MutAid: Sanger and NGS Based Integrated Pipeline for Mutation Identification, Validation and Annotation in Human Molecular Genetics

PubMed Central

Pandey, Ram Vinay; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a new pipeline called MutAid to analyze and interpret raw sequencing data produced by Sanger or several NGS sequencing platforms. It performs format conversion, base calling, quality trimming, filtering, read mapping, variant calling, variant annotation and analysis of Sanger and NGS data under a single platform. It is capable of analyzing reads from multiple patients in a single run to create a list of potential disease causing base substitutions as well as insertions and deletions. MutAid has been developed for expert and non-expert users and supports four sequencing platforms including Sanger, Illumina, 454 and Ion Torrent. Furthermore, for NGS data analysis, five read mappers including BWA, TMAP, Bowtie, Bowtie2 and GSNAP and four variant callers including GATK-HaplotypeCaller, SAMTOOLS, Freebayes and VarScan2 pipelines are supported. MutAid is freely available at https://sourceforge.net/projects/mutaid. PMID:26840129

Ensembl 2002: accommodating comparative genomics.

PubMed

Clamp, M; Andrews, D; Barker, D; Bevan, P; Cameron, G; Chen, Y; Clark, L; Cox, T; Cuff, J; Curwen, V; Down, T; Durbin, R; Eyras, E; Gilbert, J; Hammond, M; Hubbard, T; Kasprzyk, A; Keefe, D; Lehvaslaiho, H; Iyer, V; Melsopp, C; Mongin, E; Pettett, R; Potter, S; Rust, A; Schmidt, E; Searle, S; Slater, G; Smith, J; Spooner, W; Stabenau, A; Stalker, J; Stupka, E; Ureta-Vidal, A; Vastrik, I; Birney, E

2003-01-01

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

The promise and challenge of high-throughput sequencing of the antibody repertoire

PubMed Central

Georgiou, George; Ippolito, Gregory C; Beausang, John; Busse, Christian E; Wardemann, Hedda; Quake, Stephen R

2014-01-01

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories. PMID:24441474

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets.

PubMed

Hosseini, Parsa; Tremblay, Arianne; Matthews, Benjamin F; Alkharouf, Nadim W

2010-07-02

The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially designed to translate sequence data in a CASAVA-build into functional annotations while producing corresponding gene expression measurements. Achieving such analysis is executed in an ultrafast and highly efficient manner, whether the analysis be a single-read or paired-end sequencing experiment. TASE is a user-friendly and freely available application, allowing rapid analysis and annotation of any given Illumina Solexa sequencing dataset with ease.

Influenza Virus Database (IVDB): an integrated information resource and analysis platform for influenza virus research.

PubMed

Chang, Suhua; Zhang, Jiajie; Liao, Xiaoyun; Zhu, Xinxing; Wang, Dahai; Zhu, Jiang; Feng, Tao; Zhu, Baoli; Gao, George F; Wang, Jian; Yang, Huanming; Yu, Jun; Wang, Jing

2007-01-01

Frequent outbreaks of highly pathogenic avian influenza and the increasing data available for comparative analysis require a central database specialized in influenza viruses (IVs). We have established the Influenza Virus Database (IVDB) to integrate information and create an analysis platform for genetic, genomic, and phylogenetic studies of the virus. IVDB hosts complete genome sequences of influenza A virus generated by Beijing Institute of Genomics (BIG) and curates all other published IV sequences after expert annotation. Our Q-Filter system classifies and ranks all nucleotide sequences into seven categories according to sequence content and integrity. IVDB provides a series of tools and viewers for comparative analysis of the viral genomes, genes, genetic polymorphisms and phylogenetic relationships. A search system has been developed for users to retrieve a combination of different data types by setting search options. To facilitate analysis of global viral transmission and evolution, the IV Sequence Distribution Tool (IVDT) has been developed to display the worldwide geographic distribution of chosen viral genotypes and to couple genomic data with epidemiological data. The BLAST, multiple sequence alignment and phylogenetic analysis tools were integrated for online data analysis. Furthermore, IVDB offers instant access to pre-computed alignments and polymorphisms of IV genes and proteins, and presents the results as SNP distribution plots and minor allele distributions. IVDB is publicly available at http://influenza.genomics.org.cn.

Evaluation of next generation sequencing for the analysis of Eimeria communities in wildlife.

PubMed

Vermeulen, Elke T; Lott, Matthew J; Eldridge, Mark D B; Power, Michelle L

2016-05-01

Next-generation sequencing (NGS) techniques are well-established for studying bacterial communities but not yet for microbial eukaryotes. Parasite communities remain poorly studied, due in part to the lack of reliable and accessible molecular methods to analyse eukaryotic communities. We aimed to develop and evaluate a methodology to analyse communities of the protozoan parasite Eimeria from populations of the Australian marsupial Petrogale penicillata (brush-tailed rock-wallaby) using NGS. An oocyst purification method for small sample sizes and polymerase chain reaction (PCR) protocol for the 18S rRNA locus targeting Eimeria was developed and optimised prior to sequencing on the Illumina MiSeq platform. A data analysis approach was developed by modifying methods from bacterial metagenomics and utilising existing Eimeria sequences in GenBank. Operational taxonomic unit (OTU) assignment at a high similarity threshold (97%) was more accurate at assigning Eimeria contigs into Eimeria OTUs but at a lower threshold (95%) there was greater resolution between OTU consensus sequences. The assessment of two amplification PCR methods prior to Illumina MiSeq, single and nested PCR, determined that single PCR was more sensitive to Eimeria as more Eimeria OTUs were detected in single amplicons. We have developed a simple and cost-effective approach to a data analysis pipeline for community analysis of eukaryotic organisms using Eimeria communities as a model. The pipeline provides a basis for evaluation using other eukaryotic organisms and potential for diverse community analysis studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Forensic massively parallel sequencing data analysis tool: Implementation of MyFLq as a standalone web- and Illumina BaseSpace(®)-application.

PubMed

Van Neste, Christophe; Gansemans, Yannick; De Coninck, Dieter; Van Hoofstat, David; Van Criekinge, Wim; Deforce, Dieter; Van Nieuwerburgh, Filip

2015-03-01

Routine use of massively parallel sequencing (MPS) for forensic genomics is on the horizon. The last few years, several algorithms and workflows have been developed to analyze forensic MPS data. However, none have yet been tailored to the needs of the forensic analyst who does not possess an extensive bioinformatics background. We developed our previously published forensic MPS data analysis framework MyFLq (My-Forensic-Loci-queries) into an open-source, user-friendly, web-based application. It can be installed as a standalone web application, or run directly from the Illumina BaseSpace environment. In the former, laboratories can keep their data on-site, while in the latter, data from forensic samples that are sequenced on an Illumina sequencer can be uploaded to Basespace during acquisition, and can subsequently be analyzed using the published MyFLq BaseSpace application. Additional features were implemented such as an interactive graphical report of the results, an interactive threshold selection bar, and an allele length-based analysis in addition to the sequenced-based analysis. Practical use of the application is demonstrated through the analysis of four 16-plex short tandem repeat (STR) samples, showing the complementarity between the sequence- and length-based analysis of the same MPS data. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

A strategic stakeholder approach for addressing further analysis requests in whole genome sequencing research.

PubMed

Thornock, Bradley Steven O

2016-01-01

Whole genome sequencing (WGS) can be a cost-effective and efficient means of diagnosis for some children, but it also raises a number of ethical concerns. One such concern is how researchers derive and communicate results from WGS, including future requests for further analysis of stored sequences. The purpose of this paper is to think about what is at stake, and for whom, in any solution that is developed to deal with such requests. To accomplish this task, this paper will utilize stakeholder theory, a common method used in business ethics. Several scenarios that connect stakeholder concerns and WGS will also posited and analyzed. This paper concludes by developing criteria composed of a series of questions that researchers can answer in order to more effectively address requests for further analysis of stored sequences.

A Three-Dimensional Approach and Open Source Structure for the Design and Experimentation of Teaching-Learning Sequences: The Case of Friction

ERIC Educational Resources Information Center

Besson, Ugo; Borghi, Lidia; De Ambrosis, Anna; Mascheretti, Paolo

2010-01-01

We have developed a teaching-learning sequence (TLS) on friction based on a preliminary study involving three dimensions: an analysis of didactic research on the topic, an overview of usual approaches, and a critical analysis of the subject, considered also in its historical development. We found that mostly the usual presentations do not take…

De novo Transcriptome Analysis of Portunus trituberculatus Ovary and Testis by RNA-Seq: Identification of Genes Involved in Gonadal Development

PubMed Central

Meng, Xian-liang; Liu, Ping; Jia, Fu-long; Li, Jian; Gao, Bao-Quan

2015-01-01

The swimming crab Portunus trituberculatus is a commercially important crab species in East Asia countries. Gonadal development is a physiological process of great significance to the reproduction as well as commercial seed production for P. trituberculatus. However, little is currently known about the molecular mechanisms governing the developmental processes of gonads in this species. To open avenues of molecular research on P. trituberculatus gonadal development, Illumina paired-end sequencing technology was employed to develop deep-coverage transcriptome sequencing data for its gonads. Illumina sequencing generated 58,429,148 and 70,474,978 high-quality reads from the ovary and testis cDNA library, respectively. All these reads were assembled into 54,960 unigenes with an average sequence length of 879 bp, of which 12,340 unigenes (22.45% of the total) matched sequences in GenBank non-redundant database. Based on our transcriptome analysis as well as published literature, a number of candidate genes potentially involved in the regulation of gonadal development of P. trituberculatus were identified, such as FAOMeT, mPRγ, PGMRC1, PGDS, PGER4, 3β-HSD and 17β-HSDs. Differential expression analysis generated 5,919 differentially expressed genes between ovary and testis, among which many genes related to gametogenesis and several genes previously reported to be critical in differentiation and development of gonads were found, including Foxl2, Wnt4, Fst, Fem-1 and Sox9. Furthermore, 28,534 SSRs and 111,646 high-quality SNPs were identified in this transcriptome dataset. This work represents the first transcriptome analysis of P. trituberculatus gonads using the next generation sequencing technology and provides a valuable dataset for understanding molecular mechanisms controlling development of gonads and facilitating future investigation of reproductive biology in this species. The molecular markers obtained in this study will provide a fundamental basis for population genetics and functional genomics in P. trituberculatus and other closely related species. PMID:26042806

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets

PubMed Central

2010-01-01

Background The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. Findings We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. Conclusions TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially designed to translate sequence data in a CASAVA-build into functional annotations while producing corresponding gene expression measurements. Achieving such analysis is executed in an ultrafast and highly efficient manner, whether the analysis be a single-read or paired-end sequencing experiment. TASE is a user-friendly and freely available application, allowing rapid analysis and annotation of any given Illumina Solexa sequencing dataset with ease. PMID:20598141

FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

EPA Science Inventory

Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

CAFE: aCcelerated Alignment-FrEe sequence analysis.

PubMed

Lu, Yang Young; Tang, Kujin; Ren, Jie; Fuhrman, Jed A; Waterman, Michael S; Sun, Fengzhu

2017-07-03

Alignment-free genome and metagenome comparisons are increasingly important with the development of next generation sequencing (NGS) technologies. Recently developed state-of-the-art k-mer based alignment-free dissimilarity measures including CVTree, $d_2^*$ and $d_2^S$ are more computationally expensive than measures based solely on the k-mer frequencies. Here, we report a standalone software, aCcelerated Alignment-FrEe sequence analysis (CAFE), for efficient calculation of 28 alignment-free dissimilarity measures. CAFE allows for both assembled genome sequences and unassembled NGS shotgun reads as input, and wraps the output in a standard PHYLIP format. In downstream analyses, CAFE can also be used to visualize the pairwise dissimilarity measures, including dendrograms, heatmap, principal coordinate analysis and network display. CAFE serves as a general k-mer based alignment-free analysis platform for studying the relationships among genomes and metagenomes, and is freely available at https://github.com/younglululu/CAFE. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of self-compressing BLSOM for comprehensive analysis of big sequence data.

PubMed

Kikuchi, Akihito; Ikemura, Toshimichi; Abe, Takashi

2015-01-01

With the remarkable increase in genomic sequence data from various organisms, novel tools are needed for comprehensive analyses of available big sequence data. We previously developed a Batch-Learning Self-Organizing Map (BLSOM), which can cluster genomic fragment sequences according to phylotype solely dependent on oligonucleotide composition and applied to genome and metagenomic studies. BLSOM is suitable for high-performance parallel-computing and can analyze big data simultaneously, but a large-scale BLSOM needs a large computational resource. We have developed Self-Compressing BLSOM (SC-BLSOM) for reduction of computation time, which allows us to carry out comprehensive analysis of big sequence data without the use of high-performance supercomputers. The strategy of SC-BLSOM is to hierarchically construct BLSOMs according to data class, such as phylotype. The first-layer BLSOM was constructed with each of the divided input data pieces that represents the data subclass, such as phylotype division, resulting in compression of the number of data pieces. The second BLSOM was constructed with a total of weight vectors obtained in the first-layer BLSOMs. We compared SC-BLSOM with the conventional BLSOM by analyzing bacterial genome sequences. SC-BLSOM could be constructed faster than BLSOM and cluster the sequences according to phylotype with high accuracy, showing the method's suitability for efficient knowledge discovery from big sequence data.

MIPS: a database for genomes and protein sequences

PubMed Central

Mewes, H. W.; Frishman, D.; Güldener, U.; Mannhaupt, G.; Mayer, K.; Mokrejs, M.; Morgenstern, B.; Münsterkötter, M.; Rudd, S.; Weil, B.

2002-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz–Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91–93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155–158; Barker et al. (2001) Nucleic Acids Res., 29, 29–32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de). PMID:11752246

MIPS: a database for genomes and protein sequences.

PubMed

Mewes, H W; Frishman, D; Güldener, U; Mannhaupt, G; Mayer, K; Mokrejs, M; Morgenstern, B; Münsterkötter, M; Rudd, S; Weil, B

2002-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz-Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91-93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155-158; Barker et al. (2001) Nucleic Acids Res., 29, 29-32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de).

Newborn Sequencing in Genomic Medicine and Public Health

PubMed Central

Agrawal, Pankaj B.; Bailey, Donald B.; Beggs, Alan H.; Brenner, Steven E.; Brower, Amy M.; Cakici, Julie A.; Ceyhan-Birsoy, Ozge; Chan, Kee; Chen, Flavia; Currier, Robert J.; Dukhovny, Dmitry; Green, Robert C.; Harris-Wai, Julie; Holm, Ingrid A.; Iglesias, Brenda; Joseph, Galen; Kingsmore, Stephen F.; Koenig, Barbara A.; Kwok, Pui-Yan; Lantos, John; Leeder, Steven J.; Lewis, Megan A.; McGuire, Amy L.; Milko, Laura V.; Mooney, Sean D.; Parad, Richard B.; Pereira, Stacey; Petrikin, Joshua; Powell, Bradford C.; Powell, Cynthia M.; Puck, Jennifer M.; Rehm, Heidi L.; Risch, Neil; Roche, Myra; Shieh, Joseph T.; Veeraraghavan, Narayanan; Watson, Michael S.; Willig, Laurel; Yu, Timothy W.; Urv, Tiina; Wise, Anastasia L.

2017-01-01

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening. PMID:28096516

A De-Novo Genome Analysis Pipeline (DeNoGAP) for large-scale comparative prokaryotic genomics studies.

PubMed

Thakur, Shalabh; Guttman, David S

2016-06-30

Comparative analysis of whole genome sequence data from closely related prokaryotic species or strains is becoming an increasingly important and accessible approach for addressing both fundamental and applied biological questions. While there are number of excellent tools developed for performing this task, most scale poorly when faced with hundreds of genome sequences, and many require extensive manual curation. We have developed a de-novo genome analysis pipeline (DeNoGAP) for the automated, iterative and high-throughput analysis of data from comparative genomics projects involving hundreds of whole genome sequences. The pipeline is designed to perform reference-assisted and de novo gene prediction, homolog protein family assignment, ortholog prediction, functional annotation, and pan-genome analysis using a range of proven tools and databases. While most existing methods scale quadratically with the number of genomes since they rely on pairwise comparisons among predicted protein sequences, DeNoGAP scales linearly since the homology assignment is based on iteratively refined hidden Markov models. This iterative clustering strategy enables DeNoGAP to handle a very large number of genomes using minimal computational resources. Moreover, the modular structure of the pipeline permits easy updates as new analysis programs become available. DeNoGAP integrates bioinformatics tools and databases for comparative analysis of a large number of genomes. The pipeline offers tools and algorithms for annotation and analysis of completed and draft genome sequences. The pipeline is developed using Perl, BioPerl and SQLite on Ubuntu Linux version 12.04 LTS. Currently, the software package accompanies script for automated installation of necessary external programs on Ubuntu Linux; however, the pipeline should be also compatible with other Linux and Unix systems after necessary external programs are installed. DeNoGAP is freely available at https://sourceforge.net/projects/denogap/ .

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

PubMed

Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

2014-01-01

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

USDA-ARS?s Scientific Manuscript database

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

Translational genomics for analysis of complex traits in peanut and sorghum

USDA-ARS?s Scientific Manuscript database

The integration of sequencing and genotype data from natural variation studies (by whole genome resequencing [wgs] or genotype by sequencing [gbs]), transcriptome (RNA-seq) and mutant analysis (also by wgs) facilitated the development of DNA markers in the form of single nucleotide polymorphic (SNP)...

Multiplexed microsatellite recovery using massively parallel sequencing

Treesearch

T.N. Jennings; B.J. Knaus; T.D. Mullins; S.M. Haig; R.C. Cronn

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of...

Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis

NASA Astrophysics Data System (ADS)

Chen, C. H. Winston; Taranenko, N. I.; Golovlev, V. V.; Isola, N. R.; Allman, S. L.

1998-03-01

Rapid DNA sequencing and/or analysis is critically important for biomedical research. In the past, gel electrophoresis has been the primary tool to achieve DNA analysis and sequencing. However, gel electrophoresis is a time-consuming and labor-extensive process. Recently, we have developed and used laser desorption mass spectrometry (LDMS) to achieve sequencing of ss-DNA longer than 100 nucleotides. With LDMS, we succeeded in sequencing DNA in seconds instead of hours or days required by gel electrophoresis. In addition to sequencing, we also applied LDMS for the detection of DNA probes for hybridization LDMS was also used to detect short tandem repeats for forensic applications. Clinical applications for disease diagnosis such as cystic fibrosis caused by base deletion and point mutation have also been demonstrated. Experimental details will be presented in the meeting. abstract.

MIPS: a database for genomes and protein sequences.

PubMed Central

Mewes, H W; Heumann, K; Kaps, A; Mayer, K; Pfeiffer, F; Stocker, S; Frishman, D

1999-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried near Munich, Germany, develops and maintains genome oriented databases. It is commonplace that the amount of sequence data available increases rapidly, but not the capacity of qualified manual annotation at the sequence databases. Therefore, our strategy aims to cope with the data stream by the comprehensive application of analysis tools to sequences of complete genomes, the systematic classification of protein sequences and the active support of sequence analysis and functional genomics projects. This report describes the systematic and up-to-date analysis of genomes (PEDANT), a comprehensive database of the yeast genome (MYGD), a database reflecting the progress in sequencing the Arabidopsis thaliana genome (MATD), the database of assembled, annotated human EST clusters (MEST), and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database (described elsewhere in this volume). MIPS provides access through its WWW server (http://www.mips.biochem.mpg.de) to a spectrum of generic databases, including the above mentioned as well as a database of protein families (PROTFAM), the MITOP database, and the all-against-all FASTA database. PMID:9847138

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Deep Sequencing to Identify the Causes of Viral Encephalitis

PubMed Central

Chan, Benjamin K.; Wilson, Theodore; Fischer, Kael F.; Kriesel, John D.

2014-01-01

Deep sequencing allows for a rapid, accurate characterization of microbial DNA and RNA sequences in many types of samples. Deep sequencing (also called next generation sequencing or NGS) is being developed to assist with the diagnosis of a wide variety of infectious diseases. In this study, seven frozen brain samples from deceased subjects with recent encephalitis were investigated. RNA from each sample was extracted, randomly reverse transcribed and sequenced. The sequence analysis was performed in a blinded fashion and confirmed with pathogen-specific PCR. This analysis successfully identified measles virus sequences in two brain samples and herpes simplex virus type-1 sequences in three brain samples. No pathogen was identified in the other two brain specimens. These results were concordant with pathogen-specific PCR and partially concordant with prior neuropathological examinations, demonstrating that deep sequencing can accurately identify viral infections in frozen brain tissue. PMID:24699691

Evaluation of Methods for de novo Genome assembly from High-throughput Sequencing Reads Reveals Dependencies that Affect the Quality of the Results

USDA-ARS?s Scientific Manuscript database

Recent developments in high-throughput sequencing technology have made low-cost sequencing an attractive approach for many genome analysis tasks. Increasing read lengths, improving quality and the production of increasingly larger numbers of usable sequences per instrument-run continue to make whole...

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

New Tools For Understanding Microbial Diversity Using High-throughput Sequence Data

NASA Astrophysics Data System (ADS)

Knight, R.; Hamady, M.; Liu, Z.; Lozupone, C.

2007-12-01

High-throughput sequencing techniques such as 454 are straining the limits of tools traditionally used to build trees, choose OTUs, and perform other essential sequencing tasks. We have developed a workflow for phylogenetic analysis of large-scale sequence data sets that combines existing tools, such as the Arb phylogeny package and the NAST multiple sequence alignment tool, with new methods for choosing and clustering OTUs and for performing phylogenetic community analysis with UniFrac. This talk discusses the cyberinfrastructure we are developing to support the human microbiome project, and the application of these workflows to analyze very large data sets that contrast the gut microbiota with a range of physical environments. These tools will ultimately help to define core and peripheral microbiomes in a range of environments, and will allow us to understand the physical and biotic factors that contribute most to differences in microbial diversity.

Whole-genome CNV analysis: advances in computational approaches.

PubMed

Pirooznia, Mehdi; Goes, Fernando S; Zandi, Peter P

2015-01-01

Accumulating evidence indicates that DNA copy number variation (CNV) is likely to make a significant contribution to human diversity and also play an important role in disease susceptibility. Recent advances in genome sequencing technologies have enabled the characterization of a variety of genomic features, including CNVs. This has led to the development of several bioinformatics approaches to detect CNVs from next-generation sequencing data. Here, we review recent advances in CNV detection from whole genome sequencing. We discuss the informatics approaches and current computational tools that have been developed as well as their strengths and limitations. This review will assist researchers and analysts in choosing the most suitable tools for CNV analysis as well as provide suggestions for new directions in future development.

Interactive computer programs for the graphic analysis of nucleotide sequence data.

PubMed Central

Luckow, V A; Littlewood, R K; Rownd, R H

1984-01-01

A group of interactive computer programs have been developed which aid in the collection and graphical analysis of nucleotide and protein sequence data. The programs perform the following basic functions: a) enter, edit, list, and rearrange sequence data; b) permit automatic entry of nucleotide sequence data directly from an autoradiograph into the computer; c) search for restriction sites or other specified patterns and plot a linear or circular restriction map, or print their locations; d) plot base composition; e) analyze homology between sequences by plotting a two-dimensional graphic matrix; and f) aid in plotting predicted secondary structures of RNA molecules. PMID:6546437

Using Phylogenetic Analysis to Detect Market Substitution of Atlantic Salmon for Pacific Salmon: An Introductory Biology Laboratory Experiment

ERIC Educational Resources Information Center

Cline, Erica; Gogarten, Jennifer

2012-01-01

We describe a laboratory exercise developed for the cell and molecular biology quarter of a year-long majors' undergraduate introductory biology sequence. In an analysis of salmon samples collected by students in their local stores and restaurants, DNA sequencing and phylogenetic analysis were used to detect market substitution of Atlantic salmon…

Current state-of-art of STR sequencing in forensic genetics.

PubMed

Alonso, Antonio; Barrio, Pedro A; Müller, Petra; Köcher, Steffi; Berger, Burkhard; Martin, Pablo; Bodner, Martin; Willuweit, Sascha; Parson, Walther; Roewer, Lutz; Budowle, Bruce

2018-05-11

The current state of validation and implementation strategies of MPS technology for the analysis of STR markers for forensic genetics use is described, covering the topics of the current catalogue of commercial MPS-STR panels, leading MPS-platforms, and MPS-STR data analysis tools. In addition, the developmental and internal validation studies carried out to date to evaluate reliability, sensitivity, mixture analysis, concordance, and the ability to analyze challenged samples are summarized. The results of various MPS-STR population studies that showed a large number of new STR sequence variants that increase the power of discrimination in several forensically-relevant loci are also presented. Finally, various initiatives developed by several international projects and standardization (or guidelines) groups to facilitate application of MPS technology for STR marker analyses are discussed in regard to promoting a standard STR sequence nomenclature, performing population studies to detect sequence variants, and developing a universal system to translate sequence variants into a simple STR nomenclature (numbers and letters) compatible with national STR databases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments.

PubMed

Huber, Thomas; Faulkner, Geoffrey; Hugenholtz, Philip

2004-09-22

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments. Bellerophon is available as an interactive web server at http://foo.maths.uq.edu.au/~huber/bellerophon.pl

Oral Counting Sequences: A Theoretical Discussion and Analysis through the Lens of Representational Redescription

ERIC Educational Resources Information Center

Voutsina, Chronoula

2016-01-01

Empirical research has documented how children's early counting develops into an increasingly abstract process, and initial counting procedures are reified as children develop and use more sophisticated counting. In this development, the learning of different oral counting sequences that allow children to count in steps bigger than one is seen as…

Analysis of SSR information in EST resources of sugarcane

USDA-ARS?s Scientific Manuscript database

Expressed sequence tags ( ESTs) offer the opportunity to exploit single, low -copy, conserved sequence motifs for the development of simple sequence repeats ( SSRs). The total of 262 113 ESTs of sugarcane (Saccharum officinarum) in the database of NCBI were downloaded and analyzed, which resulted in...

Recent patents of nanopore DNA sequencing technology: progress and challenges.

PubMed

Zhou, Jianfeng; Xu, Bingqian

2010-11-01

DNA sequencing techniques witnessed fast development in the last decades, primarily driven by the Human Genome Project. Among the proposed new techniques, Nanopore was considered as a suitable candidate for the single DNA sequencing with ultrahigh speed and very low cost. Several fabrication and modification techniques have been developed to produce robust and well-defined nanopore devices. Many efforts have also been done to apply nanopore to analyze the properties of DNA molecules. By comparing with traditional sequencing techniques, nanopore has demonstrated its distinctive superiorities in main practical issues, such as sample preparation, sequencing speed, cost-effective and read-length. Although challenges still remain, recent researches in improving the capabilities of nanopore have shed a light to achieve its ultimate goal: Sequence individual DNA strand at single nucleotide level. This patent review briefly highlights recent developments and technological achievements for DNA analysis and sequencing at single molecule level, focusing on nanopore based methods.

Newborn Sequencing in Genomic Medicine and Public Health.

PubMed

Berg, Jonathan S; Agrawal, Pankaj B; Bailey, Donald B; Beggs, Alan H; Brenner, Steven E; Brower, Amy M; Cakici, Julie A; Ceyhan-Birsoy, Ozge; Chan, Kee; Chen, Flavia; Currier, Robert J; Dukhovny, Dmitry; Green, Robert C; Harris-Wai, Julie; Holm, Ingrid A; Iglesias, Brenda; Joseph, Galen; Kingsmore, Stephen F; Koenig, Barbara A; Kwok, Pui-Yan; Lantos, John; Leeder, Steven J; Lewis, Megan A; McGuire, Amy L; Milko, Laura V; Mooney, Sean D; Parad, Richard B; Pereira, Stacey; Petrikin, Joshua; Powell, Bradford C; Powell, Cynthia M; Puck, Jennifer M; Rehm, Heidi L; Risch, Neil; Roche, Myra; Shieh, Joseph T; Veeraraghavan, Narayanan; Watson, Michael S; Willig, Laurel; Yu, Timothy W; Urv, Tiina; Wise, Anastasia L

2017-02-01

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening. Copyright © 2017 by the American Academy of Pediatrics.

Auditory sequence analysis and phonological skill

PubMed Central

Grube, Manon; Kumar, Sukhbinder; Cooper, Freya E.; Turton, Stuart; Griffiths, Timothy D.

2012-01-01

This work tests the relationship between auditory and phonological skill in a non-selected cohort of 238 school students (age 11) with the specific hypothesis that sound-sequence analysis would be more relevant to phonological skill than the analysis of basic, single sounds. Auditory processing was assessed across the domains of pitch, time and timbre; a combination of six standard tests of literacy and language ability was used to assess phonological skill. A significant correlation between general auditory and phonological skill was demonstrated, plus a significant, specific correlation between measures of phonological skill and the auditory analysis of short sequences in pitch and time. The data support a limited but significant link between auditory and phonological ability with a specific role for sound-sequence analysis, and provide a possible new focus for auditory training strategies to aid language development in early adolescence. PMID:22951739

Comparative genomic analysis of the false killer whale (Pseudorca crassidens) LMBR1 locus.

PubMed

Kim, Dae-Won; Choi, Sang-Haeng; Kim, Ryong Nam; Kim, Sun-Hong; Paik, Sang-Gi; Nam, Seong-Hyeuk; Kim, Dong-Wook; Kim, Aeri; Kang, Aram; Park, Hong-Seog

2010-09-01

The sequencing and comparative genomic analysis of LMBR1 loci in mammals or other species, including human, would be very important in understanding evolutionary genetic changes underlying the evolution of limb development. In this regard, comparative genomic annotation of the false killer whale LMBR1 locus could shed new light on the evolution of limb development. We sequenced two false killer whale BAC clones, corresponding to 156 kb and 144 kb, respectively, harboring the tightly linked RNF32, LMBR1, and NOM1 genes. Our annotation of the false killer whale LMBR1 gene showed that it consists of 17 exons (1473 bp), in contrast to 18 exons (1596 bp) in human, and it displays 93.1% and 95.6% nucleotide and amino acid sequence similarity, respectively, compared with the human gene. In particular, we discovered that exon 10, deleted in the false killer whale LMBR1 gene, is present only in primates, and this fact strongly implies that exon 10 might be crucial in determining primate-specific limb development. ZRS and TFBS sequences have been well conserved across 11 species, suggesting that these regions could be involved in an important function of limb development and limb patterning. The neighboring gene RNF32 showed several lineage-conserved exons, such as exons 2 through 9 conserved in eutherian mammals, exons 3 through 9 conserved in mammals, and exons 5 through 9 conserved in vertebrates. The other neighboring gene, NOM1, had undergone a substitution (ATG→GTA) at the start codon, giving rise to a 36 bp shorter N-terminal sequence compared with the human sequence. Our comparative analysis of the false killer whale LMBR1 genomic locus provides important clues regarding the genetic regions that may play crucial roles in limb development and patterning.

Single-Cell Sequencing Technologies for Cardiac Stem Cell Studies.

PubMed

Liu, Tiantian; Wu, Hongjin; Wu, Shixiu; Wang, Charles

2017-11-01

Today with the rapid advancements in stem cell studies and the promising potential of using stem cells in clinical therapy, there is an increasing demand for in-depth comprehensive analysis on individual cell transcriptome and epigenome, as they play critical roles in a number of cell functions such as cell differentiation, growth, and reprogramming. The development of single-cell sequencing technologies has helped in revealing some exciting new perspectives in stem cells and regenerative medicine research. Among the various potential applications, single-cell analysis for cardiac stem cells (CSCs) holds tremendous promises in understanding the mechanisms of heart development and regeneration, which might light up the path toward cell therapy for cardiovascular diseases. This review briefly highlights the recent progresses in single-cell sequencing analysis technologies and their applications in CSC research.

Statistical properties of filtered pseudorandom digital sequences formed from the sum of maximum-length sequences

NASA Technical Reports Server (NTRS)

Wallace, G. R.; Weathers, G. D.; Graf, E. R.

1973-01-01

The statistics of filtered pseudorandom digital sequences called hybrid-sum sequences, formed from the modulo-two sum of several maximum-length sequences, are analyzed. The results indicate that a relation exists between the statistics of the filtered sequence and the characteristic polynomials of the component maximum length sequences. An analysis procedure is developed for identifying a large group of sequences with good statistical properties for applications requiring the generation of analog pseudorandom noise. By use of the analysis approach, the filtering process is approximated by the convolution of the sequence with a sum of unit step functions. A parameter reflecting the overall statistical properties of filtered pseudorandom sequences is derived. This parameter is called the statistical quality factor. A computer algorithm to calculate the statistical quality factor for the filtered sequences is presented, and the results for two examples of sequence combinations are included. The analysis reveals that the statistics of the signals generated with the hybrid-sum generator are potentially superior to the statistics of signals generated with maximum-length generators. Furthermore, fewer calculations are required to evaluate the statistics of a large group of hybrid-sum generators than are required to evaluate the statistics of the same size group of approximately equivalent maximum-length sequences.

Development of Scoring Functions for Antibody Sequence Assessment and Optimization

PubMed Central

Seeliger, Daniel

2013-01-01

Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity. PMID:24204701

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

PubMed

Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

2018-01-01

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Illuminator, a desktop program for mutation detection using short-read clonal sequencing.

PubMed

Carr, Ian M; Morgan, Joanne E; Diggle, Christine P; Sheridan, Eamonn; Markham, Alexander F; Logan, Clare V; Inglehearn, Chris F; Taylor, Graham R; Bonthron, David T

2011-10-01

Current methods for sequencing clonal populations of DNA molecules yield several gigabases of data per day, typically comprising reads of < 100 nt. Such datasets permit widespread genome resequencing and transcriptome analysis or other quantitative tasks. However, this huge capacity can also be harnessed for the resequencing of smaller (gene-sized) target regions, through the simultaneous parallel analysis of multiple subjects, using sample "tagging" or "indexing". These methods promise to have a huge impact on diagnostic mutation analysis and candidate gene testing. Here we describe a software package developed for such studies, offering the ability to resolve pooled samples carrying barcode tags and to align reads to a reference sequence using a mutation-tolerant process. The program, Illuminator, can identify rare sequence variants, including insertions and deletions, and permits interactive data analysis on standard desktop computers. It facilitates the effective analysis of targeted clonal sequencer data without dedicated computational infrastructure or specialized training. Copyright © 2011 Elsevier Inc. All rights reserved.

Occupational Sequences: Auto Engines 1. AT 121.

ERIC Educational Resources Information Center

Korb, A. W.; And Others

In an attempt to individualize an automotive course, the Vocational-Technical Division of Northern Montana College has developed Occupational Sequences for an engine rebuilding course. Occupational Sequences, a learning or teaching aid, is an analysis of numbered operations involved in engine rebuilding. Job sheets, included in the book, provide a…

Learning Portfolio Analysis and Mining for SCORM Compliant Environment

ERIC Educational Resources Information Center

Su, Jun-Ming; Tseng, Shian-Shyong; Wang, Wei; Weng, Jui-Feng; Yang, Jin Tan David; Tsai, Wen-Nung

2006-01-01

With vigorous development of the Internet, e-learning system has become more and more popular. Sharable Content Object Reference Model (SCORM) 2004 provides the Sequencing and Navigation (SN) Specification to define the course sequencing behavior, control the sequencing, selecting and delivering of course, and organize the content into a…

A Methodology for the Integration of a Mechanistic Source Term Analysis in a Probabilistic Framework for Advanced Reactors

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

Grabaskas, Dave; Brunett, Acacia J.; Bucknor, Matthew

GE Hitachi Nuclear Energy (GEH) and Argonne National Laboratory are currently engaged in a joint effort to modernize and develop probabilistic risk assessment (PRA) techniques for advanced non-light water reactors. At a high level, the primary outcome of this project will be the development of next-generation PRA methodologies that will enable risk-informed prioritization of safety- and reliability-focused research and development, while also identifying gaps that may be resolved through additional research. A subset of this effort is the development of PRA methodologies to conduct a mechanistic source term (MST) analysis for event sequences that could result in the release ofmore » radionuclides. The MST analysis seeks to realistically model and assess the transport, retention, and release of radionuclides from the reactor to the environment. The MST methods developed during this project seek to satisfy the requirements of the Mechanistic Source Term element of the ASME/ANS Non-LWR PRA standard. The MST methodology consists of separate analysis approaches for risk-significant and non-risk significant event sequences that may result in the release of radionuclides from the reactor. For risk-significant event sequences, the methodology focuses on a detailed assessment, using mechanistic models, of radionuclide release from the fuel, transport through and release from the primary system, transport in the containment, and finally release to the environment. The analysis approach for non-risk significant event sequences examines the possibility of large radionuclide releases due to events such as re-criticality or the complete loss of radionuclide barriers. This paper provides details on the MST methodology, including the interface between the MST analysis and other elements of the PRA, and provides a simplified example MST calculation for a sodium fast reactor.« less

KONAGAbase: a genomic and transcriptomic database for the diamondback moth, Plutella xylostella.

PubMed

Jouraku, Akiya; Yamamoto, Kimiko; Kuwazaki, Seigo; Urio, Masahiro; Suetsugu, Yoshitaka; Narukawa, Junko; Miyamoto, Kazuhisa; Kurita, Kanako; Kanamori, Hiroyuki; Katayose, Yuichi; Matsumoto, Takashi; Noda, Hiroaki

2013-07-09

The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM). KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers. KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.

TRDistiller: a rapid filter for enrichment of sequence datasets with proteins containing tandem repeats.

PubMed

Richard, François D; Kajava, Andrey V

2014-06-01

The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such "hidden" repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other methods. The program is available upon request. Copyright © 2014 Elsevier Inc. All rights reserved.

An automated genotyping tool for enteroviruses and noroviruses.

PubMed

Kroneman, A; Vennema, H; Deforche, K; v d Avoort, H; Peñaranda, S; Oberste, M S; Vinjé, J; Koopmans, M

2011-06-01

Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

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

Fields, C.A.

1996-06-01

The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progressmore » report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.« less

Congruence analysis of point clouds from unstable stereo image sequences

NASA Astrophysics Data System (ADS)

Jepping, C.; Bethmann, F.; Luhmann, T.

2014-06-01

This paper deals with the correction of exterior orientation parameters of stereo image sequences over deformed free-form surfaces without control points. Such imaging situation can occur, for example, during photogrammetric car crash test recordings where onboard high-speed stereo cameras are used to measure 3D surfaces. As a result of such measurements 3D point clouds of deformed surfaces are generated for a complete stereo sequence. The first objective of this research focusses on the development and investigation of methods for the detection of corresponding spatial and temporal tie points within the stereo image sequences (by stereo image matching and 3D point tracking) that are robust enough for a reliable handling of occlusions and other disturbances that may occur. The second objective of this research is the analysis of object deformations in order to detect stable areas (congruence analysis). For this purpose a RANSAC-based method for congruence analysis has been developed. This process is based on the sequential transformation of randomly selected point groups from one epoch to another by using a 3D similarity transformation. The paper gives a detailed description of the congruence analysis. The approach has been tested successfully on synthetic and real image data.

Bio++: a set of C++ libraries for sequence analysis, phylogenetics, molecular evolution and population genetics.

PubMed

Dutheil, Julien; Gaillard, Sylvain; Bazin, Eric; Glémin, Sylvain; Ranwez, Vincent; Galtier, Nicolas; Belkhir, Khalid

2006-04-04

A large number of bioinformatics applications in the fields of bio-sequence analysis, molecular evolution and population genetics typically share input/output methods, data storage requirements and data analysis algorithms. Such common features may be conveniently bundled into re-usable libraries, which enable the rapid development of new methods and robust applications. We present Bio++, a set of Object Oriented libraries written in C++. Available components include classes for data storage and handling (nucleotide/amino-acid/codon sequences, trees, distance matrices, population genetics datasets), various input/output formats, basic sequence manipulation (concatenation, transcription, translation, etc.), phylogenetic analysis (maximum parsimony, markov models, distance methods, likelihood computation and maximization), population genetics/genomics (diversity statistics, neutrality tests, various multi-locus analyses) and various algorithms for numerical calculus. Implementation of methods aims at being both efficient and user-friendly. A special concern was given to the library design to enable easy extension and new methods development. We defined a general hierarchy of classes that allow the developer to implement its own algorithms while remaining compatible with the rest of the libraries. Bio++ source code is distributed free of charge under the CeCILL general public licence from its website http://kimura.univ-montp2.fr/BioPP.

[EST-SSR identification, markers development of Ligusticum chuanxiong based on Ligusticum chuanxiong transcriptome sequences].

PubMed

Yuan, Can; Peng, Fang; Yang, Ze-Mao; Zhong, Wen-Juan; Mou, Fang-Sheng; Gong, Yi-Yun; Ji, Pei-Cheng; Pu, De-Qiang; Huang, Hai-Yan; Yang, Xiao; Zhang, Chao

2017-09-01

Ligusticum chuanxiong is a well-known traditional Chinese medicine plant. The study on its molecular markers development and germplasm resources is very important. In this study, we obtained 24 422 unigenes by assembling transcriptome sequencing reads of L. chuanxiong root. EST-SSR was detected and 4 073 SSR loci were identified. EST-SSR distribution and characteristic analysis results showed that the mono-nucleotide repeats were the main repeat types, accounting for 41.0%. In addition, the sequences containing SSR were functionally annotated in Gene Ontology (GO) and KEGG pathway and were assigned to 49 GO categories, 242 KEGG pathways, among them 2 201 sequences were annotated against Nr database. By validating 235 EST-SSRs,74 primer pairs were ultimately proved to have high quality amplification. Subsequently, genetic diversity analysis, UPGMA cluster analysis, PCoA analysis and population structure analysis of 34 L. chuanxiong germplasm resources were carried out with 74 primer pairs. In both UPGMA tree and PCoA results, L. chuanxiong resources were clustered into two groups, which are believed to be partial related to their geographical distribution. In this study, EST-SSRs in L. chuanxiong was firstly identified, and newly developed molecular markers would contribute significantly to further genetic diversity study, the purity detection, gene mapping, and molecular breeding. Copyright© by the Chinese Pharmaceutical Association.

