Terry A.; Scheetz, Todd; Webster, Gregg L.; Casavant, Thomas L.

2001-07-01

The publicly-funded effort to sequence the complete nucleotide sequence of the human genome, the Human Genome Project (HGP), has currently produced more than 93% of the 3 billion nucleotides of the human genome into a preliminary `draft' format. In addition, several valuable sources of information have been developed as direct and indirect results of the HGP. These include the sequencing of model organisms (rat, mouse, fly, and others), gene discovery projects (ESTs and full-length), and new technologies such as expression analysis and resources (micro-arrays or gene chips). These resources are invaluable for the researchers identifying the functional genes of the genome that transcribe and translate into the transcriptome and proteome, both of which potentially contain orders of magnitude more complexity than the genome itself. Preliminary analyses of this data identified approximately 30,000 - 40,000 human `genes.' However, the bulk of the effort still remains -- to identify the functional and structural elements contained within the transcriptome and proteome, and to associate function in the transcriptome and proteome to genes. A fortuitous consequence of the HGP is the existence of hundreds of databases containing biological information that may contain relevant data pertaining to the identification of disease-causing genes. The task of mining these databases for information on candidate genes is a commercial application of enormous potential. We are developing a system to acquire and mine data from specific databases to aid our efforts to identify disease genes. A high speed cluster of Linux of workstations is used to analyze sequence and perform distributed sequence alignments as part of our data mining and processing. This system has been used to mine GeneMap99 sequences within specific genomic intervals to identify potential candidate disease genes associated with Bardet-Biedle Syndrome (BBS).

Genome Structural Diversity among 31 Bordetella pertussis Isolates from Two Recent U.S. Whooping Cough Statewide Epidemics.

PubMed

Bowden, Katherine E; Weigand, Michael R; Peng, Yanhui; Cassiday, Pamela K; Sammons, Scott; Knipe, Kristen; Rowe, Lori A; Loparev, Vladimir; Sheth, Mili; Weening, Keeley; Tondella, M Lucia; Williams, Margaret M

2016-01-01

During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations. IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

PubMed

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome.

PubMed

Cox, Laura A; Glenn, Jeremy P; Spradling, Kimberly D; Nijland, Mark J; Garcia, Roy; Nathanielsz, Peter W; Ford, Stephen P

2012-06-15

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome

PubMed Central

Cox, Laura A; Glenn, Jeremy P; Spradling, Kimberly D; Nijland, Mark J; Garcia, Roy; Nathanielsz, Peter W; Ford, Stephen P

2012-01-01

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development. PMID:22508961

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae

PubMed Central

Neale, David B.; McGuire, Patrick E.; Wheeler, Nicholas C.; Stevens, Kristian A.; Crepeau, Marc W.; Cardeno, Charis; Zimin, Aleksey V.; Puiu, Daniela; Pertea, Geo M.; Sezen, U. Uzay; Casola, Claudio; Koralewski, Tomasz E.; Paul, Robin; Gonzalez-Ibeas, Daniel; Zaman, Sumaira; Cronn, Richard; Yandell, Mark; Holt, Carson; Langley, Charles H.; Yorke, James A.; Salzberg, Steven L.; Wegrzyn, Jill L.

2017-01-01

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms. PMID:28751502

What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins

PubMed Central

Hutchins, James R. A.

2014-01-01

The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry–based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set–wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery. PMID:24723265

Genome constraint through sexual reproduction: application of 4D-Genomics in reproductive biology.

PubMed

Horne, Steven D; Abdallah, Batoul Y; Stevens, Joshua B; Liu, Guo; Ye, Karen J; Bremer, Steven W; Heng, Henry H Q

2013-06-01

Assisted reproductive technologies have been used to achieve pregnancies since the first successful test tube baby was born in 1978. Infertile couples are at an increased risk for multiple miscarriages and the application of current protocols are associated with high first-trimester miscarriage rates. Among the contributing factors of these higher rates is a high incidence of fetal aneuploidy. Numerous studies support that protocols including ovulation-induction, sperm cryostorage, density-gradient centrifugation, and embryo culture can induce genome instability, but the general mechanism is less clear. Application of the genome theory and 4D-Genomics recently led to the establishment of a new paradigm for sexual reproduction; sex primarily constrains genome integrity that defines the biological system rather than just providing genetic diversity at the gene level. We therefore propose that application of assisted reproductive technologies can bypass this sexual reproduction filter as well as potentially induce additional system instability. We have previously demonstrated that a single-cell resolution genomic approach, such as spectral karyotyping to trace stochastic genome level alterations, is effective for pre- and post-natal analysis. We propose that monitoring overall genome alteration at the karyotype level alongside the application of assisted reproductive technologies will improve the efficacy of the techniques while limiting stress-induced genome instability. The development of more single-cell based cytogenomic technologies are needed in order to better understand the system dynamics associated with infertility and the potential impact that assisted reproductive technologies have on genome instability. Importantly, this approach will be useful in studying the potential for diseases to arise as a result of bypassing the filter of sexual reproduction.

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

Vaccine adjuvant technology: from mechanistic concepts to practical applications.

PubMed

Degen, Winfried G J; Jansen, Theo; Schijns, Virgil E J C

2003-04-01

Distinct types of immune responses are required for efficient elimination of different pathogens. Programming of the desired type of immune response by safe nonreplicating vaccines requires suitable vaccine adjuvants. Adjuvants largely determine the magnitude and quality of immune responses specific for the coadministered antigen. Unfortunately, rational vaccine design requiring a rational choice of vaccine adjuvant, is hampered by a lack of knowledge about the mechanism(s) of vaccine adjuvant activity. The current review addresses different critical immunological processes possibly explaining adjuvant functions. In addition, we discuss traditional vaccine adjuvant formulations and their possible mode of action. Finally, we reflect on the latest technologies for the identification of novel adjuvants using molecular analysis of immune activation and functional genomics.

Transcriptome Analysis of PA Gain and Loss of Function Mutants.

PubMed

Marco, Francisco; Carrasco, Pedro

2018-01-01

Functional genomics has become a forefront methodology for plant science thanks to the widespread development of microarray technology. While technical difficulties associated with the process of obtaining raw expression data have been diminishing, allowing the appearance of tremendous amounts of transcriptome data in different databases, a common problem using "omic" technologies remains: the interpretation of these data and the inference of its biological meaning. In order to assist to this complex task, a wide variety of software tools have been developed. In this chapter we describe our current workflow of the application of some of these analyses. We have used it to compare the transcriptome of plants with differences in their polyamine levels.

Genome editing of crops: A renewed opportunity for food security.

PubMed

Georges, Fawzy; Ray, Heather

2017-01-02

Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance.

Genome editing of crops: A renewed opportunity for food security

PubMed Central

Georges, Fawzy

2017-01-01

ABSTRACT Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance. PMID:28075688

Epigenetic modifications by Trithorax group proteins during early embryogenesis: do members of Trx-G function as maternal effect genes?

PubMed

Andreu-Vieyra, Claudia; Matzuk, Martin M

2007-02-01

Maternal effect genes encode transcripts that are expressed during oogenesis. These gene products are stored in the oocyte and become functional during resumption of meiosis and zygote genome activation, and in embryonic stem cells. To date, a few maternal effect genes have been identified in mammals. Epigenetic modifications have been shown to be important during early embryonic development and involve DNA methylation and post-translational modification of core histones. During development, two families of proteins have been shown to be involved in epigenetic changes: Trithorax group (Trx-G) and Polycomb group (Pc-G) proteins. Trx-G proteins function as transcriptional activators and have been shown to accumulate in the oocyte. Deletion of Trx-G members using conventional knockout technology results in embryonic lethality in the majority of the cases analysed to date. Recent studies using conditional knockout mice have revealed that at least one family member is necessary for zygote genome activation. We propose that other Trx-G members may also regulate embryonic genome activation and that the use of oocyte-specific deletor mouse lines will help clarify their roles in this process.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

PubMed Central

Vizeacoumar, Franco J; Arnold, Roland; Vizeacoumar, Frederick S; Chandrashekhar, Megha; Buzina, Alla; Young, Jordan T F; Kwan, Julian H M; Sayad, Azin; Mero, Patricia; Lawo, Steffen; Tanaka, Hiromasa; Brown, Kevin R; Baryshnikova, Anastasia; Mak, Anthony B; Fedyshyn, Yaroslav; Wang, Yadong; Brito, Glauber C; Kasimer, Dahlia; Makhnevych, Taras; Ketela, Troy; Datti, Alessandro; Babu, Mohan; Emili, Andrew; Pelletier, Laurence; Wrana, Jeff; Wainberg, Zev; Kim, Philip M; Rottapel, Robert; O'Brien, Catherine A; Andrews, Brenda; Boone, Charles; Moffat, Jason

2013-01-01

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model. PMID:24104479

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes.

PubMed

Zhong, Xingyu; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

2013-07-01

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

Pathway perturbations in signaling networks: Linking genotype to phenotype.

PubMed

Li, Yongsheng; McGrail, Daniel J; Latysheva, Natasha; Yi, Song; Babu, M Madan; Sahni, Nidhi

2018-05-10

Genes and gene products interact with each other to form signal transduction networks in the cell. The interactome networks are under intricate regulation in physiological conditions, but could go awry upon genome instability caused by genetic mutations. In the past decade with next-generation sequencing technologies, an increasing number of genomic mutations have been identified in a variety of disease patients and healthy individuals. As functional and systematic studies on these mutations leap forward, they begin to reveal insights into cellular homeostasis and disease mechanisms. In this review, we discuss recent advances in the field of network biology and signaling pathway perturbations upon genomic changes, and highlight the success of various omics datasets in unraveling genotype-to-phenotype relationships. Copyright © 2018 Elsevier Ltd. All rights reserved.

Using Genetically Engineered Animal Models in the Postgenomic Era to Understand Gene Function in Alcoholism

PubMed Central

Reilly, Matthew T.; Harris, R. Adron; Noronha, Antonio

2012-01-01

Over the last 50 years, researchers have made substantial progress in identifying genetic variations that underlie the complex phenotype of alcoholism. Not much is known, however, about how this genetic variation translates into altered biological function. Genetic animal models recapitulating specific characteristics of the human condition have helped elucidate gene function and the genetic basis of disease. In particular, major advances have come from the ability to manipulate genes through a variety of genetic technologies that provide an unprecedented capacity to determine gene function in the living organism and in alcohol-related behaviors. Even newer genetic-engineering technologies have given researchers the ability to control when and where a specific gene or mutation is activated or deleted, allowing investigators to narrow the role of the gene’s function to circumscribed neural pathways and across development. These technologies are important for all areas of neuroscience, and several public and private initiatives are making a new generation of genetic-engineering tools available to the scientific community at large. Finally, high-throughput “next-generation sequencing” technologies are set to rapidly increase knowledge of the genome, epigenome, and transcriptome, which, combined with genetically engineered mouse mutants, will enhance insight into biological function. All of these resources will provide deeper insight into the genetic basis of alcoholism. PMID:23134044

Using genetically engineered animal models in the postgenomic era to understand gene function in alcoholism.

PubMed

Reilly, Matthew T; Harris, R Adron; Noronha, Antonio

2012-01-01

Over the last 50 years, researchers have made substantial progress in identifying genetic variations that underlie the complex phenotype of alcoholism. Not much is known, however, about how this genetic variation translates into altered biological function. Genetic animal models recapitulating specific characteristics of the human condition have helped elucidate gene function and the genetic basis of disease. In particular, major advances have come from the ability to manipulate genes through a variety of genetic technologies that provide an unprecedented capacity to determine gene function in the living organism and in alcohol-related behaviors. Even newer genetic-engineering technologies have given researchers the ability to control when and where a specific gene or mutation is activated or deleted, allowing investigators to narrow the role of the gene's function to circumscribed neural pathways and across development. These technologies are important for all areas of neuroscience, and several public and private initiatives are making a new generation of genetic-engineering tools available to the scientific community at large. Finally, high-throughput "next-generation sequencing" technologies are set to rapidly increase knowledge of the genome, epigenome, and transcriptome, which, combined with genetically engineered mouse mutants, will enhance insight into biological function. All of these resources will provide deeper insight into the genetic basis of alcoholism.

Genome-based Modeling and Design of Metabolic Interactions in Microbial Communities

PubMed Central

Mahadevan, Radhakrishnan; Henson, Michael A.

2012-01-01

Biotechnology research is traditionally focused on individual microbial strains that are perceived to have the necessary metabolic functions, or the capability to have these functions introduced, to achieve a particular task. For many important applications, the development of such omnipotent microbes is an extremely challenging if not impossible task. By contrast, nature employs a radically different strategy based on synergistic combinations of different microbial species that collectively achieve the desired task. These natural communities have evolved to exploit the native metabolic capabilities of each species and are highly adaptive to changes in their environments. However, microbial communities have proven difficult to study due to a lack of suitable experimental and computational tools. With the advent of genome sequencing, omics technologies, bioinformatics and genome-scale modeling, researchers now have unprecedented capabilities to analyze and engineer the metabolism of microbial communities. The goal of this review is to summarize recent applications of genome-scale metabolic modeling to microbial communities. A brief introduction to lumped community models is used to motivate the need for genome-level descriptions of individual species and their metabolic interactions. The review of genome-scale models begins with static modeling approaches, which are appropriate for communities where the extracellular environment can be assumed to be time invariant or slowly varying. Dynamic extensions of the static modeling approach are described, and then applications of genome-scale models for design of synthetic microbial communities are reviewed. The review concludes with a summary of metagenomic tools for analyzing community metabolism and an outlook for future research. PMID:24688668

Genome-based Modeling and Design of Metabolic Interactions in Microbial Communities.

PubMed

Mahadevan, Radhakrishnan; Henson, Michael A

2012-01-01

Biotechnology research is traditionally focused on individual microbial strains that are perceived to have the necessary metabolic functions, or the capability to have these functions introduced, to achieve a particular task. For many important applications, the development of such omnipotent microbes is an extremely challenging if not impossible task. By contrast, nature employs a radically different strategy based on synergistic combinations of different microbial species that collectively achieve the desired task. These natural communities have evolved to exploit the native metabolic capabilities of each species and are highly adaptive to changes in their environments. However, microbial communities have proven difficult to study due to a lack of suitable experimental and computational tools. With the advent of genome sequencing, omics technologies, bioinformatics and genome-scale modeling, researchers now have unprecedented capabilities to analyze and engineer the metabolism of microbial communities. The goal of this review is to summarize recent applications of genome-scale metabolic modeling to microbial communities. A brief introduction to lumped community models is used to motivate the need for genome-level descriptions of individual species and their metabolic interactions. The review of genome-scale models begins with static modeling approaches, which are appropriate for communities where the extracellular environment can be assumed to be time invariant or slowly varying. Dynamic extensions of the static modeling approach are described, and then applications of genome-scale models for design of synthetic microbial communities are reviewed. The review concludes with a summary of metagenomic tools for analyzing community metabolism and an outlook for future research.

Genomic Data Commons and Genomic Cloud Pilots - Google Hangout

Cancer.gov

Join us for a live, moderated discussion about two NCI efforts to expand access to cancer genomics data: the Genomic Data Commons and Genomic Cloud Pilots. NCI subject matters experts will include Louis M. Staudt, M.D., Ph.D., Director Center for Cancer Genomics, Warren Kibbe, Ph.D., Director, NCI Center for Biomedical Informatics and Information Technology, and moderated by Anthony Kerlavage, Ph.D., Chief, Cancer Informatics Branch, Center for Biomedical Informatics and Information Technology. We welcome your questions before and during the Hangout on Twitter using the hashtag #AskNCI.

MEGANTE: A Web-Based System for Integrated Plant Genome Annotation

PubMed Central

Numa, Hisataka; Itoh, Takeshi

2014-01-01

The recent advancement of high-throughput genome sequencing technologies has resulted in a considerable increase in demands for large-scale genome annotation. While annotation is a crucial step for downstream data analyses and experimental studies, this process requires substantial expertise and knowledge of bioinformatics. Here we present MEGANTE, a web-based annotation system that makes plant genome annotation easy for researchers unfamiliar with bioinformatics. Without any complicated configuration, users can perform genomic sequence annotations simply by uploading a sequence and selecting the species to query. MEGANTE automatically runs several analysis programs and integrates the results to select the appropriate consensus exon–intron structures and to predict open reading frames (ORFs) at each locus. Functional annotation, including a similarity search against known proteins and a functional domain search, are also performed for the predicted ORFs. The resultant annotation information is visualized with a widely used genome browser, GBrowse. For ease of analysis, the results can be downloaded in Microsoft Excel format. All of the query sequences and annotation results are stored on the server side so that users can access their own data from virtually anywhere on the web. The current release of MEGANTE targets 24 plant species from the Brassicaceae, Fabaceae, Musaceae, Poaceae, Salicaceae, Solanaceae, Rosaceae and Vitaceae families, and it allows users to submit a sequence up to 10 Mb in length and to save up to 100 sequences with the annotation information on the server. The MEGANTE web service is available at https://megante.dna.affrc.go.jp/. PMID:24253915

NCBI prokaryotic genome annotation pipeline.

PubMed

Tatusova, Tatiana; DiCuccio, Michael; Badretdin, Azat; Chetvernin, Vyacheslav; Nawrocki, Eric P; Zaslavsky, Leonid; Lomsadze, Alexandre; Pruitt, Kim D; Borodovsky, Mark; Ostell, James

2016-08-19

Recent technological advances have opened unprecedented opportunities for large-scale sequencing and analysis of populations of pathogenic species in disease outbreaks, as well as for large-scale diversity studies aimed at expanding our knowledge across the whole domain of prokaryotes. To meet the challenge of timely interpretation of structure, function and meaning of this vast genetic information, a comprehensive approach to automatic genome annotation is critically needed. In collaboration with Georgia Tech, NCBI has developed a new approach to genome annotation that combines alignment based methods with methods of predicting protein-coding and RNA genes and other functional elements directly from sequence. A new gene finding tool, GeneMarkS+, uses the combined evidence of protein and RNA placement by homology as an initial map of annotation to generate and modify ab initio gene predictions across the whole genome. Thus, the new NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) relies more on sequence similarity when confident comparative data are available, while it relies more on statistical predictions in the absence of external evidence. The pipeline provides a framework for generation and analysis of annotation on the full breadth of prokaryotic taxonomy. For additional information on PGAP see https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ and the NCBI Handbook, https://www.ncbi.nlm.nih.gov/books/NBK174280/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

USDA-ARS?s Scientific Manuscript database

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

From Genomics to Gene Therapy: Induced Pluripotent Stem Cells Meet Genome Editing.