Collaborative development for setup, execution, sharing and analytics of complex NMR experiments.

PubMed

Irvine, Alistair G; Slynko, Vadim; Nikolaev, Yaroslav; Senthamarai, Russell R P; Pervushin, Konstantin

2014-02-01

Factory settings of NMR pulse sequences are rarely ideal for every scenario in which they are utilised. The optimisation of NMR experiments has for many years been performed locally, with implementations often specific to an individual spectrometer. Furthermore, these optimised experiments are normally retained solely for the use of an individual laboratory, spectrometer or even single user. Here we introduce a web-based service that provides a database for the deposition, annotation and optimisation of NMR experiments. The application uses a Wiki environment to enable the collaborative development of pulse sequences. It also provides a flexible mechanism to automatically generate NMR experiments from deposited sequences. Multidimensional NMR experiments of proteins and other macromolecules consume significant resources, in terms of both spectrometer time and effort required to analyse the results. Systematic analysis of simulated experiments can enable optimal allocation of NMR resources for structural analysis of proteins. Our web-based application (http://nmrplus.org) provides all the necessary information, includes the auxiliaries (waveforms, decoupling sequences etc.), for analysis of experiments by accurate numerical simulation of multidimensional NMR experiments. The online database of the NMR experiments, together with a systematic evaluation of their sensitivity, provides a framework for selection of the most efficient pulse sequences. The development of such a framework provides a basis for the collaborative optimisation of pulse sequences by the NMR community, with the benefits of this collective effort being available to the whole community. Copyright © 2013 Elsevier Inc. All rights reserved.

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

PubMed

Links, Matthew G; Chaban, Bonnie; Hemmingsen, Sean M; Muirhead, Kevin; Hill, Janet E

2013-08-15

Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

Genome-Wide Prediction and Analysis of 3D-Domain Swapped Proteins in the Human Genome from Sequence Information.

PubMed

Upadhyay, Atul Kumar; Sowdhamini, Ramanathan

2016-01-01

3D-domain swapping is one of the mechanisms of protein oligomerization and the proteins exhibiting this phenomenon have many biological functions. These proteins, which undergo domain swapping, have acquired much attention owing to their involvement in human diseases, such as conformational diseases, amyloidosis, serpinopathies, proteionopathies etc. Early realisation of proteins in the whole human genome that retain tendency to domain swap will enable many aspects of disease control management. Predictive models were developed by using machine learning approaches with an average accuracy of 78% (85.6% of sensitivity, 87.5% of specificity and an MCC value of 0.72) to predict putative domain swapping in protein sequences. These models were applied to many complete genomes with special emphasis on the human genome. Nearly 44% of the protein sequences in the human genome were predicted positive for domain swapping. Enrichment analysis was performed on the positively predicted sequences from human genome for their domain distribution, disease association and functional importance based on Gene Ontology (GO). Enrichment analysis was also performed to infer a better understanding of the functional importance of these sequences. Finally, we developed hinge region prediction, in the given putative domain swapped sequence, by using important physicochemical properties of amino acids.

Quantitative analysis of image quality for acceptance and commissioning of an MRI simulator with a semiautomatic method.

PubMed

Chen, Xinyuan; Dai, Jianrong

2018-05-01

Magnetic Resonance Imaging (MRI) simulation differs from diagnostic MRI in purpose, technical requirements, and implementation. We propose a semiautomatic method for image acceptance and commissioning for the scanner, the radiofrequency (RF) coils, and pulse sequences for an MRI simulator. The ACR MRI accreditation large phantom was used for image quality analysis with seven parameters. Standard ACR sequences with a split head coil were adopted to examine the scanner's basic performance. The performance of simulation RF coils were measured and compared using the standard sequence with different clinical diagnostic coils. We used simulation sequences with simulation coils to test the quality of image and advanced performance of the scanner. Codes and procedures were developed for semiautomatic image quality analysis. When using standard ACR sequences with a split head coil, image quality passed all ACR recommended criteria. The image intensity uniformity with a simulation RF coil decreased about 34% compared with the eight-channel diagnostic head coil, while the other six image quality parameters were acceptable. Those two image quality parameters could be improved to more than 85% by built-in intensity calibration methods. In the simulation sequences test, the contrast resolution was sensitive to the FOV and matrix settings. The geometric distortion of simulation sequences such as T1-weighted and T2-weighted images was well-controlled in the isocenter and 10 cm off-center within a range of ±1% (2 mm). We developed a semiautomatic image quality analysis method for quantitative evaluation of images and commissioning of an MRI simulator. The baseline performances of simulation RF coils and pulse sequences have been established for routine QA. © 2018 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.

GobyWeb: Simplified Management and Analysis of Gene Expression and DNA Methylation Sequencing Data

PubMed Central

Dorff, Kevin C.; Chambwe, Nyasha; Zeno, Zachary; Simi, Manuele; Shaknovich, Rita; Campagne, Fabien

2013-01-01

We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins. PMID:23936070

Designing and Evaluating Research-Based Instructional Sequences for Introducing Magnetic Fields

ERIC Educational Resources Information Center

Guisasola, Jenaro; Almudi, Jose Manuel; Ceberio, Mikel; Zubimendi, Jose Luis

2009-01-01

This study examines the didactic suitability of introducing a teaching sequence when teaching the concept of magnetic fields within introductory physics courses at the university level. This instructional sequence was designed taking into account students' common conceptions, an analysis of the course content, and the history of the development of…

Development and application of a PCR assay to detect chicken and turkey parvoviruses in commercial poultry flocks in the United States.

USDA-ARS?s Scientific Manuscript database

Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A PCR assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detect p...

Library preparation and data analysis packages for rapid genome sequencing.

PubMed

Pomraning, Kyle R; Smith, Kristina M; Bredeweg, Erin L; Connolly, Lanelle R; Phatale, Pallavi A; Freitag, Michael

2012-01-01

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.

JGI Fungal Genomics Program

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

Grigoriev, Igor V.

2011-03-14

Genomes of energy and environment fungi are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). Its key project, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts), and explores fungal diversity by means of genome sequencing and analysis. Over 50 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functionalmore » genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such 'parts' suggested by comparative genomics and functional analysis in these areas are presented here« less

Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software.

PubMed

Nakano, Shogo; Asano, Yasuhisa

2015-02-03

Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL. Sequence and biochemical analysis of the designed S-HNLs suggested that accumulation of neutral mutations occurs during the process of S-HNLs evolution from a low-activity form to a high-activity (native) form. Taken together, our results demonstrate that our software and the associated methods could be applied not only to design of complete sequences, but also to predictions of protein evolution, especially within families such as esterases and S-HNLs.

Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software

NASA Astrophysics Data System (ADS)

Nakano, Shogo; Asano, Yasuhisa

2015-02-01

Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL. Sequence and biochemical analysis of the designed S-HNLs suggested that accumulation of neutral mutations occurs during the process of S-HNLs evolution from a low-activity form to a high-activity (native) form. Taken together, our results demonstrate that our software and the associated methods could be applied not only to design of complete sequences, but also to predictions of protein evolution, especially within families such as esterases and S-HNLs.

Aircraft stress sequence development: A complex engineering process made simple

NASA Technical Reports Server (NTRS)

Schrader, K. H.; Butts, D. G.; Sparks, W. A.

1994-01-01

Development of stress sequences for critical aircraft structure requires flight measured usage data, known aircraft loads, and established relationships between aircraft flight loads and structural stresses. Resulting cycle-by-cycle stress sequences can be directly usable for crack growth analysis and coupon spectra tests. Often, an expert in loads and spectra development manipulates the usage data into a typical sequence of representative flight conditions for which loads and stresses are calculated. For a fighter/trainer type aircraft, this effort is repeated many times for each of the fatigue critical locations (FCL) resulting in expenditure of numerous engineering hours. The Aircraft Stress Sequence Computer Program (ACSTRSEQ), developed by Southwest Research Institute under contract to San Antonio Air Logistics Center, presents a unique approach for making complex technical computations in a simple, easy to use method. The program is written in Microsoft Visual Basic for the Microsoft Windows environment.

Fungal Genomics for Energy and Environment

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

Grigoriev, Igor V.

2013-03-11

Genomes of fungi relevant to energy and environment are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). One of its projects, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts) by means of genome sequencing and analysis. New chapters of the Encyclopedia can be opened with user proposals to the JGI Community Sequencing Program (CSP). Another JGI project, the 1000 fungal genomes, explores fungal diversity on genome level at scale and is open for usersmore » to nominate new species for sequencing. Over 200 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functional genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such parts suggested by comparative genomics and functional analysis in these areas are presented here.« less

Single-Cell RNA-Sequencing: Assessment of Differential Expression Analysis Methods.

PubMed

Dal Molin, Alessandra; Baruzzo, Giacomo; Di Camillo, Barbara

2017-01-01

The sequencing of the transcriptomes of single-cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types and for the study of stochastic gene expression. In recent years, various tools for analyzing single-cell RNA-sequencing data have been proposed, many of them with the purpose of performing differentially expression analysis. In this work, we compare four different tools for single-cell RNA-sequencing differential expression, together with two popular methods originally developed for the analysis of bulk RNA-sequencing data, but largely applied to single-cell data. We discuss results obtained on two real and one synthetic dataset, along with considerations about the perspectives of single-cell differential expression analysis. In particular, we explore the methods performance in four different scenarios, mimicking different unimodal or bimodal distributions of the data, as characteristic of single-cell transcriptomics. We observed marked differences between the selected methods in terms of precision and recall, the number of detected differentially expressed genes and the overall performance. Globally, the results obtained in our study suggest that is difficult to identify a best performing tool and that efforts are needed to improve the methodologies for single-cell RNA-sequencing data analysis and gain better accuracy of results.

Integrated databanks access and sequence/structure analysis services at the PBIL.

PubMed

Perrière, Guy; Combet, Christophe; Penel, Simon; Blanchet, Christophe; Thioulouse, Jean; Geourjon, Christophe; Grassot, Julien; Charavay, Céline; Gouy, Manolo; Duret, Laurent; Deléage, Gilbert

2003-07-01

The World Wide Web server of the PBIL (Pôle Bioinformatique Lyonnais) provides on-line access to sequence databanks and to many tools of nucleic acid and protein sequence analyses. This server allows to query nucleotide sequence banks in the EMBL and GenBank formats and protein sequence banks in the SWISS-PROT and PIR formats. The query engine on which our data bank access is based is the ACNUC system. It allows the possibility to build complex queries to access functional zones of biological interest and to retrieve large sequence sets. Of special interest are the unique features provided by this system to query the data banks of gene families developed at the PBIL. The server also provides access to a wide range of sequence analysis methods: similarity search programs, multiple alignments, protein structure prediction and multivariate statistics. An originality of this server is the integration of these two aspects: sequence retrieval and sequence analysis. Indeed, thanks to the introduction of re-usable lists, it is possible to perform treatments on large sets of data. The PBIL server can be reached at: http://pbil.univ-lyon1.fr.

Achieving an empathic stance: dialogical sequence analysis of a change episode.

PubMed

Tikkanen, Soile; Stiles, William B; Leiman, Mikael

2013-01-01

Abstract This study examined a client's therapeutic progress within one session of an 18-session child neurological assessment. The analysis focused on a parent-psychologist dialogue in one session of the assessment process. Dialogical sequence analysis (DSA; Leiman, 2004, 2012) was used as a micro-analytic method to examine the developing discourse. The analysis traced the mother's developing of a reflective stance toward herself and her problematic ways of interacting with her daughter, who was the client. During the dialogue, the mother began to recognize her own contribution in maintaining the problematic pattern. Her gradual acknowledgment of the child's perspective and her growing sense of the child's otherness were mediated by an observer position (third-person view) toward the problematic pattern, which allowed a flexible exchange between the perspectives of self and the other. The results demonstrate the parallel development of intrapersonal and interpersonal empathy shown previously to characterize the transition from stage 3 (problem statement/clarification) to stage 4 (understanding/insight) in the assimilation of problematic experiences sequence (Brinegar, Salvi, Stiles, & Greenberg, 2006).

Implementation of Cloud based next generation sequencing data analysis in a clinical laboratory.

PubMed

Onsongo, Getiria; Erdmann, Jesse; Spears, Michael D; Chilton, John; Beckman, Kenneth B; Hauge, Adam; Yohe, Sophia; Schomaker, Matthew; Bower, Matthew; Silverstein, Kevin A T; Thyagarajan, Bharat

2014-05-23

The introduction of next generation sequencing (NGS) has revolutionized molecular diagnostics, though several challenges remain limiting the widespread adoption of NGS testing into clinical practice. One such difficulty includes the development of a robust bioinformatics pipeline that can handle the volume of data generated by high-throughput sequencing in a cost-effective manner. Analysis of sequencing data typically requires a substantial level of computing power that is often cost-prohibitive to most clinical diagnostics laboratories. To address this challenge, our institution has developed a Galaxy-based data analysis pipeline which relies on a web-based, cloud-computing infrastructure to process NGS data and identify genetic variants. It provides additional flexibility, needed to control storage costs, resulting in a pipeline that is cost-effective on a per-sample basis. It does not require the usage of EBS disk to run a sample. We demonstrate the validation and feasibility of implementing this bioinformatics pipeline in a molecular diagnostics laboratory. Four samples were analyzed in duplicate pairs and showed 100% concordance in mutations identified. This pipeline is currently being used in the clinic and all identified pathogenic variants confirmed using Sanger sequencing further validating the software.

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

PubMed

Herold, Keith E; Rasooly, Avraham

2003-12-01

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

PubMed Central

Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

2011-01-01

Background Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. Methodology/Principal Findings We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). Conclusions The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation. PMID:21909358

HTSstation: a web application and open-access libraries for high-throughput sequencing data analysis.

PubMed

David, Fabrice P A; Delafontaine, Julien; Carat, Solenne; Ross, Frederick J; Lefebvre, Gregory; Jarosz, Yohan; Sinclair, Lucas; Noordermeer, Daan; Rougemont, Jacques; Leleu, Marion

2014-01-01

The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch.

HTSstation: A Web Application and Open-Access Libraries for High-Throughput Sequencing Data Analysis

PubMed Central

David, Fabrice P. A.; Delafontaine, Julien; Carat, Solenne; Ross, Frederick J.; Lefebvre, Gregory; Jarosz, Yohan; Sinclair, Lucas; Noordermeer, Daan; Rougemont, Jacques; Leleu, Marion

2014-01-01

The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch. PMID:24475057

Glutenite bodies sequence division of the upper Es4 in northern Minfeng zone of Dongying Sag, Bohai Bay Basin, China

NASA Astrophysics Data System (ADS)

Shao, Xupeng

2017-04-01

Glutenite bodies are widely developed in northern Minfeng zone of Dongying Sag. Their litho-electric relationship is not clear. In addition, as the conventional sequence stratigraphic research method drawbacks of involving too many subjective human factors, it has limited deepening of the regional sequence stratigraphic research. The wavelet transform technique based on logging data and the time-frequency analysis technique based on seismic data have advantages of dividing sequence stratigraphy quantitatively comparing with the conventional methods. Under the basis of the conventional sequence research method, this paper used the above techniques to divide the fourth-order sequence of the upper Es4 in northern Minfeng zone of Dongying Sag. The research shows that the wavelet transform technique based on logging data and the time-frequency analysis technique based on seismic data are essentially consistent, both of which divide sequence stratigraphy quantitatively in the frequency domain; wavelet transform technique has high resolutions. It is suitable for areas with wells. The seismic time-frequency analysis technique has wide applicability, but a low resolution. Both of the techniques should be combined; the upper Es4 in northern Minfeng zone of Dongying Sag is a complete set of third-order sequence, which can be further subdivided into 5 fourth-order sequences that has the depositional characteristics of fine-upward sequence in granularity. Key words: Dongying sag, northern Minfeng zone, wavelet transform technique, time-frequency analysis technique ,the upper Es4, sequence stratigraphy

Model-based reconstruction of synthetic promoter library in Corynebacterium glutamicum.

PubMed

Zhang, Shuanghong; Liu, Dingyu; Mao, Zhitao; Mao, Yufeng; Ma, Hongwu; Chen, Tao; Zhao, Xueming; Wang, Zhiwen

2018-05-01

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum. A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the - 10 and - 35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks. To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

PubMed Central

Tan, Le Van; Tuyen, Nguyen Thi Kim; Thanh, Tran Tan; Ngan, Tran Thuy; Van, Hoang Minh Tu; Sabanathan, Saraswathy; Van, Tran Thi My; Thanh, Le Thi My; Nguyet, Lam Anh; Geoghegan, Jemma L.; Ong, Kien Chai; Perera, David; Hang, Vu Thi Ty; Ny, Nguyen Thi Han; Anh, Nguyen To; Ha, Do Quang; Qui, Phan Tu; Viet, Do Chau; Tuan, Ha Manh; Wong, Kum Thong; Holmes, Edward C.; Chau, Nguyen Van Vinh; Thwaites, Guy; van Doorn, H. Rogier

2015-01-01

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples. PMID:25704598

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development

PubMed Central

2013-01-01

Background Cymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense. Results In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development. Conclusion RNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid. PMID:23617896

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development.

PubMed

Zhang, Jianxia; Wu, Kunlin; Zeng, Songjun; Teixeira da Silva, Jaime A; Zhao, Xiaolan; Tian, Chang-En; Xia, Haoqiang; Duan, Jun

2013-04-24

Cymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense. In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development. RNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid.

Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

PubMed

Sun, Beili; Zhou, Dongrui; Tu, Jing; Lu, Zuhong

2017-01-01

The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

PubMed Central

Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai

2015-01-01

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses. PMID:26019210

Special Focus

PubMed Central

Nawrocki, Eric P.; Burge, Sarah W.

2013-01-01

The development of RNA bioinformatic tools began more than 30 y ago with the description of the Nussinov and Zuker dynamic programming algorithms for single sequence RNA secondary structure prediction. Since then, many tools have been developed for various RNA sequence analysis problems such as homology search, multiple sequence alignment, de novo RNA discovery, read-mapping, and many more. In this issue, we have collected a sampling of reviews and original research that demonstrate some of the many ways bioinformatics is integrated with current RNA biology research. PMID:23948768

Sequence editing by Apolipoprotein B RNA-editing catalytic component-B and epidemiological surveillance of transmitted HIV-1 drug resistance

PubMed Central

Gifford, Robert J.; Rhee, Soo-Yon; Eriksson, Nicolas; Liu, Tommy F.; Kiuchi, Mark; Das, Amar K.; Shafer, Robert W.

2008-01-01

Design Promiscuous guanine (G) to adenine (A) substitutions catalysed by apolipoprotein B RNA-editing catalytic component (APOBEC) enzymes are observed in a proportion of HIV-1 sequences in vivo and can introduce artifacts into some genetic analyses. The potential impact of undetected lethal editing on genotypic estimation of transmitted drug resistance was assessed. Methods Classifiers of lethal, APOBEC-mediated editing were developed by analysis of lentiviral pol gene sequence variation and evaluated using control sets of HIV-1 sequences. The potential impact of sequence editing on genotypic estimation of drug resistance was assessed in sets of sequences obtained from 77 studies of 25 or more therapy-naive individuals, using mixture modelling approaches to determine the maximum likelihood classification of sequences as lethally edited as opposed to viable. Results Analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals using a novel classifier of lethal, APOBEC3G-mediated sequence editing, the polypeptide-like 3G (APOBEC3G)-mediated defectives (A3GD) index’, detected lethal editing in association with spurious ‘transmitted drug resistance’ in nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma. Conclusion Screening for lethally edited sequences in datasets containing a proportion of proviral DNA, such as those likely to be obtained for epidemiological surveillance of transmitted drug resistance in the developing world, can eliminate rare but potentially significant errors in genotypic estimation of transmitted drug resistance. PMID:18356601

PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

PubMed

Spahn, Philipp N; Bath, Tyler; Weiss, Ryan J; Kim, Jihoon; Esko, Jeffrey D; Lewis, Nathan E; Harismendy, Olivier

2017-11-20

Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets.

SUGAR: graphical user interface-based data refiner for high-throughput DNA sequencing.

PubMed

Sato, Yukuto; Kojima, Kaname; Nariai, Naoki; Yamaguchi-Kabata, Yumi; Kawai, Yosuke; Takahashi, Mamoru; Mimori, Takahiro; Nagasaki, Masao

2014-08-08

Next-generation sequencers (NGSs) have become one of the main tools for current biology. To obtain useful insights from the NGS data, it is essential to control low-quality portions of the data affected by technical errors such as air bubbles in sequencing fluidics. We develop a software SUGAR (subtile-based GUI-assisted refiner) which can handle ultra-high-throughput data with user-friendly graphical user interface (GUI) and interactive analysis capability. The SUGAR generates high-resolution quality heatmaps of the flowcell, enabling users to find possible signals of technical errors during the sequencing. The sequencing data generated from the error-affected regions of a flowcell can be selectively removed by automated analysis or GUI-assisted operations implemented in the SUGAR. The automated data-cleaning function based on sequence read quality (Phred) scores was applied to a public whole human genome sequencing data and we proved the overall mapping quality was improved. The detailed data evaluation and cleaning enabled by SUGAR would reduce technical problems in sequence read mapping, improving subsequent variant analysis that require high-quality sequence data and mapping results. Therefore, the software will be especially useful to control the quality of variant calls to the low population cells, e.g., cancers, in a sample with technical errors of sequencing procedures.

Metaphor as a Possible Pathway to More Formal Understanding of the Definition of Sequence Convergence

ERIC Educational Resources Information Center

Dawkins, Paul Christian

2012-01-01

This study presents how the introduction of a metaphor for sequence convergence constituted an experientially real context in which an undergraduate real analysis student developed a property-based definition of sequence convergence. I use elements from Zandieh and Rasmussen's (2010) Defining as a Mathematical Activity framework to trace the…

Transcriptome Analysis and Development of SSR Molecular Markers in Glycyrrhiza uralensis Fisch.

PubMed Central

Liu, Yaling; Zhang, Pengfei; Song, Meiling; Hou, Junling; Qing, Mei; Wang, Wenquan; Liu, Chunsheng

2015-01-01

Licorice is an important traditional Chinese medicine with clinical and industrial applications. Genetic resources of licorice are insufficient for analysis of molecular biology and genetic functions; as such, transcriptome sequencing must be conducted for functional characterization and development of molecular markers. In this study, transcriptome sequencing on the Illumina HiSeq 2500 sequencing platform generated a total of 5.41 Gb clean data. De novo assembly yielded a total of 46,641 unigenes. Comparison analysis using BLAST showed that the annotations of 29,614 unigenes were conserved. Further study revealed 773 genes related to biosynthesis of secondary metabolites of licorice, 40 genes involved in biosynthesis of the terpenoid backbone, and 16 genes associated with biosynthesis of glycyrrhizic acid. Analysis of unigenes larger than 1 Kb with a length of 11,702 nt presented 7,032 simple sequence repeats (SSR). Sixty-four of 69 randomly designed and synthesized SSR pairs were successfully amplified, 33 pairs of primers were polymorphism in in Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., Glycyrrhiza glabra L. and Glycyrrhiza pallidiflora Maxim. This study not only presents the molecular biology data of licorice but also provides a basis for genetic diversity research and molecular marker-assisted breeding of licorice. PMID:26571372

CAFE: aCcelerated Alignment-FrEe sequence analysis

PubMed Central

Lu, Yang Young; Tang, Kujin; Ren, Jie; Fuhrman, Jed A.; Waterman, Michael S.

2017-01-01

Abstract Alignment-free genome and metagenome comparisons are increasingly important with the development of next generation sequencing (NGS) technologies. Recently developed state-of-the-art k-mer based alignment-free dissimilarity measures including CVTree, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$d_2^*$\\end{document} and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$d_2^S$\\end{document} are more computationally expensive than measures based solely on the k-mer frequencies. Here, we report a standalone software, aCcelerated Alignment-FrEe sequence analysis (CAFE), for efficient calculation of 28 alignment-free dissimilarity measures. CAFE allows for both assembled genome sequences and unassembled NGS shotgun reads as input, and wraps the output in a standard PHYLIP format. In downstream analyses, CAFE can also be used to visualize the pairwise dissimilarity measures, including dendrograms, heatmap, principal coordinate analysis and network display. CAFE serves as a general k-mer based alignment-free analysis platform for studying the relationships among genomes and metagenomes, and is freely available at https://github.com/younglululu/CAFE. PMID:28472388

Application of viromics: a new approach to the understanding of viral infections in humans.

PubMed

Ramamurthy, Mageshbabu; Sankar, Sathish; Kannangai, Rajesh; Nandagopal, Balaji; Sridharan, Gopalan

2017-12-01

This review is focused at exploring the strengths of modern technology driven data compiled in the areas of virus gene sequencing, virus protein structures and their implication to viral diagnosis and therapy. The information for virome analysis (viromics) is generated by the study of viral genomes (entire nucleotide sequence) and viral genes (coding for protein). Presently, the study of viral infectious diseases in terms of etiopathogenesis and development of newer therapeutics is undergoing rapid changes. Currently, viromics relies on deep sequencing, next generation sequencing (NGS) data and public domain databases like GenBank and unique virus specific databases. Two commonly used NGS platforms: Illumina and Ion Torrent, recommend maximum fragment lengths of about 300 and 400 nucleotides for analysis respectively. Direct detection of viruses in clinical samples is now evolving using these methods. Presently, there are a considerable number of good treatment options for HBV/HIV/HCV. These viruses however show development of drug resistance. The drug susceptibility regions of the genomes are sequenced and the prediction of drug resistance is now possible from 3 public domains available on the web. This has been made possible through advances in the technology with the advent of high throughput sequencing and meta-analysis through sophisticated and easy to use software and the use of high speed computers for bioinformatics. More recently NGS technology has been improved with single-molecule real-time sequencing. Here complete long reads can be obtained with less error overcoming a limitation of the NGS which is inherently prone to software anomalies that arise in the hands of personnel without adequate training. The development in understanding the viruses in terms of their genome, pathobiology, transcriptomics and molecular epidemiology constitutes viromics. It could be stated that these developments will bring about radical changes and advancement especially in the field of antiviral therapy and diagnostic virology.

Genomic Sequencing: Assessing The Health Care System, Policy, And Big-Data Implications

PubMed Central

Phillips, Kathryn A.; Trosman, Julia; Kelley, Robin K.; Pletcher, Mark J.; Douglas, Michael P.; Weldon, Christine B.

2014-01-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a “big data” technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. PMID:25006153

Genomic sequencing: assessing the health care system, policy, and big-data implications.

PubMed

Phillips, Kathryn A; Trosman, Julia R; Kelley, Robin K; Pletcher, Mark J; Douglas, Michael P; Weldon, Christine B

2014-07-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a "big data" technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. Project HOPE—The People-to-People Health Foundation, Inc.

The need for high-quality whole-genome sequence databases in microbial forensics.

PubMed

Sjödin, Andreas; Broman, Tina; Melefors, Öjar; Andersson, Gunnar; Rasmusson, Birgitta; Knutsson, Rickard; Forsman, Mats

2013-09-01

Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

Sequence stratigraphy and high-frequency cycles: New aspects for a quantitative evaluation of the Gulf of Suez basin, Egypt

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

Nio, S.D.; Yang, C.S.; Tewfik, N.

1993-09-01

A new development in the application of sequence stratigraphic concepts in marine as well as continental basins is the recognition of high-frequency cyclic patterns in rock successions in the subsurface. Studies of six wells from the northern, central, and southern parts of the Gulf of Suez show the presence of well-preserved, high-frequency cycles with periodicities similar to the orbitally forced Malankovitch parameters. Subsurface rock successions, third-order sequences, and high-frequency cycles were compared with outcrops. After establishing the biostratigraphic framework for the above-mentioned wells, a sequence analysis was performed. Sequence boundaries and maximum flooding positions in each well were calibrated withmore » the occurrences and evaluation of the high-frequency cycles. It became obvious that there is an intimate relationship between these high-frequency Milankovitch cycles and sequence organization. In addition, a close relationship can be observed in the subsurface as well as in outcrops between high-frequency climatic changes (connected to the Milankovitch cycles) and (litho)facies variability. Quantitative evaluations of each sequence and/or systems tract can be computed with the International Geoservices' cyclicity analysis tool (MILABAR). The results are summarized in a well composite chart, rate (NAR), and ratio of preserved time. In correlations between the wells, an accuracy of 500-100 Ka can be obtained. The quantitative evaluation of the sequence and high-frequency cycle analysis gave some new aspects concerning the (litho)facies and geodynamic development during the pre- as well as the synrift stages of the Gulf of Suez Basin.« less

Genomic Encyclopedia of Fungi

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

Grigoriev, Igor

Genomes of fungi relevant to energy and environment are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). Its key project, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts), and explores fungal diversity by means of genome sequencing and analysis. Over 150 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supportedmore » by functional genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such parts suggested by comparative genomics and functional analysis in these areas are presented here.« less

Analysis of simulated image sequences from sensors for restricted-visibility operations

NASA Technical Reports Server (NTRS)

Kasturi, Rangachar

1991-01-01

A real time model of the visible output from a 94 GHz sensor, based on a radiometric simulation of the sensor, was developed. A sequence of images as seen from an aircraft as it approaches for landing was simulated using this model. Thirty frames from this sequence of 200 x 200 pixel images were analyzed to identify and track objects in the image using the Cantata image processing package within the visual programming environment provided by the Khoros software system. The image analysis operations are described.

The determination of high-resolution spatio-temporal glacier motion fields from time-lapse sequences

NASA Astrophysics Data System (ADS)

Schwalbe, Ellen; Maas, Hans-Gerd

2017-12-01

This paper presents a comprehensive method for the determination of glacier surface motion vector fields at high spatial and temporal resolution. These vector fields can be derived from monocular terrestrial camera image sequences and are a valuable data source for glaciological analysis of the motion behaviour of glaciers. The measurement concepts for the acquisition of image sequences are presented, and an automated monoscopic image sequence processing chain is developed. Motion vector fields can be derived with high precision by applying automatic subpixel-accuracy image matching techniques on grey value patterns in the image sequences. Well-established matching techniques have been adapted to the special characteristics of the glacier data in order to achieve high reliability in automatic image sequence processing, including the handling of moving shadows as well as motion effects induced by small instabilities in the camera set-up. Suitable geo-referencing techniques were developed to transform image measurements into a reference coordinate system.The result of monoscopic image sequence analysis is a dense raster of glacier surface point trajectories for each image sequence. Each translation vector component in these trajectories can be determined with an accuracy of a few centimetres for points at a distance of several kilometres from the camera. Extensive practical validation experiments have shown that motion vector and trajectory fields derived from monocular image sequences can be used for the determination of high-resolution velocity fields of glaciers, including the analysis of tidal effects on glacier movement, the investigation of a glacier's motion behaviour during calving events, the determination of the position and migration of the grounding line and the detection of subglacial channels during glacier lake outburst floods.

Genomics: The Science and Technology Behind the Human Genome Project (by Charles R. Cantor and Cassandra L. Smith)

NASA Astrophysics Data System (ADS)

Serra, Reviewed By Martin J.

2000-01-01

Genomics is one of the most rapidly expanding areas of science. This book is an outgrowth of a series of lectures given by one of the former heads (CRC) of the Human Genome Initiative. The book is designed to reach a wide audience, from biologists with little chemical or physical science background through engineers, computer scientists, and physicists with little current exposure to the chemical or biological principles of genetics. The text starts with a basic review of the chemical and biological properties of DNA. However, without either a biochemistry background or a supplemental biochemistry text, this chapter and much of the rest of the text would be difficult to digest. The second chapter is designed to put DNA into the context of the larger chromosomal unit. Specialized chromosomal structures and sequences (centromeres, telomeres) are introduced, leading to a section on chromosome organization and purification. The next 4 chapters cover the physical (hybridization, electrophoresis), chemical (polymerase chain reaction), and biological (genetic) techniques that provide the backbone of genomic analysis. These chapters cover in significant detail the fundamental principles underlying each technique and provide a firm background for the remainder of the text. Chapters 7­9 consider the need and methods for the development of physical maps. Chapter 7 primarily discusses chromosomal localization techniques, including in situ hybridization, FISH, and chromosome paintings. The next two chapters focus on the development of libraries and clones. In particular, Chapter 9 considers the limitations of current mapping and clone production. The current state and future of DNA sequencing is covered in the next three chapters. The first considers the current methods of DNA sequencing - especially gel-based methods of analysis, although other possible approaches (mass spectrometry) are introduced. Much of the chapter addresses the limitations of current methods, including analysis of error in sequencing and current bottlenecks in the sequencing effort. The next chapter describes the steps necessary to scale current technologies for the sequencing of entire genomes. Chapter 12 examines alternate methods for DNA sequencing. Initially, methods of single-molecule sequencing and sequencing by microscopy are introduced; the majority of the chapter is devoted to the development of DNA sequencing methods using chip microarrays and hybridization. The remaining chapters (13-15) consider the uses and analysis of DNA sequence information. The initial focus is on the identification of genes. Several examples are given of the use of DNA sequence information for diagnosis of inherited or infectious diseases. The sequence-specific manipulation of DNA is discussed in Chapter 14. The final chapter deals with the implications of large-scale sequencing, including methods for identifying genes and finding errors in DNA sequences, to the development of computer algorithms for the interpretation of DNA sequence information. The text figures are black and white line drawings that, although clearly done, seem a bit primitive for 1999. While I appreciated the simplicity of the drawings, many students accustomed to more colorful presentations will find them wanting. The four color figures in the center of the text seem an afterthought and add little to the text's clarity. Each chapter has a set of additional reading sources, mostly primary sources. Often, specialized topics are offset into boxes that provide clarification and amplification without cluttering the text. An appendix includes a list of the Web-based database resources. As an undergraduate instructor who has previously taught biochemistry, molecular biology, and a course on the human genome, I found many interesting tidbits and amplifications throughout the text. I would recommend this book as a text for an advanced undergraduate or beginning graduate course in genomics. Although the text works though several examples of genetic and genome analysis, additional problem/homework sets would need to be developed to ensure student comprehension. The text steers clear of the ethical implications of the Human Genome Initiative and remains true to its subtitle The Science and Technology .

RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

PubMed Central

Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; Akiyama, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin; Hazama, Makoto; Nishine, Tsutomu; Harada, Akira; Yamamoto, Rintaro; Matsumoto, Hiroyuki; Sakaguchi, Sumito; Ikegami, Takashi; Kashiwagi, Katsuya; Fujiwake, Syuji; Inoue, Kouji; Togawa, Yoshiyuki; Izawa, Masaki; Ohara, Eiji; Watahiki, Masanori; Yoneda, Yuko; Ishikawa, Tomokazu; Ozawa, Kaori; Tanaka, Takumi; Matsuura, Shuji; Kawai, Jun; Okazaki, Yasushi; Muramatsu, Masami; Inoue, Yorinao; Kira, Akira; Hayashizaki, Yoshihide

2000-01-01

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3′ end and 5′ end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month. PMID:11076861

Initial steps towards a production platform for DNA sequence analysis on the grid.

PubMed

Luyf, Angela C M; van Schaik, Barbera D C; de Vries, Michel; Baas, Frank; van Kampen, Antoine H C; Olabarriaga, Silvia D

2010-12-14

Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids are often unfriendly to novice users. In this study we reused a platform that was developed in the VL-e project for the analysis of medical images. Data transfer, workflow execution and job monitoring are operated from one graphical interface. We developed workflows for two sequence alignment tools (BLAST and BLAT) as a proof of concept. The analysis time was significantly reduced. All workflows and executables are available for the members of the Dutch Life Science Grid and the VL-e Medical virtual organizations All components are open source and can be transported to other grid infrastructures. The availability of in-house expertise and tools facilitates the usage of grid resources by new users. Our first results indicate that this is a practical, powerful and scalable solution to address the capacity and collaboration issues raised by the deployment of next generation sequencers. We currently adopt this methodology on a daily basis for DNA sequencing and other applications. More information and source code is available via http://www.bioinformaticslaboratory.nl/

DNAApp: a mobile application for sequencing data analysis

PubMed Central

Nguyen, Phi-Vu; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2014-01-01

Summary: There have been numerous applications developed for decoding and visualization of ab1 DNA sequencing files for Windows and MAC platforms, yet none exists for the increasingly popular smartphone operating systems. The ability to decode sequencing files cannot easily be carried out using browser accessed Web tools. To overcome this hurdle, we have developed a new native app called DNAApp that can decode and display ab1 sequencing file on Android and iOS. In addition to in-built analysis tools such as reverse complementation, protein translation and searching for specific sequences, we have incorporated convenient functions that would facilitate the harnessing of online Web tools for a full range of analysis. Given the high usage of Android/iOS tablets and smartphones, such bioinformatics apps would raise productivity and facilitate the high demand for analyzing sequencing data in biomedical research. Availability and implementation: The Android version of DNAApp is available in Google Play Store as ‘DNAApp’, and the iOS version is available in the App Store. More details on the app can be found at www.facebook.com/APDLab; www.bii.a-star.edu.sg/research/trd/apd.php The DNAApp user guide is available at http://tinyurl.com/DNAAppuser, and a video tutorial is available on Google Play Store and App Store, as well as on the Facebook page. Contact: samuelg@bii.a-star.edu.sg PMID:25095882

EGenBio: A Data Management System for Evolutionary Genomics and Biodiversity

PubMed Central

Nahum, Laila A; Reynolds, Matthew T; Wang, Zhengyuan O; Faith, Jeremiah J; Jonna, Rahul; Jiang, Zhi J; Meyer, Thomas J; Pollock, David D

2006-01-01

Background Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; ) to begin to address this. Description EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. Conclusion EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity. PMID:17118150

DNAApp: a mobile application for sequencing data analysis.

PubMed

Nguyen, Phi-Vu; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2014-11-15

There have been numerous applications developed for decoding and visualization of ab1 DNA sequencing files for Windows and MAC platforms, yet none exists for the increasingly popular smartphone operating systems. The ability to decode sequencing files cannot easily be carried out using browser accessed Web tools. To overcome this hurdle, we have developed a new native app called DNAApp that can decode and display ab1 sequencing file on Android and iOS. In addition to in-built analysis tools such as reverse complementation, protein translation and searching for specific sequences, we have incorporated convenient functions that would facilitate the harnessing of online Web tools for a full range of analysis. Given the high usage of Android/iOS tablets and smartphones, such bioinformatics apps would raise productivity and facilitate the high demand for analyzing sequencing data in biomedical research. The Android version of DNAApp is available in Google Play Store as 'DNAApp', and the iOS version is available in the App Store. More details on the app can be found at www.facebook.com/APDLab; www.bii.a-star.edu.sg/research/trd/apd.php The DNAApp user guide is available at http://tinyurl.com/DNAAppuser, and a video tutorial is available on Google Play Store and App Store, as well as on the Facebook page. samuelg@bii.a-star.edu.sg. © The Author 2014. Published by Oxford University Press.

Atlas2 Cloud: a framework for personal genome analysis in the cloud

PubMed Central

2012-01-01

Background Until recently, sequencing has primarily been carried out in large genome centers which have invested heavily in developing the computational infrastructure that enables genomic sequence analysis. The recent advancements in next generation sequencing (NGS) have led to a wide dissemination of sequencing technologies and data, to highly diverse research groups. It is expected that clinical sequencing will become part of diagnostic routines shortly. However, limited accessibility to computational infrastructure and high quality bioinformatic tools, and the demand for personnel skilled in data analysis and interpretation remains a serious bottleneck. To this end, the cloud computing and Software-as-a-Service (SaaS) technologies can help address these issues. Results We successfully enabled the Atlas2 Cloud pipeline for personal genome analysis on two different cloud service platforms: a community cloud via the Genboree Workbench, and a commercial cloud via the Amazon Web Services using Software-as-a-Service model. We report a case study of personal genome analysis using our Atlas2 Genboree pipeline. We also outline a detailed cost structure for running Atlas2 Amazon on whole exome capture data, providing cost projections in terms of storage, compute and I/O when running Atlas2 Amazon on a large data set. Conclusions We find that providing a web interface and an optimized pipeline clearly facilitates usage of cloud computing for personal genome analysis, but for it to be routinely used for large scale projects there needs to be a paradigm shift in the way we develop tools, in standard operating procedures, and in funding mechanisms. PMID:23134663

Atlas2 Cloud: a framework for personal genome analysis in the cloud.

PubMed

Evani, Uday S; Challis, Danny; Yu, Jin; Jackson, Andrew R; Paithankar, Sameer; Bainbridge, Matthew N; Jakkamsetti, Adinarayana; Pham, Peter; Coarfa, Cristian; Milosavljevic, Aleksandar; Yu, Fuli

2012-01-01

Until recently, sequencing has primarily been carried out in large genome centers which have invested heavily in developing the computational infrastructure that enables genomic sequence analysis. The recent advancements in next generation sequencing (NGS) have led to a wide dissemination of sequencing technologies and data, to highly diverse research groups. It is expected that clinical sequencing will become part of diagnostic routines shortly. However, limited accessibility to computational infrastructure and high quality bioinformatic tools, and the demand for personnel skilled in data analysis and interpretation remains a serious bottleneck. To this end, the cloud computing and Software-as-a-Service (SaaS) technologies can help address these issues. We successfully enabled the Atlas2 Cloud pipeline for personal genome analysis on two different cloud service platforms: a community cloud via the Genboree Workbench, and a commercial cloud via the Amazon Web Services using Software-as-a-Service model. We report a case study of personal genome analysis using our Atlas2 Genboree pipeline. We also outline a detailed cost structure for running Atlas2 Amazon on whole exome capture data, providing cost projections in terms of storage, compute and I/O when running Atlas2 Amazon on a large data set. We find that providing a web interface and an optimized pipeline clearly facilitates usage of cloud computing for personal genome analysis, but for it to be routinely used for large scale projects there needs to be a paradigm shift in the way we develop tools, in standard operating procedures, and in funding mechanisms.

SU-C-17A-02: Sirius MRI Markers for Prostate Post-Implant Assessment: MR Protocol Development

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

Lim, T; Wang, J; Kudchadker, R

Purpose: Currently, CT is used to visualize prostate brachytherapy sources, at the expense of accurate structure contouring. MRI is superior to CT for anatomical delineation, but the sources appear as voids on MRI images. Previously we have developed Sirius MRI markers (C4 Imaging) to replace spacers to assist source localization on MRI images. Here we develop an MRI pulse sequence protocol that enhances the signal of these markers to enable MRI-only post-implant prostate dosimetric analysis. Methods: To simulate a clinical scenario, a CIRS multi-modality prostate phantom was implanted with 66 markers and 86 sources. The implanted phantom was imaged onmore » both 1.5T and 3.0T GE scanners under various conditions, different pulse sequences (2D fast spin echo [FSE], 3D balanced steadystate free precession [bSSFP] and 3D fast spoiled gradient echo [FSPGR]), as well as varying amount of padding to simulate various patient sizes and associated signal fall-off from the surface coil elements. Standard FSE sequences from the current clinical protocols were also evaluated. Marker visibility, marker size, intra-marker distance, total scan time and artifacts were evaluated for various combinations of echo time, repetition time, flip angle, number of excitations, bandwidth, slice thickness and spacing, fieldof- view, frequency/phase encoding steps and frequency direction. Results: We have developed a 3D FSPGR pulse sequence that enhances marker signal and ensures the integrity of the marker shape while maintaining reasonable scan time. For patients contraindicated for 3.0T, we have also developed a similar sequence for 1.5T scanners. Signal fall-off with distance from prostate to coil can be compensated mainly by decreasing bandwidth. The markers are not visible using standard FSE sequences. FSPGR sequences are more robust for consistent marker visualization as compared to bSSFP sequences. Conclusion: The developed MRI pulse sequence protocol for Sirius MRI markers assists source localization to enable MRIonly post-implant prostate dosimetric analysis. S.J. Frank is a co-founder of C4 Imaging (manufactures the MRI markers)« less

Investigating Student Understanding for a Statistical Analysis of Two Thermally Interacting Solids

NASA Astrophysics Data System (ADS)

Loverude, Michael E.

2010-10-01

As part of an ongoing research and curriculum development project for upper-division courses in thermal physics, we have developed a sequence of tutorials in which students apply statistical methods to examine the behavior of two interacting Einstein solids. In the sequence, students begin with simple results from probability and develop a means for counting the states in a single Einstein solid. The students then consider the thermal interaction of two solids, and observe that the classical equilibrium state corresponds to the most probable distribution of energy between the two solids. As part of the development of the tutorial sequence, we have developed several assessment questions to probe student understanding of various aspects of this system. In this paper, we describe the strengths and weaknesses of student reasoning, both qualitative and quantitative, to assess the readiness of students for one tutorial in the sequence.

Pfarao: a web application for protein family analysis customized for cytoskeletal and motor proteins (CyMoBase).

PubMed

Odronitz, Florian; Kollmar, Martin

2006-11-29

Annotation of protein sequences of eukaryotic organisms is crucial for the understanding of their function in the cell. Manual annotation is still by far the most accurate way to correctly predict genes. The classification of protein sequences, their phylogenetic relation and the assignment of function involves information from various sources. This often leads to a collection of heterogeneous data, which is hard to track. Cytoskeletal and motor proteins consist of large and diverse superfamilies comprising up to several dozen members per organism. Up to date there is no integrated tool available to assist in the manual large-scale comparative genomic analysis of protein families. Pfarao (Protein Family Application for Retrieval, Analysis and Organisation) is a database driven online working environment for the analysis of manually annotated protein sequences and their relationship. Currently, the system can store and interrelate a wide range of information about protein sequences, species, phylogenetic relations and sequencing projects as well as links to literature and domain predictions. Sequences can be imported from multiple sequence alignments that are generated during the annotation process. A web interface allows to conveniently browse the database and to compile tabular and graphical summaries of its content. We implemented a protein sequence-centric web application to store, organize, interrelate, and present heterogeneous data that is generated in manual genome annotation and comparative genomics. The application has been developed for the analysis of cytoskeletal and motor proteins (CyMoBase) but can easily be adapted for any protein.

A user's guide to quantitative and comparative analysis of metagenomic datasets.

PubMed

Luo, Chengwei; Rodriguez-R, Luis M; Konstantinidis, Konstantinos T

2013-01-01

Metagenomics has revolutionized microbiological studies during the past decade and provided new insights into the diversity, dynamics, and metabolic potential of natural microbial communities. However, metagenomics still represents a field in development, and standardized tools and approaches to handle and compare metagenomes have not been established yet. An important reason accounting for the latter is the continuous changes in the type of sequencing data available, for example, long versus short sequencing reads. Here, we provide a guide to bioinformatic pipelines developed to accomplish the following tasks, focusing primarily on those developed by our team: (i) assemble a metagenomic dataset; (ii) determine the level of sequence coverage obtained and the amount of sequencing required to obtain complete coverage; (iii) identify the taxonomic affiliation of a metagenomic read or assembled contig; and (iv) determine differentially abundant genes, pathways, and species between different datasets. Most of these pipelines do not depend on the type of sequences available or can be easily adjusted to fit different types of sequences, and are freely available (for instance, through our lab Web site: http://www.enve-omics.gatech.edu/). The limitations of current approaches, as well as the computational aspects that can be further improved, will also be briefly discussed. The work presented here provides practical guidelines on how to perform metagenomic analysis of microbial communities characterized by varied levels of diversity and establishes approaches to handle the resulting data, independent of the sequencing platform employed. © 2013 Elsevier Inc. All rights reserved.

Characterizing the Grape Transcriptome. Analysis of Expressed Sequence Tags from Multiple Vitis Species and Development of a Compendium of Gene Expression during Berry Development1[w

PubMed Central

Silva, Francisco Goes da; Iandolino, Alberto; Al-Kayal, Fadi; Bohlmann, Marlene C.; Cushman, Mary Ann; Lim, Hyunju; Ergul, Ali; Figueroa, Rubi; Kabuloglu, Elif K.; Osborne, Craig; Rowe, Joan; Tattersall, Elizabeth; Leslie, Anna; Xu, Jane; Baek, JongMin; Cramer, Grant R.; Cushman, John C.; Cook, Douglas R.

2005-01-01

We report the analysis and annotation of 146,075 expressed sequence tags from Vitis species. The majority of these sequences were derived from different cultivars of Vitis vinifera, comprising an estimated 25,746 unique contig and singleton sequences that survey transcription in various tissues and developmental stages and during biotic and abiotic stress. Putatively homologous proteins were identified for over 17,752 of the transcripts, with 1,962 transcripts further subdivided into one or more Gene Ontology categories. A simple structured vocabulary, with modules for plant genotype, plant development, and stress, was developed to describe the relationship between individual expressed sequence tags and cDNA libraries; the resulting vocabulary provides query terms to facilitate data mining within the context of a relational database. As a measure of the extent to which characterized metabolic pathways were encompassed by the data set, we searched for homologs of the enzymes leading from glycolysis, through the oxidative/nonoxidative pentose phosphate pathway, and into the general phenylpropanoid pathway. Homologs were identified for 65 of these 77 enzymes, with 86% of enzymatic steps represented by paralogous genes. Differentially expressed transcripts were identified by means of a stringent believability index cutoff of ≥98.4%. Correlation analysis and two-dimensional hierarchical clustering grouped these transcripts according to similarity of expression. In the broadest analysis, 665 differentially expressed transcripts were identified across 29 cDNA libraries, representing a range of developmental and stress conditions. The groupings revealed expected associations between plant developmental stages and tissue types, with the notable exception of abiotic stress treatments. A more focused analysis of flower and berry development identified 87 differentially expressed transcripts and provides the basis for a compendium that relates gene expression and annotation to previously characterized aspects of berry development and physiology. Comparison with published results for select genes, as well as correlation analysis between independent data sets, suggests that the inferred in silico patterns of expression are likely to be an accurate representation of transcript abundance for the conditions surveyed. Thus, the combined data set reveals the in silico expression patterns for hundreds of genes in V. vinifera, the majority of which have not been previously studied within this species. PMID:16219919

Tracking B-Cell Repertoires and Clonal Histories in Normal and Malignant Lymphocytes.

PubMed

Weston-Bell, Nicola J; Cowan, Graeme; Sahota, Surinder S

2017-01-01

Methods for tracking B-cell repertoires and clonal history in normal and malignant B-cells based on immunoglobulin variable region (IGV) gene analysis have developed rapidly with the advent of massive parallel next-generation sequencing (mpNGS) protocols. mpNGS permits a depth of analysis of IGV genes not hitherto feasible, and presents challenges of bioinformatics analysis, which can be readily met by current pipelines. This strategy offers a potential resolution of B-cell usage at a depth that may capture fully the natural state, in a given biological setting. Conventional methods based on RT-PCR amplification and Sanger sequencing are also available where mpNGS is not accessible. Each method offers distinct advantages. Conventional methods for IGV gene sequencing are readily adaptable to most laboratories and provide an ease of analysis to capture salient features of B-cell use. This chapter describes two methods in detail for analysis of IGV genes, mpNGS and conventional RT-PCR with Sanger sequencing.

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr)

USDA-ARS?s Scientific Manuscript database

A Multilocus Sequence Typing (MLST) method based on allelic variation of 7 chromosomal loci was developed for characterizing genotypes within the genus Bradyrhizobium. With the method 29 distinct multilocus genotypes (GTs) were identified among 191 culture collection soybean strains. The occupancy ...

Information theory applications for biological sequence analysis.

PubMed

Vinga, Susana

2014-05-01

Information theory (IT) addresses the analysis of communication systems and has been widely applied in molecular biology. In particular, alignment-free sequence analysis and comparison greatly benefited from concepts derived from IT, such as entropy and mutual information. This review covers several aspects of IT applications, ranging from genome global analysis and comparison, including block-entropy estimation and resolution-free metrics based on iterative maps, to local analysis, comprising the classification of motifs, prediction of transcription factor binding sites and sequence characterization based on linguistic complexity and entropic profiles. IT has also been applied to high-level correlations that combine DNA, RNA or protein features with sequence-independent properties, such as gene mapping and phenotype analysis, and has also provided models based on communication systems theory to describe information transmission channels at the cell level and also during evolutionary processes. While not exhaustive, this review attempts to categorize existing methods and to indicate their relation with broader transversal topics such as genomic signatures, data compression and complexity, time series analysis and phylogenetic classification, providing a resource for future developments in this promising area.

Use of wavelet-packet transforms to develop an engineering model for multifractal characterization of mutation dynamics in pathological and nonpathological gene sequences

NASA Astrophysics Data System (ADS)

Walker, David Lee

1999-12-01

This study uses dynamical analysis to examine in a quantitative fashion the information coding mechanism in DNA sequences. This exceeds the simple dichotomy of either modeling the mechanism by comparing DNA sequence walks as Fractal Brownian Motion (fbm) processes. The 2-D mappings of the DNA sequences for this research are from Iterated Function System (IFS) (Also known as the ``Chaos Game Representation'' (CGR)) mappings of the DNA sequences. This technique converts a 1-D sequence into a 2-D representation that preserves subsequence structure and provides a visual representation. The second step of this analysis involves the application of Wavelet Packet Transforms, a recently developed technique from the field of signal processing. A multi-fractal model is built by using wavelet transforms to estimate the Hurst exponent, H. The Hurst exponent is a non-parametric measurement of the dynamism of a system. This procedure is used to evaluate gene- coding events in the DNA sequence of cystic fibrosis mutations. The H exponent is calculated for various mutation sites in this gene. The results of this study indicate the presence of anti-persistent, random walks and persistent ``sub-periods'' in the sequence. This indicates the hypothesis of a multi-fractal model of DNA information encoding warrants further consideration. This work examines the model's behavior in both pathological (mutations) and non-pathological (healthy) base pair sequences of the cystic fibrosis gene. These mutations both natural and synthetic were introduced by computer manipulation of the original base pair text files. The results show that disease severity and system ``information dynamics'' correlate. These results have implications for genetic engineering as well as in mathematical biology. They suggest that there is scope for more multi-fractal models to be developed.

Ray Wu as Fifth Business: Deconstructing collective memory in the history of DNA sequencing.

PubMed

Onaga, Lisa A

2014-06-01

The concept of 'Fifth Business' is used to analyze a minority standpoint and bring serious attention to the role of scientists who play a galvanizing role in a science but for multiple reasons appear less prominently in more common recounts of any particular development. Biochemist Ray Wu (1928-2008) published a DNA sequencing experiment in March 1970 using DNA polymerase catalysis and specific nucleotide labeling, both of which are foundational to general sequencing methods today. The scant mention of Wu's work from textbooks, research articles, and other accounts of DNA sequencing calls into question how scientific collective memory forms. This alternative history seeks to understand why a key figure in nucleic acid sequence analysis has remained less visibly connected or peripheral to solidifying narratives about the history of DNA sequencing. The study resists predictable dismissals of Wu's work in order to seriously examine the formation of his nucleic acid sequence analysis research program and how he shared his knowledge of sequencing during a period of rapid advancement in the field. An analysis of Wu's work on sequencing the cohesive ends of lambda bacteriophage in the 1960s and 1970s exemplifies how a variety of individuals and groups attempted to develop protocol for sequencing the order of nucleotide base pairs comprising DNA. This historical examination of the sociality of scientific research suggests a way to understand how Wu and others contributed to the very collective memory of DNA sequencing that Wu eventually tried to repair. The study of Wu, who was a Chinese immigrant to the United States, provides a foundation for further critical scholarship on the heterogeneous histories of Asian American bioscientists, the sociality of their scientific works, and how the resulting knowledge produced is preserved, if not evenly, in a scientific field's collective memory. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh].

PubMed

Dutta, Sutapa; Kumawat, Giriraj; Singh, Bikram P; Gupta, Deepak K; Singh, Sangeeta; Dogra, Vivek; Gaikwad, Kishor; Sharma, Tilak R; Raje, Ranjeet S; Bandhopadhya, Tapas K; Datta, Subhojit; Singh, Mahendra N; Bashasab, Fakrudin; Kulwal, Pawan; Wanjari, K B; K Varshney, Rajeev; Cook, Douglas R; Singh, Nagendra K

2011-01-20

Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping. In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥ 18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population. We developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh

PubMed Central

2011-01-01

Background Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping. Results In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population. Conclusion We developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea. PMID:21251263

[Genetic analysis of two children patients affected with CHARGE syndrome].

PubMed

Li, Guoqiang; Li, Niu; Xu, Yufei; Li, Juan; Ding, Yu; Shen, Yiping; Wang, Xiumin; Wang, Jian

2018-04-10

To analyze two Chinese pediatric patients with multiple malformations and growth and development delay. Both patients were subjected to targeted gene sequencing, and the results were analyzed with Ingenuity Variant Analysis software. Suspected pathogenic variations were verified by Sanger sequencing. High-throughput sequencing showed that both patients have carried heterozygous variants of the CHD7 gene. Patient 1 carried a nonsense mutation in exon 36 (c.7957C>T, p.Arg2653*), while patient 2 carried a nonsense mutation of exon 2 (c.718C>T, p.Gln240*). Sanger sequencing confirmed the above mutations in both patients, while their parents were of wild-type for the corresponding sites, indicating that the two mutations have happened de novo. Two patients were diagnosed with CHARGE syndrome by high-throughput sequencing.

Comparison of Burrows-Wheeler transform-based mapping algorithms used in high-throughput whole-genome sequencing: application to Illumina data for livestock genomes

USDA-ARS?s Scientific Manuscript database

Ongoing developments and cost decreases in next-generation sequencing (NGS) technologies have led to an increase in their application, which has greatly enhanced the fields of genetics and genomics. Mapping sequence reads onto a reference genome is a fundamental step in the analysis of NGS data. Eff...

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

PubMed

Zhang, Xiaoyan; Marjani, Sadie L; Hu, Zhaoyang; Weissman, Sherman M; Pan, Xinghua; Wu, Shixiu

2016-03-15

Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing. ©2016 American Association for Cancer Research.

Sequence Alignment to Predict Across Species Susceptibility ...

EPA Pesticide Factsheets

Conservation of a molecular target across species can be used as a line-of-evidence to predict the likelihood of chemical susceptibility. The web-based Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed to simplify, streamline, and quantitatively assess protein sequence/structural similarity across taxonomic groups as a means to predict relative intrinsic susceptibility. The intent of the tool is to allow for evaluation of any potential protein target, so it is amenable to variable degrees of protein characterization, depending on available information about the chemical/protein interaction and the molecular target itself. To allow for flexibility in the analysis, a layered strategy was adopted for the tool. The first level of the SeqAPASS analysis compares primary amino acid sequences to a query sequence, calculating a metric for sequence similarity (including detection of candidate orthologs), the second level evaluates sequence similarity within selected domains (e.g., ligand-binding domain, DNA binding domain), and the third level of analysis compares individual amino acid residue positions identified as being of importance for protein conformation and/or ligand binding upon chemical perturbation. Each level of the SeqAPASS analysis provides increasing evidence to apply toward rapid, screening-level assessments of probable cross species susceptibility. Such analyses can support prioritization of chemicals for further ev

Sequence-dependent modelling of local DNA bending phenomena: curvature prediction and vibrational analysis.

PubMed

Vlahovicek, K; Munteanu, M G; Pongor, S

1999-01-01

Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequence-dependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in protein-coding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http:@www.icgeb.trieste.it/dna).

Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and function.

PubMed

Baxter, Laura L; Hsu, Benjamin J; Umayam, Lowell; Wolfsberg, Tyra G; Larson, Denise M; Frith, Martin C; Kawai, Jun; Hayashizaki, Yoshihide; Carninci, Piero; Pavan, William J

2007-06-01

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

Development of an Expressed Sequence Tag (EST) Resource for Wheat (Triticum aestivum L.)

PubMed Central

Lazo, G. R.; Chao, S.; Hummel, D. D.; Edwards, H.; Crossman, C. C.; Lui, N.; Matthews, D. E.; Carollo, V. L.; Hane, D. L.; You, F. M.; Butler, G. E.; Miller, R. E.; Close, T. J.; Peng, J. H.; Lapitan, N. L. V.; Gustafson, J. P.; Qi, L. L.; Echalier, B.; Gill, B. S.; Dilbirligi, M.; Randhawa, H. S.; Gill, K. S.; Greene, R. A.; Sorrells, M. E.; Akhunov, E. D.; Dvořák, J.; Linkiewicz, A. M.; Dubcovsky, J.; Hossain, K. G.; Kalavacharla, V.; Kianian, S. F.; Mahmoud, A. A.; Miftahudin; Ma, X.-F.; Conley, E. J.; Anderson, J. A.; Pathan, M. S.; Nguyen, H. T.; McGuire, P. E.; Qualset, C. O.; Anderson, O. D.

2004-01-01

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics. PMID:15514037

miRanalyzer: a microRNA detection and analysis tool for next-generation sequencing experiments.

PubMed

Hackenberg, Michael; Sturm, Martin; Langenberger, David; Falcón-Pérez, Juan Manuel; Aransay, Ana M

2009-07-01

Next-generation sequencing allows now the sequencing of small RNA molecules and the estimation of their expression levels. Consequently, there will be a high demand of bioinformatics tools to cope with the several gigabytes of sequence data generated in each single deep-sequencing experiment. Given this scene, we developed miRanalyzer, a web server tool for the analysis of deep-sequencing experiments for small RNAs. The web server tool requires a simple input file containing a list of unique reads and its copy numbers (expression levels). Using these data, miRanalyzer (i) detects all known microRNA sequences annotated in miRBase, (ii) finds all perfect matches against other libraries of transcribed sequences and (iii) predicts new microRNAs. The prediction of new microRNAs is an especially important point as there are many species with very few known microRNAs. Therefore, we implemented a highly accurate machine learning algorithm for the prediction of new microRNAs that reaches AUC values of 97.9% and recall values of up to 75% on unseen data. The web tool summarizes all the described steps in a single output page, which provides a comprehensive overview of the analysis, adding links to more detailed output pages for each analysis module. miRanalyzer is available at http://web.bioinformatics.cicbiogune.es/microRNA/.

Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression.

PubMed

Manchado, Manuel; Infante, Carlos; Asensio, Esther; Cañavate, Jose Pedro; Douglas, Susan E

2007-07-03

Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture.

Advances in high throughput DNA sequence data compression.

PubMed

Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz

2016-06-01

Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted.

Extensive characterization of Tupaia belangeri neuropeptidome using an integrated mass spectrometric approach.

PubMed

Petruzziello, Filomena; Fouillen, Laetitia; Wadensten, Henrik; Kretz, Robert; Andren, Per E; Rainer, Gregor; Zhang, Xiaozhe

2012-02-03

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

CloVR-ITS: Automated internal transcribed spacer amplicon sequence analysis pipeline for the characterization of fungal microbiota

PubMed Central

2013-01-01

Background Besides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms. Results Here we introduce CloVR-ITS, an automated pipeline for comparative analysis of internal transcribed spacer (ITS) pyrosequences amplified from metagenomic DNA isolates and representing fungal species. This pipeline performs a variety of steps similar to those commonly used for 16S rRNA amplicon sequence analysis, including preprocessing for quality, chimera detection, clustering of sequences into operational taxonomic units (OTUs), taxonomic assignment (at class, order, family, genus, and species levels) and statistical analysis of sample groups of interest based on user-provided information. Using ITS amplicon pyrosequencing data from a previous human gastric fluid study, we demonstrate the utility of CloVR-ITS for fungal microbiota analysis and provide runtime and cost examples, including analysis of extremely large datasets on the cloud. We show that the largest fractions of reads from the stomach fluid samples were assigned to Dothideomycetes, Saccharomycetes, Agaricomycetes and Sordariomycetes but that all samples were dominated by sequences that could not be taxonomically classified. Representatives of the Candida genus were identified in all samples, most notably C. quercitrusa, while sequence reads assigned to the Aspergillus genus were only identified in a subset of samples. CloVR-ITS is made available as a pre-installed, automated, and portable software pipeline for cloud-friendly execution as part of the CloVR virtual machine package (http://clovr.org). Conclusion The CloVR-ITS pipeline provides fungal microbiota analysis that can be complementary to bacterial 16S rRNA and total metagenome sequence analysis allowing for more comprehensive studies of environmental and host-associated microbial communities. PMID:24451270

Environmental microbiology through the lens of high-throughput DNA sequencing: synopsis of current platforms and bioinformatics approaches.

PubMed

Logares, Ramiro; Haverkamp, Thomas H A; Kumar, Surendra; Lanzén, Anders; Nederbragt, Alexander J; Quince, Christopher; Kauserud, Håvard

2012-10-01

The incursion of High-Throughput Sequencing (HTS) in environmental microbiology brings unique opportunities and challenges. HTS now allows a high-resolution exploration of the vast taxonomic and metabolic diversity present in the microbial world, which can provide an exceptional insight on global ecosystem functioning, ecological processes and evolution. This exploration has also economic potential, as we will have access to the evolutionary innovation present in microbial metabolisms, which could be used for biotechnological development. HTS is also challenging the research community, and the current bottleneck is present in the data analysis side. At the moment, researchers are in a sequence data deluge, with sequencing throughput advancing faster than the computer power needed for data analysis. However, new tools and approaches are being developed constantly and the whole process could be depicted as a fast co-evolution between sequencing technology, informatics and microbiologists. In this work, we examine the most popular and recently commercialized HTS platforms as well as bioinformatics methods for data handling and analysis used in microbial metagenomics. This non-exhaustive review is intended to serve as a broad state-of-the-art guide to researchers expanding into this rapidly evolving field. Copyright © 2012 Elsevier B.V. All rights reserved.

Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of Gossypium hirsutum L

PubMed Central

Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

2013-01-01

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species. PMID:24146870

A Concise Atlas of Thyroid Cancer Next-Generation Sequencing Panel ThyroSeq v.2

PubMed Central

Alsina, Jorge; Alsina, Raul; Gulec, Seza

2017-01-01

The next-generation sequencing technology allows high out-put genomic analysis. An innovative assay in thyroid cancer, ThyroSeq® was developed for targeted mutation detection by next generation sequencing technology in fine needle aspiration and tissue samples. ThyroSeq v.2 next generation sequencing panel offers simultaneous sequencing and detection in >1000 hotspots of 14 thyroid cancer-related genes and for 42 types of gene fusions known to occur in thyroid cancer. ThyroSeq is being increasingly used to further narrow the indeterminate category defined by cytology for thyroid nodules. From a surgical perspective, genomic profiling also provides prognostic and predictive information and closely relates to determination of surgical strategy. Both the genomic analysis technology and the informatics for the cancer genome data base are rapidly developing. In this paper, we have gathered existing information on the thyroid cancer-related genes involved in the initiation and progression of thyroid cancer. Our goal is to assemble a glossary for the current ThyroSeq genomic panel that can help elucidate the role genomics play in thyroid cancer oncogenesis. PMID:28117295

Next-Generation Technologies for Multiomics Approaches Including Interactome Sequencing

PubMed Central

Ohashi, Hiroyuki; Miyamoto-Sato, Etsuko

2015-01-01

The development of high-speed analytical techniques such as next-generation sequencing and microarrays allows high-throughput analysis of biological information at a low cost. These techniques contribute to medical and bioscience advancements and provide new avenues for scientific research. Here, we outline a variety of new innovative techniques and discuss their use in omics research (e.g., genomics, transcriptomics, metabolomics, proteomics, and interactomics). We also discuss the possible applications of these methods, including an interactome sequencing technology that we developed, in future medical and life science research. PMID:25649523

Selected Insights from Application of Whole Genome Sequencing for Outbreak Investigations

PubMed Central

Le, Vien Thi Minh; Diep, Binh An

2014-01-01

Purpose of review The advent of high-throughput whole genome sequencing has the potential to revolutionize the conduct of outbreak investigation. Because of its ultimate pathogen strain resolution, whole genome sequencing could augment traditional epidemiologic investigations of infectious disease outbreaks. Recent findings The combination of whole genome sequencing and intensive epidemiologic analysis provided new insights on the sources and transmission dynamics of large-scale epidemics caused by Escherichia coli and Vibrio cholerae, nosocomial outbreaks caused by methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, and Mycobacterium abscessus, community-centered outbreaks caused by Mycobacterium tuberculosis, and natural disaster-associated outbreak caused by environmentally acquired molds. Summary When combined with traditional epidemiologic investigation, whole genome sequencing has proven useful for elucidating sources and transmission dynamics of disease outbreaks. Development of a fully automated bioinformatics pipeline for analysis of whole genome sequence data is much needed to make this powerful tool more widely accessible. PMID:23856896

Pulseq-Graphical Programming Interface: Open source visual environment for prototyping pulse sequences and integrated magnetic resonance imaging algorithm development.

PubMed

Ravi, Keerthi Sravan; Potdar, Sneha; Poojar, Pavan; Reddy, Ashok Kumar; Kroboth, Stefan; Nielsen, Jon-Fredrik; Zaitsev, Maxim; Venkatesan, Ramesh; Geethanath, Sairam

2018-03-11

To provide a single open-source platform for comprehensive MR algorithm development inclusive of simulations, pulse sequence design and deployment, reconstruction, and image analysis. We integrated the "Pulseq" platform for vendor-independent pulse programming with Graphical Programming Interface (GPI), a scientific development environment based on Python. Our integrated platform, Pulseq-GPI, permits sequences to be defined visually and exported to the Pulseq file format for execution on an MR scanner. For comparison, Pulseq files using either MATLAB only ("MATLAB-Pulseq") or Python only ("Python-Pulseq") were generated. We demonstrated three fundamental sequences on a 1.5 T scanner. Execution times of the three variants of implementation were compared on two operating systems. In vitro phantom images indicate equivalence with the vendor supplied implementations and MATLAB-Pulseq. The examples demonstrated in this work illustrate the unifying capability of Pulseq-GPI. The execution times of all the three implementations were fast (a few seconds). The software is capable of user-interface based development and/or command line programming. The tool demonstrated here, Pulseq-GPI, integrates the open-source simulation, reconstruction and analysis capabilities of GPI Lab with the pulse sequence design and deployment features of Pulseq. Current and future work includes providing an ISMRMRD interface and incorporating Specific Absorption Ratio and Peripheral Nerve Stimulation computations. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of Genomic Simple Sequence Repeats (SSR) by Enrichment Libraries in Date Palm.

PubMed

Al-Faifi, Sulieman A; Migdadi, Hussein M; Algamdi, Salem S; Khan, Mohammad Altaf; Al-Obeed, Rashid S; Ammar, Megahed H; Jakse, Jerenj

2017-01-01

Development of highly informative markers such as simple sequence repeats (SSR) for cultivar identification and germplasm characterization and management is essential for date palms genetic studies. The present study documents the development of SSR markers and assesses genetic relationships of commonly grown date palm (Phoenix dactylifera L.) cultivars in different geographical regions of Saudi Arabia. A total of 93 novel simple sequence repeat (SSR) markers were screened for their ability to detect polymorphism in date palm. Around 71% of genomic SSRs are dinucleotide, 25% trinucleotide, 3% tetranucleotide, and 1% pentanucleotide motives and show 100% polymorphism. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis illustrates that cultivars trend to group according to their class of maturity, region of cultivation, and fruit color. Analysis of molecular variations (AMOVA) reveals genetic variation among and within cultivars of 27% and 73%, respectively, according to the geographical distribution of the cultivars. Developed microsatellite markers are of additional value to date palm characterization, tools which can be used by researchers in population genetics, cultivar identification, as well as genetic resource exploration and management. The cultivars tested exhibited a significant amount of genetic diversity and could be suitable for successful breeding programs. Genomic sequences generated from this study are available at the National Center for Biotechnology Information (NCBI), Sequence Read Archive (Accession numbers. LIBGSS_039019).

Fueling the Future with Fungal Genomes

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

Grigoriev, Igor V.

2014-10-27

Genomes of fungi relevant to energy and environment are in focus of the JGI Fungal Genomic Program. One of its projects, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts and pathogens) and biorefinery processes (cellulose degradation and sugar fermentation) by means of genome sequencing and analysis. New chapters of the Encyclopedia can be opened with user proposals to the JGI Community Science Program (CSP). Another JGI project, the 1000 fungal genomes, explores fungal diversity on genome level at scale and is open for users to nominate new species for sequencing. Over 400 fungal genomes have beenmore » sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functional genomics will lead to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such ‘parts’ suggested by comparative genomics and functional analysis in these areas are presented here.« less

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

A computational proposal for designing structured RNA pools for in vitro selection of RNAs.

PubMed

Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

2007-04-01

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

Recurrence time statistics: versatile tools for genomic DNA sequence analysis.

PubMed

Cao, Yinhe; Tung, Wen-Wen; Gao, J B

2004-01-01

With the completion of the human and a few model organisms' genomes, and the genomes of many other organisms waiting to be sequenced, it has become increasingly important to develop faster computational tools which are capable of easily identifying the structures and extracting features from DNA sequences. One of the more important structures in a DNA sequence is repeat-related. Often they have to be masked before protein coding regions along a DNA sequence are to be identified or redundant expressed sequence tags (ESTs) are to be sequenced. Here we report a novel recurrence time based method for sequence analysis. The method can conveniently study all kinds of periodicity and exhaustively find all repeat-related features from a genomic DNA sequence. An efficient codon index is also derived from the recurrence time statistics, which has the salient features of being largely species-independent and working well on very short sequences. Efficient codon indices are key elements of successful gene finding algorithms, and are particularly useful for determining whether a suspected EST belongs to a coding or non-coding region. We illustrate the power of the method by studying the genomes of E. coli, the yeast S. cervisivae, the nematode worm C. elegans, and the human, Homo sapiens. Computationally, our method is very efficient. It allows us to carry out analysis of genomes on the whole genomic scale by a PC.

DraGnET: Software for storing, managing and analyzing annotated draft genome sequence data

PubMed Central

2010-01-01

Background New "next generation" DNA sequencing technologies offer individual researchers the ability to rapidly generate large amounts of genome sequence data at dramatically reduced costs. As a result, a need has arisen for new software tools for storage, management and analysis of genome sequence data. Although bioinformatic tools are available for the analysis and management of genome sequences, limitations still remain. For example, restrictions on the submission of data and use of these tools may be imposed, thereby making them unsuitable for sequencing projects that need to remain in-house or proprietary during their initial stages. Furthermore, the availability and use of next generation sequencing in industrial, governmental and academic environments requires biologist to have access to computational support for the curation and analysis of the data generated; however, this type of support is not always immediately available. Results To address these limitations, we have developed DraGnET (Draft Genome Evaluation Tool). DraGnET is an open source web application which allows researchers, with no experience in programming and database management, to setup their own in-house projects for storing, retrieving, organizing and managing annotated draft and complete genome sequence data. The software provides a web interface for the use of BLAST, allowing users to perform preliminary comparative analysis among multiple genomes. We demonstrate the utility of DraGnET for performing comparative genomics on closely related bacterial strains. Furthermore, DraGnET can be further developed to incorporate additional tools for more sophisticated analyses. Conclusions DraGnET is designed for use either by individual researchers or as a collaborative tool available through Internet (or Intranet) deployment. For genome projects that require genome sequencing data to initially remain proprietary, DraGnET provides the means for researchers to keep their data in-house for analysis using local programs or until it is made publicly available, at which point it may be uploaded to additional analysis software applications. The DraGnET home page is available at http://www.dragnet.cvm.iastate.edu and includes example files for examining the functionalities, a link for downloading the DraGnET setup package and a link to the DraGnET source code hosted with full documentation on SourceForge. PMID:20175920

PGen: large-scale genomic variations analysis workflow and browser in SoyKB.

PubMed

Liu, Yang; Khan, Saad M; Wang, Juexin; Rynge, Mats; Zhang, Yuanxun; Zeng, Shuai; Chen, Shiyuan; Maldonado Dos Santos, Joao V; Valliyodan, Babu; Calyam, Prasad P; Merchant, Nirav; Nguyen, Henry T; Xu, Dong; Joshi, Trupti

2016-10-06

With the advances in next-generation sequencing (NGS) technology and significant reductions in sequencing costs, it is now possible to sequence large collections of germplasm in crops for detecting genome-scale genetic variations and to apply the knowledge towards improvements in traits. To efficiently facilitate large-scale NGS resequencing data analysis of genomic variations, we have developed "PGen", an integrated and optimized workflow using the Extreme Science and Engineering Discovery Environment (XSEDE) high-performance computing (HPC) virtual system, iPlant cloud data storage resources and Pegasus workflow management system (Pegasus-WMS). The workflow allows users to identify single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), perform SNP annotations and conduct copy number variation analyses on multiple resequencing datasets in a user-friendly and seamless way. We have developed both a Linux version in GitHub ( https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow ) and a web-based implementation of the PGen workflow integrated within the Soybean Knowledge Base (SoyKB), ( http://soykb.org/Pegasus/index.php ). Using PGen, we identified 10,218,140 single-nucleotide polymorphisms (SNPs) and 1,398,982 indels from analysis of 106 soybean lines sequenced at 15X coverage. 297,245 non-synonymous SNPs and 3330 copy number variation (CNV) regions were identified from this analysis. SNPs identified using PGen from additional soybean resequencing projects adding to 500+ soybean germplasm lines in total have been integrated. These SNPs are being utilized for trait improvement using genotype to phenotype prediction approaches developed in-house. In order to browse and access NGS data easily, we have also developed an NGS resequencing data browser ( http://soykb.org/NGS_Resequence/NGS_index.php ) within SoyKB to provide easy access to SNP and downstream analysis results for soybean researchers. PGen workflow has been optimized for the most efficient analysis of soybean data using thorough testing and validation. This research serves as an example of best practices for development of genomics data analysis workflows by integrating remote HPC resources and efficient data management with ease of use for biological users. PGen workflow can also be easily customized for analysis of data in other species.

Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.)

PubMed Central

2011-01-01

Background Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea. Results A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme. Conclusion In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea. PMID:21447154

Development of Pineapple Microsatellite Markers and Germplasm Genetic Diversity Analysis

PubMed Central

Tong, Helin; Chen, You; Wang, Jingyi; Chen, Yeyuan; Sun, Guangming; He, Junhu; Wu, Yaoting

2013-01-01

Two methods were used to develop pineapple microsatellite markers. Genomic library-based SSR development: using selectively amplified microsatellite assay, 86 sequences were generated from pineapple genomic library. 91 (96.8%) of the 94 Simple Sequence Repeat (SSR) loci were dinucleotide repeats (39 AC/GT repeats and 52 GA/TC repeats, accounting for 42.9% and 57.1%, resp.), and the other three were mononucleotide repeats. Thirty-six pairs of SSR primers were designed; 24 of them generated clear bands of expected sizes, and 13 of them showed polymorphism. EST-based SSR development: 5659 pineapple EST sequences obtained from NCBI were analyzed; among 1397 nonredundant EST sequences, 843 were found containing 1110 SSR loci (217 of them contained more than one SSR locus). Frequency of SSRs in pineapple EST sequences is 1SSR/3.73 kb, and 44 types were found. Mononucleotide, dinucleotide, and trinucleotide repeats dominate, accounting for 95.6% in total. AG/CT and AGC/GCT were the dominant type of dinucleotide and trinucleotide repeats, accounting for 83.5% and 24.1%, respectively. Thirty pairs of primers were designed for each of randomly selected 30 sequences; 26 of them generated clear and reproducible bands, and 22 of them showed polymorphism. Eighteen pairs of primers obtained by the one or the other of the two methods above that showed polymorphism were selected to carry out germplasm genetic diversity analysis for 48 breeds of pineapple; similarity coefficients of these breeds were between 0.59 and 1.00, and they can be divided into four groups accordingly. Amplification products of five SSR markers were extracted and sequenced, corresponding repeat loci were found and locus mutations are mainly in copy number of repeats and base mutations in the flanking region. PMID:24024187

Analysis of plant microbe interactions in the era of next generation sequencing technologies

PubMed Central

Knief, Claudia

2014-01-01

Next generation sequencing (NGS) technologies have impressively accelerated research in biological science during the last years by enabling the production of large volumes of sequence data to a drastically lower price per base, compared to traditional sequencing methods. The recent and ongoing developments in the field allow addressing research questions in plant-microbe biology that were not conceivable just a few years ago. The present review provides an overview of NGS technologies and their usefulness for the analysis of microorganisms that live in association with plants. Possible limitations of the different sequencing systems, in particular sources of errors and bias, are critically discussed and methods are disclosed that help to overcome these shortcomings. A focus will be on the application of NGS methods in metagenomic studies, including the analysis of microbial communities by amplicon sequencing, which can be considered as a targeted metagenomic approach. Different applications of NGS technologies are exemplified by selected research articles that address the biology of the plant associated microbiota to demonstrate the worth of the new methods. PMID:24904612

Use of tuf Sequences for Genus-Specific PCR Detection and Phylogenetic Analysis of 28 Streptococcal Species

PubMed Central

Picard, François J.; Ke, Danbing; Boudreau, Dominique K.; Boissinot, Maurice; Huletsky, Ann; Richard, Dave; Ouellette, Marc; Roy, Paul H.; Bergeron, Michel G.

2004-01-01

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence. PMID:15297518

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge.

PubMed

Brownstein, Catherine A; Beggs, Alan H; Homer, Nils; Merriman, Barry; Yu, Timothy W; Flannery, Katherine C; DeChene, Elizabeth T; Towne, Meghan C; Savage, Sarah K; Price, Emily N; Holm, Ingrid A; Luquette, Lovelace J; Lyon, Elaine; Majzoub, Joseph; Neupert, Peter; McCallie, David; Szolovits, Peter; Willard, Huntington F; Mendelsohn, Nancy J; Temme, Renee; Finkel, Richard S; Yum, Sabrina W; Medne, Livija; Sunyaev, Shamil R; Adzhubey, Ivan; Cassa, Christopher A; de Bakker, Paul I W; Duzkale, Hatice; Dworzyński, Piotr; Fairbrother, William; Francioli, Laurent; Funke, Birgit H; Giovanni, Monica A; Handsaker, Robert E; Lage, Kasper; Lebo, Matthew S; Lek, Monkol; Leshchiner, Ignaty; MacArthur, Daniel G; McLaughlin, Heather M; Murray, Michael F; Pers, Tune H; Polak, Paz P; Raychaudhuri, Soumya; Rehm, Heidi L; Soemedi, Rachel; Stitziel, Nathan O; Vestecka, Sara; Supper, Jochen; Gugenmus, Claudia; Klocke, Bernward; Hahn, Alexander; Schubach, Max; Menzel, Mortiz; Biskup, Saskia; Freisinger, Peter; Deng, Mario; Braun, Martin; Perner, Sven; Smith, Richard J H; Andorf, Janeen L; Huang, Jian; Ryckman, Kelli; Sheffield, Val C; Stone, Edwin M; Bair, Thomas; Black-Ziegelbein, E Ann; Braun, Terry A; Darbro, Benjamin; DeLuca, Adam P; Kolbe, Diana L; Scheetz, Todd E; Shearer, Aiden E; Sompallae, Rama; Wang, Kai; Bassuk, Alexander G; Edens, Erik; Mathews, Katherine; Moore, Steven A; Shchelochkov, Oleg A; Trapane, Pamela; Bossler, Aaron; Campbell, Colleen A; Heusel, Jonathan W; Kwitek, Anne; Maga, Tara; Panzer, Karin; Wassink, Thomas; Van Daele, Douglas; Azaiez, Hela; Booth, Kevin; Meyer, Nic; Segal, Michael M; Williams, Marc S; Tromp, Gerard; White, Peter; Corsmeier, Donald; Fitzgerald-Butt, Sara; Herman, Gail; Lamb-Thrush, Devon; McBride, Kim L; Newsom, David; Pierson, Christopher R; Rakowsky, Alexander T; Maver, Aleš; Lovrečić, Luca; Palandačić, Anja; Peterlin, Borut; Torkamani, Ali; Wedell, Anna; Huss, Mikael; Alexeyenko, Andrey; Lindvall, Jessica M; Magnusson, Måns; Nilsson, Daniel; Stranneheim, Henrik; Taylan, Fulya; Gilissen, Christian; Hoischen, Alexander; van Bon, Bregje; Yntema, Helger; Nelen, Marcel; Zhang, Weidong; Sager, Jason; Zhang, Lu; Blair, Kathryn; Kural, Deniz; Cariaso, Michael; Lennon, Greg G; Javed, Asif; Agrawal, Saloni; Ng, Pauline C; Sandhu, Komal S; Krishna, Shuba; Veeramachaneni, Vamsi; Isakov, Ofer; Halperin, Eran; Friedman, Eitan; Shomron, Noam; Glusman, Gustavo; Roach, Jared C; Caballero, Juan; Cox, Hannah C; Mauldin, Denise; Ament, Seth A; Rowen, Lee; Richards, Daniel R; San Lucas, F Anthony; Gonzalez-Garay, Manuel L; Caskey, C Thomas; Bai, Yu; Huang, Ying; Fang, Fang; Zhang, Yan; Wang, Zhengyuan; Barrera, Jorge; Garcia-Lobo, Juan M; González-Lamuño, Domingo; Llorca, Javier; Rodriguez, Maria C; Varela, Ignacio; Reese, Martin G; De La Vega, Francisco M; Kiruluta, Edward; Cargill, Michele; Hart, Reece K; Sorenson, Jon M; Lyon, Gholson J; Stevenson, David A; Bray, Bruce E; Moore, Barry M; Eilbeck, Karen; Yandell, Mark; Zhao, Hongyu; Hou, Lin; Chen, Xiaowei; Yan, Xiting; Chen, Mengjie; Li, Cong; Yang, Can; Gunel, Murat; Li, Peining; Kong, Yong; Alexander, Austin C; Albertyn, Zayed I; Boycott, Kym M; Bulman, Dennis E; Gordon, Paul M K; Innes, A Micheil; Knoppers, Bartha M; Majewski, Jacek; Marshall, Christian R; Parboosingh, Jillian S; Sawyer, Sarah L; Samuels, Mark E; Schwartzentruber, Jeremy; Kohane, Isaac S; Margulies, David M

2014-03-25

There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

PubMed Central

2014-01-01

Background There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. Results A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. Conclusions The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups. PMID:24667040

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

PubMed Central

Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

2011-01-01

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.