PubMed

Hotta, Akitsu; Yamanaka, Shinya

2015-01-01

The advent of induced pluripotent stem (iPS) cells has opened up numerous avenues of opportunity for cell therapy, including the initiation in September 2014 of the first human clinical trial to treat dry age-related macular degeneration. In parallel, advances in genome-editing technologies by site-specific nucleases have dramatically improved our ability to edit endogenous genomic sequences at targeted sites of interest. In fact, clinical trials have already begun to implement this technology to control HIV infection. Genome editing in iPS cells is a powerful tool and enables researchers to investigate the intricacies of the human genome in a dish. In the near future, the groundwork laid by such an approach may expand the possibilities of gene therapy for treating congenital disorders. In this review, we summarize the exciting progress being made in the utilization of genomic editing technologies in pluripotent stem cells and discuss remaining challenges toward gene therapy applications.

Genome editing in pluripotent stem cells: research and therapeutic applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deleidi, Michela, E-mail: michela.deleidi@dzne.de; Hertie Institute for Clinical Brain Research, University of Tübingen; Yu, Cong

Recent progress in human pluripotent stem cell (hPSC) and genome editing technologies has opened up new avenues for the investigation of human biology in health and disease as well as the development of therapeutic applications. Gene editing approaches with programmable nucleases have been successfully established in hPSCs and applied to study gene function, develop novel animal models and perform genetic and chemical screens. Several studies now show the successful editing of disease-linked alleles in somatic and patient-derived induced pluripotent stem cells (iPSCs) as well as in animal models. Importantly, initial clinical trials have shown the safety of programmable nucleases formore » ex vivo somatic gene therapy. In this context, the unlimited proliferation potential and the pluripotent properties of iPSCs may offer advantages for gene targeting approaches. However, many technical and safety issues still need to be addressed before genome-edited iPSCs are translated into the clinical setting. Here, we provide an overview of the available genome editing systems and discuss opportunities and perspectives for their application in basic research and clinical practice, with a particular focus on hPSC based research and gene therapy approaches. Finally, we discuss recent research on human germline genome editing and its social and ethical implications. - Highlights: • Programmable nucleases have proven efficient and specific for genome editing in human pluripotent stem cells (hPSCs). • Genome edited hPSCs can be employed to study gene function in health and disease as well as drug and chemical screens. • Genome edited hPSCs hold great promise for ex vivo gene therapy approaches. • Technical and safety issues should be first addressed to advance the clinical use of gene-edited hPSCs.« less

Producing genome structure populations with the dynamic and automated PGS software.

PubMed

Hua, Nan; Tjong, Harianto; Shin, Hanjun; Gong, Ke; Zhou, Xianghong Jasmine; Alber, Frank

2018-05-01

Chromosome conformation capture technologies such as Hi-C are widely used to investigate the spatial organization of genomes. Because genome structures can vary considerably between individual cells of a population, interpreting ensemble-averaged Hi-C data can be challenging, in particular for long-range and interchromosomal interactions. We pioneered a probabilistic approach for the generation of a population of distinct diploid 3D genome structures consistent with all the chromatin-chromatin interaction probabilities from Hi-C experiments. Each structure in the population is a physical model of the genome in 3D. Analysis of these models yields new insights into the causes and the functional properties of the genome's organization in space and time. We provide a user-friendly software package, called PGS, which runs on local machines (for practice runs) and high-performance computing platforms. PGS takes a genome-wide Hi-C contact frequency matrix, along with information about genome segmentation, and produces an ensemble of 3D genome structures entirely consistent with the input. The software automatically generates an analysis report, and provides tools to extract and analyze the 3D coordinates of specific domains. Basic Linux command-line knowledge is sufficient for using this software. A typical running time of the pipeline is ∼3 d with 300 cores on a computer cluster to generate a population of 1,000 diploid genome structures at topological-associated domain (TAD)-level resolution.

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

PubMed

O'Duibhir, Eoghan; Carragher, Neil O; Pollard, Steven M

2017-04-01

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Supporting community annotation and user collaboration in the integrated microbial genomes (IMG) system

DOE PAGES

Chen, I-Min A.; Markowitz, Victor M.; Palaniappan, Krishna; ...

2016-04-26

Background: The exponential growth of genomic data from next generation technologies renders traditional manual expert curation effort unsustainable. Many genomic systems have included community annotation tools to address the problem. Most of these systems adopted a "Wiki-based" approach to take advantage of existing wiki technologies, but encountered obstacles in issues such as usability, authorship recognition, information reliability and incentive for community participation. Results: Here, we present a different approach, relying on tightly integrated method rather than "Wiki-based" method, to support community annotation and user collaboration in the Integrated Microbial Genomes (IMG) system. The IMG approach allows users to use existingmore » IMG data warehouse and analysis tools to add gene, pathway and biosynthetic cluster annotations, to analyze/reorganize contigs, genes and functions using workspace datasets, and to share private user annotations and workspace datasets with collaborators. We show that the annotation effort using IMG can be part of the research process to overcome the user incentive and authorship recognition problems thus fostering collaboration among domain experts. The usability and reliability issues are addressed by the integration of curated information and analysis tools in IMG, together with DOE Joint Genome Institute (JGI) expert review. Conclusion: By incorporating annotation operations into IMG, we provide an integrated environment for users to perform deeper and extended data analysis and annotation in a single system that can lead to publications and community knowledge sharing as shown in the case studies.« less

Supporting community annotation and user collaboration in the integrated microbial genomes (IMG) system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, I-Min A.; Markowitz, Victor M.; Palaniappan, Krishna

Background: The exponential growth of genomic data from next generation technologies renders traditional manual expert curation effort unsustainable. Many genomic systems have included community annotation tools to address the problem. Most of these systems adopted a "Wiki-based" approach to take advantage of existing wiki technologies, but encountered obstacles in issues such as usability, authorship recognition, information reliability and incentive for community participation. Results: Here, we present a different approach, relying on tightly integrated method rather than "Wiki-based" method, to support community annotation and user collaboration in the Integrated Microbial Genomes (IMG) system. The IMG approach allows users to use existingmore » IMG data warehouse and analysis tools to add gene, pathway and biosynthetic cluster annotations, to analyze/reorganize contigs, genes and functions using workspace datasets, and to share private user annotations and workspace datasets with collaborators. We show that the annotation effort using IMG can be part of the research process to overcome the user incentive and authorship recognition problems thus fostering collaboration among domain experts. The usability and reliability issues are addressed by the integration of curated information and analysis tools in IMG, together with DOE Joint Genome Institute (JGI) expert review. Conclusion: By incorporating annotation operations into IMG, we provide an integrated environment for users to perform deeper and extended data analysis and annotation in a single system that can lead to publications and community knowledge sharing as shown in the case studies.« less

DNA modification and functional delivery into human cells using Escherichia coli DH10B

PubMed Central

Narayanan, Kumaran; Warburton, Peter E.

2003-01-01

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ∼200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies. PMID:12711696

Potential pitfalls of CRISPR/Cas9-mediated genome editing.

PubMed

Peng, Rongxue; Lin, Guigao; Li, Jinming

2016-04-01

Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012. © 2015 FEBS.

Competitive Genomic Screens of Barcoded Yeast Libraries

PubMed Central

Urbanus, Malene; Proctor, Michael; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

2011-01-01

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. PMID:21860376

Genome-editing Technologies for Gene and Cell Therapy.

PubMed

Maeder, Morgan L; Gersbach, Charles A

2016-03-01

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

Genome-editing Technologies for Gene and Cell Therapy

PubMed Central

Maeder, Morgan L; Gersbach, Charles A

2016-01-01

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed. PMID:26755333

The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data.

PubMed

Clarke, Laura; Fairley, Susan; Zheng-Bradley, Xiangqun; Streeter, Ian; Perry, Emily; Lowy, Ernesto; Tassé, Anne-Marie; Flicek, Paul

2017-01-04

The International Genome Sample Resource (IGSR; http://www.internationalgenome.org) expands in data type and population diversity the resources from the 1000 Genomes Project. IGSR represents the largest open collection of human variation data and provides easy access to these resources. IGSR was established in 2015 to maintain and extend the 1000 Genomes Project data, which has been widely used as a reference set of human variation and by researchers developing analysis methods. IGSR has mapped all of the 1000 Genomes sequence to the newest human reference (GRCh38), and will release updated variant calls to ensure maximal usefulness of the existing data. IGSR is collecting new structural variation data on the 1000 Genomes samples from long read sequencing and other technologies, and will collect relevant functional data into a single comprehensive resource. IGSR is extending coverage with new populations sequenced by collaborating groups. Here, we present the new data and analysis that IGSR has made available. We have also introduced a new data portal that increases discoverability of our data-previously only browseable through our FTP site-by focusing on particular samples, populations or data sets of interest. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human Genomic Loci Important in Common Infectious Diseases: Role of High-Throughput Sequencing and Genome-Wide Association Studies

PubMed Central

Sserwadda, Ivan; Amujal, Marion; Namatovu, Norah

2018-01-01

HIV/AIDS, tuberculosis (TB), and malaria are 3 major global public health threats that undermine development in many resource-poor settings. Recently, the notion that positive selection during epidemics or longer periods of exposure to common infectious diseases may have had a major effect in modifying the constitution of the human genome is being interrogated at a large scale in many populations around the world. This positive selection from infectious diseases increases power to detect associations in genome-wide association studies (GWASs). High-throughput sequencing (HTS) has transformed both the management of infectious diseases and continues to enable large-scale functional characterization of host resistance/susceptibility alleles and loci; a paradigm shift from single candidate gene studies. Application of genome sequencing technologies and genomics has enabled us to interrogate the host-pathogen interface for improving human health. Human populations are constantly locked in evolutionary arms races with pathogens; therefore, identification of common infectious disease-associated genomic variants/markers is important in therapeutic, vaccine development, and screening susceptible individuals in a population. This review describes a range of host-pathogen genomic loci that have been associated with disease susceptibility and resistant patterns in the era of HTS. We further highlight potential opportunities for these genetic markers. PMID:29755620

Superior cross-species reference genes: a blueberry case study

USDA-ARS?s Scientific Manuscript database

The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the applic...