PubMed

Canver, Matthew C; Haeussler, Maximilian; Bauer, Daniel E; Orkin, Stuart H; Sanjana, Neville E; Shalem, Ophir; Yuan, Guo-Cheng; Zhang, Feng; Concordet, Jean-Paul; Pinello, Luca

2018-05-01

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

Open Reading Frame Phylogenetic Analysis on the Cloud

PubMed Central

2013-01-01

Phylogenetic analysis has become essential in researching the evolutionary relationships between viruses. These relationships are depicted on phylogenetic trees, in which viruses are grouped based on sequence similarity. Viral evolutionary relationships are identified from open reading frames rather than from complete sequences. Recently, cloud computing has become popular for developing internet-based bioinformatics tools. Biocloud is an efficient, scalable, and robust bioinformatics computing service. In this paper, we propose a cloud-based open reading frame phylogenetic analysis service. The proposed service integrates the Hadoop framework, virtualization technology, and phylogenetic analysis methods to provide a high-availability, large-scale bioservice. In a case study, we analyze the phylogenetic relationships among Norovirus. Evolutionary relationships are elucidated by aligning different open reading frame sequences. The proposed platform correctly identifies the evolutionary relationships between members of Norovirus. PMID:23671843

Attomole-level Genomics with Single-molecule Direct DNA, cDNA and RNA Sequencing Technologies.

PubMed

Ozsolak, Fatih

2016-01-01

With the introduction of next-generation sequencing (NGS) technologies in 2005, the domination of microarrays in genomics quickly came to an end due to NGS's superior technical performance and cost advantages. By enabling genetic analysis capabilities that were not possible previously, NGS technologies have started to play an integral role in all areas of biomedical research. This chapter outlines the low-quantity DNA and cDNA sequencing capabilities and applications developed with the Helicos single molecule DNA sequencing technology.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

HTSeq--a Python framework to work with high-throughput sequencing data.

PubMed

Anders, Simon; Pyl, Paul Theodor; Huber, Wolfgang

2015-01-15

A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed. We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes. HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq. © The Author 2014. Published by Oxford University Press.

Hepatitis C Virus Antigenic Convergence

PubMed Central

Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury

2012-01-01

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

PubMed Central

2014-01-01

Background Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome. Results To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks. Conclusions The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface. PMID:24646187

DELIMINATE--a fast and efficient method for loss-less compression of genomic sequences: sequence analysis.

PubMed

Mohammed, Monzoorul Haque; Dutta, Anirban; Bose, Tungadri; Chadaram, Sudha; Mande, Sharmila S

2012-10-01

An unprecedented quantity of genome sequence data is currently being generated using next-generation sequencing platforms. This has necessitated the development of novel bioinformatics approaches and algorithms that not only facilitate a meaningful analysis of these data but also aid in efficient compression, storage, retrieval and transmission of huge volumes of the generated data. We present a novel compression algorithm (DELIMINATE) that can rapidly compress genomic sequence data in a loss-less fashion. Validation results indicate relatively higher compression efficiency of DELIMINATE when compared with popular general purpose compression algorithms, namely, gzip, bzip2 and lzma. Linux, Windows and Mac implementations (both 32 and 64-bit) of DELIMINATE are freely available for download at: http://metagenomics.atc.tcs.com/compression/DELIMINATE. sharmila@atc.tcs.com Supplementary data are available at Bioinformatics online.

Pfarao: a web application for protein family analysis customized for cytoskeletal and motor proteins (CyMoBase)

PubMed Central

Odronitz, Florian; Kollmar, Martin

2006-01-01

Background Annotation of protein sequences of eukaryotic organisms is crucial for the understanding of their function in the cell. Manual annotation is still by far the most accurate way to correctly predict genes. The classification of protein sequences, their phylogenetic relation and the assignment of function involves information from various sources. This often leads to a collection of heterogeneous data, which is hard to track. Cytoskeletal and motor proteins consist of large and diverse superfamilies comprising up to several dozen members per organism. Up to date there is no integrated tool available to assist in the manual large-scale comparative genomic analysis of protein families. Description Pfarao (Protein Family Application for Retrieval, Analysis and Organisation) is a database driven online working environment for the analysis of manually annotated protein sequences and their relationship. Currently, the system can store and interrelate a wide range of information about protein sequences, species, phylogenetic relations and sequencing projects as well as links to literature and domain predictions. Sequences can be imported from multiple sequence alignments that are generated during the annotation process. A web interface allows to conveniently browse the database and to compile tabular and graphical summaries of its content. Conclusion We implemented a protein sequence-centric web application to store, organize, interrelate, and present heterogeneous data that is generated in manual genome annotation and comparative genomics. The application has been developed for the analysis of cytoskeletal and motor proteins (CyMoBase) but can easily be adapted for any protein. PMID:17134497

Bacterial community comparisons by taxonomy-supervised analysis independent of sequence alignment and clustering

PubMed Central

Sul, Woo Jun; Cole, James R.; Jesus, Ederson da C.; Wang, Qiong; Farris, Ryan J.; Fish, Jordan A.; Tiedje, James M.

2011-01-01

High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. PMID:21873204

[Genome-scale sequence data processing and epigenetic analysis of DNA methylation].

PubMed

Wang, Ting-Zhang; Shan, Gao; Xu, Jian-Hong; Xue, Qing-Zhong

2013-06-01

A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under-stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive sequences, such as transposable elements. In this paper, we introduce the preprocessing steps of DNA methylation data, by which cytosine (C) and guanine (G) in the reference sequence are transferred to thymine (T) and adenine (A), and cytosine in reads is transferred to thymine, respectively. We also comprehensively review the main content of the DNA methylation analysis on the genomic scale: (1) the cytosine methylation under the context of different sequences; (2) the distribution of genomic methylcytosine; (3) DNA methylation context and the preference for the nucleotides; (4) DNA- protein interaction sites of DNA methylation; (5) degree of methylation of cytosine in the different structural elements of genes. DNA methylation analysis technique provides a powerful tool for the epigenome study in human and other species, and genes and environment interaction, and founds the theoretical basis for further development of disease diagnostics and therapeutics in human.

Automated sequence analysis and editing software for HIV drug resistance testing.

PubMed

Struck, Daniel; Wallis, Carole L; Denisov, Gennady; Lambert, Christine; Servais, Jean-Yves; Viana, Raquel V; Letsoalo, Esrom; Bronze, Michelle; Aitken, Sue C; Schuurman, Rob; Stevens, Wendy; Schmit, Jean Claude; Rinke de Wit, Tobias; Perez Bercoff, Danielle

2012-05-01

Access to antiretroviral treatment in resource-limited-settings is inevitably paralleled by the emergence of HIV drug resistance. Monitoring treatment efficacy and HIV drugs resistance testing are therefore of increasing importance in resource-limited settings. Yet low-cost technologies and procedures suited to the particular context and constraints of such settings are still lacking. The ART-A (Affordable Resistance Testing for Africa) consortium brought together public and private partners to address this issue. To develop an automated sequence analysis and editing software to support high throughput automated sequencing. The ART-A Software was designed to automatically process and edit ABI chromatograms or FASTA files from HIV-1 isolates. The ART-A Software performs the basecalling, assigns quality values, aligns query sequences against a set reference, infers a consensus sequence, identifies the HIV type and subtype, translates the nucleotide sequence to amino acids and reports insertions/deletions, premature stop codons, ambiguities and mixed calls. The results can be automatically exported to Excel to identify mutations. Automated analysis was compared to manual analysis using a panel of 1624 PR-RT sequences generated in 3 different laboratories. Discrepancies between manual and automated sequence analysis were 0.69% at the nucleotide level and 0.57% at the amino acid level (668,047 AA analyzed), and discordances at major resistance mutations were recorded in 62 cases (4.83% of differences, 0.04% of all AA) for PR and 171 (6.18% of differences, 0.03% of all AA) cases for RT. The ART-A Software is a time-sparing tool for pre-analyzing HIV and viral quasispecies sequences in high throughput laboratories and highlighting positions requiring attention. Copyright © 2012 Elsevier B.V. All rights reserved.

Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat

PubMed Central

2012-01-01

Background Bread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. Results The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29%) were 3A-specific and compared to 17 (18%) for 96 SSRs. Conclusion This work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping. PMID:22559868

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis

PubMed Central

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described. PMID:28542338

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.

PubMed

Young, Brian; King, Jonathan L; Budowle, Bruce; Armogida, Luigi

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

The Use of Weighted Graphs for Large-Scale Genome Analysis

PubMed Central

Zhou, Fang; Toivonen, Hannu; King, Ross D.

2014-01-01

There is an acute need for better tools to extract knowledge from the growing flood of sequence data. For example, thousands of complete genomes have been sequenced, and their metabolic networks inferred. Such data should enable a better understanding of evolution. However, most existing network analysis methods are based on pair-wise comparisons, and these do not scale to thousands of genomes. Here we propose the use of weighted graphs as a data structure to enable large-scale phylogenetic analysis of networks. We have developed three types of weighted graph for enzymes: taxonomic (these summarize phylogenetic importance), isoenzymatic (these summarize enzymatic variety/redundancy), and sequence-similarity (these summarize sequence conservation); and we applied these types of weighted graph to survey prokaryotic metabolism. To demonstrate the utility of this approach we have compared and contrasted the large-scale evolution of metabolism in Archaea and Eubacteria. Our results provide evidence for limits to the contingency of evolution. PMID:24619061

The European Classical Swine Fever Virus Database: Blueprint for a Pathogen-Specific Sequence Database with Integrated Sequence Analysis Tools

PubMed Central

Postel, Alexander; Schmeiser, Stefanie; Zimmermann, Bernd; Becher, Paul

2016-01-01

Molecular epidemiology has become an indispensable tool in the diagnosis of diseases and in tracing the infection routes of pathogens. Due to advances in conventional sequencing and the development of high throughput technologies, the field of sequence determination is in the process of being revolutionized. Platforms for sharing sequence information and providing standardized tools for phylogenetic analyses are becoming increasingly important. The database (DB) of the European Union (EU) and World Organisation for Animal Health (OIE) Reference Laboratory for classical swine fever offers one of the world’s largest semi-public virus-specific sequence collections combined with a module for phylogenetic analysis. The classical swine fever (CSF) DB (CSF-DB) became a valuable tool for supporting diagnosis and epidemiological investigations of this highly contagious disease in pigs with high socio-economic impacts worldwide. The DB has been re-designed and now allows for the storage and analysis of traditionally used, well established genomic regions and of larger genomic regions including complete viral genomes. We present an application example for the analysis of highly similar viral sequences obtained in an endemic disease situation and introduce the new geographic “CSF Maps” tool. The concept of this standardized and easy-to-use DB with an integrated genetic typing module is suited to serve as a blueprint for similar platforms for other human or animal viruses. PMID:27827988

Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut.

PubMed

Cheng, Yunqing; Liu, Jianfeng; Zhang, Huidi; Wang, Ju; Zhao, Yixin; Geng, Wanting

2015-01-01

A high ratio of blank fruit in hazelnut (Corylus heterophylla Fisch) is a very common phenomenon that causes serious yield losses in northeast China. The development of blank fruit in the Corylus genus is known to be associated with embryo abortion. However, little is known about the molecular mechanisms responsible for embryo abortion during the nut development stage. Genomic information for C. heterophylla Fisch is not available; therefore, data related to transcriptome and gene expression profiling of developing and abortive ovules are needed. In this study, de novo transcriptome sequencing and RNA-seq analysis were conducted using short-read sequencing technology (Illumina HiSeq 2000). The results of the transcriptome assembly analysis revealed genetic information that was associated with the fruit development stage. Two digital gene expression libraries were constructed, one for a full (normally developing) ovule and one for an empty (abortive) ovule. Transcriptome sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. These results were annotated using the public databases NR, NT, Swiss-Prot, KEGG, COG, and GO. Using digital gene expression profiling, gene expression differences in developing and abortive ovules were identified. A total of 1,637 and 715 unigenes were significantly upregulated and downregulated, respectively, in abortive ovules, compared with developing ovules. Quantitative real-time polymerase chain reaction analysis was used in order to verify the differential expression of some genes. The transcriptome and digital gene expression profiling data of normally developing and abortive ovules in hazelnut provide exhaustive information that will improve our understanding of the molecular mechanisms of abortive ovule formation in hazelnut.

High-Performance Integrated Virtual Environment (HIVE) Tools and Applications for Big Data Analysis.

PubMed

Simonyan, Vahan; Mazumder, Raja

2014-09-30

The High-performance Integrated Virtual Environment (HIVE) is a high-throughput cloud-based infrastructure developed for the storage and analysis of genomic and associated biological data. HIVE consists of a web-accessible interface for authorized users to deposit, retrieve, share, annotate, compute and visualize Next-generation Sequencing (NGS) data in a scalable and highly efficient fashion. The platform contains a distributed storage library and a distributed computational powerhouse linked seamlessly. Resources available through the interface include algorithms, tools and applications developed exclusively for the HIVE platform, as well as commonly used external tools adapted to operate within the parallel architecture of the system. HIVE is composed of a flexible infrastructure, which allows for simple implementation of new algorithms and tools. Currently, available HIVE tools include sequence alignment and nucleotide variation profiling tools, metagenomic analyzers, phylogenetic tree-building tools using NGS data, clone discovery algorithms, and recombination analysis algorithms. In addition to tools, HIVE also provides knowledgebases that can be used in conjunction with the tools for NGS sequence and metadata analysis.

Integrated systems analysis reveals a molecular network underlying autism spectrum disorders

PubMed Central

Li, Jingjing; Shi, Minyi; Ma, Zhihai; Zhao, Shuchun; Euskirchen, Ghia; Ziskin, Jennifer; Urban, Alexander; Hallmayer, Joachim; Snyder, Michael

2014-01-01

Autism is a complex disease whose etiology remains elusive. We integrated previously and newly generated data and developed a systems framework involving the interactome, gene expression and genome sequencing to identify a protein interaction module with members strongly enriched for autism candidate genes. Sequencing of 25 patients confirmed the involvement of this module in autism, which was subsequently validated using an independent cohort of over 500 patients. Expression of this module was dichotomized with a ubiquitously expressed subcomponent and another subcomponent preferentially expressed in the corpus callosum, which was significantly affected by our identified mutations in the network center. RNA-sequencing of the corpus callosum from patients with autism exhibited extensive gene mis-expression in this module, and our immunochemical analysis showed that the human corpus callosum is predominantly populated by oligodendrocyte cells. Analysis of functional genomic data further revealed a significant involvement of this module in the development of oligodendrocyte cells in mouse brain. Our analysis delineates a natural network involved in autism, helps uncover novel candidate genes for this disease and improves our understanding of its molecular pathology. PMID:25549968

High-Performance Integrated Virtual Environment (HIVE) Tools and Applications for Big Data Analysis

PubMed Central

Simonyan, Vahan; Mazumder, Raja

2014-01-01

The High-performance Integrated Virtual Environment (HIVE) is a high-throughput cloud-based infrastructure developed for the storage and analysis of genomic and associated biological data. HIVE consists of a web-accessible interface for authorized users to deposit, retrieve, share, annotate, compute and visualize Next-generation Sequencing (NGS) data in a scalable and highly efficient fashion. The platform contains a distributed storage library and a distributed computational powerhouse linked seamlessly. Resources available through the interface include algorithms, tools and applications developed exclusively for the HIVE platform, as well as commonly used external tools adapted to operate within the parallel architecture of the system. HIVE is composed of a flexible infrastructure, which allows for simple implementation of new algorithms and tools. Currently, available HIVE tools include sequence alignment and nucleotide variation profiling tools, metagenomic analyzers, phylogenetic tree-building tools using NGS data, clone discovery algorithms, and recombination analysis algorithms. In addition to tools, HIVE also provides knowledgebases that can be used in conjunction with the tools for NGS sequence and metadata analysis. PMID:25271953

Development of a Green Fluorescent Protein-Based Laboratory Curriculum

ERIC Educational Resources Information Center

Larkin, Patrick D.; Hartberg, Yasha

2005-01-01

A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and…

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

PubMed

Ufnar, Jennifer A; Ufnar, David F; Wang, Shiao Y; Ellender, R D

2007-08-01

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes▿

PubMed Central

Ufnar, Jennifer A.; Ufnar, David F.; Wang, Shiao Y.; Ellender, R. D.

2007-01-01

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6 g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker. PMID:17586669

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

The Transcriptome Analysis and Comparison Explorer--T-ACE: a platform-independent, graphical tool to process large RNAseq datasets of non-model organisms.

PubMed

Philipp, E E R; Kraemer, L; Mountfort, D; Schilhabel, M; Schreiber, S; Rosenstiel, P

2012-03-15

Next generation sequencing (NGS) technologies allow a rapid and cost-effective compilation of large RNA sequence datasets in model and non-model organisms. However, the storage and analysis of transcriptome information from different NGS platforms is still a significant bottleneck, leading to a delay in data dissemination and subsequent biological understanding. Especially database interfaces with transcriptome analysis modules going beyond mere read counts are missing. Here, we present the Transcriptome Analysis and Comparison Explorer (T-ACE), a tool designed for the organization and analysis of large sequence datasets, and especially suited for transcriptome projects of non-model organisms with little or no a priori sequence information. T-ACE offers a TCL-based interface, which accesses a PostgreSQL database via a php-script. Within T-ACE, information belonging to single sequences or contigs, such as annotation or read coverage, is linked to the respective sequence and immediately accessible. Sequences and assigned information can be searched via keyword- or BLAST-search. Additionally, T-ACE provides within and between transcriptome analysis modules on the level of expression, GO terms, KEGG pathways and protein domains. Results are visualized and can be easily exported for external analysis. We developed T-ACE for laboratory environments, which have only a limited amount of bioinformatics support, and for collaborative projects in which different partners work on the same dataset from different locations or platforms (Windows/Linux/MacOS). For laboratories with some experience in bioinformatics and programming, the low complexity of the database structure and open-source code provides a framework that can be customized according to the different needs of the user and transcriptome project.

Characterisation and Next-generation Sequencing Analysis of Unknown Arboviruses

DTIC Science & Technology

2012-09-01

on the development of real- time PCR detection assays for Vibrio cholerae, a water-borne bacterium responsible for severe enteric disease. From...specific sequence [22]. The length of time from harvesting virus to generating samples that are ready for sequencing takes about two weeks, which is a...two viruses, and on day 4 post infection significant and widespread cytopathic effect was observed. The viruses were harvested by ultracentrifugation

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

Integrated analysis of 454 and Illumina transcriptomic sequencing characterizes carbon flux and energy source for fatty acid synthesis in developing Lindera glauca fruits for woody biodiesel.

PubMed

Lin, Zixin; An, Jiyong; Wang, Jia; Niu, Jun; Ma, Chao; Wang, Libing; Yuan, Guanshen; Shi, Lingling; Liu, Lili; Zhang, Jinsong; Zhang, Zhixiang; Qi, Ji; Lin, Shanzhi

2017-01-01

Lindera glauca fruit with high quality and quantity of oil has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of carbon flux and energy source for oil biosynthesis in developing fruits is still unknown. To better develop fruit oils of L. glauca as woody biodiesel, a combination of two different sequencing platforms (454 and Illumina) and qRT-PCR analysis was used to define a minimal reference transcriptome of developing L. glauca fruits, and to construct carbon and energy metabolic model for regulation of carbon partitioning and energy supply for FA biosynthesis and oil accumulation. We first analyzed the dynamic patterns of growth tendency, oil content, FA compositions, biodiesel properties, and the contents of ATP and pyridine nucleotide of L. glauca fruits from seven different developing stages. Comprehensive characterization of transcriptome of the developing L. glauca fruit was performed using a combination of two different next-generation sequencing platforms, of which three representative fruit samples (50, 125, and 150 DAF) and one mixed sample from seven developing stages were selected for Illumina and 454 sequencing, respectively. The unigenes separately obtained from long and short reads (201, and 259, respectively, in total) were reconciled using TGICL software, resulting in a total of 60,031 unigenes (mean length = 1061.95 bp) to describe a transcriptome for developing L. glauca fruits. Notably, 198 genes were annotated for photosynthesis, sucrose cleavage, carbon allocation, metabolite transport, acetyl-CoA formation, oil synthesis, and energy metabolism, among which some specific transporters, transcription factors, and enzymes were identified to be implicated in carbon partitioning and energy source for oil synthesis by an integrated analysis of transcriptomic sequencing and qRT-PCR. Importantly, the carbon and energy metabolic model was well established for oil biosynthesis of developing L. glauca fruits, which could help to reveal the molecular regulatory mechanism of the increased oil production in developing fruits. This study presents for the first time the application of an integrated two different sequencing analyses (Illumina and 454) and qRT-PCR detection to define a minimal reference transcriptome for developing L. glauca fruits, and to elucidate the molecular regulatory mechanism of carbon flux control and energy provision for oil synthesis. Our results will provide a valuable resource for future fundamental and applied research on the woody biodiesel plants.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

PubMed

Dan, Tong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Menghe, Bilige; Zhang, Heping; Sun, Zhihong

2015-05-20

Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.

Identification of MHC class I sequences in Chinese-origin rhesus macaques

PubMed Central

Karl, Julie A.; Wiseman, Roger W.; Campbell, Kevin J.; Blasky, Alex J.; Hughes, Austin L.; Ferguson, Betsy; Read, Daniel S.

2010-01-01

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population. PMID:18097659

GAP Final Technical Report 12-14-04

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

Andrew J. Bordner, PhD, Senior Research Scientist

2004-12-14

The Genomics Annotation Platform (GAP) was designed to develop new tools for high throughput functional annotation and characterization of protein sequences and structures resulting from genomics and structural proteomics, benchmarking and application of those tools. Furthermore, this platform integrated the genomic scale sequence and structural analysis and prediction tools with the advanced structure prediction and bioinformatics environment of ICM. The development of GAP was primarily oriented towards the annotation of new biomolecular structures using both structural and sequence data. Even though the amount of protein X-ray crystal data is growing exponentially, the volume of sequence data is growing even moremore » rapidly. This trend was exploited by leveraging the wealth of sequence data to provide functional annotation for protein structures. The additional information provided by GAP is expected to assist the majority of the commercial users of ICM, who are involved in drug discovery, in identifying promising drug targets as well in devising strategies for the rational design of therapeutics directed at the protein of interest. The GAP also provided valuable tools for biochemistry education, and structural genomics centers. In addition, GAP incorporates many novel prediction and analysis methods not available in other molecular modeling packages. This development led to signing the first Molsoft agreement in the structural genomics annotation area with the University of oxford Structural Genomics Center. This commercial agreement validated the Molsoft efforts under the GAP project and provided the basis for further development of the large scale functional annotation platform.« less

Genomic resources for songbird research and their use in characterizing gene expression during brain development

PubMed Central

Li, XiaoChing; Wang, Xiu-Jie; Tannenhauser, Jonathan; Podell, Sheila; Mukherjee, Piali; Hertel, Moritz; Biane, Jeremy; Masuda, Shoko; Nottebohm, Fernando; Gaasterland, Terry

2007-01-01

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3′ ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30–50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis. PMID:17426146

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

PubMed

Barboza, Karina; Beretta, Vanesa; Kozub, Perla C; Salinas, Cecilia; Morgenfeld, Mauro M; Galmarini, Claudio R; Cavagnaro, Pablo F

2018-04-28

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

Learning Progressions and Teaching Sequences: A Review and Analysis

ERIC Educational Resources Information Center

Duschl, Richard; Maeng, Seungho; Sezen, Asli

2011-01-01

Our paper is an analytical review of the design, development and reporting of learning progressions and teaching sequences. Research questions are: (1) what criteria are being used to propose a "hypothetical learning progression/trajectory" and (2) what measurements/evidence are being used to empirically define and refine a "hypothetical learning…

Gene expression analysis of flax seed development

PubMed Central

2011-01-01

Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well. PMID:21529361

CFGP: a web-based, comparative fungal genomics platform.

PubMed

Park, Jongsun; Park, Bongsoo; Jung, Kyongyong; Jang, Suwang; Yu, Kwangyul; Choi, Jaeyoung; Kong, Sunghyung; Park, Jaejin; Kim, Seryun; Kim, Hyojeong; Kim, Soonok; Kim, Jihyun F; Blair, Jaime E; Lee, Kwangwon; Kang, Seogchan; Lee, Yong-Hwan

2008-01-01

Since the completion of the Saccharomyces cerevisiae genome sequencing project in 1996, the genomes of over 80 fungal species have been sequenced or are currently being sequenced. Resulting data provide opportunities for studying and comparing fungal biology and evolution at the genome level. To support such studies, the Comparative Fungal Genomics Platform (CFGP; http://cfgp.snu.ac.kr), a web-based multifunctional informatics workbench, was developed. The CFGP comprises three layers, including the basal layer, middleware and the user interface. The data warehouse in the basal layer contains standardized genome sequences of 65 fungal species. The middleware processes queries via six analysis tools, including BLAST, ClustalW, InterProScan, SignalP 3.0, PSORT II and a newly developed tool named BLASTMatrix. The BLASTMatrix permits the identification and visualization of genes homologous to a query across multiple species. The Data-driven User Interface (DUI) of the CFGP was built on a new concept of pre-collecting data and post-executing analysis instead of the 'fill-in-the-form-and-press-SUBMIT' user interfaces utilized by most bioinformatics sites. A tool termed Favorite, which supports the management of encapsulated sequence data and provides a personalized data repository to users, is another novel feature in the DUI.

Next-generation sequencing analysis of the ARMS2 gene in Turkish exudative age-related macular degeneration patients.

PubMed

Bardak, H; Gunay, M; Ercalik, Y; Bardak, Y; Ozbas, H; Bagci, O

2017-01-23

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It is a complex disease with both genetic and environmental risk factors. To improve clinical management of this condition, it is important to develop risk assessment and prevention strategies for environmental influences, and establish a more effective treatment approach. The aim of the present study was to investigate age-related maculopathy susceptibility protein 2 (ARMS2) gene sequences among Turkish patients with exudative AMD. In addition to 39 advanced exudative AMD patients, 250 healthy individuals for whom exome sequencing data were available were included as a control group. Patients with a history of known environmental and systemic AMD risk factors were excluded. Genomic DNA was isolated from peripheral blood and analyzed using next-generation sequencing. All coding exons of the ARMS2 gene were assessed. Three different ARMS2 sequence variations (rs10490923, rs2736911, and rs10490924) were identified in both the patient and control group. Within the control group, two further ARMS2 gene variants (rs7088128 and rs36213074) were also detected. Logistic regression analysis revealed a relationship between the rs10490924 polymorphism and AMD in the Turkish population.

Distinguishing thrust sequences in gravity-driven fold and thrust belts

NASA Astrophysics Data System (ADS)

Alsop, G. I.; Weinberger, R.; Marco, S.

2018-04-01

Piggyback or foreland-propagating thrust sequences, where younger thrusts develop in the footwalls of existing thrusts, are generally assumed to be the typical order of thrust development in most orogenic settings. However, overstep or 'break-back' sequences, where later thrusts develop above and in the hangingwalls of earlier thrusts, may potentially form during cessation of movement in gravity-driven mass transport deposits (MTDs). In this study, we provide a detailed outcrop-based analysis of such an overstep thrust sequence developed in an MTD in the southern Dead Sea Basin. Evidence that may be used to discriminate overstep thrusting from piggyback thrust sequences within the gravity-driven fold and thrust belt includes upright folds and forethrusts that are cut by younger overlying thrusts. Backthrusts form ideal markers that are also clearly offset and cut by overlying younger forethrusts. Portions of the basal detachment to the thrust system are folded and locally imbricated in footwall synclines below forethrust ramps, and these geometries also support an overstep sequence. However, new 'short-cut' basal detachments develop below these synclines, indicating that movement continued on the basal detachment rather than it being abandoned as in classic overstep sequences. Further evidence for 'synchronous thrusting', where movement on more than one thrust occurs at the same time, is provided by displacement patterns on sequences of thrust ramp imbricates that systematically increases downslope towards the toe of the MTD. Older thrusts that initiate downslope in the broadly overstep sequence continue to move and therefore accrue greater displacements during synchronous thrusting. Our study provides a template to help distinguish different thrust sequences in both orogenic settings and gravity-driven surficial systems, with displacement patterns potentially being imaged in seismic sections across offshore MTDs.

Ensembl 2004.

PubMed

Birney, E; Andrews, D; Bevan, P; Caccamo, M; Cameron, G; Chen, Y; Clarke, L; Coates, G; Cox, T; Cuff, J; Curwen, V; Cutts, T; Down, T; Durbin, R; Eyras, E; Fernandez-Suarez, X M; Gane, P; Gibbins, B; Gilbert, J; Hammond, M; Hotz, H; Iyer, V; Kahari, A; Jekosch, K; Kasprzyk, A; Keefe, D; Keenan, S; Lehvaslaiho, H; McVicker, G; Melsopp, C; Meidl, P; Mongin, E; Pettett, R; Potter, S; Proctor, G; Rae, M; Searle, S; Slater, G; Smedley, D; Smith, J; Spooner, W; Stabenau, A; Stalker, J; Storey, R; Ureta-Vidal, A; Woodwark, C; Clamp, M; Hubbard, T

2004-01-01

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organize biology around the sequences of large genomes. It is a comprehensive and integrated source of annotation of large genome sequences, available via interactive website, web services or flat files. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. The facilities of the system range from sequence analysis to data storage and visualization and installations exist around the world both in companies and at academic sites. With a total of nine genome sequences available from Ensembl and more genomes to follow, recent developments have focused mainly on closer integration between genomes and external data.

The convergence of the order sequence and the solution function sequence on fractional partial differential equation

NASA Astrophysics Data System (ADS)

Rusyaman, E.; Parmikanti, K.; Chaerani, D.; Asefan; Irianingsih, I.

2018-03-01

One of the application of fractional ordinary differential equation is related to the viscoelasticity, i.e., a correlation between the viscosity of fluids and the elasticity of solids. If the solution function develops into function with two or more variables, then its differential equation must be changed into fractional partial differential equation. As the preliminary study for two variables viscoelasticity problem, this paper discusses about convergence analysis of function sequence which is the solution of the homogenous fractional partial differential equation. The method used to solve the problem is Homotopy Analysis Method. The results show that if given two real number sequences (αn) and (βn) which converge to α and β respectively, then the solution function sequences of fractional partial differential equation with order (αn, βn) will also converge to the solution function of fractional partial differential equation with order (α, β).

GWFASTA: server for FASTA search in eukaryotic and microbial genomes.

PubMed

Issac, Biju; Raghava, G P S

2002-09-01

Similarity searches are a powerful method for solving important biological problems such as database scanning, evolutionary studies, gene prediction, and protein structure prediction. FASTA is a widely used sequence comparison tool for rapid database scanning. Here we describe the GWFASTA server that was developed to assist the FASTA user in similarity searches against partially and/or completely sequenced genomes. GWFASTA consists of more than 60 microbial genomes, eight eukaryote genomes, and proteomes of annotatedgenomes. Infact, it provides the maximum number of databases for similarity searching from a single platform. GWFASTA allows the submission of more than one sequence as a single query for a FASTA search. It also provides integrated post-processing of FASTA output, including compositional analysis of proteins, multiple sequences alignment, and phylogenetic analysis. Furthermore, it summarizes the search results organism-wise for prokaryotes and chromosome-wise for eukaryotes. Thus, the integration of different tools for sequence analyses makes GWFASTA a powerful toolfor biologists.

Comparative Analysis of Expressed Genes from Cacao Meristems Infected by Moniliophthora perniciosa

PubMed Central

Gesteira, Abelmon S.; Micheli, Fabienne; Carels, Nicolas; Da Silva, Aline C.; Gramacho, Karina P.; Schuster, Ivan; Macêdo, Joci N.; Pereira, Gonçalo A. G.; Cascardo, Júlio C. M.

2007-01-01

Background and Aims Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao–Moniliophthora perniciosa interaction. Methods Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao–M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5′ end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. Key Results A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. Conclusions As far as is known this is the first EST resource from the cacao–M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches broom, and as a source of polymorphism for molecular marker development and marker-assisted selection. PMID:17557832

TaxI: a software tool for DNA barcoding using distance methods

PubMed Central

Steinke, Dirk; Vences, Miguel; Salzburger, Walter; Meyer, Axel

2005-01-01

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding. PMID:16214755

Genomic Sequence Variation Markup Language (GSVML).

PubMed

Nakaya, Jun; Kimura, Michio; Hiroi, Kaei; Ido, Keisuke; Yang, Woosung; Tanaka, Hiroshi

2010-02-01

With the aim of making good use of internationally accumulated genomic sequence variation data, which is increasing rapidly due to the explosive amount of genomic research at present, the development of an interoperable data exchange format and its international standardization are necessary. Genomic Sequence Variation Markup Language (GSVML) will focus on genomic sequence variation data and human health applications, such as gene based medicine or pharmacogenomics. We developed GSVML through eight steps, based on case analysis and domain investigations. By focusing on the design scope to human health applications and genomic sequence variation, we attempted to eliminate ambiguity and to ensure practicability. We intended to satisfy the requirements derived from the use case analysis of human-based clinical genomic applications. Based on database investigations, we attempted to minimize the redundancy of the data format, while maximizing the data covering range. We also attempted to ensure communication and interface ability with other Markup Languages, for exchange of omics data among various omics researchers or facilities. The interface ability with developing clinical standards, such as the Health Level Seven Genotype Information model, was analyzed. We developed the human health-oriented GSVML comprising variation data, direct annotation, and indirect annotation categories; the variation data category is required, while the direct and indirect annotation categories are optional. The annotation categories contain omics and clinical information, and have internal relationships. For designing, we examined 6 cases for three criteria as human health application and 15 data elements for three criteria as data formats for genomic sequence variation data exchange. The data format of five international SNP databases and six Markup Languages and the interface ability to the Health Level Seven Genotype Model in terms of 317 items were investigated. GSVML was developed as a potential data exchanging format for genomic sequence variation data exchange focusing on human health applications. The international standardization of GSVML is necessary, and is currently underway. GSVML can be applied to enhance the utilization of genomic sequence variation data worldwide by providing a communicable platform between clinical and research applications. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch.

PubMed

Kaplan, Oktay I; Berber, Burak; Hekim, Nezih; Doluca, Osman

2016-11-02

Many studies show that short non-coding sequences are widely conserved among regulatory elements. More and more conserved sequences are being discovered since the development of next generation sequencing technology. A common approach to identify conserved sequences with regulatory roles relies on topological changes such as hairpin formation at the DNA or RNA level. G-quadruplexes, non-canonical nucleic acid topologies with little established biological roles, are increasingly considered for conserved regulatory element discovery. Since the tertiary structure of G-quadruplexes is strongly dependent on the loop sequence which is disregarded by the generally accepted algorithm, we hypothesized that G-quadruplexes with similar topology and, indirectly, similar interaction patterns, can be determined using phylogenetic clustering based on differences in the loop sequences. Phylogenetic analysis of 52 G-quadruplex forming sequences in the Escherichia coli genome revealed two conserved G-quadruplex motifs with a potential regulatory role. Further analysis revealed that both motifs tend to form hairpins and G quadruplexes, as supported by circular dichroism studies. The phylogenetic analysis as described in this work can greatly improve the discovery of functional G-quadruplex structures and may explain unknown regulatory patterns. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Illumina MiSeq Sequencing for Preliminary Analysis of Microbiome Causing Primary Endodontic Infections in Egypt

PubMed Central

Azab, Marwa Mohamed; Fayyad, Dalia Mukhtar

2018-01-01

The use of high throughput next generation technologies has allowed more comprehensive analysis than traditional Sanger sequencing. The specific aim of this study was to investigate the microbial diversity of primary endodontic infections using Illumina MiSeq sequencing platform in Egyptian patients. Samples were collected from 19 patients in Suez Canal University Hospital (Endodontic Department) using sterile # 15K file and paper points. DNA was extracted using Mo Bio power soil DNA isolation extraction kit followed by PCR amplification and agarose gel electrophoresis. The microbiome was characterized on the basis of the V3 and V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. MOTHUR software was used in sequence filtration and analysis of sequenced data. A total of 1858 operational taxonomic units at 97% similarity were assigned to 26 phyla, 245 families, and 705 genera. Four main phyla Firmicutes, Bacteroidetes, Proteobacteria, and Synergistetes were predominant in all samples. At genus level, Prevotella, Bacillus, Porphyromonas, Streptococcus, and Bacteroides were the most abundant. Illumina MiSeq platform sequencing can be used to investigate oral microbiome composition of endodontic infections. Elucidating the ecology of endodontic infections is a necessary step in developing effective intracanal antimicrobials. PMID:29849646

HPV-QUEST: A highly customized system for automated HPV sequence analysis capable of processing Next Generation sequencing data set.

PubMed

Yin, Li; Yao, Jiqiang; Gardner, Brent P; Chang, Kaifen; Yu, Fahong; Goodenow, Maureen M

2012-01-01

Next Generation sequencing (NGS) applied to human papilloma viruses (HPV) can provide sensitive methods to investigate the molecular epidemiology of multiple type HPV infection. Currently a genotyping system with a comprehensive collection of updated HPV reference sequences and a capacity to handle NGS data sets is lacking. HPV-QUEST was developed as an automated and rapid HPV genotyping system. The web-based HPV-QUEST subtyping algorithm was developed using HTML, PHP, Perl scripting language, and MYSQL as the database backend. HPV-QUEST includes a database of annotated HPV reference sequences with updated nomenclature covering 5 genuses, 14 species and 150 mucosal and cutaneous types to genotype blasted query sequences. HPV-QUEST processes up to 10 megabases of sequences within 1 to 2 minutes. Results are reported in html, text and excel formats and display e-value, blast score, and local and coverage identities; provide genus, species, type, infection site and risk for the best matched reference HPV sequence; and produce results ready for additional analyses.

Reconstructing evolutionary trees in parallel for massive sequences.

PubMed

Zou, Quan; Wan, Shixiang; Zeng, Xiangxiang; Ma, Zhanshan Sam

2017-12-14

Building the evolutionary trees for massive unaligned DNA sequences is challenging and crucial. However, reconstructing evolutionary tree for ultra-large sequences is hard. Massive multiple sequence alignment is also challenging and time/space consuming. Hadoop and Spark are developed recently, which bring spring light for the classical computational biology problems. In this paper, we tried to solve the multiple sequence alignment and evolutionary reconstruction in parallel. HPTree, which is developed in this paper, can deal with big DNA sequence files quickly. It works well on the >1GB files, and gets better performance than other evolutionary reconstruction tools. Users could use HPTree for reonstructing evolutioanry trees on the computer clusters or cloud platform (eg. Amazon Cloud). HPTree could help on population evolution research and metagenomics analysis. In this paper, we employ the Hadoop and Spark platform and design an evolutionary tree reconstruction software tool for unaligned massive DNA sequences. Clustering and multiple sequence alignment are done in parallel. Neighbour-joining model was employed for the evolutionary tree building. We opened our software together with source codes via http://lab.malab.cn/soft/HPtree/ .

Next Generation Sequencing Technologies: The Doorway to the Unexplored Genomics of Non-Model Plants

PubMed Central

Unamba, Chibuikem I. N.; Nag, Akshay; Sharma, Ram K.

2015-01-01

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping. PMID:26734016

Mapping-by-sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat.

PubMed

Gardiner, Laura-Jayne; Bansept-Basler, Pauline; Olohan, Lisa; Joynson, Ryan; Brenchley, Rachel; Hall, Neil; O'Sullivan, Donal M; Hall, Anthony

2016-08-01

Previously we extended the utility of mapping-by-sequencing by combining it with sequence capture and mapping sequence data to pseudo-chromosomes that were organized using wheat-Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L. identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping-by-synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110 MB NimbleGen capture probe set was used to enrich and sequence a doubled haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo-chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo-chromosomes allow us to demonstrate the application of mapping-by-sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub-genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus - defining a smaller genic region than was previously possible; associate the interval with one wheat sub-genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user-friendly community resource for phenotype mapping. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Googling DNA sequences on the World Wide Web.

PubMed

Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean.

PubMed

Voelker, T A; Staswick, P; Chrispeels, M J

1986-12-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained.

Single-Cell RNA Sequencing of Glioblastoma Cells.

PubMed

Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

2018-01-01

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection

PubMed Central

Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike

2018-01-01

ABSTRACT Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection. PMID:29564396

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

PubMed

Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike; Khan, Arifa S

2018-01-01

Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection.

SONAR: A High-Throughput Pipeline for Inferring Antibody Ontogenies from Longitudinal Sequencing of B Cell Transcripts.

PubMed

Schramm, Chaim A; Sheng, Zizhang; Zhang, Zhenhai; Mascola, John R; Kwong, Peter D; Shapiro, Lawrence

2016-01-01

The rapid advance of massively parallel or next-generation sequencing technologies has made possible the characterization of B cell receptor repertoires in ever greater detail, and these developments have triggered a proliferation of software tools for processing and annotating these data. Of especial interest, however, is the capability to track the development of specific antibody lineages across time, which remains beyond the scope of most current programs. We have previously reported on the use of techniques such as inter- and intradonor analysis and CDR3 tracing to identify transcripts related to an antibody of interest. Here, we present Software for the Ontogenic aNalysis of Antibody Repertoires (SONAR), capable of automating both general repertoire analysis and specialized techniques for investigating specific lineages. SONAR annotates next-generation sequencing data, identifies transcripts in a lineage of interest, and tracks lineage development across multiple time points. SONAR also generates figures, such as identity-divergence plots and longitudinal phylogenetic "birthday" trees, and provides interfaces to other programs such as DNAML and BEAST. SONAR can be downloaded as a ready-to-run Docker image or manually installed on a local machine. In the latter case, it can also be configured to take advantage of a high-performance computing cluster for the most computationally intensive steps, if available. In summary, this software provides a useful new tool for the processing of large next-generation sequencing datasets and the ontogenic analysis of neutralizing antibody lineages. SONAR can be found at https://github.com/scharch/SONAR, and the Docker image can be obtained from https://hub.docker.com/r/scharch/sonar/.

Onco-STS: a web-based laboratory information management system for sample and analysis tracking in oncogenomic experiments.

PubMed

Gavrielides, Mike; Furney, Simon J; Yates, Tim; Miller, Crispin J; Marais, Richard

2014-01-01

Whole genomes, whole exomes and transcriptomes of tumour samples are sequenced routinely to identify the drivers of cancer. The systematic sequencing and analysis of tumour samples, as well other oncogenomic experiments, necessitates the tracking of relevant sample information throughout the investigative process. These meta-data of the sequencing and analysis procedures include information about the samples and projects as well as the sequencing centres, platforms, data locations, results locations, alignments, analysis specifications and further information relevant to the experiments. The current work presents a sample tracking system for oncogenomic studies (Onco-STS) to store these data and make them easily accessible to the researchers who work with the samples. The system is a web application, which includes a database and a front-end web page that allows the remote access, submission and updating of the sample data in the database. The web application development programming framework Grails was used for the development and implementation of the system. The resulting Onco-STS solution is efficient, secure and easy to use and is intended to replace the manual data handling of text records. Onco-STS allows simultaneous remote access to the system making collaboration among researchers more effective. The system stores both information on the samples in oncogenomic studies and details of the analyses conducted on the resulting data. Onco-STS is based on open-source software, is easy to develop and can be modified according to a research group's needs. Hence it is suitable for laboratories that do not require a commercial system.

DNA-encoded chemistry: enabling the deeper sampling of chemical space.

PubMed

Goodnow, Robert A; Dumelin, Christoph E; Keefe, Anthony D

2017-02-01

DNA-encoded chemical library technologies are increasingly being adopted in drug discovery for hit and lead generation. DNA-encoded chemistry enables the exploration of chemical spaces four to five orders of magnitude more deeply than is achievable by traditional high-throughput screening methods. Operation of this technology requires developing a range of capabilities including aqueous synthetic chemistry, building block acquisition, oligonucleotide conjugation, large-scale molecular biological transformations, selection methodologies, PCR, sequencing, sequence data analysis and the analysis of large chemistry spaces. This Review provides an overview of the development and applications of DNA-encoded chemistry, highlighting the challenges and future directions for the use of this technology.

PAQ: Partition Analysis of Quasispecies.

PubMed

Baccam, P; Thompson, R J; Fedrigo, O; Carpenter, S; Cornette, J L

2001-01-01

The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.

Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization.

PubMed

Song, B K; Pan, M Z; Lau, Y L; Wan, K L

2014-07-29

Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.

Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region▿

PubMed Central

Steer, Penelope A.; Kirkpatrick, Naomi C.; O'Rourke, Denise; Noormohammadi, Amir H.

2009-01-01

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens. PMID:19036935

Secure and robust cloud computing for high-throughput forensic microsatellite sequence analysis and databasing.

PubMed

Bailey, Sarah F; Scheible, Melissa K; Williams, Christopher; Silva, Deborah S B S; Hoggan, Marina; Eichman, Christopher; Faith, Seth A

2017-11-01

Next-generation Sequencing (NGS) is a rapidly evolving technology with demonstrated benefits for forensic genetic applications, and the strategies to analyze and manage the massive NGS datasets are currently in development. Here, the computing, data storage, connectivity, and security resources of the Cloud were evaluated as a model for forensic laboratory systems that produce NGS data. A complete front-to-end Cloud system was developed to upload, process, and interpret raw NGS data using a web browser dashboard. The system was extensible, demonstrating analysis capabilities of autosomal and Y-STRs from a variety of NGS instrumentation (Illumina MiniSeq and MiSeq, and Oxford Nanopore MinION). NGS data for STRs were concordant with standard reference materials previously characterized with capillary electrophoresis and Sanger sequencing. The computing power of the Cloud was implemented with on-demand auto-scaling to allow multiple file analysis in tandem. The system was designed to store resulting data in a relational database, amenable to downstream sample interpretations and databasing applications following the most recent guidelines in nomenclature for sequenced alleles. Lastly, a multi-layered Cloud security architecture was tested and showed that industry standards for securing data and computing resources were readily applied to the NGS system without disadvantageous effects for bioinformatic analysis, connectivity or data storage/retrieval. The results of this study demonstrate the feasibility of using Cloud-based systems for secured NGS data analysis, storage, databasing, and multi-user distributed connectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Shotgun metagenomic data streams: surfing without fear

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

Berendzen, Joel R

2010-12-06

Timely information about bio-threat prevalence, consequence, propagation, attribution, and mitigation is needed to support decision-making, both routinely and in a crisis. One DNA sequencer can stream 25 Gbp of information per day, but sampling strategies and analysis techniques are needed to turn raw sequencing power into actionable knowledge. Shotgun metagenomics can enable biosurveillance at the level of a single city, hospital, or airplane. Metagenomics characterizes viruses and bacteria from complex environments such as soil, air filters, or sewage. Unlike targeted-primer-based sequencing, shotgun methods are not blind to sequences that are truly novel, and they can measure absolute prevalence. Shotgun metagenomicmore » sampling can be non-invasive, efficient, and inexpensive while being informative. We have developed analysis techniques for shotgun metagenomic sequencing that rely upon phylogenetic signature patterns. They work by indexing local sequence patterns in a manner similar to web search engines. Our methods are laptop-fast and favorable scaling properties ensure they will be sustainable as sequencing methods grow. We show examples of application to soil metagenomic samples.« less

De novo transcriptomic analysis of cowpea (Vigna unguiculata L. Walp.) for genic SSR marker development.

PubMed

Chen, Honglin; Wang, Lixia; Liu, Xiaoyan; Hu, Liangliang; Wang, Suhua; Cheng, Xuzhen

2017-07-11

Cowpea [Vigna unguiculata (L.) Walp.] is one of the most important legumes in tropical and semi-arid regions. However, there is relatively little genomic information available for genetic research on and breeding of cowpea. The objectives of this study were to analyse the cowpea transcriptome and develop genic molecular markers for future genetic studies of this genus. Approximately 54 million high-quality cDNA sequence reads were obtained from cowpea based on Illumina paired-end sequencing technology and were de novo assembled to generate 47,899 unigenes with an N50 length of 1534 bp. Sequence similarity analysis revealed 36,289 unigenes (75.8%) with significant similarity to known proteins in the non-redundant (Nr) protein database, 23,471 unigenes (49.0%) with BLAST hits in the Swiss-Prot database, and 20,654 unigenes (43.1%) with high similarity in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Further analysis identified 5560 simple sequence repeats (SSRs) as potential genic molecular markers. Validating a random set of 500 SSR markers yielded 54 polymorphic markers among 32 cowpea accessions. This transcriptomic analysis of cowpea provided a valuable set of genomic data for characterizing genes with important agronomic traits in Vigna unguiculata and a new set of genic SSR markers for further genetic studies and breeding in cowpea and related Vigna species.

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

PubMed Central

Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

2016-01-01

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

QuickNGS elevates Next-Generation Sequencing data analysis to a new level of automation.

PubMed

Wagle, Prerana; Nikolić, Miloš; Frommolt, Peter

2015-07-01

Next-Generation Sequencing (NGS) has emerged as a widely used tool in molecular biology. While time and cost for the sequencing itself are decreasing, the analysis of the massive amounts of data remains challenging. Since multiple algorithmic approaches for the basic data analysis have been developed, there is now an increasing need to efficiently use these tools to obtain results in reasonable time. We have developed QuickNGS, a new workflow system for laboratories with the need to analyze data from multiple NGS projects at a time. QuickNGS takes advantage of parallel computing resources, a comprehensive back-end database, and a careful selection of previously published algorithmic approaches to build fully automated data analysis workflows. We demonstrate the efficiency of our new software by a comprehensive analysis of 10 RNA-Seq samples which we can finish in only a few minutes of hands-on time. The approach we have taken is suitable to process even much larger numbers of samples and multiple projects at a time. Our approach considerably reduces the barriers that still limit the usability of the powerful NGS technology and finally decreases the time to be spent before proceeding to further downstream analysis and interpretation of the data.

GenomeFingerprinter: the genome fingerprint and the universal genome fingerprint analysis for systematic comparative genomics.

PubMed

Ai, Yuncan; Ai, Hannan; Meng, Fanmei; Zhao, Lei

2013-01-01

No attention has been paid on comparing a set of genome sequences crossing genetic components and biological categories with far divergence over large size range. We define it as the systematic comparative genomics and aim to develop the methodology. First, we create a method, GenomeFingerprinter, to unambiguously produce a set of three-dimensional coordinates from a sequence, followed by one three-dimensional plot and six two-dimensional trajectory projections, to illustrate the genome fingerprint of a given genome sequence. Second, we develop a set of concepts and tools, and thereby establish a method called the universal genome fingerprint analysis (UGFA). Particularly, we define the total genetic component configuration (TGCC) (including chromosome, plasmid, and phage) for describing a strain as a systematic unit, the universal genome fingerprint map (UGFM) of TGCC for differentiating strains as a universal system, and the systematic comparative genomics (SCG) for comparing a set of genomes crossing genetic components and biological categories. Third, we construct a method of quantitative analysis to compare two genomes by using the outcome dataset of genome fingerprint analysis. Specifically, we define the geometric center and its geometric mean for a given genome fingerprint map, followed by the Euclidean distance, the differentiate rate, and the weighted differentiate rate to quantitatively describe the difference between two genomes of comparison. Moreover, we demonstrate the applications through case studies on various genome sequences, giving tremendous insights into the critical issues in microbial genomics and taxonomy. We have created a method, GenomeFingerprinter, for rapidly computing, geometrically visualizing, intuitively comparing a set of genomes at genome fingerprint level, and hence established a method called the universal genome fingerprint analysis, as well as developed a method of quantitative analysis of the outcome dataset. These have set up the methodology of systematic comparative genomics based on the genome fingerprint analysis.

Multiplexed microsatellite recovery using massively parallel sequencing

USGS Publications Warehouse

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

Molecular Analysis of Dehalococcoides 16S Ribosomal DNA from Chloroethene-Contaminated Sites throughout North America and Europe

PubMed Central

Hendrickson, Edwin R.; Payne, Jo Ann; Young, Roslyn M.; Starr, Mark G.; Perry, Michael P.; Fahnestock, Stephen; Ellis, David E.; Ebersole, Richard C.

2002-01-01

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes. PMID:11823182

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

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

Zhou, Xiaofan; Peris, David; Kominek, Jacek

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

DOE PAGES

Zhou, Xiaofan; Peris, David; Kominek, Jacek; ...

2016-09-16

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

Developmental staging of male murine embryonic gonad by SAGE analysis

PubMed Central

Lee, Tin-Lap; Li, Yunmin; Alba, Diana; Vong, Queenie P.; Wu, Shao-Ming; Baxendale, Vanessa; Rennert, Owen M.; Lau, Yun-Fai Chris; Chan, Wai-Yee

2012-01-01

Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets. PMID:19376482

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

Exploiting EST databases for the development and characterisation of 3425 gene-tagged CISP markers in biofuel crop sugarcane and their transferability in cereals and orphan tropical grasses.

PubMed

Chandra, Amaresh; Jain, Radha; Solomon, Sushil; Shrivastava, Shiksha; Roy, Ajoy K

2013-02-04

Sugarcane is an important cash crop, providing 70% of the global raw sugar as well as raw material for biofuel production. Genetic analysis is hindered in sugarcane because of its large and complex polyploid genome and lack of sufficiently informative gene-tagged markers. Modern genomics has produced large amount of ESTs, which can be exploited to develop molecular markers based on comparative analysis with EST datasets of related crops and whole rice genome sequence, and accentuate their cross-technical functionality in orphan crops like tropical grasses. Utilising 246,180 Saccharum officinarum EST sequences vis-à-vis its comparative analysis with ESTs of sorghum and barley and the whole rice genome sequence, we have developed 3425 novel gene-tagged markers - namely, conserved-intron scanning primers (CISP) - using the web program GeMprospector. Rice orthologue annotation results indicated homology of 1096 sequences with expressed proteins, 491 with hypothetical proteins. The remaining 1838 were miscellaneous in nature. A total of 367 primer-pairs were tested in diverse panel of samples. The data indicate amplification of 41% polymorphic bands leading to 0.52 PIC and 3.50 MI with a set of sugarcane varieties and Saccharum species. In addition, a moderate technical functionality of a set of such markers with orphan tropical grasses (22%) and fodder cum cereal oat (33%) is observed. Developed gene-tagged CISP markers exhibited considerable technical functionality with varieties of sugarcane and unexplored species of tropical grasses. These markers would thus be particularly useful in identifying the economical traits in sugarcane and developing conservation strategies for orphan tropical grasses.

Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis.

PubMed

Suzuki, Masaharu; Ketterling, Matthew G; McCarty, Donald R

2005-09-01

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

BioPig: Developing Cloud Computing Applications for Next-Generation Sequence Analysis

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

Bhatia, Karan; Wang, Zhong

Next Generation sequencing is producing ever larger data sizes with a growth rate outpacing Moore's Law. The data deluge has made many of the current sequenceanalysis tools obsolete because they do not scale with data. Here we present BioPig, a collection of cloud computing tools to scale data analysis and management. Pig is aflexible data scripting language that uses Apache's Hadoop data structure and map reduce framework to process very large data files in parallel and combine the results.BioPig extends Pig with capability with sequence analysis. We will show the performance of BioPig on a variety of bioinformatics tasks, includingmore » screeningsequence contaminants, Illumina QA/QC, and gene discovery from metagenome data sets using the Rumen metagenome as an example.« less

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

PubMed

Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

2017-02-01

Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry.

PubMed

Higgins, Sean A; Savage, David F

2018-01-09

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

Elements of Mathematics, Book O: Intuitive Background. Chapter 1, Operational Systems.

ERIC Educational Resources Information Center

Exner, Robert; And Others

The sixteen chapters of this book provide the core material for the Elements of Mathematics Program, a secondary sequence developed for highly motivated students with strong verbal abilities. The sequence is based on a functional-relational approach to mathematics teaching, and emphasizes teaching by analysis of real-life situations. This text is…

Elements of Mathematics, Book O: Intuitive Background. Chapter 5, Mappings.

ERIC Educational Resources Information Center

Exner, Robert; And Others

The sixteen chapters of this book provide the core material for the Elements of Mathematics Program, a secondary sequence developed for highly motivated students with strong verbal abilities. The sequence is based on a functional-relational approach to mathematics teaching, and emphasizes teaching by analysis of real-life situations. This text is…

Emergence of Tables as First-Graders Cope with Modelling Tasks

ERIC Educational Resources Information Center

Peled, Irit; Keisar, Einav

2015-01-01

In this action research, first-graders were challenged to cope with a sequence of modelling tasks involving an analysis of given situations and choices of mathematical tools. In the course of the sequence, they underwent a change in the nature of their problem-solving processes and developed modelling competencies. Moreover, during the task…

Validity Evidence in Scale Development: The Application of Cross Validation and Classification-Sequencing Validation

ERIC Educational Resources Information Center

Acar, Tu¨lin

2014-01-01

In literature, it has been observed that many enhanced criteria are limited by factor analysis techniques. Besides examinations of statistical structure and/or psychological structure, such validity studies as cross validation and classification-sequencing studies should be performed frequently. The purpose of this study is to examine cross…

Elements of Mathematics, Book O: Intuitive Background. Chapter 2, The Integers.

ERIC Educational Resources Information Center

Exner, Robert; And Others

The sixteen chapters of this book provide the core materials for the Elements of Mathematics Program, a secondary sequence developed for highly motivated students with strong verbal abilities. The sequence is based on a functional-relational approach to mathematics teaching, and emphasizes teaching by analysis of real-life situations. This text is…

Reads2Type: a web application for rapid microbial taxonomy identification.

PubMed

Saputra, Dhany; Rasmussen, Simon; Larsen, Mette V; Haddad, Nizar; Sperotto, Maria Maddalena; Aarestrup, Frank M; Lund, Ole; Sicheritz-Pontén, Thomas

2015-11-25

Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale. In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

deepTools: a flexible platform for exploring deep-sequencing data.

PubMed

Ramírez, Fidel; Dündar, Friederike; Diehl, Sarah; Grüning, Björn A; Manke, Thomas

2014-07-01

We present a Galaxy based web server for processing and visualizing deeply sequenced data. The web server's core functionality consists of a suite of newly developed tools, called deepTools, that enable users with little bioinformatic background to explore the results of their sequencing experiments in a standardized setting. Users can upload pre-processed files with continuous data in standard formats and generate heatmaps and summary plots in a straight-forward, yet highly customizable manner. In addition, we offer several tools for the analysis of files containing aligned reads and enable efficient and reproducible generation of normalized coverage files. As a modular and open-source platform, deepTools can easily be expanded and customized to future demands and developments. The deepTools webserver is freely available at http://deeptools.ie-freiburg.mpg.de and is accompanied by extensive documentation and tutorials aimed at conveying the principles of deep-sequencing data analysis. The web server can be used without registration. deepTools can be installed locally either stand-alone or as part of Galaxy. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multi-Donor Longitudinal Antibody Repertoire Sequencing Reveals the Existence of Public Antibody Clonotypes in HIV-1 Infection.

PubMed

Setliff, Ian; McDonnell, Wyatt J; Raju, Nagarajan; Bombardi, Robin G; Murji, Amyn A; Scheepers, Cathrine; Ziki, Rutendo; Mynhardt, Charissa; Shepherd, Bryan E; Mamchak, Alusha A; Garrett, Nigel; Karim, Salim Abdool; Mallal, Simon A; Crowe, James E; Morris, Lynn; Georgiev, Ivelin S

2018-06-13

Characterization of single antibody lineages within infected individuals has provided insights into the development of Env-specific antibodies. However, a systems-level understanding of the humoral response against HIV-1 is limited. Here, we interrogated the antibody repertoires of multiple HIV-infected donors from an infection-naive state through acute and chronic infection using next-generation sequencing. This analysis revealed the existence of "public" antibody clonotypes that were shared among multiple HIV-infected individuals. The HIV-1 reactivity for representative antibodies from an identified public clonotype shared by three donors was confirmed. Furthermore, a meta-analysis of publicly available antibody repertoire sequencing datasets revealed antibodies with high sequence identity to known HIV-reactive antibodies, even in repertoires that were reported to be HIV naive. The discovery of public antibody clonotypes in HIV-infected individuals represents an avenue of significant potential for better understanding antibody responses to HIV-1 infection, as well as for clonotype-specific vaccine development. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

PubMed Central

Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

2004-01-01

Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants.

PubMed

Rodovalho, Cynara M; Ferro, Milene; Fonseca, Fernando Pp; Antonio, Erik A; Guilherme, Ivan R; Henrique-Silva, Flávio; Bacci, Maurício

2011-06-17

Leafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America. The analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity. The generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters.

Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants

PubMed Central

2011-01-01

Background Leafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America. Results The analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity. Conclusion The generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters. PMID:21682882

Overlap and diversity in antimicrobial peptide databases: compiling a non-redundant set of sequences.

PubMed

Aguilera-Mendoza, Longendri; Marrero-Ponce, Yovani; Tellez-Ibarra, Roberto; Llorente-Quesada, Monica T; Salgado, Jesús; Barigye, Stephen J; Liu, Jun

2015-08-01

The large variety of antimicrobial peptide (AMP) databases developed to date are characterized by a substantial overlap of data and similarity of sequences. Our goals are to analyze the levels of redundancy for all available AMP databases and use this information to build a new non-redundant sequence database. For this purpose, a new software tool is introduced. A comparative study of 25 AMP databases reveals the overlap and diversity among them and the internal diversity within each database. The overlap analysis shows that only one database (Peptaibol) contains exclusive data, not present in any other, whereas all sequences in the LAMP_Patent database are included in CAMP_Patent. However, the majority of databases have their own set of unique sequences, as well as some overlap with other databases. The complete set of non-duplicate sequences comprises 16 990 cases, which is almost half of the total number of reported peptides. On the other hand, the diversity analysis identifies the most and least diverse databases and proves that all databases exhibit some level of redundancy. Finally, we present a new parallel-free software, named Dover Analyzer, developed to compute the overlap and diversity between any number of databases and compile a set of non-redundant sequences. These results are useful for selecting or building a suitable representative set of AMPs, according to specific needs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

DUK - A Fast and Efficient Kmer Based Sequence Matching Tool

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

Li, Mingkun; Copeland, Alex; Han, James

2011-03-21

A new tool, DUK, is developed to perform matching task. Matching is to find whether a query sequence partially or totally matches given reference sequences or not. Matching is similar to alignment. Indeed many traditional analysis tasks like contaminant removal use alignment tools. But for matching, there is no need to know which bases of a query sequence matches which position of a reference sequence, it only need know whether there exists a match or not. This subtle difference can make matching task much faster than alignment. DUK is accurate, versatile, fast, and has efficient memory usage. It uses Kmermore » hashing method to index reference sequences and Poisson model to calculate p-value. DUK is carefully implemented in C++ in object oriented design. The resulted classes can also be used to develop other tools quickly. DUK have been widely used in JGI for a wide range of applications such as contaminant removal, organelle genome separation, and assembly refinement. Many real applications and simulated dataset demonstrate its power.« less

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions.

PubMed

Coelho Graça, Didia; Hartmer, Ralf; Jabs, Wolfgang; Beris, Photis; Clerici, Lorella; Stoermer, Carsten; Samii, Kaveh; Hochstrasser, Denis; Tsybin, Yury O; Scherl, Alexander; Lescuyer, Pierre

2015-04-01

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an (A)γ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

mtDNAmanager: a Web-based tool for the management and quality analysis of mitochondrial DNA control-region sequences

PubMed Central

Lee, Hwan Young; Song, Injee; Ha, Eunho; Cho, Sung-Bae; Yang, Woo Ick; Shin, Kyoung-Jin

2008-01-01

Background For the past few years, scientific controversy has surrounded the large number of errors in forensic and literature mitochondrial DNA (mtDNA) data. However, recent research has shown that using mtDNA phylogeny and referring to known mtDNA haplotypes can be useful for checking the quality of sequence data. Results We developed a Web-based bioinformatics resource "mtDNAmanager" that offers a convenient interface supporting the management and quality analysis of mtDNA sequence data. The mtDNAmanager performs computations on mtDNA control-region sequences to estimate the most-probable mtDNA haplogroups and retrieves similar sequences from a selected database. By the phased designation of the most-probable haplogroups (both expected and estimated haplogroups), mtDNAmanager enables users to systematically detect errors whilst allowing for confirmation of the presence of clear key diagnostic mutations and accompanying mutations. The query tools of mtDNAmanager also facilitate database screening with two options of "match" and "include the queried nucleotide polymorphism". In addition, mtDNAmanager provides Web interfaces for users to manage and analyse their own data in batch mode. Conclusion The mtDNAmanager will provide systematic routines for mtDNA sequence data management and analysis via easily accessible Web interfaces, and thus should be very useful for population, medical and forensic studies that employ mtDNA analysis. mtDNAmanager can be accessed at . PMID:19014619

HEP Computing Tools, Grid and Supercomputers for Genome Sequencing Studies

NASA Astrophysics Data System (ADS)

De, K.; Klimentov, A.; Maeno, T.; Mashinistov, R.; Novikov, A.; Poyda, A.; Tertychnyy, I.; Wenaus, T.

2017-10-01

PanDA - Production and Distributed Analysis Workload Management System has been developed to address ATLAS experiment at LHC data processing and analysis challenges. Recently PanDA has been extended to run HEP scientific applications on Leadership Class Facilities and supercomputers. The success of the projects to use PanDA beyond HEP and Grid has drawn attention from other compute intensive sciences such as bioinformatics. Recent advances of Next Generation Genome Sequencing (NGS) technology led to increasing streams of sequencing data that need to be processed, analysed and made available for bioinformaticians worldwide. Analysis of genomes sequencing data using popular software pipeline PALEOMIX can take a month even running it on the powerful computer resource. In this paper we will describe the adaptation the PALEOMIX pipeline to run it on a distributed computing environment powered by PanDA. To run pipeline we split input files into chunks which are run separately on different nodes as separate inputs for PALEOMIX and finally merge output file, it is very similar to what it done by ATLAS to process and to simulate data. We dramatically decreased the total walltime because of jobs (re)submission automation and brokering within PanDA. Using software tools developed initially for HEP and Grid can reduce payload execution time for Mammoths DNA samples from weeks to days.

Experiences Building Globus Genomics: A Next-Generation Sequencing Analysis Service using Galaxy, Globus, and Amazon Web Services

PubMed Central

Madduri, Ravi K.; Sulakhe, Dinanath; Lacinski, Lukasz; Liu, Bo; Rodriguez, Alex; Chard, Kyle; Dave, Utpal J.; Foster, Ian T.

2014-01-01

We describe Globus Genomics, a system that we have developed for rapid analysis of large quantities of next-generation sequencing (NGS) genomic data. This system achieves a high degree of end-to-end automation that encompasses every stage of data analysis including initial data retrieval from remote sequencing centers or storage (via the Globus file transfer system); specification, configuration, and reuse of multi-step processing pipelines (via the Galaxy workflow system); creation of custom Amazon Machine Images and on-demand resource acquisition via a specialized elastic provisioner (on Amazon EC2); and efficient scheduling of these pipelines over many processors (via the HTCondor scheduler). The system allows biomedical researchers to perform rapid analysis of large NGS datasets in a fully automated manner, without software installation or a need for any local computing infrastructure. We report performance and cost results for some representative workloads. PMID:25342933

Experiences Building Globus Genomics: A Next-Generation Sequencing Analysis Service using Galaxy, Globus, and Amazon Web Services.

PubMed

Madduri, Ravi K; Sulakhe, Dinanath; Lacinski, Lukasz; Liu, Bo; Rodriguez, Alex; Chard, Kyle; Dave, Utpal J; Foster, Ian T

2014-09-10

We describe Globus Genomics, a system that we have developed for rapid analysis of large quantities of next-generation sequencing (NGS) genomic data. This system achieves a high degree of end-to-end automation that encompasses every stage of data analysis including initial data retrieval from remote sequencing centers or storage (via the Globus file transfer system); specification, configuration, and reuse of multi-step processing pipelines (via the Galaxy workflow system); creation of custom Amazon Machine Images and on-demand resource acquisition via a specialized elastic provisioner (on Amazon EC2); and efficient scheduling of these pipelines over many processors (via the HTCondor scheduler). The system allows biomedical researchers to perform rapid analysis of large NGS datasets in a fully automated manner, without software installation or a need for any local computing infrastructure. We report performance and cost results for some representative workloads.

MetaDP: a comprehensive web server for disease prediction of 16S rRNA metagenomic datasets.

PubMed

Xu, Xilin; Wu, Aiping; Zhang, Xinlei; Su, Mingming; Jiang, Taijiao; Yuan, Zhe-Ming

2016-01-01

High-throughput sequencing-based metagenomics has garnered considerable interest in recent years. Numerous methods and tools have been developed for the analysis of metagenomic data. However, it is still a daunting task to install a large number of tools and complete a complicated analysis, especially for researchers with minimal bioinformatics backgrounds. To address this problem, we constructed an automated software named MetaDP for 16S rRNA sequencing data analysis, including data quality control, operational taxonomic unit clustering, diversity analysis, and disease risk prediction modeling. Furthermore, a support vector machine-based prediction model for intestinal bowel syndrome (IBS) was built by applying MetaDP to microbial 16S sequencing data from 108 children. The success of the IBS prediction model suggests that the platform may also be applied to other diseases related to gut microbes, such as obesity, metabolic syndrome, or intestinal cancer, among others (http://metadp.cn:7001/).

Morphological identification and COI barcodes of adult flies help determine species identities of chironomid larvae (Diptera, Chironomidae).

PubMed

Failla, A J; Vasquez, A A; Hudson, P; Fujimoto, M; Ram, J L

2016-02-01

Establishing reliable methods for the identification of benthic chironomid communities is important due to their significant contribution to biomass, ecology and the aquatic food web. Immature larval specimens are more difficult to identify to species level by traditional morphological methods than their fully developed adult counterparts, and few keys are available to identify the larval species. In order to develop molecular criteria to identify species of chironomid larvae, larval and adult chironomids from Western Lake Erie were subjected to both molecular and morphological taxonomic analysis. Mitochondrial cytochrome c oxidase I (COI) barcode sequences of 33 adults that were identified to species level by morphological methods were grouped with COI sequences of 189 larvae in a neighbor-joining taxon-ID tree. Most of these larvae could be identified only to genus level by morphological taxonomy (only 22 of the 189 sequenced larvae could be identified to species level). The taxon-ID tree of larval sequences had 45 operational taxonomic units (OTUs, defined as clusters with >97% identity or individual sequences differing from nearest neighbors by >3%; supported by analysis of all larval pairwise differences), of which seven could be identified to species or 'species group' level by larval morphology. Reference sequences from the GenBank and BOLD databases assigned six larval OTUs with presumptive species level identifications and confirmed one previously assigned species level identification. Sequences from morphologically identified adults in the present study grouped with and further classified the identity of 13 larval OTUs. The use of morphological identification and subsequent DNA barcoding of adult chironomids proved to be beneficial in revealing possible species level identifications of larval specimens. Sequence data from this study also contribute to currently inadequate public databases relevant to the Great Lakes region, while the neighbor-joining analysis reported here describes the application and confirmation of a useful tool that can accelerate identification and bioassessment of chironomid communities.

Morphological identification and COI barcodes of adult flies help determine species identities of chironomid larvae (Diptera, Chironomidae)

USGS Publications Warehouse

Failla, Andrew Joseph; Vasquez, Adrian Amelio; Hudson, Patrick L.; Fujimoto, Masanori; Ram, Jeffrey L.

2016-01-01

Establishing reliable methods for the identification of benthic chironomid communities is important due to their significant contribution to biomass, ecology and the aquatic food web. Immature larval specimens are more difficult to identify to species level by traditional morphological methods than their fully developed adult counterparts, and few keys are available to identify the larval species. In order to develop molecular criteria to identify species of chironomid larvae, larval and adult chironomids from Western Lake Erie were subjected to both molecular and morphological taxonomic analysis. Mitochondrial cytochrome c oxidase I (COI) barcode sequences of 33 adults that were identified to species level by morphological methods were grouped with COI sequences of 189 larvae in a neighbor-joining taxon-ID tree. Most of these larvae could be identified only to genus level by morphological taxonomy (only 22 of the 189 sequenced larvae could be identified to species level). The taxon-ID tree of larval sequences had 45 operational taxonomic units (OTUs, defined as clusters with >97% identity or individual sequences differing from nearest neighbors by >3%; supported by analysis of all larval pairwise differences), of which seven could be identified to species or ‘species group’ level by larval morphology. Reference sequences from the GenBank and BOLD databases assigned six larval OTUs with presumptive species level identifications and confirmed one previously assigned species level identification. Sequences from morphologically identified adults in the present study grouped with and further classified the identity of 13 larval OTUs. The use of morphological identification and subsequent DNA barcoding of adult chironomids proved to be beneficial in revealing possible species level identifications of larval specimens. Sequence data from this study also contribute to currently inadequate public databases relevant to the Great Lakes region, while the neighbor-joining analysis reported here describes the application and confirmation of a useful tool that can accelerate identification and bioassesment of chironomid communities.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing.

PubMed

Jäger, Marten; Ott, Claus-Eric; Grünhagen, Johannes; Hecht, Jochen; Schell, Hanna; Mundlos, Stefan; Duda, Georg N; Robinson, Peter N; Lienau, Jasmin

2011-03-24

The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

PubMed Central

2011-01-01

Background The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Results Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Conclusions Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism. PMID:21435219

Identification of Human Lineage-Specific Transcriptional Coregulators Enabled by a Glossary of Binding Modules and Tunable Genomic Backgrounds.

PubMed

Mariani, Luca; Weinand, Kathryn; Vedenko, Anastasia; Barrera, Luis A; Bulyk, Martha L

2017-09-27

Transcription factors (TFs) control cellular processes by binding specific DNA motifs to modulate gene expression. Motif enrichment analysis of regulatory regions can identify direct and indirect TF binding sites. Here, we created a glossary of 108 non-redundant TF-8mer "modules" of shared specificity for 671 metazoan TFs from publicly available and new universal protein binding microarray data. Analysis of 239 ENCODE TF chromatin immunoprecipitation sequencing datasets and associated RNA sequencing profiles suggest the 8mer modules are more precise than position weight matrices in identifying indirect binding motifs and their associated tethering TFs. We also developed GENRE (genomically equivalent negative regions), a tunable tool for construction of matched genomic background sequences for analysis of regulatory regions. GENRE outperformed four state-of-the-art approaches to background sequence construction. We used our TF-8mer glossary and GENRE in the analysis of the indirect binding motifs for the co-occurrence of tethering factors, suggesting novel TF-TF interactions. We anticipate that these tools will aid in elucidating tissue-specific gene-regulatory programs. Copyright © 2017 Elsevier Inc. All rights reserved.

VisRseq: R-based visual framework for analysis of sequencing data

PubMed Central

2015-01-01

Background Several tools have been developed to enable biologists to perform initial browsing and exploration of sequencing data. However the computational tool set for further analyses often requires significant computational expertise to use and many of the biologists with the knowledge needed to interpret these data must rely on programming experts. Results We present VisRseq, a framework for analysis of sequencing datasets that provides a computationally rich and accessible framework for integrative and interactive analyses without requiring programming expertise. We achieve this aim by providing R apps, which offer a semi-auto generated and unified graphical user interface for computational packages in R and repositories such as Bioconductor. To address the interactivity limitation inherent in R libraries, our framework includes several native apps that provide exploration and brushing operations as well as an integrated genome browser. The apps can be chained together to create more powerful analysis workflows. Conclusions To validate the usability of VisRseq for analysis of sequencing data, we present two case studies performed by our collaborators and report their workflow and insights. PMID:26328469

VisRseq: R-based visual framework for analysis of sequencing data.

PubMed

Younesy, Hamid; Möller, Torsten; Lorincz, Matthew C; Karimi, Mohammad M; Jones, Steven J M

2015-01-01

Several tools have been developed to enable biologists to perform initial browsing and exploration of sequencing data. However the computational tool set for further analyses often requires significant computational expertise to use and many of the biologists with the knowledge needed to interpret these data must rely on programming experts. We present VisRseq, a framework for analysis of sequencing datasets that provides a computationally rich and accessible framework for integrative and interactive analyses without requiring programming expertise. We achieve this aim by providing R apps, which offer a semi-auto generated and unified graphical user interface for computational packages in R and repositories such as Bioconductor. To address the interactivity limitation inherent in R libraries, our framework includes several native apps that provide exploration and brushing operations as well as an integrated genome browser. The apps can be chained together to create more powerful analysis workflows. To validate the usability of VisRseq for analysis of sequencing data, we present two case studies performed by our collaborators and report their workflow and insights.

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1991-01-21

bisphosphate carboxylase ( RubisCO ) from symbiotic bacteria of various origins, b) To continue methods development for 16S rRNA sequencing from symbionts in...frozen and badly preserved specimens, and c) To use these new techniques to sequence 16s DNA from a variety of symbionts a) RubisCO We have cloned the...gene coding for RubisCO from the sulfur oxidixing symbiont of the gastropod Alvinochoncha hessleri. Nucleotide sequence analysis of the cloned fragment

Development of a Prognostic Marker for Lung Cancer Using Analysis of Tumor Evolution

DTIC Science & Technology

2017-08-01

SUPPLEMENTARY NOTES 14. ABSTRACT The goal of this project is to sequence the exomes of single tumor cells from tumors in order to construct evolutionary trees...dissociation, tumor cell isolation, whole genome amplification, and exome sequencing. We have begun to sequence the exomes of single cells and to...of populations, the evolution of tumor cells within a tumor can be diagrammed on a phylogenetic tree. The more diverse a tumor’s phylogenetic tree

Assessing the genetic diversity of Cu resistance in mine tailings through high-throughput recovery of full-length copA genes

PubMed Central

Li, Xiaofang; Zhu, Yong-Guan; Shaban, Babak; Bruxner, Timothy J. C.; Bond, Philip L.; Huang, Longbin

2015-01-01

Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses. PMID:26286020

Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. and Nakai) constructed using whole-genome re-sequencing data

PubMed Central

Liu, Shi; Gao, Peng; Zhu, Qianglong; Luan, Feishi; Davis, Angela R.; Wang, Xiaolu

2016-01-01

Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). This study detected and converted SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon, for linkage map construction and quantitative trait locus (QTL) analysis. Two inbred lines, Cream of Saskatchewan (COS) and LSW-177 had been re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. 88.7% and 78.5% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. Comparative assembled genome data analysis provided 225,693 and 19,268 SNPs and indels between the two materials. 532 pairs of CAPS markers were designed with 16 restriction enzymes, among which 271 pairs of primers gave distinct bands of the expected length and polymorphic bands, via PCR and enzyme digestion, with a polymorphic rate of 50.94%. Using the new CAPS markers, an initial CAPS-based genetic linkage map was constructed with the F2 population, spanning 1836.51 cM with 11 linkage groups and 301 markers. 12 QTLs were detected related to fruit flesh color, length, width, shape index, and brix content. These newly CAPS markers will be a valuable resource for breeding programs and genetic studies of watermelon. PMID:27162496

Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

NASA Astrophysics Data System (ADS)

Shepard, Jason R. E.

2009-05-01

The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach.

PubMed

Algama, Manjula; Tasker, Edward; Williams, Caitlin; Parslow, Adam C; Bryson-Richardson, Robert J; Keith, Jonathan M

2017-03-27

Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Pathway analysis with next-generation sequencing data.

PubMed

Zhao, Jinying; Zhu, Yun; Boerwinkle, Eric; Xiong, Momiao

2015-04-01

Although pathway analysis methods have been developed and successfully applied to association studies of common variants, the statistical methods for pathway-based association analysis of rare variants have not been well developed. Many investigators observed highly inflated false-positive rates and low power in pathway-based tests of association of rare variants. The inflated false-positive rates and low true-positive rates of the current methods are mainly due to their lack of ability to account for gametic phase disequilibrium. To overcome these serious limitations, we develop a novel statistic that is based on the smoothed functional principal component analysis (SFPCA) for pathway association tests with next-generation sequencing data. The developed statistic has the ability to capture position-level variant information and account for gametic phase disequilibrium. By intensive simulations, we demonstrate that the SFPCA-based statistic for testing pathway association with either rare or common or both rare and common variants has the correct type 1 error rates. Also the power of the SFPCA-based statistic and 22 additional existing statistics are evaluated. We found that the SFPCA-based statistic has a much higher power than other existing statistics in all the scenarios considered. To further evaluate its performance, the SFPCA-based statistic is applied to pathway analysis of exome sequencing data in the early-onset myocardial infarction (EOMI) project. We identify three pathways significantly associated with EOMI after the Bonferroni correction. In addition, our preliminary results show that the SFPCA-based statistic has much smaller P-values to identify pathway association than other existing methods.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

PubMed

Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

2018-01-01

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

USGS Publications Warehouse

Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

1999-01-01

We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

PubMed

Khanna, Namita; Ghosh, Ananta Kumar; Huntemann, Marcel; Deshpande, Shweta; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Pagani, Ioanna; Pati, Amrita; Pitluck, Sam; Nolan, Matt; Woyke, Tanja; Teshima, Hazuki; Chertkov, Olga; Daligault, Hajnalka; Davenport, Karen; Gu, Wei; Munk, Christine; Zhang, Xiaojing; Bruce, David; Detter, Chris; Xu, Yan; Quintana, Beverly; Reitenga, Krista; Kunde, Yulia; Green, Lance; Erkkila, Tracy; Han, Cliff; Brambilla, Evelyne-Marie; Lang, Elke; Klenk, Hans-Peter; Goodwin, Lynne; Chain, Patrick; Das, Debabrata

2013-12-20

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

GAMES identifies and annotates mutations in next-generation sequencing projects.