[Genome editing ~Principle and possibility of a novel genetic engineering technology. Basic principles of genome editing.

PubMed

Yamamoto, Takashi

Programmable site-specific nuclease mediated-genome editing is an emerging biotechnology for precise manipulation of target genes. In genome editing, gene-knockout as well as gene-knockin are possible in various organisms and cultured cells. CRISPR-Cas9, which was developed in 2012, is a convenient and efficient programmable site-specific nuclease and the use spreads around the world rapidly. For this, it is important for the progress of life science research to introduce the genome editing technology.

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer.

PubMed

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph; Aury, Jean-Marc

2017-02-01

Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. © The Author 2017. Published by Oxford University Press.

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

PubMed Central

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph

2017-01-01

Abstract Background: Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Results: Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Conclusion: Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. PMID:28369459

CRISPR Genome Engineering for Human Pluripotent Stem Cell Research

PubMed Central

Chaterji, Somali; Ahn, Eun Hyun; Kim, Deok-Ho

2017-01-01

The emergence of targeted and efficient genome editing technologies, such as repurposed bacterial programmable nucleases (e.g., CRISPR-Cas systems), has abetted the development of cell engineering approaches. Lessons learned from the development of RNA-interference (RNA-i) therapies can spur the translation of genome editing, such as those enabling the translation of human pluripotent stem cell engineering. In this review, we discuss the opportunities and the challenges of repurposing bacterial nucleases for genome editing, while appreciating their roles, primarily at the epigenomic granularity. First, we discuss the evolution of high-precision, genome editing technologies, highlighting CRISPR-Cas9. They exist in the form of programmable nucleases, engineered with sequence-specific localizing domains, and with the ability to revolutionize human stem cell technologies through precision targeting with greater on-target activities. Next, we highlight the major challenges that need to be met prior to bench-to-bedside translation, often learning from the path-to-clinic of complementary technologies, such as RNA-i. Finally, we suggest potential bioinformatics developments and CRISPR delivery vehicles that can be deployed to circumvent some of the challenges confronting genome editing technologies en route to the clinic. PMID:29158838

Identifying the Future Needs for Long-Term USDA Efforts in Agricultural Animal Genomics

PubMed Central

Green, R. D.; Qureshi, M. A.; Long, J. A.; Burfening, P.J.; Hamernik, D.L.

2007-01-01

Agricultural animal research has been immensely successful over the past century in developing technology and methodologies that have dramatically enhanced production efficiency of the beef, dairy, swine, poultry, sheep, and aquaculture industries. In the past two decades, molecular biology has changed the face of agricultural animal research, primarily in the arena of genomics and the relatively new offshoot areas of functional genomics, proteomics, transcriptomics, metabolomics and metagenomics. Publication of genetic and physical genome maps in the past 15 years has given rise to the possibility of being able finally to understand the molecular nature of the genetic component of phenotypic variation. While quantitative geneticists have been remarkably successful in improving production traits, genomic technology holds potential for being able to lead to more accurate and rapid animal improvement, especially for phenotypic traits that are difficult to measure. Recently, the agricultural research community has been able to capitalize on the infrastructure built by the human genome project by sequencing two of the major livestock genomes (Gallus domesticus and Bos Taurus). The 2005 calendar year is truly unprecedented in the history of agricultural animal research since draft genome sequences were completed for chickens and cattle. In addition, sequencing the swine and equine genome was initiated in early 2006. We now have in place a powerful toolbox for understanding the genetic variation underlying economically important and complex phenotypes. Over the past few years, new challenges have emerged for animal agriculture. Enhancements in production efficiency have not come without some negative side effects on animal well-being and longevity in production environments, including losses in reproductive efficiency, increased stress susceptibility, increased animal waste issues, and increased susceptibility to animal metabolic and infectious diseases. When considered in concert with societal concerns in the areas of natural resource conservation and protection, animal welfare, and food safety, it is clear that publicly supported agricultural research must be focused on enhancing the functionality and well-being of livestock and poultry in environmentally neutral production systems in the future. Realizing the great potential for animal genomics to address these and other issues, a workshop was convened by the U. S. Department of Agriculture (USDA) in Washington, DC in September of 2004. The workshop was entitled “Charting the Road Map for Long Term USDA Efforts in Agricultural Animal Genomics”. This paper summarizes the proceedings of the workshop and the resulting recommendations. The need for a cohesive, comprehensive long-term plan for all of USDA's research efforts in animal genomics was evident at the workshop, requiring further integration of the efforts of the USDA's Cooperative State Research, Education, and Extension Service (CSREES) and the USDA's Agricultural Research Service (ARS) to achieve the greatest return on investment. PMID:17384737

Cloud Based Resource for Data Hosting, Visualization and Analysis Using UCSC Cancer Genomics Browser | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The Cancer Analysis Virtual Machine (CAVM) project will leverage cloud technology, the UCSC Cancer Genomics Browser, and the Galaxy analysis workflow system to provide investigators with a flexible, scalable platform for hosting, visualizing and analyzing their own genomic data.

Loss of stomach, loss of appetite? Sequencing of the ballan wrasse (Labrus bergylta) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

PubMed

Lie, Kai K; Tørresen, Ole K; Solbakken, Monica Hongrø; Rønnestad, Ivar; Tooming-Klunderud, Ave; Nederbragt, Alexander J; Jentoft, Sissel; Sæle, Øystein

2018-03-06

The ballan wrasse (Labrus bergylta) belongs to a large teleost family containing more than 600 species showing several unique evolutionary traits such as lack of stomach and hermaphroditism. Agastric fish are found throughout the teleost phylogeny, in quite diverse and unrelated lineages, indicating stomach loss has occurred independently multiple times in the course of evolution. By assembling the ballan wrasse genome and transcriptome we aimed to determine the genetic basis for its digestive system function and appetite regulation. Among other, this knowledge will aid the formulation of aquaculture diets that meet the nutritional needs of agastric species. Long and short read sequencing technologies were combined to generate a ballan wrasse genome of 805 Mbp. Analysis of the genome and transcriptome assemblies confirmed the absence of genes that code for proteins involved in gastric function. The gene coding for the appetite stimulating protein ghrelin was also absent in wrasse. Gene synteny mapping identified several appetite-controlling genes and their paralogs previously undescribed in fish. Transcriptome profiling along the length of the intestine found a declining expression gradient from the anterior to the posterior, and a distinct expression profile in the hind gut. We showed gene loss has occurred for all known genes related to stomach function in the ballan wrasse, while the remaining functions of the digestive tract appear intact. The results also show appetite control in ballan wrasse has undergone substantial changes. The loss of ghrelin suggests that other genes, such as motilin, may play a ghrelin like role. The wrasse genome offers novel insight in to the evolutionary traits of this large family. As the stomach plays a major role in protein digestion, the lack of genes related to stomach digestion in wrasse suggests it requires formulated diets with higher levels of readily digestible protein than those for gastric species.

Social and Communicative Functions of Informed Consent Forms in East Asia and Beyond

PubMed Central

Yoshizawa, Go; Sasongko, Teguh H.; Ho, Chih-Hsing; Kato, Kazuto

2017-01-01

The recent research and technology development in medical genomics has raised new issues that are profoundly different from those encountered in traditional clinical research for which informed consent was developed. Global initiatives for international collaboration and public participation in genomics research now face an increasing demand for new forms of informed consent which reflect local contexts. This article analyzes informed consent forms (ICFs) for genomic research formulated by four selected research programs and institutes in East Asia – the Medical Genome Science Program in Japan, Universiti Sains Malaysia Human Research Ethics Committee in Malaysia, and the Taiwan Biobank and the Taipei Medical University- Joint Institutional Review Board in Taiwan. The comparative text analysis highlights East Asian contexts as distinct from other regions by identifying communicative and social functions of consent forms. The communicative functions include re-contact options and offering interactive support for research participants, and setting opportunities for family or community engagement in the consent process. This implies that informed consent cannot be validated solely with the completion of a consent form at the initial stage of the research, and informed consent templates can facilitate interactions between researchers and participants through (even before and after) the research process. The social functions consist of informing participants of possible social risks that include genetic discrimination, sample and data sharing, and highlighting the role of ethics committees. Although international ethics harmonization and the subsequent coordination of consent forms may be necessary to maintain the quality and consistency of consent process for data-intensive international research, it is also worth paying more attention to the local values and different settings that exist where research participants are situated for research in medical genomics. More than simply tools to gain consent from research participants, ICFs function rather as a device of social communication between research communities and civic communities in liaison with intermediary agents like ethics committees, genetic counselors, and public biobanks and databases. PMID:28775738

Social and Communicative Functions of Informed Consent Forms in East Asia and Beyond.