PubMed

Sana, Maria Elena; Iascone, Maria; Marchetti, Daniela; Palatini, Jeff; Galasso, Marco; Volinia, Stefano

2011-01-01

Next-generation sequencing (NGS) methods have the potential for changing the landscape of biomedical science, but at the same time pose several problems in analysis and interpretation. Currently, there are many commercial and public software packages that analyze NGS data. However, the limitations of these applications include output which is insufficiently annotated and of difficult functional comprehension to end users. We developed GAMES (Genomic Analysis of Mutations Extracted by Sequencing), a pipeline aiming to serve as an efficient middleman between data deluge and investigators. GAMES attains multiple levels of filtering and annotation, such as aligning the reads to a reference genome, performing quality control and mutational analysis, integrating results with genome annotations and sorting each mismatch/deletion according to a range of parameters. Variations are matched to known polymorphisms. The prediction of functional mutations is achieved by using different approaches. Overall GAMES enables an effective complexity reduction in large-scale DNA-sequencing projects. GAMES is available free of charge to academic users and may be obtained from http://aqua.unife.it/GAMES.

WHAM!: a web-based visualization suite for user-defined analysis of metagenomic shotgun sequencing data.

PubMed

Devlin, Joseph C; Battaglia, Thomas; Blaser, Martin J; Ruggles, Kelly V

2018-06-25

Exploration of large data sets, such as shotgun metagenomic sequence or expression data, by biomedical experts and medical professionals remains as a major bottleneck in the scientific discovery process. Although tools for this purpose exist for 16S ribosomal RNA sequencing analysis, there is a growing but still insufficient number of user-friendly interactive visualization workflows for easy data exploration and figure generation. The development of such platforms for this purpose is necessary to accelerate and streamline microbiome laboratory research. We developed the Workflow Hub for Automated Metagenomic Exploration (WHAM!) as a web-based interactive tool capable of user-directed data visualization and statistical analysis of annotated shotgun metagenomic and metatranscriptomic data sets. WHAM! includes exploratory and hypothesis-based gene and taxa search modules for visualizing differences in microbial taxa and gene family expression across experimental groups, and for creating publication quality figures without the need for command line interface or in-house bioinformatics. WHAM! is an interactive and customizable tool for downstream metagenomic and metatranscriptomic analysis providing a user-friendly interface allowing for easy data exploration by microbiome and ecological experts to facilitate discovery in multi-dimensional and large-scale data sets.

Epigenetics of prostate cancer.

PubMed

McKee, Tawnya C; Tricoli, James V

2015-01-01

The introduction of novel technologies that can be applied to the investigation of the molecular underpinnings of human cancer has allowed for new insights into the mechanisms associated with tumor development and progression. They have also advanced the diagnosis, prognosis and treatment of cancer. These technologies include microarray and other analysis methods for the generation of large-scale gene expression data on both mRNA and miRNA, next-generation DNA sequencing technologies utilizing a number of platforms to perform whole genome, whole exome, or targeted DNA sequencing to determine somatic mutational differences and gene rearrangements, and a variety of proteomic analysis platforms including liquid chromatography/mass spectrometry (LC/MS) analysis to survey alterations in protein profiles in tumors. One other important advancement has been our current ability to survey the methylome of human tumors in a comprehensive fashion through the use of sequence-based and array-based methylation analysis (Bock et al., Nat Biotechnol 28:1106-1114, 2010; Harris et al., Nat Biotechnol 28:1097-1105, 2010). The focus of this chapter is to present and discuss the evidence for key genes involved in prostate tumor development, progression, or resistance to therapy that are regulated by methylation-induced silencing.

Transcriptome analysis in Concholepas concholepas (Gastropoda, Muricidae): mining and characterization of new genomic and molecular markers.

PubMed

Cárdenas, Leyla; Sánchez, Roland; Gomez, Daniela; Fuenzalida, Gonzalo; Gallardo-Escárate, Cristián; Tanguy, Arnaud

2011-09-01

The marine gastropod Concholepas concholepas, locally known as the "loco", is the main target species of the benthonic Chilean fisheries. Genetic and genomic tools are necessary to study the genome of this species in order to understand the molecular basis of its development, growth, and other key traits to improve the management strategies and to identify local adaptation to prevent loss of biodiversity. Here, we use pyrosequencing technologies to generate the first transcriptomic database from adult specimens of the loco. After trimming, a total of 140,756 Expressed Sequence Tag sequences were achieved. Clustering and assembly analysis identified 19,219 contigs and 105,435 singleton sequences. BlastN analysis showed a significant identity with Expressed Sequence Tags of different gastropod species available in public databases. Similarly, BlastX results showed that only 895 out of the total 124,654 had significant hits and may represent novel genes for marine gastropods. From this database, simple sequence repeat motifs were also identified and a total of 38 primer pairs were designed and tested to assess their potential as informative markers and to investigate their cross-species amplification in different related gastropod species. This dataset represents the first publicly available 454 data for a marine gastropod endemic to the southeastern Pacific coast, providing a valuable transcriptomic resource for future efforts of gene discovery and development of functional markers in other marine gastropods. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses

PubMed Central

KIKUCHI, Kohta; SASAKI, Keisuke; AKIZAWA, Hiroki; TSUKAHARA, Hayato; BAI, Hanako; TAKAHASHI, Masashi; NAMBO, Yasuo; HATA, Hiroshi; KAWAHARA, Manabu

2017-01-01

Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5ʹ- and 3ʹ-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses. PMID:29151450

Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

PubMed Central

2012-01-01

Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison. PMID:22908993

[Development of Staphylococcus Haemolyticus multilocus sequencing scheme and its use for molecular-epidemiologic analysis of strains isolated in hospitals in Russian federation in 2009-2010].

PubMed

Voronina, O L; Kunda, M S; Dmitrenko, O A; Lunin, V G; Gintsburg, A L

2011-01-01

Development of Staphylococcus haemolyticus strain typing method based on multilocus sequencing for resolving problems of molecular epidemiology. 102 strains of coagulase negative staphylococci (CNS) isolated in hospitals of various specialization in N. Novgorod and Moscow were studied. Species identification of strain was performed by using tuf gene fragment sequencing, S. haemolyticus strain differentiation--by MLST results. eBURST approach was used for cluster analysis of MLST data; structural changes in tagatose-6-phosphate kinase were studied by using InterProScan platform and SWISS-MODEL site programs; MLST scheme gene allele variability analysis was performed by using MEGA4.0 program package. In the 102 strains sampled CNS was detected in 28 strains of the S. haemolyticus species. The MLST scheme developed for the first time for S. haemolyticus including mvaK, rphE, tphK, gtr, arcC, triA, aroE genes allowed the differentiation of the sampled strains by 11 genotypes. Strains with ST 3, 8, 6, 1, 4, 5 and 11 differed by highest epidemiologic significance. Cluster and phylogenetic analysis of the data obtained showed a high adaptive ability of the nosocomial S. haemolyticus strains. Multiresistance to antibacterial preparations was detected in the analyzed strains. The MLST method developed was effective in the differentiation of S. haemolyticus strains that circulate in hospitals and threaten both neonates and hospitalized adult patients.

Quantifying the relationship between sequence and three-dimensional structure conservation in RNA

PubMed Central

2010-01-01

Background In recent years, the number of available RNA structures has rapidly grown reflecting the increased interest on RNA biology. Similarly to the studies carried out two decades ago for proteins, which gave the fundamental grounds for developing comparative protein structure prediction methods, we are now able to quantify the relationship between sequence and structure conservation in RNA. Results Here we introduce an all-against-all sequence- and three-dimensional (3D) structure-based comparison of a representative set of RNA structures, which have allowed us to quantitatively confirm that: (i) there is a measurable relationship between sequence and structure conservation that weakens for alignments resulting in below 60% sequence identity, (ii) evolution tends to conserve more RNA structure than sequence, and (iii) there is a twilight zone for RNA homology detection. Discussion The computational analysis here presented quantitatively describes the relationship between sequence and structure for RNA molecules and defines a twilight zone region for detecting RNA homology. Our work could represent the theoretical basis and limitations for future developments in comparative RNA 3D structure prediction. PMID:20550657

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles

PubMed Central

2011-01-01

Background Cultivated watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues. Results We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development. Conclusion We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology. PMID:21936920

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles.

PubMed

Guo, Shaogui; Liu, Jingan; Zheng, Yi; Huang, Mingyun; Zhang, Haiying; Gong, Guoyi; He, Hongju; Ren, Yi; Zhong, Silin; Fei, Zhangjun; Xu, Yong

2011-09-21

Cultivated watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues. We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development. We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.

Comparative Genome Sequence Analysis of the Bpa/Str Region in Mouse and Man

PubMed Central

Mallon, A.-M.; Platzer, M.; Bate, R.; Gloeckner, G.; Botcherby, M.R.M.; Nordsiek, G.; Strivens, M.A.; Kioschis, P.; Dangel, A.; Cunningham, D.; Straw, R.N.A.; Weston, P.; Gilbert, M.; Fernando, S.; Goodall, K.; Hunter, G.; Greystrong, J.S.; Clarke, D.; Kimberley, C.; Goerdes, M.; Blechschmidt, K.; Rump, A.; Hinzmann, B.; Mundy, C.R.; Miller, W.; Poustka, A.; Herman, G.E.; Rhodes, M.; Denny, P.; Rosenthal, A.; Brown, S.D.M.

2000-01-01

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions. [The sequence data described in this paper have been submitted to EMBL under the following accession nos.: Mouse Genomic Sequence: Mouse contig A (AL021127), Mouse contig B (AL049866), BAC41M10 (AL136328), PAC303O11(AL136329). Human Genomic Sequence: Human contig 1 (U82671, U82670), Human contig 2 (U82695).] PMID:10854409

BCM Search Launcher--an integrated interface to molecular biology data base search and analysis services available on the World Wide Web.

PubMed

Smith, R F; Wiese, B A; Wojzynski, M K; Davison, D B; Worley, K C

1996-05-01

The BCM Search Launcher is an integrated set of World Wide Web (WWW) pages that organize molecular biology-related search and analysis services available on the WWW by function, and provide a single point of entry for related searches. The Protein Sequence Search Page, for example, provides a single sequence entry form for submitting sequences to WWW servers that offer remote access to a variety of different protein sequence search tools, including BLAST, FASTA, Smith-Waterman, BEAUTY, PROSITE, and BLOCKS searches. Other Launch pages provide access to (1) nucleic acid sequence searches, (2) multiple and pair-wise sequence alignments, (3) gene feature searches, (4) protein secondary structure prediction, and (5) miscellaneous sequence utilities (e.g., six-frame translation). The BCM Search Launcher also provides a mechanism to extend the utility of other WWW services by adding supplementary hypertext links to results returned by remote servers. For example, links to the NCBI's Entrez data base and to the Sequence Retrieval System (SRS) are added to search results returned by the NCBI's WWW BLAST server. These links provide easy access to auxiliary information, such as Medline abstracts, that can be extremely helpful when analyzing BLAST data base hits. For new or infrequent users of sequence data base search tools, we have preset the default search parameters to provide the most informative first-pass sequence analysis possible. We have also developed a batch client interface for Unix and Macintosh computers that allows multiple input sequences to be searched automatically as a background task, with the results returned as individual HTML documents directly to the user's system. The BCM Search Launcher and batch client are available on the WWW at URL http:@gc.bcm.tmc.edu:8088/search-launcher.html.

[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.

RefSeq microbial genomes database: new representation and annotation strategy.

PubMed

Tatusova, Tatiana; Ciufo, Stacy; Fedorov, Boris; O'Neill, Kathleen; Tolstoy, Igor

2014-01-01

The source of the microbial genomic sequences in the RefSeq collection is the set of primary sequence records submitted to the International Nucleotide Sequence Database public archives. These can be accessed through the Entrez search and retrieval system at http://www.ncbi.nlm.nih.gov/genome. Next-generation sequencing has enabled researchers to perform genomic sequencing at rates that were unimaginable in the past. Microbial genomes can now be sequenced in a matter of hours, which has led to a significant increase in the number of assembled genomes deposited in the public archives. This huge increase in DNA sequence data presents new challenges for the annotation, analysis and visualization bioinformatics tools. New strategies have been developed for the annotation and representation of reference genomes and sequence variations derived from population studies and clinical outbreaks.

Genome sequencing and analysis of the model grass Brachypodium distachyon

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

Yang, Xiaohan; Kalluri, Udaya C; Tuskan, Gerald A

Three subfamilies of grasses, the Ehrhartoideae, Panicoideae and Pooideae, provide the bulk of human nutrition and are poised to become major sources of renewable energy. Here we describe the genome sequence of the wild grass Brachypodium distachyon (Brachypodium), which is, to our knowledge, the first member of the Pooideae subfamily to be sequenced. Comparison of the Brachypodium, rice and sorghum genomes shows a precise history of genome evolution across a broad diversity of the grasses, and establishes a template for analysis of the large genomes of economically important pooid grasses such as wheat. The high-quality genome sequence, coupled with easemore » of cultivation and transformation, small size and rapid life cycle, will help Brachypodium reach its potential as an important model system for developing new energy and food crops.« less

Next Generation Sequencing Technology and Genomewide Data Analysis: Perspectives for Retinal Research

PubMed Central

Chaitankar, Vijender; Karakülah, Gökhan; Ratnapriya, Rinki; Giuste, Felipe O.; Brooks, Matthew J.; Swaroop, Anand

2016-01-01

The advent of high throughput next generation sequencing (NGS) has accelerated the pace of discovery of disease-associated genetic variants and genomewide profiling of expressed sequences and epigenetic marks, thereby permitting systems-based analyses of ocular development and disease. Rapid evolution of NGS and associated methodologies presents significant challenges in acquisition, management, and analysis of large data sets and for extracting biologically or clinically relevant information. Here we illustrate the basic design of commonly used NGS-based methods, specifically whole exome sequencing, transcriptome, and epigenome profiling, and provide recommendations for data analyses. We briefly discuss systems biology approaches for integrating multiple data sets to elucidate gene regulatory or disease networks. While we provide examples from the retina, the NGS guidelines reviewed here are applicable to other tissues/cell types as well. PMID:27297499

The Ensembl genome database project.

PubMed

Hubbard, T; Barker, D; Birney, E; Cameron, G; Chen, Y; Clark, L; Cox, T; Cuff, J; Curwen, V; Down, T; Durbin, R; Eyras, E; Gilbert, J; Hammond, M; Huminiecki, L; Kasprzyk, A; Lehvaslaiho, H; Lijnzaad, P; Melsopp, C; Mongin, E; Pettett, R; Pocock, M; Potter, S; Rust, A; Schmidt, E; Searle, S; Slater, G; Smith, J; Spooner, W; Stabenau, A; Stalker, J; Stupka, E; Ureta-Vidal, A; Vastrik, I; Clamp, M

2002-01-01

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

Diazotrophic Community Structure and Function in Two Successional Stages of Biological Soil Crusts from the Colorado Plateau and Chihuahuan Desert

USGS Publications Warehouse

Yeager, C.M.; Kornosky, J.L.; Housman, D.C.; Grote, E.E.; Belnap, J.; Kuske, C.R.

2004-01-01

The objective of this study was to characterize the community structure and activity of N2-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of nifH sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to Nostoc spp. and that the composition of N2-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of nifH sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a Microcoleus vaginatus-dominated, poorly developed crust to mature crusts harboring a greater percentage of Nostoc and Scytonema spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N2-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by Microcoleus vaginatus.

Draft versus finished sequence data for DNA and protein diagnostic signature development

PubMed Central

Gardner, Shea N.; Lam, Marisa W.; Smith, Jason R.; Torres, Clinton L.; Slezak, Tom R.

2005-01-01

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10−3–10−5 (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. PMID:16243783

CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database

PubMed Central

Jia, Baofeng; Raphenya, Amogelang R.; Alcock, Brian; Waglechner, Nicholas; Guo, Peiyao; Tsang, Kara K.; Lago, Briony A.; Dave, Biren M.; Pereira, Sheldon; Sharma, Arjun N.; Doshi, Sachin; Courtot, Mélanie; Lo, Raymond; Williams, Laura E.; Frye, Jonathan G.; Elsayegh, Tariq; Sardar, Daim; Westman, Erin L.; Pawlowski, Andrew C.; Johnson, Timothy A.; Brinkman, Fiona S.L.; Wright, Gerard D.; McArthur, Andrew G.

2017-01-01

The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis. PMID:27789705

Computational annotation of genes differentially expressed along olive fruit development

PubMed Central

Galla, Giulio; Barcaccia, Gianni; Ramina, Angelo; Collani, Silvio; Alagna, Fiammetta; Baldoni, Luciana; Cultrera, Nicolò GM; Martinelli, Federico; Sebastiani, Luca; Tonutti, Pietro

2009-01-01

Background Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software. Results mRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all known KEGG (Kyoto Encyclopaedia of Genes and Genomes) metabolic pathways for characterizing and positioning retrieved EST records. The integration of the olive sequence datasets within the MapMan platform for microarray analysis allowed the identification of specific biosynthetic pathways useful for the definition of key functional categories in time course analyses for gene groups. Conclusion The bioinformatic annotation of all gene sequences was useful to shed light on metabolic pathways and transcriptional aspects related to carbohydrates, fatty acids, secondary metabolites, transcription factors and hormones as well as response to biotic and abiotic stresses throughout olive drupe development. These results represent a first step toward both functional genomics and systems biology research for understanding the gene functions and regulatory networks in olive fruit growth and ripening. PMID:19852839

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

PubMed Central

Kamps, Rick; Brandão, Rita D.; van den Bosch, Bianca J.; Paulussen, Aimee D. C.; Xanthoulea, Sofia; Blok, Marinus J.; Romano, Andrea

2017-01-01

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided. PMID:28146134

A short review of variants calling for single-cell-sequencing data with applications.

PubMed

Wei, Zhuohui; Shu, Chang; Zhang, Changsheng; Huang, Jingying; Cai, Hongmin

2017-11-01

The field of single-cell sequencing is fleetly expanding, and many techniques have been developed in the past decade. With this technology, biologists can study not only the heterogeneity between two adjacent cells in the same tissue or organ, but also the evolutionary relationships and degenerative processes in a single cell. Calling variants is the main purpose in analyzing single cell sequencing (SCS) data. Currently, some popular methods used for bulk-cell-sequencing data analysis are tailored directly to be applied in dealing with SCS data. However, SCS requires an extra step of genome amplification to accumulate enough quantity for satisfying sequencing needs. The amplification yields large biases and thus raises challenge for using the bulk-cell-sequencing methods. In order to provide guidance for the development of specialized analyzed methods as well as using currently developed tools for SNS, this paper aims to bridge the gap. In this paper, we firstly introduced two popular genome amplification methods and compared their capabilities. Then we introduced a few popular models for calling single-nucleotide polymorphisms and copy-number variations. Finally, break-through applications of SNS were summarized to demonstrate its potential in researching cell evolution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Clustering of Genetically Defined Allele Classes in the Caenorhabditis elegans DAF-2 Insulin/IGF-1 Receptor

PubMed Central

Patel, Dhaval S.; Garza-Garcia, Acely; Nanji, Manoj; McElwee, Joshua J.; Ackerman, Daniel; Driscoll, Paul C.; Gems, David

2008-01-01

The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology. PMID:18245374

Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development.

PubMed

Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Alves de Brito, Cristiana Ferreira

2011-01-01

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.

Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

PubMed Central

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-01-01

Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

PubMed

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-12-27

Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

PubMed Central

Prada, Anne E.

2014-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis has been implemented for Cystic Fibrosis (CF) carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD). Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM) curve analysis, allele-specific PCR (AS-PCR) and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing. PMID:25071991

Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

PubMed Central

Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

2014-01-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

PubMed

Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

2014-12-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

A Toolkit for bulk PCR-based marker design from next-generation sequence data: application for development of a framework linkage map in bulb onion (Allium cepa L.)

PubMed Central

2012-01-01

Background Although modern sequencing technologies permit the ready detection of numerous DNA sequence variants in any organisms, converting such information to PCR-based genetic markers is hampered by a lack of simple, scalable tools. Onion is an example of an under-researched crop with a complex, heterozygous genome where genome-based research has previously been hindered by limited sequence resources and genetic markers. Results We report the development of generic tools for large-scale web-based PCR-based marker design in the Galaxy bioinformatics framework, and their application for development of next-generation genetics resources in a wide cross of bulb onion (Allium cepa L.). Transcriptome sequence resources were developed for the homozygous doubled-haploid bulb onion line ‘CUDH2150’ and the genetically distant Indian landrace ‘Nasik Red’, using 454™ sequencing of normalised cDNA libraries of leaf and shoot. Read mapping of ‘Nasik Red’ reads onto ‘CUDH2150’ assemblies revealed 16836 indel and SNP polymorphisms that were mined for portable PCR-based marker development. Tools for detection of restriction polymorphisms and primer set design were developed in BioPython and adapted for use in the Galaxy workflow environment, enabling large-scale and targeted assay design. Using PCR-based markers designed with these tools, a framework genetic linkage map of over 800cM spanning all chromosomes was developed in a subset of 93 F2 progeny from a very large F2 family developed from the ‘Nasik Red’ x ‘CUDH2150’ inter-cross. The utility of tools and genetic resources developed was tested by designing markers to transcription factor-like polymorphic sequences. Bin mapping these markers using a subset of 10 progeny confirmed the ability to place markers within 10 cM bins, enabling increased efficiency in marker assignment and targeted map refinement. The major genetic loci conditioning red bulb colour (R) and fructan content (Frc) were located on this map by QTL analysis. Conclusions The generic tools developed for the Galaxy environment enable rapid development of sets of PCR assays targeting sequence variants identified from Illumina and 454 sequence data. They enable non-specialist users to validate and exploit large volumes of next-generation sequence data using basic equipment. PMID:23157543

A toolkit for bulk PCR-based marker design from next-generation sequence data: application for development of a framework linkage map in bulb onion (Allium cepa L.).

PubMed

Baldwin, Samantha; Revanna, Roopashree; Thomson, Susan; Pither-Joyce, Meeghan; Wright, Kathryn; Crowhurst, Ross; Fiers, Mark; Chen, Leshi; Macknight, Richard; McCallum, John A

2012-11-19

Although modern sequencing technologies permit the ready detection of numerous DNA sequence variants in any organisms, converting such information to PCR-based genetic markers is hampered by a lack of simple, scalable tools. Onion is an example of an under-researched crop with a complex, heterozygous genome where genome-based research has previously been hindered by limited sequence resources and genetic markers. We report the development of generic tools for large-scale web-based PCR-based marker design in the Galaxy bioinformatics framework, and their application for development of next-generation genetics resources in a wide cross of bulb onion (Allium cepa L.). Transcriptome sequence resources were developed for the homozygous doubled-haploid bulb onion line 'CUDH2150' and the genetically distant Indian landrace 'Nasik Red', using 454™ sequencing of normalised cDNA libraries of leaf and shoot. Read mapping of 'Nasik Red' reads onto 'CUDH2150' assemblies revealed 16836 indel and SNP polymorphisms that were mined for portable PCR-based marker development. Tools for detection of restriction polymorphisms and primer set design were developed in BioPython and adapted for use in the Galaxy workflow environment, enabling large-scale and targeted assay design. Using PCR-based markers designed with these tools, a framework genetic linkage map of over 800cM spanning all chromosomes was developed in a subset of 93 F(2) progeny from a very large F(2) family developed from the 'Nasik Red' x 'CUDH2150' inter-cross. The utility of tools and genetic resources developed was tested by designing markers to transcription factor-like polymorphic sequences. Bin mapping these markers using a subset of 10 progeny confirmed the ability to place markers within 10 cM bins, enabling increased efficiency in marker assignment and targeted map refinement. The major genetic loci conditioning red bulb colour (R) and fructan content (Frc) were located on this map by QTL analysis. The generic tools developed for the Galaxy environment enable rapid development of sets of PCR assays targeting sequence variants identified from Illumina and 454 sequence data. They enable non-specialist users to validate and exploit large volumes of next-generation sequence data using basic equipment.

Prediction of the translocon-mediated membrane insertion free energies of protein sequences.

PubMed

Park, Yungki; Helms, Volkhard

2008-05-15

Helical membrane proteins (HMPs) play crucial roles in a variety of cellular processes. Unlike water-soluble proteins, HMPs need not only to fold but also get inserted into the membrane to be fully functional. This process of membrane insertion is mediated by the translocon complex. Thus, it is of great interest to develop computational methods for predicting the translocon-mediated membrane insertion free energies of protein sequences. We have developed Membrane Insertion (MINS), a novel sequence-based computational method for predicting the membrane insertion free energies of protein sequences. A benchmark test gives a correlation coefficient of 0.74 between predicted and observed free energies for 357 known cases, which corresponds to a mean unsigned error of 0.41 kcal/mol. These results are significantly better than those obtained by traditional hydropathy analysis. Moreover, the ability of MINS to reasonably predict membrane insertion free energies of protein sequences allows for effective identification of transmembrane (TM) segments. Subsequently, MINS was applied to predict the membrane insertion free energies of 316 TM segments found in known structures. An in-depth analysis of the predicted free energies reveals a number of interesting findings about the biogenesis and structural stability of HMPs. A web server for MINS is available at http://service.bioinformatik.uni-saarland.de/mins

Isolation and Identification of Pathogenicity Mutant of Curvularia lunata via Restriction Enzyme-Mediated Integration.

PubMed

Wang, Y J; Liu, T; Hou, J M; Zuo, Y H

2013-09-01

In this report, 156 hygromycin-resistant mutants were generated via restriction enzyme-mediated insertional (REMI) mutagenesis. All mutants were subjected to a bioassay on detached leaves. Five mutants (T4, T39, T71, T91, and T135) showed reduced symptom development, whereas one mutant (T120) did not exhibit any symptoms on the leaves compared with the wild type. The pathogenicity of these mutants was further assayed through the spray inoculation of whole seedlings. The results demonstrated that the pathogenicity of the T4, T39, T71, T91, and T135 mutants was reduced, whereas the T120 mutant lost its pathogenicity. Southern blot analysis revealed that the plasmids were inserted at different sites in the genome with different copy numbers. Flanking sequences approximately 550, 860, and 150 bp were obtained from T7, T91, and T120, respectively through plasmids rescue. Sequence analysis of the flanking sequences from T7 and T91 showed no homology to any known sequences in GenBank. The flanking sequence from the T120 mutant was highly homologous to MAPKK kinases, which regulates sexual/asexual development, melanization, pathogenicity from Cochliobolus heterostrophus. These results indicate that REMI and plasmids rescue have great potential for finding pathogenicity genes.

CFGP: a web-based, comparative fungal genomics platform

PubMed Central

Park, Jongsun; Park, Bongsoo; Jung, Kyongyong; Jang, Suwang; Yu, Kwangyul; Choi, Jaeyoung; Kong, Sunghyung; Park, Jaejin; Kim, Seryun; Kim, Hyojeong; Kim, Soonok; Kim, Jihyun F.; Blair, Jaime E.; Lee, Kwangwon; Kang, Seogchan; Lee, Yong-Hwan

2008-01-01

Since the completion of the Saccharomyces cerevisiae genome sequencing project in 1996, the genomes of over 80 fungal species have been sequenced or are currently being sequenced. Resulting data provide opportunities for studying and comparing fungal biology and evolution at the genome level. To support such studies, the Comparative Fungal Genomics Platform (CFGP; http://cfgp.snu.ac.kr), a web-based multifunctional informatics workbench, was developed. The CFGP comprises three layers, including the basal layer, middleware and the user interface. The data warehouse in the basal layer contains standardized genome sequences of 65 fungal species. The middleware processes queries via six analysis tools, including BLAST, ClustalW, InterProScan, SignalP 3.0, PSORT II and a newly developed tool named BLASTMatrix. The BLASTMatrix permits the identification and visualization of genes homologous to a query across multiple species. The Data-driven User Interface (DUI) of the CFGP was built on a new concept of pre-collecting data and post-executing analysis instead of the ‘fill-in-the-form-and-press-SUBMIT’ user interfaces utilized by most bioinformatics sites. A tool termed Favorite, which supports the management of encapsulated sequence data and provides a personalized data repository to users, is another novel feature in the DUI. PMID:17947331

SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects.

PubMed

Dereeper, Alexis; Nicolas, Stéphane; Le Cunff, Loïc; Bacilieri, Roberto; Doligez, Agnès; Peros, Jean-Pierre; Ruiz, Manuel; This, Patrice

2011-05-05

High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data. In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats. Our experiments on grapevine genetic projects showed that SNiPlay allows geneticists to rapidly obtain advanced results in several key research areas of plant genetic diversity. Both the management and treatment of large amounts of SNP data are rendered considerably easier for end-users through automation and integration. Current developments are taking into account new advances in high-throughput technologies.SNiPlay is available at: http://sniplay.cirad.fr/.

Genomic insight into the common carp (Cyprinus carpio) genome by sequencing analysis of BAC-end sequences

PubMed Central

2011-01-01

Background Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively underdeveloped. BAC end sequences (BES) are important resources for genome research on BAC-anchored genetic marker development, linkage map and physical map integration, and whole genome sequence assembling and scaffolding. Result To develop such valuable resources in common carp (Cyprinus carpio), a total of 40,224 BAC clones were sequenced on both ends, generating 65,720 clean BES with an average read length of 647 bp after sequence processing, representing 42,522,168 bp or 2.5% of common carp genome. The first survey of common carp genome was conducted with various bioinformatics tools. The common carp genome contains over 17.3% of repetitive elements with GC content of 36.8% and 518 transposon ORFs. To identify and develop BAC-anchored microsatellite markers, a total of 13,581 microsatellites were detected from 10,355 BES. The coding region of 7,127 genes were recognized from 9,443 BES on 7,453 BACs, with 1,990 BACs have genes on both ends. To evaluate the similarity to the genome of closely related zebrafish, BES of common carp were aligned against zebrafish genome. A total of 39,335 BES of common carp have conserved homologs on zebrafish genome which demonstrated the high similarity between zebrafish and common carp genomes, indicating the feasibility of comparative mapping between zebrafish and common carp once we have physical map of common carp. Conclusion BAC end sequences are great resources for the first genome wide survey of common carp. The repetitive DNA was estimated to be approximate 28% of common carp genome, indicating the higher complexity of the genome. Comparative analysis had mapped around 40,000 BES to zebrafish genome and established over 3,100 microsyntenies, covering over 50% of the zebrafish genome. BES of common carp are tremendous tools for comparative mapping between the two closely related species, zebrafish and common carp, which should facilitate both structural and functional genome analysis in common carp. PMID:21492448

Sequence analysis of the L protein of the Ebola 2014 outbreak: Insight into conserved regions and mutations.

PubMed

Ayub, Gohar; Waheed, Yasir

2016-06-01

The 2014 Ebola outbreak was one of the largest that have occurred; it started in Guinea and spread to Nigeria, Liberia and Sierra Leone. Phylogenetic analysis of the current virus species indicated that this outbreak is the result of a divergent lineage of the Zaire ebolavirus. The L protein of Ebola virus (EBOV) is the catalytic subunit of the RNA‑dependent RNA polymerase complex, which, with VP35, is key for the replication and transcription of viral RNA. Earlier sequence analysis demonstrated that the L protein of all non‑segmented negative‑sense (NNS) RNA viruses consists of six domains containing conserved functional motifs. The aim of the present study was to analyze the presence of these motifs in 2014 EBOV isolates, highlight their function and how they may contribute to the overall pathogenicity of the isolates. For this purpose, 81 2014 EBOV L protein sequences were aligned with 475 other NNS RNA viruses, including Paramyxoviridae and Rhabdoviridae viruses. Phylogenetic analysis of all EBOV outbreak L protein sequences was also performed. Analysis of the amino acid substitutions in the 2014 EBOV outbreak was conducted using sequence analysis. The alignment demonstrated the presence of previously conserved motifs in the 2014 EBOV isolates and novel residues. Notably, all the mutations identified in the 2014 EBOV isolates were tolerant, they were pathogenic with certain examples occurring within previously determined functional conserved motifs, possibly altering viral pathogenicity, replication and virulence. The phylogenetic analysis demonstrated that all sequences with the exception of the 2014 EBOV sequences were clustered together. The 2014 EBOV outbreak has acquired a great number of mutations, which may explain the reasons behind this unprecedented outbreak. Certain residues critical to the function of the polymerase remain conserved and may be targets for the development of antiviral therapeutic agents.

Bicycle: a bioinformatics pipeline to analyze bisulfite sequencing data.

PubMed

Graña, Osvaldo; López-Fernández, Hugo; Fdez-Riverola, Florentino; González Pisano, David; Glez-Peña, Daniel

2018-04-15

High-throughput sequencing of bisulfite-converted DNA is a technique used to measure DNA methylation levels. Although a considerable number of computational pipelines have been developed to analyze such data, none of them tackles all the peculiarities of the analysis together, revealing limitations that can force the user to manually perform additional steps needed for a complete processing of the data. This article presents bicycle, an integrated, flexible analysis pipeline for bisulfite sequencing data. Bicycle analyzes whole genome bisulfite sequencing data, targeted bisulfite sequencing data and hydroxymethylation data. To show how bicycle overtakes other available pipelines, we compared them on a defined number of features that are summarized in a table. We also tested bicycle with both simulated and real datasets, to show its level of performance, and compared it to different state-of-the-art methylation analysis pipelines. Bicycle is publicly available under GNU LGPL v3.0 license at http://www.sing-group.org/bicycle. Users can also download a customized Ubuntu LiveCD including bicycle and other bisulfite sequencing data pipelines compared here. In addition, a docker image with bicycle and its dependencies, which allows a straightforward use of bicycle in any platform (e.g. Linux, OS X or Windows), is also available. ograna@cnio.es or dgpena@uvigo.es. Supplementary data are available at Bioinformatics online.

Task analysis in curriculum design: a hierarchically sequenced introductory mathematics curriculum1

PubMed Central

Resnick, Lauren B.; Wang, Margaret C.; Kaplan, Jerome

1973-01-01

A method of systematic task analysis is applied to the problem of designing a sequence of learning objectives that will provide an optimal match for the child's natural sequence of acquisition of mathematical skills and concepts. The authors begin by proposing an operational definition of the number concept in the form of a set of behaviors which, taken together, permit the inference that the child has an abstract concept of “number”. These are the “objectives” of the curriculum. Each behavior in the defining set is then subjected to an analysis that identifies hypothesized components of skilled performance and prerequisites for learning these components. On the basis of these analyses, specific sequences of learning objectives are proposed. The proposed sequences are hypothesized to be those that will best facilitate learning, by maximizing transfer from earlier to later objectives. Relevant literature on early learning and cognitive development is considered in conjunction with the analyses and the resulting sequences. The paper concludes with a discussion of the ways in which the curriculum can be implemented and studied in schools. Examples of data on individual children are presented, and the use of such data for improving the curriculum itself, as well as for examining the effects of other treatment variables, is considered. PMID:16795452

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PubMed

Retterer, Kyle; Scuffins, Julie; Schmidt, Daniel; Lewis, Rachel; Pineda-Alvarez, Daniel; Stafford, Amanda; Schmidt, Lindsay; Warren, Stephanie; Gibellini, Federica; Kondakova, Anastasia; Blair, Amanda; Bale, Sherri; Matyakhina, Ludmila; Meck, Jeanne; Aradhya, Swaroop; Haverfield, Eden

2015-08-01

Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

PubMed

Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

2012-01-01

Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate pathogen-specific drug targets identification which will increase their success and durability as drugs developed against them have less chance to develop resistances and adverse impact on environment. The server is freely available at http://117.211.115.67/UDT/main.html. The standalone application (source codes) is available at http://www.bioinformatics.org/ftp/pub/bioinfojuit/UDT.rar.

NGSPanPipe: A Pipeline for Pan-genome Identification in Microbial Strains from Experimental Reads.

PubMed

Kulsum, Umay; Kapil, Arti; Singh, Harpreet; Kaur, Punit

2018-01-01

Recent advancements in sequencing technologies have decreased both time span and cost for sequencing the whole bacterial genome. High-throughput Next-Generation Sequencing (NGS) technology has led to the generation of enormous data concerning microbial populations publically available across various repositories. As a consequence, it has become possible to study and compare the genomes of different bacterial strains within a species or genus in terms of evolution, ecology and diversity. Studying the pan-genome provides insights into deciphering microevolution, global composition and diversity in virulence and pathogenesis of a species. It can also assist in identifying drug targets and proposing vaccine candidates. The effective analysis of these large genome datasets necessitates the development of robust tools. Current methods to develop pan-genome do not support direct input of raw reads from the sequencer machine but require preprocessing of reads as an assembled protein/gene sequence file or the binary matrix of orthologous genes/proteins. We have designed an easy-to-use integrated pipeline, NGSPanPipe, which can directly identify the pan-genome from short reads. The output from the pipeline is compatible with other pan-genome analysis tools. We evaluated our pipeline with other methods for developing pan-genome, i.e. reference-based assembly and de novo assembly using simulated reads of Mycobacterium tuberculosis. The single script pipeline (pipeline.pl) is applicable for all bacterial strains. It integrates multiple in-house Perl scripts and is freely accessible from https://github.com/Biomedinformatics/NGSPanPipe .

SONAR: A High-Throughput Pipeline for Inferring Antibody Ontogenies from Longitudinal Sequencing of B Cell Transcripts

PubMed Central

Schramm, Chaim A.; Sheng, Zizhang; Zhang, Zhenhai; Mascola, John R.; Kwong, Peter D.; Shapiro, Lawrence

2016-01-01

The rapid advance of massively parallel or next-generation sequencing technologies has made possible the characterization of B cell receptor repertoires in ever greater detail, and these developments have triggered a proliferation of software tools for processing and annotating these data. Of especial interest, however, is the capability to track the development of specific antibody lineages across time, which remains beyond the scope of most current programs. We have previously reported on the use of techniques such as inter- and intradonor analysis and CDR3 tracing to identify transcripts related to an antibody of interest. Here, we present Software for the Ontogenic aNalysis of Antibody Repertoires (SONAR), capable of automating both general repertoire analysis and specialized techniques for investigating specific lineages. SONAR annotates next-generation sequencing data, identifies transcripts in a lineage of interest, and tracks lineage development across multiple time points. SONAR also generates figures, such as identity–divergence plots and longitudinal phylogenetic “birthday” trees, and provides interfaces to other programs such as DNAML and BEAST. SONAR can be downloaded as a ready-to-run Docker image or manually installed on a local machine. In the latter case, it can also be configured to take advantage of a high-performance computing cluster for the most computationally intensive steps, if available. In summary, this software provides a useful new tool for the processing of large next-generation sequencing datasets and the ontogenic analysis of neutralizing antibody lineages. SONAR can be found at https://github.com/scharch/SONAR, and the Docker image can be obtained from https://hub.docker.com/r/scharch/sonar/. PMID:27708645

Easy-to-use phylogenetic analysis system for hepatitis B virus infection.

PubMed

Sugiyama, Masaya; Inui, Ayano; Shin-I, Tadasu; Komatsu, Haruki; Mukaide, Motokazu; Masaki, Naohiko; Murata, Kazumoto; Ito, Kiyoaki; Nakanishi, Makoto; Fujisawa, Tomoo; Mizokami, Masashi

2011-10-01

  The molecular phylogenetic analysis has been broadly applied to clinical and virological study. However, the appropriate settings and application of calculation parameters are difficult for non-specialists of molecular genetics. In the present study, the phylogenetic analysis tool was developed for the easy determination of genotypes and transmission route.   A total of 23 patients of 10 families infected with hepatitis B virus (HBV) were enrolled and expected to undergo intrafamilial transmission. The extracted HBV DNA were amplified and sequenced in a region of the S gene.   The software to automatically classify query sequence was constructed and installed on the Hepatitis Virus Database (HVDB). Reference sequences were retrieved from HVDB, which contained major genotypes from A to H. Multiple-alignments using CLUSTAL W were performed before the genetic distance matrix was calculated with the six-parameter method. The phylogenetic tree was output by the neighbor-joining method. User interface using WWW-browser was also developed for intuitive control. This system was named as the easy-to-use phylogenetic analysis system (E-PAS). Twenty-three sera of 10 families were analyzed to evaluate E-PAS. The queries obtained from nine families were genotype C and were located in one cluster per family. However, one patient of a family was classified into the cluster different from her family, suggesting that E-PAS detected the sample distinct from that of her family on the transmission route.   The E-PAS to output phylogenetic tree was developed since requisite material was sequence data only. E-PAS could expand to determine HBV genotypes as well as transmission routes. © 2011 The Japan Society of Hepatology.

Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis).

PubMed

Huang, Jinqiang; Li, Yongjuan; Shao, Changwei; Wang, Na; Chen, Songlin

2017-06-20

The nanos gene encodes an RNA-binding zinc finger protein, which is required in the development and maintenance of germ cells. However, there is very limited information about nanos in flatfish, which impedes its application in fish breeding. In this study, we report the molecular cloning, characterization and functional analysis of the 3'-untranslated region of the nanos gene (Csnanos) from half-smooth tongue sole (Cynoglossus semilaevis), which is an economically important flatfish in China. The 1233-bp cDNA sequence, 1709-bp genomic sequence and flanking sequences (2.8-kb 5'- and 1.6-kb 3'-flanking regions) of Csnanos were cloned and characterized. Sequence analysis revealed that CsNanos shares low homology with Nanos in other species, but the zinc finger domain of CsNanos is highly similar. Phylogenetic analysis indicated that CsNanos belongs to the Nanos2 subfamily. Csnanos expression was widely detected in various tissues, but the expression level was higher in testis and ovary. During early development and sex differentiation, Csnanos expression exhibited a clear sexually dimorphic pattern, suggesting its different roles in the migration and differentiation of primordial germ cells (PGCs). Higher expression levels of Csnanos mRNA in normal females and males than in neomales indicated that the nanos gene may play key roles in maintaining the differentiation of gonad. Moreover, medaka PGCs were successfully labeled by the microinjection of synthesized mRNA consisting of green fluorescence protein and the 3'-untranslated region of Csnanos. These findings provide new insights into nanos gene expression and function, and lay the foundation for further study of PGC development and applications in tongue sole breeding. Copyright © 2017 Elsevier B.V. All rights reserved.