PubMed

Yoshizawa, Go; Sasongko, Teguh H; Ho, Chih-Hsing; Kato, Kazuto

2017-01-01

The recent research and technology development in medical genomics has raised new issues that are profoundly different from those encountered in traditional clinical research for which informed consent was developed. Global initiatives for international collaboration and public participation in genomics research now face an increasing demand for new forms of informed consent which reflect local contexts. This article analyzes informed consent forms (ICFs) for genomic research formulated by four selected research programs and institutes in East Asia - the Medical Genome Science Program in Japan, Universiti Sains Malaysia Human Research Ethics Committee in Malaysia, and the Taiwan Biobank and the Taipei Medical University- Joint Institutional Review Board in Taiwan. The comparative text analysis highlights East Asian contexts as distinct from other regions by identifying communicative and social functions of consent forms. The communicative functions include re-contact options and offering interactive support for research participants, and setting opportunities for family or community engagement in the consent process. This implies that informed consent cannot be validated solely with the completion of a consent form at the initial stage of the research, and informed consent templates can facilitate interactions between researchers and participants through (even before and after) the research process. The social functions consist of informing participants of possible social risks that include genetic discrimination, sample and data sharing, and highlighting the role of ethics committees. Although international ethics harmonization and the subsequent coordination of consent forms may be necessary to maintain the quality and consistency of consent process for data-intensive international research, it is also worth paying more attention to the local values and different settings that exist where research participants are situated for research in medical genomics. More than simply tools to gain consent from research participants, ICFs function rather as a device of social communication between research communities and civic communities in liaison with intermediary agents like ethics committees, genetic counselors, and public biobanks and databases.

A blueprint for a Human Epigenome Project: the AACR Human Epigenome Workshop.

PubMed

Jones, Peter A; Martienssen, Robert

2005-12-15

Epigenetic processes control the packaging and function of the human genome and contribute to normal and pathologic states, including cancer. The time is ripe to undertake an international effort to identify all the chemical changes and relationships between chromatin constituents that provide function to the genetic code. A timely workshop of leading experts, convened by the American Association for Cancer Research (AACR), confirmed that the technology is at hand to begin defining human epigenomes at high resolution.

Next-Generation Sequencing: The Translational Medicine Approach from “Bench to Bedside to Population”

PubMed Central

Beigh, Mohammad Muzafar

2016-01-01

Humans have predicted the relationship between heredity and diseases for a long time. Only in the beginning of the last century, scientists begin to discover the connotations between different genes and disease phenotypes. Recent trends in next-generation sequencing (NGS) technologies have brought a great momentum in biomedical research that in turn has remarkably augmented our basic understanding of human biology and its associated diseases. State-of-the-art next generation biotechnologies have started making huge strides in our current understanding of mechanisms of various chronic illnesses like cancers, metabolic disorders, neurodegenerative anomalies, etc. We are experiencing a renaissance in biomedical research primarily driven by next generation biotechnologies like genomics, transcriptomics, proteomics, metabolomics, lipidomics etc. Although genomic discoveries are at the forefront of next generation omics technologies, however, their implementation into clinical arena had been painstakingly slow mainly because of high reaction costs and unavailability of requisite computational tools for large-scale data analysis. However rapid innovations and steadily lowering cost of sequence-based chemistries along with the development of advanced bioinformatics tools have lately prompted launching and implementation of large-scale massively parallel genome sequencing programs in different fields ranging from medical genetics, infectious biology, agriculture sciences etc. Recent advances in large-scale omics-technologies is bringing healthcare research beyond the traditional “bench to bedside” approach to more of a continuum that will include improvements, in public healthcare and will be primarily based on predictive, preventive, personalized, and participatory medicine approach (P4). Recent large-scale research projects in genetic and infectious disease biology have indicated that massively parallel whole-genome/whole-exome sequencing, transcriptome analysis, and other functional genomic tools can reveal large number of unique functional elements and/or markers that otherwise would be undetected by traditional sequencing methodologies. Therefore, latest trends in the biomedical research is giving birth to the new branch in medicine commonly referred to as personalized and/or precision medicine. Developments in the post-genomic era are believed to completely restructure the present clinical pattern of disease prevention and treatment as well as methods of diagnosis and prognosis. The next important step in the direction of the precision/personalized medicine approach should be its early adoption in clinics for future medical interventions. Consequently, in coming year’s next generation biotechnologies will reorient medical practice more towards disease prediction and prevention approaches rather than curing them at later stages of their development and progression, even at wider population level(s) for general public healthcare system. PMID:28930123

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells.

PubMed

Kelbauskas, Laimonas; Ashili, Shashaanka P; Lee, Kristen B; Zhu, Haixin; Tian, Yanqing; Meldrum, Deirdre R

2018-03-12

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)-a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)-a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

PubMed Central

Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

2014-01-01

Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

PubMed Central

Chi, Shen; Weiss, Arthur; Wang, Haopeng

2016-01-01

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

Metabolomic strategies to map functions of metabolic pathways.

PubMed

Mulvihill, Melinda M; Nomura, Daniel K

2014-08-01

Genome sequencing efforts have revealed a strikingly large number of unannotated and uncharacterized genes that fall into metabolic enzymes classes, likely indicating that our current knowledge of biochemical pathways in normal physiology, let alone in disease states, remains largely incomplete. This realization presents a daunting challenge for post-genomic-era scientists in deciphering the biochemical and (patho)physiological roles of these enzymes and their metabolites and metabolic networks. This is further complicated by many recent studies showing a rewiring of normal metabolic networks in disease states to give rise to unique pathophysiological functions of enzymes, metabolites, and metabolic pathways. This review focuses on recent discoveries made using metabolic mapping technologies to uncover novel pathways and metabolite-mediated posttranslational modifications and epigenetic alterations and their impact on physiology and disease. Copyright © 2014 the American Physiological Society.

Protein Function Prediction: Problems and Pitfalls.

PubMed

Pearson, William R

2015-09-03

The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified proteins. Here, we highlight some of the challenges in functional inference from sequence similarity. Investigators can improve the accuracy of function prediction by (1) being conservative about the evolutionary distance to a protein of known function; (2) considering the ambiguous meaning of "functional similarity," and (3) being aware of the limitations of annotations in functional databases. Protein function prediction does not offer "one-size-fits-all" solutions. Prediction strategies work better when the idiosyncrasies of function and functional annotation are better understood. Copyright © 2015 John Wiley & Sons, Inc.

The mitochondrial genome in embryo technologies.

PubMed

Hiendleder, S; Wolf, E

2003-08-01

The mammalian mitochondrial genome encodes for 37 genes which are involved in a broad range of cellular functions. The mitochondrial DNA (mtDNA) molecule is commonly assumed to be inherited through oocyte cytoplasm in a clonal manner, and apparently species-specific mechanisms have evolved to eliminate the contribution of sperm mitochondria after natural fertilization. However, recent evidence for paternal mtDNA inheritance in embryos and offspring questions the general validity of this model, particularly in the context of assisted reproduction and embryo biotechnology. In addition to normal mt DNA haplotype variation, oocytes and spermatozoa show remarkable differences in mtDNA content and may be affected by inherited or acquired mtDNA aberrations. All these parameters have been correlated with gamete quality and reproductive success rates. Nuclear transfer (NT) technology provides experimental models for studying interactions between nuclear and mitochondrial genomes. Recent studies demonstrated (i) a significant effect of mtDNA haplotype or other maternal cytoplasmic factors on the efficiency of NT; (ii) phenotypic differences between transmitochondrial clones pointing to functionally relevant nuclear-cytoplasmic interactions; and (iii) neutral or non-neutral selection of mtDNA haplotypes in heteroplasmic conditions. Mitochondria form a dynamic reticulum, enabling complementation of mitochondrial components and possibly mixing of different mtDNA populations in heteroplasmic individuals. Future directions of research on mtDNA in the context of reproductive biotechnology range from the elimination of adverse effects of artificial heteroplasmy, e.g. created by ooplasm transfer, to engineering of optimized constellations of nuclear and cytoplasmic genes for the production of superior livestock.

Hybrid genome assembly and annotation of Paenibacillus pasadenensis strain R16 reveals insights on endophytic life style and antifungal activity

PubMed Central

Passera, Alessandro; Marcolungo, Luca; Brasca, Milena; Quaglino, Fabio; Cantaloni, Chiara; Delledonne, Massimo

2018-01-01

Bacteria of the Paenibacillus genus are becoming important in many fields of science, including agriculture, for their positive effects on the health of plants. However, there are little information available on this genus compared to other bacteria (such as Bacillus or Pseudomonas), especially when considering genomic information. Sequencing the genomes of plant-beneficial bacteria is a crucial step to identify the genetic elements underlying the adaptation to life inside a plant host and, in particular, which of these features determine the differences between a helpful microorganism and a pathogenic one. In this study, we have characterized the genome of Paenibacillus pasadenensis, strain R16, recently investigated for its antifungal activities and plant-associated features. An hybrid assembly approach was used integrating the very precise reads obtained by Illumina technology and long fragments acquired with Oxford Nanopore Technology (ONT) sequencing. De novo genome assembly based solely on Illumina reads generated a relatively fragmented assembly of 5.72 Mbp in 99 ungapped sequences with an N50 length of 544 Kbp; hybrid assembly, integrating Illumina and ONT reads, improved the assembly quality, generating a genome of 5.75 Mbp, organized in 6 contigs with an N50 length of 3.4 Mbp. Annotation of the latter genome identified 4987 coding sequences, of which 1610 are hypothetical proteins. Enrichment analysis identified pathways of particular interest for the endophyte biology, including the chitin-utilization pathway and the incomplete siderophore pathway which hints at siderophore parasitism. In addition the analysis led to the identification of genes for the production of terpenes, as for example farnesol, that was hypothesized as the main antifungal molecule produced by the strain. The functional analysis on the genome confirmed several plant-associated, plant-growth promotion, and biocontrol traits of strain R16, thus adding insights in the genetic bases of these complex features, and of the Paenibacillus genus in general. PMID:29351296

Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations

PubMed Central

2017-01-01

Recent advances in next-generation sequencing approaches have revolutionized our understanding of transcriptional expression in diverse systems. However, measurements of transcription do not necessarily reflect gene translation, the process of ultimate importance in understanding cellular function. To circumvent this limitation, biochemical tagging of ribosome subunits to isolate ribosome-associated mRNA has been developed. However, this approach, called TRAP, lacks quantitative resolution compared to a superior technology, ribosome profiling. Here, we report the development of an optimized ribosome profiling approach in Drosophila. We first demonstrate successful ribosome profiling from a specific tissue, larval muscle, with enhanced resolution compared to conventional TRAP approaches. We next validate the ability of this technology to define genome-wide translational regulation. This technology is leveraged to test the relative contributions of transcriptional and translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function at the neuromuscular junction. Surprisingly, we find no evidence that significant changes in the transcription or translation of specific genes are necessary to enable retrograde homeostatic signaling, implying that post-translational mechanisms ultimately gate instructive retrograde communication. Finally, we show that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this development and validation of tissue-specific ribosome profiling enables sensitive and specific analysis of translation in Drosophila. PMID:29194454

[Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology].

PubMed

Meng, Fanrong; Zhao, Dan; Zhou, Qinghua; Liu, Zhe

2018-05-20

It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology. In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR. The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue. The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.

Towards a complete map of the human long non-coding RNA transcriptome.

PubMed

Uszczynska-Ratajczak, Barbara; Lagarde, Julien; Frankish, Adam; Guigó, Roderic; Johnson, Rory

2018-05-23

Gene maps, or annotations, enable us to navigate the functional landscape of our genome. They are a resource upon which virtually all studies depend, from single-gene to genome-wide scales and from basic molecular biology to medical genetics. Yet present-day annotations suffer from trade-offs between quality and size, with serious but often unappreciated consequences for downstream studies. This is particularly true for long non-coding RNAs (lncRNAs), which are poorly characterized compared to protein-coding genes. Long-read sequencing technologies promise to improve current annotations, paving the way towards a complete annotation of lncRNAs expressed throughout a human lifetime.

Management of Incidental Findings in the Era of Next-generation Sequencing

PubMed Central

Blackburn, Heather L.; Schroeder, Bradley; Turner, Clesson; Shriver, Craig D.; Ellsworth, Darrell L.; Ellsworth, Rachel E.

2015-01-01

Next-generation sequencing (NGS) technologies allow for the generation of whole exome or whole genome sequencing data, which can be used to identify novel genetic alterations associated with defined phenotypes or to expedite discovery of functional variants for improved patient care. Because this robust technology has the ability to identify all mutations within a genome, incidental findings (IF)- genetic alterations associated with conditions or diseases unrelated to the patient’s present condition for which current tests are being performed- may have important clinical ramifications. The current debate among genetic scientists and clinicians focuses on the following questions: 1) should any IF be disclosed to patients, and 2) which IF should be disclosed – actionable mutations, variants of unknown significance, or all IF? Policies for disclosure of IF are being developed for when and how to convey these findings and whether adults, minors, or individuals unable to provide consent have the right to refuse receipt of IF. In this review, we detail current NGS technology platforms, discuss pressing issues regarding disclosure of IF, and how IF are currently being handled in prenatal, pediatric, and adult patients. PMID:26069456

The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation.

PubMed

Mavromatis, Konstantinos; Land, Miriam L; Brettin, Thomas S; Quest, Daniel J; Copeland, Alex; Clum, Alicia; Goodwin, Lynne; Woyke, Tanja; Lapidus, Alla; Klenk, Hans Peter; Cottingham, Robert W; Kyrpides, Nikos C

2012-01-01

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation. In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis. These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).

The emerging genomics and systems biology research lead to systems genomics studies.

PubMed

Yang, Mary Qu; Yoshigoe, Kenji; Yang, William; Tong, Weida; Qin, Xiang; Dunker, A; Chen, Zhongxue; Arbania, Hamid R; Liu, Jun S; Niemierko, Andrzej; Yang, Jack Y

2014-01-01

Synergistically integrating multi-layer genomic data at systems level not only can lead to deeper insights into the molecular mechanisms related to disease initiation and progression, but also can guide pathway-based biomarker and drug target identification. With the advent of high-throughput next-generation sequencing technologies, sequencing both DNA and RNA has generated multi-layer genomic data that can provide DNA polymorphism, non-coding RNA, messenger RNA, gene expression, isoform and alternative splicing information. Systems biology on the other hand studies complex biological systems, particularly systematic study of complex molecular interactions within specific cells or organisms. Genomics and molecular systems biology can be merged into the study of genomic profiles and implicated biological functions at cellular or organism level. The prospectively emerging field can be referred to as systems genomics or genomic systems biology. The Mid-South Bioinformatics Centre (MBC) and Joint Bioinformatics Ph.D. Program of University of Arkansas at Little Rock and University of Arkansas for Medical Sciences are particularly interested in promoting education and research advancement in this prospectively emerging field. Based on past investigations and research outcomes, MBC is further utilizing differential gene and isoform/exon expression from RNA-seq and co-regulation from the ChiP-seq specific for different phenotypes in combination with protein-protein interactions, and protein-DNA interactions to construct high-level gene networks for an integrative genome-phoneme investigation at systems biology level.

Jatropha curcas, a biofuel crop: functional genomics for understanding metabolic pathways and genetic improvement.

PubMed

Maghuly, Fatemeh; Laimer, Margit

2013-10-01

Jatropha curcas is currently attracting much attention as an oilseed crop for biofuel, as Jatropha can grow under climate and soil conditions that are unsuitable for food production. However, little is known about Jatropha, and there are a number of challenges to be overcome. In fact, Jatropha has not really been domesticated; most of the Jatropha accessions are toxic, which renders the seedcake unsuitable for use as animal feed. The seeds of Jatropha contain high levels of polyunsaturated fatty acids, which negatively impact the biofuel quality. Fruiting of Jatropha is fairly continuous, thus increasing costs of harvesting. Therefore, before starting any improvement program using conventional or molecular breeding techniques, understanding gene function and the genome scale of Jatropha are prerequisites. This review presents currently available and relevant information on the latest technologies (genomics, transcriptomics, proteomics and metabolomics) to decipher important metabolic pathways within Jatropha, such as oil and toxin synthesis. Further, it discusses future directions for biotechnological approaches in Jatropha breeding and improvement. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimizing high performance computing workflow for protein functional annotation.

PubMed

Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

2014-09-10

Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data.

Optimizing high performance computing workflow for protein functional annotation

PubMed Central

Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

2014-01-01

Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data. PMID:25313296

Scientific Advances with Aspergillus Species that Are Used for Food and Biotech Applications.

PubMed

Biesebeke, Rob Te; Record, Erik

2008-01-01

Yeast and filamentous fungi have been used for centuries in diverse biotechnological processes. Fungal fermentation technology is traditionally used in relation to food production, such as for bread, beer, cheese, sake and soy sauce. Last century, the industrial application of yeast and filamentous fungi expanded rapidly, with excellent examples such as purified enzymes and secondary metabolites (e.g. antibiotics), which are used in a wide range of food as well as non-food industries. Research on protein and/or metabolite secretion by fungal species has focused on identifying bottlenecks in (post-) transcriptional regulation of protein production, metabolic rerouting, morphology and the transit of proteins through the secretion pathway. In past years, genome sequencing of some fungi (e.g. Aspergillus oryzae, Aspergillus niger) has been completed. The available genome sequences have enabled identification of genes and functionally important regions of the genome. This has directed research to focus on a post-genomics era in which transcriptomics, proteomics and metabolomics methodologies will help to explore the scientific relevance and industrial application of fungal genome sequences.

CRISPR-Cas9 Toolkit for Actinomycete Genome Editing.

PubMed

Tong, Yaojun; Robertsen, Helene Lunde; Blin, Kai; Weber, Tilmann; Lee, Sang Yup

2018-01-01

Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes.

Quantifying on- and off-target genome editing.

PubMed

Hendel, Ayal; Fine, Eli J; Bao, Gang; Porteus, Matthew H

2015-02-01

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. While the ability to make precise and controlled changes at specified sites throughout the genome has grown tremendously in recent years, we still lack a comprehensive and standardized battery of assays for measuring the different genome editing outcomes created at endogenous genomic loci. Here we review the existing assays for quantifying on- and off-target genome editing and describe their utility in advancing the technology. We also highlight unmet assay needs for quantifying on- and off-target genome editing outcomes and discuss their importance for the genome editing field. Copyright © 2014 Elsevier Ltd. All rights reserved.

Emerging Role of CRISPR/Cas9 Technology for MicroRNAs Editing in Cancer Research.

PubMed

Aquino-Jarquin, Guillermo

2017-12-15

MicroRNAs (miRNA) are small, noncoding RNA molecules with a master role in the regulation of important tasks in different critical processes of cancer pathogenesis. Because there are different miRNAs implicated in all the stages of cancer, for example, functioning as oncogenes, this makes these small molecules suitable targets for cancer diagnosis and therapy. RNA-mediated interference has been one major approach for sequence-specific regulation of gene expression in eukaryotic organisms. Recently, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, first identified in bacteria and archaea as an adaptive immune response to invading genetic material, has been explored as a sequence-specific molecular tool for editing genomic sequences for basic research in life sciences and for therapeutic purposes. There is growing evidence that small noncoding RNAs, including miRNAs, can be targeted by the CRISPR/Cas9 system despite their lacking an open reading frame to evaluate functional loss. Thus, CRISPR/Cas9 technology represents a novel gene-editing strategy with compelling robustness, specificity, and stability for the modification of miRNA expression. Here, I summarize key features of current knowledge of genomic editing by CRISPR/Cas9 technology as a feasible strategy for globally interrogating miRNA gene function and miRNA-based therapeutic intervention. Alternative emerging strategies for nonviral delivery of CRISPR/Cas9 core components into human cells in a clinical context are also analyzed critically. Cancer Res; 77(24); 6812-7. ©2017 AACR . ©2017 American Association for Cancer Research.

Home - The Cancer Genome Atlas - Cancer Genome - TCGA

Cancer.gov

The Cancer Genome Atlas (TCGA) is a comprehensive and coordinated effort to accelerate our understanding of the molecular basis of cancer through the application of genome analysis technologies, including large-scale genome sequencing.

Genome Maps, a new generation genome browser.

PubMed

Medina, Ignacio; Salavert, Francisco; Sanchez, Rubén; de Maria, Alejandro; Alonso, Roberto; Escobar, Pablo; Bleda, Marta; Dopazo, Joaquín

2013-07-01

Genome browsers have gained importance as more genomes and related genomic information become available. However, the increase of information brought about by new generation sequencing technologies is, at the same time, causing a subtle but continuous decrease in the efficiency of conventional genome browsers. Here, we present Genome Maps, a genome browser that implements an innovative model of data transfer and management. The program uses highly efficient technologies from the new HTML5 standard, such as scalable vector graphics, that optimize workloads at both server and client sides and ensure future scalability. Thus, data management and representation are entirely carried out by the browser, without the need of any Java Applet, Flash or other plug-in technology installation. Relevant biological data on genes, transcripts, exons, regulatory features, single-nucleotide polymorphisms, karyotype and so forth, are imported from web services and are available as tracks. In addition, several DAS servers are already included in Genome Maps. As a novelty, this web-based genome browser allows the local upload of huge genomic data files (e.g. VCF or BAM) that can be dynamically visualized in real time at the client side, thus facilitating the management of medical data affected by privacy restrictions. Finally, Genome Maps can easily be integrated in any web application by including only a few lines of code. Genome Maps is an open source collaborative initiative available in the GitHub repository (https://github.com/compbio-bigdata-viz/genome-maps). Genome Maps is available at: http://www.genomemaps.org.

Genome Maps, a new generation genome browser

PubMed Central

Medina, Ignacio; Salavert, Francisco; Sanchez, Rubén; de Maria, Alejandro; Alonso, Roberto; Escobar, Pablo; Bleda, Marta; Dopazo, Joaquín

2013-01-01

Genome browsers have gained importance as more genomes and related genomic information become available. However, the increase of information brought about by new generation sequencing technologies is, at the same time, causing a subtle but continuous decrease in the efficiency of conventional genome browsers. Here, we present Genome Maps, a genome browser that implements an innovative model of data transfer and management. The program uses highly efficient technologies from the new HTML5 standard, such as scalable vector graphics, that optimize workloads at both server and client sides and ensure future scalability. Thus, data management and representation are entirely carried out by the browser, without the need of any Java Applet, Flash or other plug-in technology installation. Relevant biological data on genes, transcripts, exons, regulatory features, single-nucleotide polymorphisms, karyotype and so forth, are imported from web services and are available as tracks. In addition, several DAS servers are already included in Genome Maps. As a novelty, this web-based genome browser allows the local upload of huge genomic data files (e.g. VCF or BAM) that can be dynamically visualized in real time at the client side, thus facilitating the management of medical data affected by privacy restrictions. Finally, Genome Maps can easily be integrated in any web application by including only a few lines of code. Genome Maps is an open source collaborative initiative available in the GitHub repository (https://github.com/compbio-bigdata-viz/genome-maps). Genome Maps is available at: http://www.genomemaps.org. PMID:23748955

Genome Sequencing of Steroid Producing Bacteria Using Ion Torrent Technology and a Reference Genome.

PubMed

Sola-Landa, Alberto; Rodríguez-García, Antonio; Barreiro, Carlos; Pérez-Redondo, Rosario

2017-01-01

The Next-Generation Sequencing technology has enormously eased the bacterial genome sequencing and several tens of thousands of genomes have been sequenced during the last 10 years. Most of the genome projects are published as draft version, however, for certain applications the complete genome sequence is required.In this chapter, we describe the strategy that allowed the complete genome sequencing of Mycobacterium neoaurum NRRL B-3805, an industrial strain exploited for steroid production, using Ion Torrent sequencing reads and the genome of a close strain as the reference. This protocol can be applied to analyze the genetic variations between closely related strains; for example, to elucidate the point mutations between a parental strain and a random mutagenesis-derived mutant.

Status of duckweed genomics and transcriptomics.

PubMed

Wang, W; Messing, J

2015-01-01

Duckweeds belong to the smallest flowering plants that undergo fast vegetative growth in an aquatic environment. They are commonly used in wastewater treatment and animal feed. Whereas duckweeds have been studied at the biochemical level, their reduced morphology and wide environmental adaption had not been subjected to molecular analysis until recently. Here, we review the progress that has been made in using a DNA barcode system and the sequences of chloroplast and mitochondrial genomes to identify duckweed species at the species or population level. We also review analysis of the nuclear genome sequence of Spirodela that provides new insights into fundamental biological questions. Indeed, reduced gene families and missing genes are consistent with its compact morphogenesis, aquatic floating and suppression of juvenile-to-adult transition. Furthermore, deep RNA sequencing of Spirodela at the onset of dormancy and Landoltia in exposure of nutrient deficiency illustrate the molecular network for environmental adaption and stress response, constituting major progress towards a post-genome sequencing phase, where further functional genomic details can be explored. Rapid advances in sequencing technologies could continue to promote a proliferation of genome sequences for additional ecotypes as well as for other duckweed species. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

The complete chloroplast genome sequence of Actinidia arguta using the PacBio RS II platform

PubMed Central

Lin, Miaomiao; Qi, Xiujuan; Chen, Jinyong; Sun, Leiming; Zhong, Yunpeng; Fang, Jinbao; Hu, Chungen

2018-01-01

Actinidia arguta is the most basal species in a phylogenetically and economically important genus in the family Actinidiaceae. To better understand the molecular basis of the Actinidia arguta chloroplast (cp), we sequenced the complete cp genome from A. arguta using Illumina and PacBio RS II sequencing technologies. The cp genome from A. arguta was 157,611 bp in length and composed of a pair of 24,232 bp inverted repeats (IRs) separated by a 20,463 bp small single copy region (SSC) and an 88,684 bp large single copy region (LSC). Overall, the cp genome contained 113 unique genes. The cp genomes from A. arguta and three other Actinidia species from GenBank were subjected to a comparative analysis. Indel mutation events and high frequencies of base substitution were identified, and the accD and ycf2 genes showed a high degree of variation within Actinidia. Forty-seven simple sequence repeats (SSRs) and 155 repetitive structures were identified, further demonstrating the rapid evolution in Actinidia. The cp genome analysis and the identification of variable loci provide vital information for understanding the evolution and function of the chloroplast and for characterizing Actinidia population genetics. PMID:29795601

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

PubMed

Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

2015-10-01

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms. © 2015 The Author(s).

Sockeye: A 3D Environment for Comparative Genomics

PubMed Central

Montgomery, Stephen B.; Astakhova, Tamara; Bilenky, Mikhail; Birney, Ewan; Fu, Tony; Hassel, Maik; Melsopp, Craig; Rak, Marcin; Robertson, A. Gordon; Sleumer, Monica; Siddiqui, Asim S.; Jones, Steven J.M.

2004-01-01

Comparative genomics techniques are used in bioinformatics analyses to identify the structural and functional properties of DNA sequences. As the amount of available sequence data steadily increases, the ability to perform large-scale comparative analyses has become increasingly relevant. In addition, the growing complexity of genomic feature annotation means that new approaches to genomic visualization need to be explored. We have developed a Java-based application called Sockeye that uses three-dimensional (3D) graphics technology to facilitate the visualization of annotation and conservation across multiple sequences. This software uses the Ensembl database project to import sequence and annotation information from several eukaryotic species. A user can additionally import their own custom sequence and annotation data. Individual annotation objects are displayed in Sockeye by using custom 3D models. Ensembl-derived and imported sequences can be analyzed by using a suite of multiple and pair-wise alignment algorithms. The results of these comparative analyses are also displayed in the 3D environment of Sockeye. By using the Java3D API to visualize genomic data in a 3D environment, we are able to compactly display cross-sequence comparisons. This provides the user with a novel platform for visualizing and comparing genomic feature organization. PMID:15123592

Getting a Grip on Complexes

PubMed Central

Nie, Yan; Viola, Cristina; Bieniossek, Christoph; Trowitzsch, Simon; Vijay-achandran, Lakshmi Sumitra; Chaillet, Maxime; Garzoni, Frederic; Berger, Imre

2009-01-01

We are witnessing tremendous advances in our understanding of the organization of life. Complete genomes are being deciphered with ever increasing speed and accuracy, thereby setting the stage for addressing the entire gene product repertoire of cells, towards understanding whole biological systems. Advances in bioinformatics and mass spectrometric techniques have revealed the multitude of interactions present in the proteome. Multiprotein complexes are emerging as a paramount cornerstone of biological activity, as many proteins appear to participate, stably or transiently, in large multisubunit assemblies. Analysis of the architecture of these assemblies and their manifold interactions is imperative for understanding their function at the molecular level. Structural genomics efforts have fostered the development of many technologies towards achieving the throughput required for studying system-wide single proteins and small interaction motifs at high resolution. The present shift in focus towards large multiprotein complexes, in particular in eukaryotes, now calls for a likewise concerted effort to develop and provide new technologies that are urgently required to produce in quality and quantity the plethora of multiprotein assemblies that form the complexome, and to routinely study their structure and function at the molecular level. Current efforts towards this objective are summarized and reviewed in this contribution. PMID:20514218

Whole genome sequencing of a begomovirus-resistant tomato inbred reveals introgressions from wild Solanum species

USDA-ARS?s Scientific Manuscript database

The low cost of next generation sequencing (NGS) technology and the availability of a large number of well annotated plant genomes has made sequencing technology useful to breeding programs. With the published high quality tomato reference genome of the processing cultivar Heinz 1706, we can now uti...

Novel technologies in doubled haploid line development.

PubMed

Ren, Jiaojiao; Wu, Penghao; Trampe, Benjamin; Tian, Xiaolong; Lübberstedt, Thomas; Chen, Shaojiang

2017-11-01

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter- and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere-mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

PubMed Central

2011-01-01

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

Geminiviruses for biotechnology: the art of parasite taming.

PubMed

Lozano-Durán, Rosa

2016-04-01

Viruses are intracellular pathogens that have evolved efficient strategies for replication and expression of their proteins in the host cells. Geminiviruses - plant viruses with small circular single-stranded DNA genomes - effectively manipulate plant cell processes for viral functions, entailing great potential for biotechnological applications. This potentiality has been realized in the form of protein expression and gene-silencing vectors, and, more recently, vectors for genome editing - a technology that these viruses seem particularly well-suited to facilitate. This insight offers an overview of the biological properties of geminiviruses, with emphasis on those leveraging development of geminivirus-based replicons. It illustrates the basis for engineering geminivirus-based replicons and their applications. Furthermore, it discusses the reported use and future perspectives of geminivirus-based replicons for genome editing. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

PubMed Central

Hall, Robyn N.; Meers, Joanne; Fowler, Elizabeth; Mahony, Timothy

2012-01-01

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses. PMID:22470833

[Artificial Intelligence in Drug Discovery].

PubMed

Fujiwara, Takeshi; Kamada, Mayumi; Okuno, Yasushi

2018-04-01

According to the increase of data generated from analytical instruments, application of artificial intelligence(AI)technology in medical field is indispensable. In particular, practical application of AI technology is strongly required in "genomic medicine" and "genomic drug discovery" that conduct medical practice and novel drug development based on individual genomic information. In our laboratory, we have been developing a database to integrate genome data and clinical information obtained by clinical genome analysis and a computational support system for clinical interpretation of variants using AI. In addition, with the aim of creating new therapeutic targets in genomic drug discovery, we have been also working on the development of a binding affinity prediction system for mutated proteins and drugs by molecular dynamics simulation using supercomputer "Kei". We also have tackled for problems in a drug virtual screening. Our developed AI technology has successfully generated virtual compound library, and deep learning method has enabled us to predict interaction between compound and target protein.

Precision genome engineering in lactic acid bacteria

PubMed Central

2014-01-01

Innovative new genome engineering technologies for manipulating chromosomes have appeared in the last decade. One of these technologies, recombination mediated genetic engineering (recombineering) allows for precision DNA engineering of chromosomes and plasmids in Escherichia coli. Single-stranded DNA recombineering (SSDR) allows for the generation of subtle mutations without the need for selection and without leaving behind any foreign DNA. In this review we discuss the application of SSDR technology in lactic acid bacteria, with an emphasis on key factors that were critical to move this technology from E. coli into Lactobacillus reuteri and Lactococcus lactis. We also provide a blueprint for how to proceed if one is attempting to establish SSDR technology in a lactic acid bacterium. The emergence of CRISPR-Cas technology in genome engineering and its potential application to enhancing SSDR in lactic acid bacteria is discussed. The ability to perform precision genome engineering in medically and industrially important lactic acid bacteria will allow for the genetic improvement of strains without compromising safety. PMID:25185700

PATtyFams: Protein families for the microbial genomes in the PATRIC database

DOE PAGES

Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; ...

2016-02-08

The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

How may targeted proteomics complement genomic data in breast cancer?

PubMed

Guerin, Mathilde; Gonçalves, Anthony; Toiron, Yves; Baudelet, Emilie; Audebert, Stéphane; Boyer, Jean-Baptiste; Borg, Jean-Paul; Camoin, Luc

2017-01-01

Breast cancer (BC) is the most common female cancer in the world and was recently deconstructed in different molecular entities. Although most of the recent assays to characterize tumors at the molecular level are genomic-based, proteins are the actual executors of cellular functions and represent the vast majority of targets for anticancer drugs. Accumulated data has demonstrated an important level of quantitative and qualitative discrepancies between genomic/transcriptomic alterations and their protein counterparts, mostly related to the large number of post-translational modifications. Areas covered: This review will present novel proteomics technologies such as Reverse Phase Protein Array (RPPA) or mass-spectrometry (MS) based approaches that have emerged and that could progressively replace old-fashioned methods (e.g. immunohistochemistry, ELISA, etc.) to validate proteins as diagnostic, prognostic or predictive biomarkers, and eventually monitor them in the routine practice. Expert commentary: These different targeted proteomic approaches, able to complement genomic data in BC and characterize tumors more precisely, will permit to go through a more personalized treatment for each patient and tumor.

Therapeutic applications of CRISPR RNA-guided genome editing.

PubMed

Koo, Taeyoung; Kim, Jin-Soo

2017-01-01

The rapid development of programmable nuclease-based genome editing technologies has enabled targeted gene disruption and correction both in vitro and in vivo This revolution opens up the possibility of precise genome editing at target genomic sites to modulate gene function in animals and plants. Among several programmable nucleases, the type II clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) system has progressed remarkably in recent years, leading to its widespread use in research, medicine and biotechnology. In particular, CRISPR-Cas9 shows highly efficient gene editing activity for therapeutic purposes in systems ranging from patient stem cells to animal models. However, the development of therapeutic approaches and delivery methods remains a great challenge for biomedical applications. Herein, we review therapeutic applications that use the CRISPR-Cas9 system and discuss the possibilities and challenges ahead. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

PubMed

Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P; Swayze, Eric E; Bennett, C Frank; Cleveland, Don W

2015-12-22

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

PubMed Central

Jensen, Michael K

2018-01-01

Abstract The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies. PMID:29726937

PATtyFams: Protein families for the microbial genomes in the PATRIC database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.

The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

A Roadmap for Functional Structural Variants in the Soybean Genome

PubMed Central

Anderson, Justin E.; Kantar, Michael B.; Kono, Thomas Y.; Fu, Fengli; Stec, Adrian O.; Song, Qijian; Cregan, Perry B.; Specht, James E.; Diers, Brian W.; Cannon, Steven B.; McHale, Leah K.; Stupar, Robert M.

2014-01-01

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits. PMID:24855315

Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

PubMed Central

Lu, Jia; Zhao, Chen; Zhao, Yingze; Zhang, Jingfang; Zhang, Yue; Chen, Li; Han, Qiyuan; Ying, Yue; Peng, Shuai; Ai, Runna; Wang, Yu

2018-01-01

Abstract Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes. PMID:29237052