Elements of Mathematics, Book O: Intuitive Background. Chapter 14, Geometry: Similitudes, Coordinates, and Trigonometry.

ERIC Educational Resources Information Center

Exner, Robert; And Others

The sixteen chapters of this book provide the core material for the Elements of Mathematics Program, a secondary sequence developed for highly motivated students with strong verbal abilities. The sequence is based on a functional-relational approach to mathematics teaching, and emphasizes teaching by analysis of real-life situations. This text is…

Cloning of the poly(ADP-ribose) Gene from Rat Liver.

DTIC Science & Technology

1986-09-24

Levinson, Ph.D. (Cetus Corp., Berkeley). 5. Amino acid analysis done in UCSF Bioanal. Lab. TABLE OF CONTENTS Page METHOD I...TABLE I ............. ............................... ... 12 Proteolytic degradation, isolation of peptide and amino acid sequences...technique developed for enzyme quantitation in biological materials. The amino- acid sequence of the enzyme has so far been determined because the amino

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

PubMed

Hebert, Paul D N; Dewaard, Jeremy R; Zakharov, Evgeny V; Prosser, Sean W J; Sones, Jayme E; McKeown, Jaclyn T A; Mantle, Beth; La Salle, John

2013-01-01

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

In-silico mining, type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS-LRR regions of finger millet with rice.

PubMed

Kalyana Babu, B; Pandey, Dinesh; Agrawal, P K; Sood, Salej; Kumar, Anil

2014-05-01

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS-LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS-LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

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

Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric

2010-03-23

Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities tomore » known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.« less

SeqHBase: a big data toolset for family based sequencing data analysis.

PubMed

He, Min; Person, Thomas N; Hebbring, Scott J; Heinzen, Ethan; Ye, Zhan; Schrodi, Steven J; McPherson, Elizabeth W; Lin, Simon M; Peissig, Peggy L; Brilliant, Murray H; O'Rawe, Jason; Robison, Reid J; Lyon, Gholson J; Wang, Kai

2015-04-01

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies are increasingly used to identify disease-contributing mutations in human genomic studies. It can be a significant challenge to process such data, especially when a large family or cohort is sequenced. Our objective was to develop a big data toolset to efficiently manipulate genome-wide variants, functional annotations and coverage, together with conducting family based sequencing data analysis. Hadoop is a framework for reliable, scalable, distributed processing of large data sets using MapReduce programming models. Based on Hadoop and HBase, we developed SeqHBase, a big data-based toolset for analysing family based sequencing data to detect de novo, inherited homozygous, or compound heterozygous mutations that may contribute to disease manifestations. SeqHBase takes as input BAM files (for coverage at every site), variant call format (VCF) files (for variant calls) and functional annotations (for variant prioritisation). We applied SeqHBase to a 5-member nuclear family and a 10-member 3-generation family with WGS data, as well as a 4-member nuclear family with WES data. Analysis times were almost linearly scalable with number of data nodes. With 20 data nodes, SeqHBase took about 5 secs to analyse WES familial data and approximately 1 min to analyse WGS familial data. These results demonstrate SeqHBase's high efficiency and scalability, which is necessary as WGS and WES are rapidly becoming standard methods to study the genetics of familial disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM)☆

PubMed Central

Parson, Walther; Strobl, Christina; Huber, Gabriela; Zimmermann, Bettina; Gomes, Sibylle M.; Souto, Luis; Fendt, Liane; Delport, Rhena; Langit, Reina; Wootton, Sharon; Lagacé, Robert; Irwin, Jodi

2013-01-01

Insights into the human mitochondrial phylogeny have been primarily achieved by sequencing full mitochondrial genomes (mtGenomes). In forensic genetics (partial) mtGenome information can be used to assign haplotypes to their phylogenetic backgrounds, which may, in turn, have characteristic geographic distributions that would offer useful information in a forensic case. In addition and perhaps even more relevant in the forensic context, haplogroup-specific patterns of mutations form the basis for quality control of mtDNA sequences. The current method for establishing (partial) mtDNA haplotypes is Sanger-type sequencing (STS), which is laborious, time-consuming, and expensive. With the emergence of Next Generation Sequencing (NGS) technologies, the body of available mtDNA data can potentially be extended much more quickly and cost-efficiently. Customized chemistries, laboratory workflows and data analysis packages could support the community and increase the utility of mtDNA analysis in forensics. We have evaluated the performance of mtGenome sequencing using the Personal Genome Machine (PGM) and compared the resulting haplotypes directly with conventional Sanger-type sequencing. A total of 64 mtGenomes (>1 million bases) were established that yielded high concordance with the corresponding STS haplotypes (<0.02% differences). About two-thirds of the differences were observed in or around homopolymeric sequence stretches. In addition, the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of alignment software would be desirable to facilitate the application of NGS in mtDNA forensic genetics. PMID:23948325

Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth

PubMed Central

Marcy, Yann; Ouverney, Cleber; Bik, Elisabeth M.; Lösekann, Tina; Ivanova, Natalia; Martin, Hector Garcia; Szeto, Ernest; Platt, Darren; Hugenholtz, Philip; Relman, David A.; Quake, Stephen R.

2007-01-01

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community. PMID:17620602

Draft Genome Sequence of the Yeast Starmerella bombicola NBRC10243, a Producer of Sophorolipids, Glycolipid Biosurfactants

PubMed Central

Matsuzawa, Tomohiko; Koike, Hideaki; Saika, Azusa; Fukuoka, Tokuma; Sato, Shun; Habe, Hiroshi; Kitamoto, Dai

2015-01-01

The yeast Starmerella bombicola NBRC10243 is an excellent producer of sophorolipids (SLs) from various feedstocks. Here, we report the draft genome sequence of S. bombicola NBRC10243. Analysis of the sequence may provide insight into the properties of this yeast that make it superior for use in the production of functional glycolipids and biomolecules, leading to the further development of S. bombicola NBRC10243 for industrial applications. PMID:25814600

Genome sequence of the white koji mold Aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu.

PubMed

Futagami, Taiki; Mori, Kazuki; Yamashita, Ayaka; Wada, Shotaro; Kajiwara, Yasuhiro; Takashita, Hideharu; Omori, Toshiro; Takegawa, Kaoru; Tashiro, Kosuke; Kuhara, Satoru; Goto, Masatoshi

2011-11-01

The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications.

CBrowse: a SAM/BAM-based contig browser for transcriptome assembly visualization and analysis.

PubMed

Li, Pei; Ji, Guoli; Dong, Min; Schmidt, Emily; Lenox, Douglas; Chen, Liangliang; Liu, Qi; Liu, Lin; Zhang, Jie; Liang, Chun

2012-09-15

To address the impending need for exploring rapidly increased transcriptomics data generated for non-model organisms, we developed CBrowse, an AJAX-based web browser for visualizing and analyzing transcriptome assemblies and contigs. Designed in a standard three-tier architecture with a data pre-processing pipeline, CBrowse is essentially a Rich Internet Application that offers many seamlessly integrated web interfaces and allows users to navigate, sort, filter, search and visualize data smoothly. The pre-processing pipeline takes the contig sequence file in FASTA format and its relevant SAM/BAM file as the input; detects putative polymorphisms, simple sequence repeats and sequencing errors in contigs and generates image, JSON and database-compatible CSV text files that are directly utilized by different web interfaces. CBowse is a generic visualization and analysis tool that facilitates close examination of assembly quality, genetic polymorphisms, sequence repeats and/or sequencing errors in transcriptome sequencing projects. CBrowse is distributed under the GNU General Public License, available at http://bioinfolab.muohio.edu/CBrowse/ liangc@muohio.edu or liangc.mu@gmail.com; glji@xmu.edu.cn Supplementary data are available at Bioinformatics online.

A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

PubMed

Ohshima, Chihiro; Takahashi, Hajime; Iwakawa, Ai; Kuda, Takashi; Kimura, Bon

2017-07-17

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future. Copyright © 2017. Published by Elsevier B.V.

Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome

PubMed Central

2011-01-01

Background One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. Results The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. Conclusions This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak. PMID:21645357

Sequence Search and Comparative Genomic Analysis of SUMO-Activating Enzymes Using CoGe.

PubMed

Carretero-Paulet, Lorenzo; Albert, Victor A

2016-01-01

The growing number of genome sequences completed during the last few years has made necessary the development of bioinformatics tools for the easy access and retrieval of sequence data, as well as for downstream comparative genomic analyses. Some of these are implemented as online platforms that integrate genomic data produced by different genome sequencing initiatives with data mining tools as well as various comparative genomic and evolutionary analysis possibilities.Here, we use the online comparative genomics platform CoGe ( http://www.genomevolution.org/coge/ ) (Lyons and Freeling. Plant J 53:661-673, 2008; Tang and Lyons. Front Plant Sci 3:172, 2012) (1) to retrieve the entire complement of orthologous and paralogous genes belonging to the SUMO-Activating Enzymes 1 (SAE1) gene family from a set of species representative of the Brassicaceae plant eudicot family with genomes fully sequenced, and (2) to investigate the history, timing, and molecular mechanisms of the gene duplications driving the evolutionary expansion and functional diversification of the SAE1 family in Brassicaceae.

Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

PubMed Central

Lin, Jinke; Kudrna, Dave; Wing, Rod A.

2011-01-01

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers. PMID:21234344

Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2.

PubMed

Dong, Lanlan; Zhou, Simin; He, Yuan; Jia, Yan; Bai, Qunhua; Deng, Peng; Gao, Jieying; Li, Yingli; Xiao, Hong

2018-05-01

This study is to investigate the genome sequence of Serratia sp. S2. The genomic DNA of Serratia sp. S2 was extracted and the sequencing library was constructed. The sequencing was carried out by Illumina 2000 and complete genomic sequences were obtained. Gene function annotation and bioinformatics analysis were performed by comparing with the known databases. The genome size of Serratia sp. S2 was 5,604,115 bp and the G+C content was 57.61%. There were 5373 protein coding genes, and 3732, 3614, and 3942 genes were respectively annotated into the GO, KEGG, and COG databases. There were 12 genes related to chromium metabolism in the Serratia sp. S2 genome. The whole genome sequence of Serratia sp. S2 is submitted to the GenBank database with gene accession number of LNRP00000000. Our findings may provide theoretical basis for the subsequent development of new biotechnology to repair environmental chromium pollution.

BioPig: a Hadoop-based analytic toolkit for large-scale sequence data.

PubMed

Nordberg, Henrik; Bhatia, Karan; Wang, Kai; Wang, Zhong

2013-12-01

The recent revolution in sequencing technologies has led to an exponential growth of sequence data. As a result, most of the current bioinformatics tools become obsolete as they fail to scale with data. To tackle this 'data deluge', here we introduce the BioPig sequence analysis toolkit as one of the solutions that scale to data and computation. We built BioPig on the Apache's Hadoop MapReduce system and the Pig data flow language. Compared with traditional serial and MPI-based algorithms, BioPig has three major advantages: first, BioPig's programmability greatly reduces development time for parallel bioinformatics applications; second, testing BioPig with up to 500 Gb sequences demonstrates that it scales automatically with size of data; and finally, BioPig can be ported without modification on many Hadoop infrastructures, as tested with Magellan system at National Energy Research Scientific Computing Center and the Amazon Elastic Compute Cloud. In summary, BioPig represents a novel program framework with the potential to greatly accelerate data-intensive bioinformatics analysis.

B-MIC: An Ultrafast Three-Level Parallel Sequence Aligner Using MIC.

PubMed

Cui, Yingbo; Liao, Xiangke; Zhu, Xiaoqian; Wang, Bingqiang; Peng, Shaoliang

2016-03-01

Sequence alignment is the central process for sequence analysis, where mapping raw sequencing data to reference genome. The large amount of data generated by NGS is far beyond the process capabilities of existing alignment tools. Consequently, sequence alignment becomes the bottleneck of sequence analysis. Intensive computing power is required to address this challenge. Intel recently announced the MIC coprocessor, which can provide massive computing power. The Tianhe-2 is the world's fastest supercomputer now equipped with three MIC coprocessors each compute node. A key feature of sequence alignment is that different reads are independent. Considering this property, we proposed a MIC-oriented three-level parallelization strategy to speed up BWA, a widely used sequence alignment tool, and developed our ultrafast parallel sequence aligner: B-MIC. B-MIC contains three levels of parallelization: firstly, parallelization of data IO and reads alignment by a three-stage parallel pipeline; secondly, parallelization enabled by MIC coprocessor technology; thirdly, inter-node parallelization implemented by MPI. In this paper, we demonstrate that B-MIC outperforms BWA by a combination of those techniques using Inspur NF5280M server and the Tianhe-2 supercomputer. To the best of our knowledge, B-MIC is the first sequence alignment tool to run on Intel MIC and it can achieve more than fivefold speedup over the original BWA while maintaining the alignment precision.

DNA Clutch Probes for Circulating Tumor DNA Analysis.

PubMed

Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

2016-08-31

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

Endosymbiotic Microbiota of the Bamboo Pseudococcid Antonina crawii (Insecta, Homoptera)

PubMed Central

Fukatsu, Takema; Nikoh, Naruo

2000-01-01

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the γ subdivision of the division Proteobacteria (γ-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a β-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The γ- and β-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the γ- and β-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the β-Proteobacteria symbiont was detected in all five pseudococcids, the γ-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii. PMID:10653730

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data

PubMed Central

Watson, Christopher M.; Crinnion, Laura A.; Gurgel‐Gianetti, Juliana; Harrison, Sally M.; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F.; Pena, Sergio D. J.; Bonthron, David T.

2015-01-01

ABSTRACT Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease‐causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome‐wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution. PMID:26037133

MIPS: analysis and annotation of proteins from whole genomes

PubMed Central

Mewes, H. W.; Amid, C.; Arnold, R.; Frishman, D.; Güldener, U.; Mannhaupt, G.; Münsterkötter, M.; Pagel, P.; Strack, N.; Stümpflen, V.; Warfsmann, J.; Ruepp, A.

2004-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein–protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de). PMID:14681354

MIPS: analysis and annotation of proteins from whole genomes.

PubMed

Mewes, H W; Amid, C; Arnold, R; Frishman, D; Güldener, U; Mannhaupt, G; Münsterkötter, M; Pagel, P; Strack, N; Stümpflen, V; Warfsmann, J; Ruepp, A

2004-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein-protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de).

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

PubMed Central

Blanchard, Adam M.; Egan, Sharon A.; Emes, Richard D.; Warry, Andrew; Leigh, James A.

2016-01-01

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance. PMID:27826289

De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube.

PubMed

Iaria, Domenico; Chiappetta, Adriana; Muzzalupo, Innocenzo

2016-01-01

In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca(2+) binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

The technology and biology of single-cell RNA sequencing.

PubMed

Kolodziejczyk, Aleksandra A; Kim, Jong Kyoung; Svensson, Valentine; Marioni, John C; Teichmann, Sarah A

2015-05-21

The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

NABIC: A New Access Portal to Search, Visualize, and Share Agricultural Genomics Data.

PubMed

Seol, Young-Joo; Lee, Tae-Ho; Park, Dong-Suk; Kim, Chang-Kug

2016-01-01

The National Agricultural Biotechnology Information Center developed an access portal to search, visualize, and share agricultural genomics data with a focus on South Korean information and resources. The portal features an agricultural biotechnology database containing a wide range of omics data from public and proprietary sources. We collected 28.4 TB of data from 162 agricultural organisms, with 10 types of omics data comprising next-generation sequencing sequence read archive, genome, gene, nucleotide, DNA chip, expressed sequence tag, interactome, protein structure, molecular marker, and single-nucleotide polymorphism datasets. Our genomic resources contain information on five animals, seven plants, and one fungus, which is accessed through a genome browser. We also developed a data submission and analysis system as a web service, with easy-to-use functions and cutting-edge algorithms, including those for handling next-generation sequencing data.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

A 1,681-locus consensus genetic map of cultivated cucumber including 67 NB-LRR resistance gene homolog and ten gene loci

PubMed Central

2013-01-01

Background Cucumber is an important vegetable crop that is susceptible to many pathogens, but no disease resistance (R) genes have been cloned. The availability of whole genome sequences provides an excellent opportunity for systematic identification and characterization of the nucleotide binding and leucine-rich repeat (NB-LRR) type R gene homolog (RGH) sequences in the genome. Cucumber has a very narrow genetic base making it difficult to construct high-density genetic maps. Development of a consensus map by synthesizing information from multiple segregating populations is a method of choice to increase marker density. As such, the objectives of the present study were to identify and characterize NB-LRR type RGHs, and to develop a high-density, integrated cucumber genetic-physical map anchored with RGH loci. Results From the Gy14 draft genome, 70 NB-containing RGHs were identified and characterized. Most RGHs were in clusters with uneven distribution across seven chromosomes. In silico analysis indicated that all 70 RGHs had EST support for gene expression. Phylogenetic analysis classified 58 RGHs into two clades: CNL and TNL. Comparative analysis revealed high-degree sequence homology and synteny in chromosomal locations of these RGH members between the cucumber and melon genomes. Fifty-four molecular markers were developed to delimit 67 of the 70 RGHs, which were integrated into a genetic map through linkage analysis. A 1,681-locus cucumber consensus map including 10 gene loci and spanning 730.0 cM in seven linkage groups was developed by integrating three component maps with a bin-mapping strategy. Physically, 308 scaffolds with 193.2 Mbp total DNA sequences were anchored onto this consensus map that covered 52.6% of the 367 Mbp cucumber genome. Conclusions Cucumber contains relatively few NB-LRR RGHs that are clustered and unevenly distributed in the genome. All RGHs seem to be transcribed and shared significant sequence homology and synteny with the melon genome suggesting conservation of these RGHs in the Cucumis lineage. The 1,681-locus consensus genetic-physical map developed and the RGHs identified and characterized herein are valuable genomics resources that may have many applications such as quantitative trait loci identification, map-based gene cloning, association mapping, marker-assisted selection, as well as assembly of a more complete cucumber genome. PMID:23531125

SeqCompress: an algorithm for biological sequence compression.

PubMed

Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz; Bajwa, Hassan

2014-10-01

The growth of Next Generation Sequencing technologies presents significant research challenges, specifically to design bioinformatics tools that handle massive amount of data efficiently. Biological sequence data storage cost has become a noticeable proportion of total cost in the generation and analysis. Particularly increase in DNA sequencing rate is significantly outstripping the rate of increase in disk storage capacity, which may go beyond the limit of storage capacity. It is essential to develop algorithms that handle large data sets via better memory management. This article presents a DNA sequence compression algorithm SeqCompress that copes with the space complexity of biological sequences. The algorithm is based on lossless data compression and uses statistical model as well as arithmetic coding to compress DNA sequences. The proposed algorithm is compared with recent specialized compression tools for biological sequences. Experimental results show that proposed algorithm has better compression gain as compared to other existing algorithms. Copyright © 2014 Elsevier Inc. All rights reserved.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

PubMed

Zackay, Arie; Steinhoff, Christine

2010-12-15

Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

PubMed Central

2010-01-01

Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

PubMed

Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Mu-Chiou; Wang, Li-Tin; Huang, Lina; Lee, Fwu-Ling

2012-10-01

To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing. Copyright © 2012 Society of Chemical Industry.

Comparative genome analysis in the integrated microbial genomes (IMG) system.

PubMed

Markowitz, Victor M; Kyrpides, Nikos C

2007-01-01

Comparative genome analysis is critical for the effective exploration of a rapidly growing number of complete and draft sequences for microbial genomes. The Integrated Microbial Genomes (IMG) system (img.jgi.doe.gov) has been developed as a community resource that provides support for comparative analysis of microbial genomes in an integrated context. IMG allows users to navigate the multidimensional microbial genome data space and focus their analysis on a subset of genes, genomes, and functions of interest. IMG provides graphical viewers, summaries, and occurrence profile tools for comparing genes, pathways, and functions (terms) across specific genomes. Genes can be further examined using gene neighborhoods and compared with sequence alignment tools.

Verification of nonlinear dynamic structural test results by combined image processing and acoustic analysis

NASA Astrophysics Data System (ADS)

Tene, Yair; Tene, Noam; Tene, G.

1993-08-01

An interactive data fusion methodology of video, audio, and nonlinear structural dynamic analysis for potential application in forensic engineering is presented. The methodology was developed and successfully demonstrated in the analysis of heavy transportable bridge collapse during preparation for testing. Multiple bridge elements failures were identified after the collapse, including fracture, cracks and rupture of high performance structural materials. Videotape recording by hand held camcorder was the only source of information about the collapse sequence. The interactive data fusion methodology resulted in extracting relevant information form the videotape and from dynamic nonlinear structural analysis, leading to full account of the sequence of events during the bridge collapse.

De Novo Sequencing and Characterization of the Floral Transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae)

PubMed Central

Li, De-Zhu; Guo, Zhen-Hua

2012-01-01

Background Transcriptome sequencing can be used to determine gene sequences and transcript abundance in non-model species, and the advent of next-generation sequencing (NGS) technologies has greatly decreased the cost and time required for this process. Transcriptome data are especially desirable in bamboo species, as certain members constitute an economically and culturally important group of mostly semelparous plants with remarkable flowering features, yet little bamboo genomic research has been performed. Here we present, for the first time, extensive sequence and transcript abundance data for the floral transcriptome of a key bamboo species, Dendrocalamus latiflorus, obtained using the Illumina GAII sequencing platform. Our further goal was to identify patterns of gene expression during bamboo flower development. Results Approximately 96 million sequencing reads were generated and assembled de novo, yielding 146,395 high quality unigenes with an average length of 461 bp. Of these, 80,418 were identified as putative homologs of annotated sequences in the public protein databases, of which 290 were associated with the floral transition and 47 were related to flower development. Digital abundance analysis identified 26,529 transcripts differentially enriched between two developmental stages, young flower buds and older developing flowers. Unigenes found at each stage were categorized according to their putative functional categories. These sequence and putative function data comprise a resource for future investigation of the floral transition and flower development in bamboo species. Conclusions Our results present the first broad survey of a bamboo floral transcriptome. Although it will be necessary to validate the functions carried out by these genes, these results represent a starting point for future functional research on D. latiflorus and related species. PMID:22916120

A weighted U-statistic for genetic association analyses of sequencing data.

PubMed

Wei, Changshuai; Li, Ming; He, Zihuai; Vsevolozhskaya, Olga; Schaid, Daniel J; Lu, Qing

2014-12-01

With advancements in next-generation sequencing technology, a massive amount of sequencing data is generated, which offers a great opportunity to comprehensively investigate the role of rare variants in the genetic etiology of complex diseases. Nevertheless, the high-dimensional sequencing data poses a great challenge for statistical analysis. The association analyses based on traditional statistical methods suffer substantial power loss because of the low frequency of genetic variants and the extremely high dimensionality of the data. We developed a Weighted U Sequencing test, referred to as WU-SEQ, for the high-dimensional association analysis of sequencing data. Based on a nonparametric U-statistic, WU-SEQ makes no assumption of the underlying disease model and phenotype distribution, and can be applied to a variety of phenotypes. Through simulation studies and an empirical study, we showed that WU-SEQ outperformed a commonly used sequence kernel association test (SKAT) method when the underlying assumptions were violated (e.g., the phenotype followed a heavy-tailed distribution). Even when the assumptions were satisfied, WU-SEQ still attained comparable performance to SKAT. Finally, we applied WU-SEQ to sequencing data from the Dallas Heart Study (DHS), and detected an association between ANGPTL 4 and very low density lipoprotein cholesterol. © 2014 WILEY PERIODICALS, INC.

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects.

PubMed

Pardo, Carolina E; Carr, Ian M; Hoffman, Christopher J; Darst, Russell P; Markham, Alexander F; Bonthron, David T; Kladde, Michael P

2011-01-01

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

PubMed

Lindblad-Toh, Kerstin; Wade, Claire M; Mikkelsen, Tarjei S; Karlsson, Elinor K; Jaffe, David B; Kamal, Michael; Clamp, Michele; Chang, Jean L; Kulbokas, Edward J; Zody, Michael C; Mauceli, Evan; Xie, Xiaohui; Breen, Matthew; Wayne, Robert K; Ostrander, Elaine A; Ponting, Chris P; Galibert, Francis; Smith, Douglas R; DeJong, Pieter J; Kirkness, Ewen; Alvarez, Pablo; Biagi, Tara; Brockman, William; Butler, Jonathan; Chin, Chee-Wye; Cook, April; Cuff, James; Daly, Mark J; DeCaprio, David; Gnerre, Sante; Grabherr, Manfred; Kellis, Manolis; Kleber, Michael; Bardeleben, Carolyne; Goodstadt, Leo; Heger, Andreas; Hitte, Christophe; Kim, Lisa; Koepfli, Klaus-Peter; Parker, Heidi G; Pollinger, John P; Searle, Stephen M J; Sutter, Nathan B; Thomas, Rachael; Webber, Caleb; Baldwin, Jennifer; Abebe, Adal; Abouelleil, Amr; Aftuck, Lynne; Ait-Zahra, Mostafa; Aldredge, Tyler; Allen, Nicole; An, Peter; Anderson, Scott; Antoine, Claudel; Arachchi, Harindra; Aslam, Ali; Ayotte, Laura; Bachantsang, Pasang; Barry, Andrew; Bayul, Tashi; Benamara, Mostafa; Berlin, Aaron; Bessette, Daniel; Blitshteyn, Berta; Bloom, Toby; Blye, Jason; Boguslavskiy, Leonid; Bonnet, Claude; Boukhgalter, Boris; Brown, Adam; Cahill, Patrick; Calixte, Nadia; Camarata, Jody; Cheshatsang, Yama; Chu, Jeffrey; Citroen, Mieke; Collymore, Alville; Cooke, Patrick; Dawoe, Tenzin; Daza, Riza; Decktor, Karin; DeGray, Stuart; Dhargay, Norbu; Dooley, Kimberly; Dooley, Kathleen; Dorje, Passang; Dorjee, Kunsang; Dorris, Lester; Duffey, Noah; Dupes, Alan; Egbiremolen, Osebhajajeme; Elong, Richard; Falk, Jill; Farina, Abderrahim; Faro, Susan; Ferguson, Diallo; Ferreira, Patricia; Fisher, Sheila; FitzGerald, Mike; Foley, Karen; Foley, Chelsea; Franke, Alicia; Friedrich, Dennis; Gage, Diane; Garber, Manuel; Gearin, Gary; Giannoukos, Georgia; Goode, Tina; Goyette, Audra; Graham, Joseph; Grandbois, Edward; Gyaltsen, Kunsang; Hafez, Nabil; Hagopian, Daniel; Hagos, Birhane; Hall, Jennifer; Healy, Claire; Hegarty, Ryan; Honan, Tracey; Horn, Andrea; Houde, Nathan; Hughes, Leanne; Hunnicutt, Leigh; Husby, M; Jester, Benjamin; Jones, Charlien; Kamat, Asha; Kanga, Ben; Kells, Cristyn; Khazanovich, Dmitry; Kieu, Alix Chinh; Kisner, Peter; Kumar, Mayank; Lance, Krista; Landers, Thomas; Lara, Marcia; Lee, William; Leger, Jean-Pierre; Lennon, Niall; Leuper, Lisa; LeVine, Sarah; Liu, Jinlei; Liu, Xiaohong; Lokyitsang, Yeshi; Lokyitsang, Tashi; Lui, Annie; Macdonald, Jan; Major, John; Marabella, Richard; Maru, Kebede; Matthews, Charles; McDonough, Susan; Mehta, Teena; Meldrim, James; Melnikov, Alexandre; Meneus, Louis; Mihalev, Atanas; Mihova, Tanya; Miller, Karen; Mittelman, Rachel; Mlenga, Valentine; Mulrain, Leonidas; Munson, Glen; Navidi, Adam; Naylor, Jerome; Nguyen, Tuyen; Nguyen, Nga; Nguyen, Cindy; Nguyen, Thu; Nicol, Robert; Norbu, Nyima; Norbu, Choe; Novod, Nathaniel; Nyima, Tenchoe; Olandt, Peter; O'Neill, Barry; O'Neill, Keith; Osman, Sahal; Oyono, Lucien; Patti, Christopher; Perrin, Danielle; Phunkhang, Pema; Pierre, Fritz; Priest, Margaret; Rachupka, Anthony; Raghuraman, Sujaa; Rameau, Rayale; Ray, Verneda; Raymond, Christina; Rege, Filip; Rise, Cecil; Rogers, Julie; Rogov, Peter; Sahalie, Julie; Settipalli, Sampath; Sharpe, Theodore; Shea, Terrance; Sheehan, Mechele; Sherpa, Ngawang; Shi, Jianying; Shih, Diana; Sloan, Jessie; Smith, Cherylyn; Sparrow, Todd; Stalker, John; Stange-Thomann, Nicole; Stavropoulos, Sharon; Stone, Catherine; Stone, Sabrina; Sykes, Sean; Tchuinga, Pierre; Tenzing, Pema; Tesfaye, Senait; Thoulutsang, Dawa; Thoulutsang, Yama; Topham, Kerri; Topping, Ira; Tsamla, Tsamla; Vassiliev, Helen; Venkataraman, Vijay; Vo, Andy; Wangchuk, Tsering; Wangdi, Tsering; Weiand, Michael; Wilkinson, Jane; Wilson, Adam; Yadav, Shailendra; Yang, Shuli; Yang, Xiaoping; Young, Geneva; Yu, Qing; Zainoun, Joanne; Zembek, Lisa; Zimmer, Andrew; Lander, Eric S

2005-12-08

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

Analyzing ion distributions around DNA: sequence-dependence of potassium ion distributions from microsecond molecular dynamics

PubMed Central

Pasi, Marco; Maddocks, John H.; Lavery, Richard

2015-01-01

Microsecond molecular dynamics simulations of B-DNA oligomers carried out in an aqueous environment with a physiological salt concentration enable us to perform a detailed analysis of how potassium ions interact with the double helix. The oligomers studied contain all 136 distinct tetranucleotides and we are thus able to make a comprehensive analysis of base sequence effects. Using a recently developed curvilinear helicoidal coordinate method we are able to analyze the details of ion populations and densities within the major and minor grooves and in the space surrounding DNA. The results show higher ion populations than have typically been observed in earlier studies and sequence effects that go beyond the nature of individual base pairs or base pair steps. We also show that, in some special cases, ion distributions converge very slowly and, on a microsecond timescale, do not reflect the symmetry of the corresponding base sequence. PMID:25662221

Top-down analysis of protein samples by de novo sequencing techniques

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

Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.

MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. Themore » former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns.« less

Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

PubMed

Siju, S; Dhanya, K; Syamkumar, S; Sasikumar, B; Sheeja, T E; Bhat, A I; Parthasarathy, V A

2010-02-01

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.

Development of 23 novel polymorphic EST-SSR markers for the endangered relict conifer Metasequoia glyptostroboides.

PubMed

Jin, Yuqing; Bi, Quanxin; Guan, Wenbin; Mao, Jian-Feng

2015-09-01

Metasequoia glyptostroboides is an endangered relict conifer species endemic to China. In this study, expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed using transcriptome mining for future genetic and functional studies. We collected 97,565 unigene sequences generated by 454 pyrosequencing. A bioinformatics analysis identified 2087 unique and putative microsatellites, from which 96 novel microsatellite markers were developed. Fifty-three of the 96 primer sets successfully amplified clear fragments of the expected sizes; 23 of those loci were polymorphic. The number of alleles per locus ranged from two to eight, with an average of three, and the observed and expected heterozygosity values ranged from 0 to 1.0 and 0.117 to 0.813, respectively. These microsatellite loci will enrich the genetic resources to develop functional studies and conservation strategies for this endangered relict species.

Development of 23 novel polymorphic EST-SSR markers for the endangered relict conifer Metasequoia glyptostroboides1

PubMed Central

Jin, Yuqing; Bi, Quanxin; Guan, Wenbin; Mao, Jian-Feng

2015-01-01

Premise of the study: Metasequoia glyptostroboides is an endangered relict conifer species endemic to China. In this study, expressed sequence tag–simple sequence repeat (EST-SSR) markers were developed using transcriptome mining for future genetic and functional studies. Methods and Results: We collected 97,565 unigene sequences generated by 454 pyrosequencing. A bioinformatics analysis identified 2087 unique and putative microsatellites, from which 96 novel microsatellite markers were developed. Fifty-three of the 96 primer sets successfully amplified clear fragments of the expected sizes; 23 of those loci were polymorphic. The number of alleles per locus ranged from two to eight, with an average of three, and the observed and expected heterozygosity values ranged from 0 to 1.0 and 0.117 to 0.813, respectively. Conclusions: These microsatellite loci will enrich the genetic resources to develop functional studies and conservation strategies for this endangered relict species. PMID:26421250

Process development for automated solar cell and module production. Task 4: Automated array assembly

NASA Technical Reports Server (NTRS)

1980-01-01

A process sequence which can be used in conjunction with automated equipment for the mass production of solar cell modules for terrestrial use was developed. The process sequence was then critically analyzed from a technical and economic standpoint to determine the technological readiness of certain process steps for implementation. The steps receiving analysis were: back contact metallization, automated cell array layup/interconnect, and module edge sealing. For automated layup/interconnect, both hard automation and programmable automation (using an industrial robot) were studied. The programmable automation system was then selected for actual hardware development.

Unique BK virus non-coding control region (NCCR) variants in hematopoietic stem cell transplant recipients with and without hemorrhagic cystitis.

PubMed

Carr, Michael J; McCormack, Grace P; Mutton, Ken J; Crowley, Brendan

2006-04-01

Hematopoietic stem cell transplant recipients frequently develop BK virus (BKV)-associated hemorrhagic cystitis, which coincides with BK viruria. However, the precise role of BKV in the etiology of hemorrhagic cystitis in hematopoietic stem cell transplant recipients remains unclear, since approximately 50% of all such adult transplant recipients excrete BKV, yet do not develop this clinical condition. In the present study, BKV were analyzed to determine if mutations in the non-coding control region (NCCR), and specific BKV sub-types defined by sequence analysis of major capsid protein VP1, were associated with development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. The regions encoding VP1 and NCCRs of BKV in urine samples collected from 15 hematopoietic stem cell transplant recipients with hemorrhagic cystitis and 20 without this illness were amplified and sequenced. Sequence variations in the NCCRs of BKV were identified in urine samples from those with and without hemorrhagic cystitis. Furthermore, five unique sequence variations within transcription factor binding sites in the canonical NCCR, O-P-Q-R-S, were identified, representing new BKV variants from a population of cloned quasi-species obtained from patients with and without hemorrhagic cystitis. Thirty-five BKV VP1 sequences were analyzed by phylogenetic analysis but no specific BKV sub-type was associated with hemorrhagic cystitis. Five previously unrecognized naturally occurring variants of the BKV are described which involve amplifications, deletions, and rearrangements of the archetypal BKV NCCRs in individuals with and without hemorrhagic cystitis. Architectural rearrangements in the NCCRs of BKV did not appear to be a prerequisite for development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. Copyright 2006 Wiley-Liss, Inc.

Insights into the fold organization of TIM barrel from interaction energy based structure networks.

PubMed

Vijayabaskar, M S; Vishveshwara, Saraswathi

2012-01-01

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

Overcoming bias and systematic errors in next generation sequencing data.

PubMed

Taub, Margaret A; Corrada Bravo, Hector; Irizarry, Rafael A

2010-12-10

Considerable time and effort has been spent in developing analysis and quality assessment methods to allow the use of microarrays in a clinical setting. As is the case for microarrays and other high-throughput technologies, data from new high-throughput sequencing technologies are subject to technological and biological biases and systematic errors that can impact downstream analyses. Only when these issues can be readily identified and reliably adjusted for will clinical applications of these new technologies be feasible. Although much work remains to be done in this area, we describe consistently observed biases that should be taken into account when analyzing high-throughput sequencing data. In this article, we review current knowledge about these biases, discuss their impact on analysis results, and propose solutions.

GenomePeek—an online tool for prokaryotic genome and metagenome analysis

DOE PAGES

McNair, Katelyn; Edwards, Robert A.

2015-06-16

As increases in prokaryotic sequencing take place, a method to quickly and accurately analyze this data is needed. Previous tools are mainly designed for metagenomic analysis and have limitations; such as long runtimes and significant false positive error rates. The online tool GenomePeek (edwards.sdsu.edu/GenomePeek) was developed to analyze both single genome and metagenome sequencing files, quickly and with low error rates. GenomePeek uses a sequence assembly approach where reads to a set of conserved genes are extracted, assembled and then aligned against the highly specific reference database. GenomePeek was found to be faster than traditional approaches while still keeping errormore » rates low, as well as offering unique data visualization options.« less

Facies analysis and sequence stratigraphic framework of upper Campanian strata (Neslen and Mount Garfield formations, Bluecastle Tongue of the Castlegate sandstone, and Mancos shale), Eastern Book cliffs, Colorado and Utah

USGS Publications Warehouse

Kirschbaum, Mark A.; Hettinger, Robert D.

2004-01-01

Facies and sequence-stratigraphic analysis identifies six high-resolution sequences within upper Campanian strata across about 120 miles of the Book Cliffs in western Colorado and eastern Utah. The six sequences are named after prominent sandstone units and include, in ascending order, upper Sego sequence, Neslen sequence, Corcoran sequence, Buck Canyon/lower Cozzette sequence, upper Cozzette sequence, and Cozzette/Rollins sequence. A seventh sequence, the Bluecastle sequence, is present in the extreme western part of the study area. Facies analysis documents deepening- and shallowing- upward successions, parasequence stacking patterns, downlap in subsurface cross sections, facies dislocations, basinward shifts in facies, and truncation of strata.All six sequences display major incision into shoreface deposits of the Sego Sandstone and sandstones of the Corcoran and Cozzette Members of the Mount Garfield Formation. The incised surfaces represent sequence-boundary unconformities that allowed bypass of sediment to lowstand shorelines that are either attached to the older highstand shorelines or are detached from the older highstand shorelines and located southeast of the main study area. The sequence boundary unconformities represent valley incisions that were cut during successive lowstands of relative sea level. The overlying valley-fill deposits generally consist of tidally influenced strata deposited during an overall base level rise. Transgressive surfaces can be traced or projected over, or locally into, estuarine deposits above and landward of their associated shoreface deposits. Maximum flooding surfaces can be traced or projected landward from offshore strata into, or above, coastal-plain deposits. With the exception of the Cozzette/Rollins sequence, the majority of coal-bearing coastal-plain strata was deposited before maximum flooding and is therefore within the transgressive systems tracts. Maximum flooding was followed by strong progradation of parasequences and low preservation potential of coastal-plain strata within the highstand systems tract. The large incised valleys, lack of transgressive retrogradational parasequences, strong progradational nature of highstand parasequences, and low preservation of coastal-plain strata in the highstand systems tracts argue for relatively low accommodation space during deposition of the Sego, Corcoran, and Cozzette sequences. The Buck Canyon/Cozzette and Cozzette/Rollins sequences contrast with other sequences in that the preservation of retrogradational parasequences and the development of large estuaries coincident with maximum flooding indicate a relative increase in accommodation space during deposition of these strata. Following maximum flooding, the Buck Canyon/Cozzette sequence follows the pattern of the other sequences, but the Cozzette/Rollins sequence exhibits a contrasting offlapping pattern with development of offshore clinoforms that downlap and eventually parallel its maximum flooding surface. This highstand systems tract preserves a thick coal-bearing section where the Rollins Sandstone Member of the Mount Garfield Formation parasequences prograde out of the study area, stepping up as much as 800 ft stratigraphically over a distance of about 90 miles. This progradational stacking pattern indicates a higher accommodation space and increased sedimentation rate compared to the previous sequences.

Motion video analysis using planar parallax

NASA Astrophysics Data System (ADS)

Sawhney, Harpreet S.

1994-04-01

Motion and structure analysis in video sequences can lead to efficient descriptions of objects and their motions. Interesting events in videos can be detected using such an analysis--for instance independent object motion when the camera itself is moving, figure-ground segregation based on the saliency of a structure compared to its surroundings. In this paper we present a method for 3D motion and structure analysis that uses a planar surface in the environment as a reference coordinate system to describe a video sequence. The motion in the video sequence is described as the motion of the reference plane, and the parallax motion of all the non-planar components of the scene. It is shown how this method simplifies the otherwise hard general 3D motion analysis problem. In addition, a natural coordinate system in the environment is used to describe the scene which can simplify motion based segmentation. This work is a part of an ongoing effort in our group towards video annotation and analysis for indexing and retrieval. Results from a demonstration system being developed are presented.

Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.).

PubMed

Bushakra, Jill M; Lewers, Kim S; Staton, Margaret E; Zhebentyayeva, Tetyana; Saski, Christopher A

2015-10-26

Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed sequence tags (ESTs) are a source of SSRs that can be used to develop markers to facilitate plant breeding and for more basic research across genera and higher plant orders. Leaf and meristem tissue from 'Heritage' red raspberry (Rubus idaeus) and 'Bristol' black raspberry (R. occidentalis) were utilized for RNA extraction. After conversion to cDNA and library construction, ESTs were sequenced, quality verified, assembled and scanned for SSRs.  Primers flanking the SSRs were designed and a subset tested for amplification, polymorphism and transferability across species. ESTs containing SSRs were functionally annotated using the GenBank non-redundant (nr) database and further classified using the gene ontology database. To accelerate development of EST-SSRs in the genus Rubus (Rosaceae), 1149 and 2358 cDNA sequences were generated from red raspberry and black raspberry, respectively. The cDNA sequences were screened using rigorous filtering criteria which resulted in the identification of 121 and 257 SSR loci for red and black raspberry, respectively. Primers were designed from the surrounding sequences resulting in 131 and 288 primer pairs, respectively, as some sequences contained more than one SSR locus. Sequence analysis revealed that the SSR-containing genes span a diversity of functions and share more sequence identity with strawberry genes than with other Rosaceous species. This resource of Rubus-specific, gene-derived markers will facilitate the construction of linkage maps composed of transferable markers for studying and manipulating important traits in this economically important genus.

IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing

PubMed Central

Deonovic, Benjamin; Wang, Yunhao; Weirather, Jason; Wang, Xiu-Jie; Au, Kin Fai

2017-01-01

Abstract Allele-specific expression (ASE) is a fundamental problem in studying gene regulation and diploid transcriptome profiles, with two key challenges: (i) haplotyping and (ii) estimation of ASE at the gene isoform level. Existing ASE analysis methods are limited by a dependence on haplotyping from laborious experiments or extra genome/family trio data. In addition, there is a lack of methods for gene isoform level ASE analysis. We developed a tool, IDP-ASE, for full ASE analysis. By innovative integration of Third Generation Sequencing (TGS) long reads with Second Generation Sequencing (SGS) short reads, the accuracy of haplotyping and ASE quantification at the gene and gene isoform level was greatly improved as demonstrated by the gold standard data GM12878 data and semi-simulation data. In addition to methodology development, applications of IDP-ASE to human embryonic stem cells and breast cancer cells indicate that the imbalance of ASE and non-uniformity of gene isoform ASE is widespread, including tumorigenesis relevant genes and pluripotency markers. These results show that gene isoform expression and allele-specific expression cooperate to provide high diversity and complexity of gene regulation and expression, highlighting the importance of studying ASE at the gene isoform level. Our study provides a robust bioinformatics solution to understand ASE using RNA sequencing data only. PMID:27899656

High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in high plants. However, the diversity of miRNAs and their roles in floral development in Japanese apricot (Prunus mume Sieb. et Zucc) remains largely unexplored. Imperfect flowers with pistil abortion seriously decrease production yields. To understand the role of miRNAs in pistil development, pistil development-related miRNAs were identified by Solexa sequencing in Japanese apricot. Results Solexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22,561,972 and 24,952,690 reads were sequenced from two small RNA libraries constructed from perfect and imperfect flower buds, respectively. Sixty-one known miRNAs, belonging to 24 families, were identified. Comparative profiling revealed that seven known miRNAs exhibited significant differential expression between perfect and imperfect flower buds. A total of 61 potentially novel miRNAs/new members of known miRNA families were also identified by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that six potentially novel miRNAs were differentially expressed between perfect and imperfect flower buds. Target predictions of the 13 differentially expressed miRNAs resulted in 212 target genes. Gene ontology (GO) annotation revealed that high-ranking miRNA target genes are those implicated in the developmental process, the regulation of transcription and response to stress. Conclusions This study represents the first comparative identification of miRNAomes between perfect and imperfect Japanese apricot flowers. Seven known miRNAs and six potentially novel miRNAs associated with pistil development were identified, using high-throughput sequencing of small RNAs. The findings, both computationally and experimentally, provide valuable information for further functional characterisation of miRNAs associated with pistil development in plants. PMID:22863067

Insights into phylogeny, sex function and age of Fragaria based on whole chloroplast genome sequencing

Treesearch

Wambui Njunguna; Aaron Liston; Richard Cronn; Tia-Lynn Ashman; Nahla Bassil

2013-01-01

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria...

Construction of a high-density genetic map by specific locus amplified fragment sequencing (SLAF-seq) and its application to Quantitative Trait Loci (QTL) analysis for boll weight in upland cotton (Gossypium hirsutum.).

PubMed

Zhang, Zhen; Shang, Haihong; Shi, Yuzhen; Huang, Long; Li, Junwen; Ge, Qun; Gong, Juwu; Liu, Aiying; Chen, Tingting; Wang, Dan; Wang, Yanling; Palanga, Koffi Kibalou; Muhammad, Jamshed; Li, Weijie; Lu, Quanwei; Deng, Xiaoying; Tan, Yunna; Song, Weiwu; Cai, Juan; Li, Pengtao; Rashid, Harun or; Gong, Wankui; Yuan, Youlu

2016-04-11

Upland Cotton (Gossypium hirsutum) is one of the most important worldwide crops it provides natural high-quality fiber for the industrial production and everyday use. Next-generation sequencing is a powerful method to identify single nucleotide polymorphism markers on a large scale for the construction of a high-density genetic map for quantitative trait loci mapping. In this research, a recombinant inbred lines population developed from two upland cotton cultivars 0-153 and sGK9708 was used to construct a high-density genetic map through the specific locus amplified fragment sequencing method. The high-density genetic map harbored 5521 single nucleotide polymorphism markers which covered a total distance of 3259.37 cM with an average marker interval of 0.78 cM without gaps larger than 10 cM. In total 18 quantitative trait loci of boll weight were identified as stable quantitative trait loci and were detected in at least three out of 11 environments and explained 4.15-16.70 % of the observed phenotypic variation. In total, 344 candidate genes were identified within the confidence intervals of these stable quantitative trait loci based on the cotton genome sequence. These genes were categorized based on their function through gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and eukaryotic orthologous groups analysis. This research reported the first high-density genetic map for Upland Cotton (Gossypium hirsutum) with a recombinant inbred line population using single nucleotide polymorphism markers developed by specific locus amplified fragment sequencing. We also identified quantitative trait loci of boll weight across 11 environments and identified candidate genes within the quantitative trait loci confidence intervals. The results of this research would provide useful information for the next-step work including fine mapping, gene functional analysis, pyramiding breeding of functional genes as well as marker-assisted selection.

Characterization of microRNAs Expressed during Secondary Wall Biosynthesis in Acacia mangium

PubMed Central

Ong, Seong Siang; Wickneswari, Ratnam

2012-01-01

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants. PMID:23251324

Microfluidic single-cell whole-transcriptome sequencing.

PubMed

Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

2014-05-13

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

[Review of Second Generation Sequencing and Its Application in Forensic Genetics].

PubMed

Zhang, S H; Bian, Y N; Zhao, Q; Li, C T

2016-08-01

The rapid development of second generation sequencing （SGS） within the past few years has led to the increasement of data throughput and read length while at the same time brought down substantially the sequencing cost. This made new breakthrough in the area of biology and ushered the forensic genetics into a new era. Based on the history of sequencing application in forensic genetics, this paper reviews the importance of sequencing technologies for genetic marker detection. The application status and potential of SGS in forensic genetics are discussed based on the already explored SGS platforms of Roche, Illumina and Life Technologies. With these platforms, DNA markers （SNP, STR）, RNA markers （mRNA, microRNA） and whole mtDNA can be sequenced. However, development and validation of application kits, maturation of analysis software, connection to the existing databases and the possible ethical issues occurred with big data will be the key factors that determine whether this technology can substitute or supplement PCR-CE, the mature technology, and be widely used for cases detection. Copyright© by the Editorial Department of Journal of Forensic Medicine.

Genome-wide gene–gene interaction analysis for next-generation sequencing

PubMed Central

Zhao, Jinying; Zhu, Yun; Xiong, Momiao

2016-01-01

The critical barrier in interaction analysis for next-generation sequencing (NGS) data is that the traditional pairwise interaction analysis that is suitable for common variants is difficult to apply to rare variants because of their prohibitive computational time, large number of tests and low power. The great challenges for successful detection of interactions with NGS data are (1) the demands in the paradigm of changes in interaction analysis; (2) severe multiple testing; and (3) heavy computations. To meet these challenges, we shift the paradigm of interaction analysis between two SNPs to interaction analysis between two genomic regions. In other words, we take a gene as a unit of analysis and use functional data analysis techniques as dimensional reduction tools to develop a novel statistic to collectively test interaction between all possible pairs of SNPs within two genome regions. By intensive simulations, we demonstrate that the functional logistic regression for interaction analysis has the correct type 1 error rates and higher power to detect interaction than the currently used methods. The proposed method was applied to a coronary artery disease dataset from the Wellcome Trust Case Control Consortium (WTCCC) study and the Framingham Heart Study (FHS) dataset, and the early-onset myocardial infarction (EOMI) exome sequence datasets with European origin from the NHLBI's Exome Sequencing Project. We discovered that 6 of 27 pairs of significantly interacted genes in the FHS were replicated in the independent WTCCC study and 24 pairs of significantly interacted genes after applying Bonferroni correction in the EOMI study. PMID:26173972

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

Breath-held MR Cholangiopancreatography (MRCP) using a 3D Dixon fat–water separated balanced steady state free precession sequence

PubMed Central

Glockner, James F.; Saranathan, Manojkumar; Bayram, Ersin; Lee, Christine U.

2014-01-01

A novel 3D breath-held Dixon fat–water separated balanced steady state free precession (b-SSFP) sequence for MR cholangiopancreatography (MRCP) is described and its potential clinical utility assessed in a series of patients. The main motivation is to develop a robust breath-held alternative to the respiratory gated 3D Fast Spin Echo (FSE) sequence, the current clinical sequence of choice for MRCP. Respiratory gated acquisitions are susceptible to motion artifacts and blurring in patients with significant diaphragmatic drift, erratic respiratory rhythms or sleep apnea. A two point Dixon fat–water separation scheme was developed which eliminates signal loss arising from B0 inhomogeneity effects and minimizes artifacts from perturbation of the b-SSFP steady state. Preliminary results from qualitative analysis of 49 patients demonstrate robust performance of the 3D Dixon b-SSFP sequence with diagnostic image quality acquired in a 20–24 s breath-hold. PMID:23876262

Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers.

PubMed

Varshney, Rajeev K; Chen, Wenbin; Li, Yupeng; Bharti, Arvind K; Saxena, Rachit K; Schlueter, Jessica A; Donoghue, Mark T A; Azam, Sarwar; Fan, Guangyi; Whaley, Adam M; Farmer, Andrew D; Sheridan, Jaime; Iwata, Aiko; Tuteja, Reetu; Penmetsa, R Varma; Wu, Wei; Upadhyaya, Hari D; Yang, Shiaw-Pyng; Shah, Trushar; Saxena, K B; Michael, Todd; McCombie, W Richard; Yang, Bicheng; Zhang, Gengyun; Yang, Huanming; Wang, Jun; Spillane, Charles; Cook, Douglas R; May, Gregory D; Xu, Xun; Jackson, Scott A

2011-11-06

Pigeonpea is an important legume food crop grown primarily by smallholder farmers in many semi-arid tropical regions of the world. We used the Illumina next-generation sequencing platform to generate 237.2 Gb of sequence, which along with Sanger-based bacterial artificial chromosome end sequences and a genetic map, we assembled into scaffolds representing 72.7% (605.78 Mb) of the 833.07 Mb pigeonpea genome. Genome analysis predicted 48,680 genes for pigeonpea and also showed the potential role that certain gene families, for example, drought tolerance-related genes, have played throughout the domestication of pigeonpea and the evolution of its ancestors. Although we found a few segmental duplication events, we did not observe the recent genome-wide duplication events observed in soybean. This reference genome sequence will facilitate the identification of the genetic basis of agronomically important traits, and accelerate the development of improved pigeonpea varieties that could improve food security in many developing countries.

The Impact of Normalization Methods on RNA-Seq Data Analysis

PubMed Central

Zyprych-Walczak, J.; Szabelska, A.; Handschuh, L.; Górczak, K.; Klamecka, K.; Figlerowicz, M.; Siatkowski, I.

2015-01-01

High-throughput sequencing technologies, such as the Illumina Hi-seq, are powerful new tools for investigating a wide range of biological and medical problems. Massive and complex data sets produced by the sequencers create a need for development of statistical and computational methods that can tackle the analysis and management of data. The data normalization is one of the most crucial steps of data processing and this process must be carefully considered as it has a profound effect on the results of the analysis. In this work, we focus on a comprehensive comparison of five normalization methods related to sequencing depth, widely used for transcriptome sequencing (RNA-seq) data, and their impact on the results of gene expression analysis. Based on this study, we suggest a universal workflow that can be applied for the selection of the optimal normalization procedure for any particular data set. The described workflow includes calculation of the bias and variance values for the control genes, sensitivity and specificity of the methods, and classification errors as well as generation of the diagnostic plots. Combining the above information facilitates the selection of the most appropriate normalization method for the studied data sets and determines which methods can be used interchangeably. PMID:26176014

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean

PubMed Central

Voelker, Toni A.; Staswick, Paul; Chrispeels, Maarten J.

1986-01-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained. ImagesFig. 5. PMID:16453730

Blast2GO goes grid: developing a grid-enabled prototype for functional genomics analysis.

PubMed

Aparicio, G; Götz, S; Conesa, A; Segrelles, D; Blanquer, I; García, J M; Hernandez, V; Robles, M; Talon, M

2006-01-01

The vast amount in complexity of data generated in Genomic Research implies that new dedicated and powerful computational tools need to be developed to meet their analysis requirements. Blast2GO (B2G) is a bioinformatics tool for Gene Ontology-based DNA or protein sequence annotation and function-based data mining. The application has been developed with the aim of affering an easy-to-use tool for functional genomics research. Typical B2G users are middle size genomics labs carrying out sequencing, ETS and microarray projects, handling datasets up to several thousand sequences. In the current version of B2G. The power and analytical potential of both annotation and function data-mining is somehow restricted to the computational power behind each particular installation. In order to be able to offer the possibility of an enhanced computational capacity within this bioinformatics application, a Grid component is being developed. A prototype has been conceived for the particular problem of speeding up the Blast searches to obtain fast results for large datasets. Many efforts have been done in the literature concerning the speeding up of Blast searches, but few of them deal with the use of large heterogeneous production Grid Infrastructures. These are the infrastructures that could reach the largest number of resources and the best load balancing for data access. The Grid Service under development will analyse requests based on the number of sequences, splitting them accordingly to the available resources. Lower-level computation will be performed through MPIBLAST. The software architecture is based on the WSRF standard.

Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis

PubMed Central

Sun, Zhifu; Cunningham, Julie; Slager, Susan; Kocher, Jean-Pierre

2015-01-01

Bisulfite treatment-based methylation microarray (mainly Illumina 450K Infinium array) and next-generation sequencing (reduced representation bisulfite sequencing, Agilent SureSelect Human Methyl-Seq, NimbleGen SeqCap Epi CpGiant or whole-genome bisulfite sequencing) are commonly used for base resolution DNA methylome research. Although multiple tools and methods have been developed and used for the data preprocessing and analysis, confusions remains for these platforms including how and whether the 450k array should be normalized; which platform should be used to better fit researchers’ needs; and which statistical models would be more appropriate for differential methylation analysis. This review presents the commonly used platforms and compares the pros and cons of each in methylome profiling. We then discuss approaches to study design, data normalization, bias correction and model selection for differentially methylated individual CpGs and regions. PMID:26366945

Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

PubMed

Wrzeszczynski, Kazimierz O; Frank, Mayu O; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A; Moore Vogel, Julia L; Bruce, Jeffrey N; Lassman, Andrew B; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V; Zody, Michael C; Jobanputra, Vaidehi; Royyuru, Ajay K; Darnell, Robert B

2017-08-01

To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. NCT02725684.

Methods, Tools and Current Perspectives in Proteogenomics *

PubMed Central

Ruggles, Kelly V.; Krug, Karsten; Wang, Xiaojing; Clauser, Karl R.; Wang, Jing; Payne, Samuel H.; Fenyö, David; Zhang, Bing; Mani, D. R.

2017-01-01

With combined technological advancements in high-throughput next-generation sequencing and deep mass spectrometry-based proteomics, proteogenomics, i.e. the integrative analysis of proteomic and genomic data, has emerged as a new research field. Early efforts in the field were focused on improving protein identification using sample-specific genomic and transcriptomic sequencing data. More recently, integrative analysis of quantitative measurements from genomic and proteomic studies have identified novel insights into gene expression regulation, cell signaling, and disease. Many methods and tools have been developed or adapted to enable an array of integrative proteogenomic approaches and in this article, we systematically classify published methods and tools into four major categories, (1) Sequence-centric proteogenomics; (2) Analysis of proteogenomic relationships; (3) Integrative modeling of proteogenomic data; and (4) Data sharing and visualization. We provide a comprehensive review of methods and available tools in each category and highlight their typical applications. PMID:28456751

High-Throughput Single-Cell RNA Sequencing and Data Analysis.

PubMed

Sagar; Herman, Josip Stefan; Pospisilik, John Andrew; Grün, Dominic

2018-01-01

Understanding biological systems at a single cell resolution may reveal several novel insights which remain masked by the conventional population-based techniques providing an average readout of the behavior of cells. Single-cell transcriptome sequencing holds the potential to identify novel cell types and characterize the cellular composition of any organ or tissue in health and disease. Here, we describe a customized high-throughput protocol for single-cell RNA-sequencing (scRNA-seq) combining flow cytometry and a nanoliter-scale robotic system. Since scRNA-seq requires amplification of a low amount of endogenous cellular RNA, leading to substantial technical noise in the dataset, downstream data filtering and analysis require special care. Therefore, we also briefly describe in-house state-of-the-art data analysis algorithms developed to identify cellular subpopulations including rare cell types as well as to derive lineage trees by ordering the identified subpopulations of cells along the inferred differentiation trajectories.

Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery1

PubMed Central

Gardner, Elliot M.; Johnson, Matthew G.; Ragone, Diane; Wickett, Norman J.; Zerega, Nyree J. C.

2016-01-01

Premise of the study: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. Methods and Results: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. Conclusions: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes. PMID:27437173

Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.

PubMed

Chatzidimopoulos, Michael; Ganopoulos, Ioannis; Vellios, Evangelos; Madesis, Panagiotis; Tsaftaris, Athanasios; Pappas, Athanassios C

2014-11-01

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

In silico analysis of expressed sequence tags from Trichostrongylus vitrinus (Nematoda): comparison of the automated ESTExplorer workflow platform with conventional database searches.

PubMed

Nagaraj, Shivashankar H; Gasser, Robin B; Nisbet, Alasdair J; Ranganathan, Shoba

2008-01-01

The analysis of expressed sequence tags (EST) offers a rapid and cost effective approach to elucidate the transcriptome of an organism, but requires several computational methods for assembly and annotation. Researchers frequently analyse each step manually, which is laborious and time consuming. We have recently developed ESTExplorer, a semi-automated computational workflow system, in order to achieve the rapid analysis of EST datasets. In this study, we evaluated EST data analysis for the parasitic nematode Trichostrongylus vitrinus (order Strongylida) using ESTExplorer, compared with database matching alone. We functionally annotated 1776 ESTs obtained via suppressive-subtractive hybridisation from T. vitrinus, an important parasitic trichostrongylid of small ruminants. Cluster and comparative genomic analyses of the transcripts using ESTExplorer indicated that 290 (41%) sequences had homologues in Caenorhabditis elegans, 329 (42%) in parasitic nematodes, 202 (28%) in organisms other than nematodes, and 218 (31%) had no significant match to any sequence in the current databases. Of the C. elegans homologues, 90 were associated with 'non-wildtype' double-stranded RNA interference (RNAi) phenotypes, including embryonic lethality, maternal sterility, sterile progeny, larval arrest and slow growth. We could functionally classify 267 (38%) sequences using the Gene Ontologies (GO) and establish pathway associations for 230 (33%) sequences using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Further examination of this EST dataset revealed a number of signalling molecules, proteases, protease inhibitors, enzymes, ion channels and immune-related genes. In addition, we identified 40 putative secreted proteins that could represent potential candidates for developing novel anthelmintics or vaccines. We further compared the automated EST sequence annotations, using ESTExplorer, with database search results for individual T. vitrinus ESTs. ESTExplorer reliably and rapidly annotated 301 ESTs, with pathway and GO information, eliminating 60 low quality hits from database searches. We evaluated the efficacy of ESTExplorer in analysing EST data, and demonstrate that computational tools can be used to accelerate the process of gene discovery in EST sequencing projects. The present study has elucidated sets of relatively conserved and potentially novel genes for biological investigation, and the annotated EST set provides further insight into the molecular biology of T. vitrinus, towards the identification of novel drug targets.

Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.).

PubMed

Aramrak, Attawan; Kidwell, Kimberlee K; Steber, Camille M; Burke, Ian C

2015-10-23

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized. Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring "Louise'. Development of genome-specific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis. The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7 % identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100 % identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7 % identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice). The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance.

SIMPLEX: Cloud-Enabled Pipeline for the Comprehensive Analysis of Exome Sequencing Data

PubMed Central

Fischer, Maria; Snajder, Rene; Pabinger, Stephan; Dander, Andreas; Schossig, Anna; Zschocke, Johannes; Trajanoski, Zlatko; Stocker, Gernot

2012-01-01

In recent studies, exome sequencing has proven to be a successful screening tool for the identification of candidate genes causing rare genetic diseases. Although underlying targeted sequencing methods are well established, necessary data handling and focused, structured analysis still remain demanding tasks. Here, we present a cloud-enabled autonomous analysis pipeline, which comprises the complete exome analysis workflow. The pipeline combines several in-house developed and published applications to perform the following steps: (a) initial quality control, (b) intelligent data filtering and pre-processing, (c) sequence alignment to a reference genome, (d) SNP and DIP detection, (e) functional annotation of variants using different approaches, and (f) detailed report generation during various stages of the workflow. The pipeline connects the selected analysis steps, exposes all available parameters for customized usage, performs required data handling, and distributes computationally expensive tasks either on a dedicated high-performance computing infrastructure or on the Amazon cloud environment (EC2). The presented application has already been used in several research projects including studies to elucidate the role of rare genetic diseases. The pipeline is continuously tested and is publicly available under the GPL as a VirtualBox or Cloud image at http://simplex.i-med.ac.at; additional supplementary data is provided at http://www.icbi.at/exome. PMID:22870267

Alignment of high-throughput sequencing data inside in-memory databases.

PubMed

Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

2014-01-01

In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

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

Lapidus, Alla L.

From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly ofmore » whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.« less

Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

PubMed Central

Amexis, Georgios; Oeth, Paul; Abel, Kenneth; Ivshina, Anna; Pelloquin, Francois; Cantor, Charles R.; Braun, Andreas; Chumakov, Konstantin

2001-01-01

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest. PMID:11593021

Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria.

PubMed

Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne; Harmsen, Hermie J M; Welling, Gjalt W; Bos, Nicolaas A

2002-11-01

Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria'. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

A genomewide survey of basic helix–loop–helix factors in Drosophila

PubMed Central

Moore, Adrian W.; Barbel, Sandra; Jan, Lily Yeh; Jan, Yuh Nung

2000-01-01

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm. PMID:10973473

Benzofurazane as a new redox label for electrochemical detection of DNA: towards multipotential redox coding of DNA bases.

PubMed

Balintová, Jana; Plucnara, Medard; Vidláková, Pavlína; Pohl, Radek; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

2013-09-16

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane-modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three-potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Image sequence analysis workstation for multipoint motion analysis

NASA Astrophysics Data System (ADS)

Mostafavi, Hassan

1990-08-01

This paper describes an application-specific engineering workstation designed and developed to analyze motion of objects from video sequences. The system combines the software and hardware environment of a modem graphic-oriented workstation with the digital image acquisition, processing and display techniques. In addition to automation and Increase In throughput of data reduction tasks, the objective of the system Is to provide less invasive methods of measurement by offering the ability to track objects that are more complex than reflective markers. Grey level Image processing and spatial/temporal adaptation of the processing parameters is used for location and tracking of more complex features of objects under uncontrolled lighting and background conditions. The applications of such an automated and noninvasive measurement tool include analysis of the trajectory and attitude of rigid bodies such as human limbs, robots, aircraft in flight, etc. The system's key features are: 1) Acquisition and storage of Image sequences by digitizing and storing real-time video; 2) computer-controlled movie loop playback, freeze frame display, and digital Image enhancement; 3) multiple leading edge tracking in addition to object centroids at up to 60 fields per second from both live input video or a stored Image sequence; 4) model-based estimation and tracking of the six degrees of freedom of a rigid body: 5) field-of-view and spatial calibration: 6) Image sequence and measurement data base management; and 7) offline analysis software for trajectory plotting and statistical analysis.

Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions.

PubMed

Senol Cali, Damla; Kim, Jeremie S; Ghose, Saugata; Alkan, Can; Mutlu, Onur

2018-04-02

Nanopore sequencing technology has the potential to render other sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for nanopore sequence analysis are of critical importance, as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for nanopore sequence analysis to understand their advantages, disadvantages and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we (1) analyze the multiple steps and the associated tools in the genome assembly pipeline using nanopore sequence data, and (2) provide guidelines for determining the appropriate tools for each step. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of nanopore sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, to overcome the high error rates of the nanopore sequencing technology.

Phylogenomics from Whole Genome Sequences Using aTRAM.

PubMed

Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P

2017-09-01

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

On-Line Detection and Segmentation of Sports Motions Using a Wearable Sensor.

PubMed

Kim, Woosuk; Kim, Myunggyu

2018-03-19

In sports motion analysis, observation is a prerequisite for understanding the quality of motions. This paper introduces a novel approach to detect and segment sports motions using a wearable sensor for supporting systematic observation. The main goal is, for convenient analysis, to automatically provide motion data, which are temporally classified according to the phase definition. For explicit segmentation, a motion model is defined as a sequence of sub-motions with boundary states. A sequence classifier based on deep neural networks is designed to detect sports motions from continuous sensor inputs. The evaluation on two types of motions (soccer kicking and two-handed ball throwing) verifies that the proposed method is successful for the accurate detection and segmentation of sports motions. By developing a sports motion analysis system using the motion model and the sequence classifier, we show that the proposed method is useful for observation of sports motions by automatically providing relevant motion data for analysis.

Statistical framework for detection of genetically modified organisms based on Next Generation Sequencing.

PubMed

Willems, Sander; Fraiture, Marie-Alice; Deforce, Dieter; De Keersmaecker, Sigrid C J; De Loose, Marc; Ruttink, Tom; Herman, Philippe; Van Nieuwerburgh, Filip; Roosens, Nancy

2016-02-01

Because the number and diversity of genetically modified (GM) crops has significantly increased, their analysis based on real-time PCR (qPCR) methods is becoming increasingly complex and laborious. While several pioneers already investigated Next Generation Sequencing (NGS) as an alternative to qPCR, its practical use has not been assessed for routine analysis. In this study a statistical framework was developed to predict the number of NGS reads needed to detect transgene sequences, to prove their integration into the host genome and to identify the specific transgene event in a sample with known composition. This framework was validated by applying it to experimental data from food matrices composed of pure GM rice, processed GM rice (noodles) or a 10% GM/non-GM rice mixture, revealing some influential factors. Finally, feasibility of NGS for routine analysis of GM crops was investigated by applying the framework to samples commonly encountered in routine analysis of GM crops. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular analysis of Toxoplasma gondii Surface Antigen 1 (SAG1) gene cloned from Toxoplasma gondii DNA isolated from Javanese acute toxoplasmosis

NASA Astrophysics Data System (ADS)

Haryati, Sri; Agung Prasetyo, Afiono; Sari, Yulia; Dharmawan, Ruben

2018-05-01

Toxoplasma gondii Surface Antigen 1 (SAG1) is often used as a diagnostic tool due to its immunodominant-specific as antigen. However, data of the Toxoplasma gondii SAG1 protein from Indonesian isolate is limited. To study the protein, genomic DNA was isolated from a Javanese acute toxoplasmosis blood samples patient. A complete coding sequence of Toxoplasma gondii SAG1 was cloned and inserted into an Escherichia coli expression plasmid and sequenced. The sequencing results were subjected to bioinformatics analysis. The Toxoplasma gondii SAG1 complete coding sequences were successfully cloned. Physicochemical analysis revealed the 336 aa of SAG1 had 34.7 kDa of weight. The isoelectric point and aliphatic index were 8.4 and 78.4, respectively. The N-terminal methionine half-life in Escherichia coli was more than 10 hours. The antigenicity, secondary structure, and identification of the HLA binding motifs also had been discussed. The results of this study would contribute information about Toxoplasma gondii SAG1 and benefits for further works willing to develop diagnostic and therapeutic strategies against the parasite.

BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of variants in chromatin remodeling genes.

PubMed

Hasumi, Hisashi; Furuya, Mitsuko; Tatsuno, Kenji; Yamamoto, Shogo; Baba, Masaya; Hasumi, Yukiko; Isono, Yasuhiro; Suzuki, Kae; Jikuya, Ryosuke; Otake, Shinji; Muraoka, Kentaro; Osaka, Kimito; Hayashi, Narihiko; Makiyama, Kazuhide; Miyoshi, Yasuhide; Kondo, Keiichi; Nakaigawa, Noboru; Kawahara, Takashi; Izumi, Koji; Teranishi, Junichi; Yumura, Yasushi; Uemura, Hiroji; Nagashima, Yoji; Metwalli, Adam R; Schmidt, Laura S; Aburatani, Hiroyuki; Linehan, W Marston; Yao, Masahiro

2018-05-14

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using LC/MS and GC/MS. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming towards upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics which are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

A Three-Dimensional Approach and Open Source Structure for the Design and Experimentation of Teaching-Learning Sequences: The case of friction

NASA Astrophysics Data System (ADS)

Besson, Ugo; Borghi, Lidia; De Ambrosis, Anna; Mascheretti, Paolo

2010-07-01

We have developed a teaching-learning sequence (TLS) on friction based on a preliminary study involving three dimensions: an analysis of didactic research on the topic, an overview of usual approaches, and a critical analysis of the subject, considered also in its historical development. We found that mostly the usual presentations do not take into account the complexity of friction as it emerges from scientific research, may reinforce some inaccurate students' conceptions, and favour a limited vision of friction phenomena. The TLS we propose begins by considering a wide range of friction phenomena to favour an initial motivation and a broader view of the topic and then develops a path of interrelated observations, experiments, and theoretical aspects. It proposes the use of structural models, involving visual representations and stimulating intuition, aimed at helping students build mental models of friction mechanisms. To facilitate the reproducibility in school contexts, the sequence is designed as an open source structure, with a core of contents, conceptual correlations and methodological choices, and a cloud of elements that can be re-designed by teachers. The sequence has been tested in teacher education and in upper secondary school, and has shown positive results in overcoming student difficulties and stimulating richer reasoning based on the structural models we suggested. The proposed path has modified the teachers' view of the topic, producing a motivation to change their traditional presentations. The open structure of the sequence has facilitated its implementation by teachers in school in coherence with the rationale of the proposal.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

PubMed

Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

2014-09-01

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV. © 2014 Poultry Science Association Inc.

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

PubMed

Dubey, Anuja; Farmer, Andrew; Schlueter, Jessica; Cannon, Steven B; Abernathy, Brian; Tuteja, Reetu; Woodward, Jimmy; Shah, Trushar; Mulasmanovic, Benjamin; Kudapa, Himabindu; Raju, Nikku L; Gothalwal, Ragini; Pande, Suresh; Xiao, Yongli; Town, Chris D; Singh, Nagendra K; May, Gregory D; Jackson, Scott; Varshney, Rajeev K

2011-06-01

This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

tropiTree: An NGS-Based EST-SSR Resource for 24 Tropical Tree Species

PubMed Central

Russell, Joanne R.; Hedley, Peter E.; Cardle, Linda; Dancey, Siobhan; Morris, Jenny; Booth, Allan; Odee, David; Mwaura, Lucy; Omondi, William; Angaine, Peter; Machua, Joseph; Muchugi, Alice; Milne, Iain; Kindt, Roeland; Jamnadass, Ramni; Dawson, Ian K.

2014-01-01

The development of genetic tools for non-model organisms has been hampered by cost, but advances in next-generation sequencing (NGS) have created new opportunities. In ecological research, this raises the prospect for developing molecular markers to simultaneously study important genetic processes such as gene flow in multiple non-model plant species within complex natural and anthropogenic landscapes. Here, we report the use of bar-coded multiplexed paired-end Illumina NGS for the de novo development of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers at low cost for a range of 24 tree species. Each chosen tree species is important in complex tropical agroforestry systems where little is currently known about many genetic processes. An average of more than 5,000 EST-SSRs was identified for each of the 24 sequenced species, whereas prior to analysis 20 of the species had fewer than 100 nucleotide sequence citations. To make results available to potential users in a suitable format, we have developed an open-access, interactive online database, tropiTree (http://bioinf.hutton.ac.uk/tropiTree), which has a range of visualisation and search facilities, and which is a model for the efficient presentation and application of NGS data. PMID:25025376

Transcriptomic analysis of Prunus domestica undergoing hypersensitive response to plum pox virus infection.

PubMed

Rodamilans, Bernardo; San León, David; Mühlberger, Louisa; Candresse, Thierry; Neumüller, Michael; Oliveros, Juan Carlos; García, Juan Antonio

2014-01-01

Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named 'Jojo', develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected 'Jojo' trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR≤0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

PubMed Central

Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J.; Rohan, Thomas; Ben-Dov, Iddo Z.

2017-01-01

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens. PMID:28335433

RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA

PubMed Central

Wright, Imogen A.; Travers, Simon A.

2014-01-01

The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. PMID:24861618

On site DNA barcoding by nanopore sequencing

PubMed Central

Menegon, Michele; Cantaloni, Chiara; Rodriguez-Prieto, Ana; Centomo, Cesare; Abdelfattah, Ahmed; Rossato, Marzia; Bernardi, Massimo; Xumerle, Luciano; Loader, Simon; Delledonne, Massimo

2017-01-01

Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities. PMID:28977016

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

PubMed

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Comparison of Next-Generation Sequencing Systems

PubMed Central

Liu, Lin; Li, Yinhu; Li, Siliang; Hu, Ni; He, Yimin; Pong, Ray; Lin, Danni; Lu, Lihua; Law, Maggie

2012-01-01

With fast development and wide applications of next-generation sequencing (NGS) technologies, genomic sequence information is within reach to aid the achievement of goals to decode life mysteries, make better crops, detect pathogens, and improve life qualities. NGS systems are typically represented by SOLiD/Ion Torrent PGM from Life Sciences, Genome Analyzer/HiSeq 2000/MiSeq from Illumina, and GS FLX Titanium/GS Junior from Roche. Beijing Genomics Institute (BGI), which possesses the world's biggest sequencing capacity, has multiple NGS systems including 137 HiSeq 2000, 27 SOLiD, one Ion Torrent PGM, one MiSeq, and one 454 sequencer. We have accumulated extensive experience in sample handling, sequencing, and bioinformatics analysis. In this paper, technologies of these systems are reviewed, and first-hand data from extensive experience is summarized and analyzed to discuss the advantages and specifics associated with each sequencing system. At last, applications of NGS are summarized. PMID:22829749

dictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond.

PubMed

Stajdohar, Miha; Rosengarten, Rafael D; Kokosar, Janez; Jeran, Luka; Blenkus, Domen; Shaulsky, Gad; Zupan, Blaz

2017-06-02

Dictyostelium discoideum, a soil-dwelling social amoeba, is a model for the study of numerous biological processes. Research in the field has benefited mightily from the adoption of next-generation sequencing for genomics and transcriptomics. Dictyostelium biologists now face the widespread challenges of analyzing and exploring high dimensional data sets to generate hypotheses and discovering novel insights. We present dictyExpress (2.0), a web application designed for exploratory analysis of gene expression data, as well as data from related experiments such as Chromatin Immunoprecipitation sequencing (ChIP-Seq). The application features visualization modules that include time course expression profiles, clustering, gene ontology enrichment analysis, differential expression analysis and comparison of experiments. All visualizations are interactive and interconnected, such that the selection of genes in one module propagates instantly to visualizations in other modules. dictyExpress currently stores the data from over 800 Dictyostelium experiments and is embedded within a general-purpose software framework for management of next-generation sequencing data. dictyExpress allows users to explore their data in a broader context by reciprocal linking with dictyBase-a repository of Dictyostelium genomic data. In addition, we introduce a companion application called GenBoard, an intuitive graphic user interface for data management and bioinformatics analysis. dictyExpress and GenBoard enable broad adoption of next generation sequencing based inquiries by the Dictyostelium research community. Labs without the means to undertake deep sequencing projects can mine the data available to the public. The entire information flow, from raw sequence data to hypothesis testing, can be accomplished in an efficient workspace. The software framework is generalizable and represents a useful approach for any research community. To encourage more wide usage, the backend is open-source, available for extension and further development by bioinformaticians and data scientists.

StatsDB: platform-agnostic storage and understanding of next generation sequencing run metrics

PubMed Central

Ramirez-Gonzalez, Ricardo H.; Leggett, Richard M.; Waite, Darren; Thanki, Anil; Drou, Nizar; Caccamo, Mario; Davey, Robert

2014-01-01

Modern sequencing platforms generate enormous quantities of data in ever-decreasing amounts of time. Additionally, techniques such as multiplex sequencing allow one run to contain hundreds of different samples. With such data comes a significant challenge to understand its quality and to understand how the quality and yield are changing across instruments and over time. As well as the desire to understand historical data, sequencing centres often have a duty to provide clear summaries of individual run performance to collaborators or customers. We present StatsDB, an open-source software package for storage and analysis of next generation sequencing run metrics. The system has been designed for incorporation into a primary analysis pipeline, either at the programmatic level or via integration into existing user interfaces. Statistics are stored in an SQL database and APIs provide the ability to store and access the data while abstracting the underlying database design. This abstraction allows simpler, wider querying across multiple fields than is possible by the manual steps and calculation required to dissect individual reports, e.g. ”provide metrics about nucleotide bias in libraries using adaptor barcode X, across all runs on sequencer A, within the last month”. The software is supplied with modules for storage of statistics from FastQC, a commonly used tool for analysis of sequence reads, but the open nature of the database schema means it can be easily adapted to other tools. Currently at The Genome Analysis Centre (TGAC), reports are accessed through our LIMS system or through a standalone GUI tool, but the API and supplied examples make it easy to develop custom reports and to interface with other packages. PMID:24627795

A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4.

PubMed

Prins, Theo W; Scholtens, Ingrid M J; Bak, Arno W; van Dijk, Jeroen P; Voorhuijzen, Marleen M; Laurensse, Emile J; Kok, Esther J

2016-12-15

During routine monitoring for GMOs in food in the Netherlands, papaya-containing food supplements were found positive for the genetically modified (GM) elements P-35S and T-nos. The goal of this study was to identify the unknown and EU unauthorised GM papaya event(s). A screening strategy was applied using additional GM screening elements including a newly developed PRSV coat protein PCR. The detected PRSV coat protein PCR product was sequenced and the nucleotide sequence showed identity to PRSV YK strains indigenous to China and Taiwan. The GM events 16-0-1 and 18-2-4 could be identified by amplifying and sequencing events-specific sequences. Further analyses showed that both papaya event 16-0-1 and event 18-2-4 were transformed with the same construct. For use in routine analysis, derived TaqMan qPCR methods for events 16-0-1 and 18-2-4 were developed. Event 16-0-1 was detected in all samples tested whereas event 18-2-4 was detected in one sample. This study presents a strategy for combining information from different sources (literature, patent databases) and novel sequence data to identify unknown GM papaya events. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-throughput physical mapping of chromosomes using automated in situ hybridization.

PubMed

George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

2012-06-28

Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

Helicos BioSciences.

PubMed

Milos, Patrice

2008-04-01

Helicos BioSciences Corporation is a life sciences company developing revolutionary new single molecule sequencing technology to provide the path to the US$1000 genome. True Single Molecule Sequencing (tSMS) will drive advancements in pharmacogenomics that can enable a better understanding of an individual's susceptibility to disease, develop more effective disease diagnoses and differentiate response to disease therapies. During 2007, genome-wide disease-association studies, the encylopedia of DNA elements (ENCODE) and the published genome sequence of two individuals have revealed human genome variation far more extensive than originally believed. These also demonstrated that common variations explain only a fraction of the genetic basis of disease. Therefore, the capability to understand an individual genome is critical in setting the foundation for the next great revolution in healthcare. Helicos is committed to this vision and will provide cost-effective genome sequencing and comprehensive analysis of the transcribed genome that can unlock the era of personalized healthcare.

Computational tool for the early screening of monoclonal antibodies for their viscosities

PubMed Central

Agrawal, Neeraj J; Helk, Bernhard; Kumar, Sandeep; Mody, Neil; Sathish, Hasige A.; Samra, Hardeep S.; Buck, Patrick M; Li, Li; Trout, Bernhardt L

2016-01-01

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures). The SCM tool has been extensively validated at 3 different organizations, and has proved successful in correctly identifying highly viscous antibodies. As a quantitative tool, SCM is amenable to high-throughput automated analysis, and can be effectively implemented during the antibody screening or engineering phase for the selection of low-viscosity antibodies. PMID:26399600

NABIC: A New Access Portal to Search, Visualize, and Share Agricultural Genomics Data

PubMed Central

Seol, Young-Joo; Lee, Tae-Ho; Park, Dong-Suk; Kim, Chang-Kug

2016-01-01

The National Agricultural Biotechnology Information Center developed an access portal to search, visualize, and share agricultural genomics data with a focus on South Korean information and resources. The portal features an agricultural biotechnology database containing a wide range of omics data from public and proprietary sources. We collected 28.4 TB of data from 162 agricultural organisms, with 10 types of omics data comprising next-generation sequencing sequence read archive, genome, gene, nucleotide, DNA chip, expressed sequence tag, interactome, protein structure, molecular marker, and single-nucleotide polymorphism datasets. Our genomic resources contain information on five animals, seven plants, and one fungus, which is accessed through a genome browser. We also developed a data submission and analysis system as a web service, with easy-to-use functions and cutting-edge algorithms, including those for handling next-generation sequencing data. PMID:26848255

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives.

PubMed

Zhao, Min; Wang, Qingguo; Wang, Quan; Jia, Peilin; Zhao, Zhongming

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

PubMed Central

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development. PMID:24564169

Genome Sequence of the White Koji Mold Aspergillus kawachii IFO 4308, Used for Brewing the Japanese Distilled Spirit Shochu

PubMed Central

Futagami, Taiki; Mori, Kazuki; Yamashita, Ayaka; Wada, Shotaro; Kajiwara, Yasuhiro; Takashita, Hideharu; Omori, Toshiro; Takegawa, Kaoru; Tashiro, Kosuke; Kuhara, Satoru; Goto, Masatoshi

2011-01-01

The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications. PMID:22045919

HUNT: launch of a full-length cDNA database from the Helix Research Institute.

PubMed

Yudate, H T; Suwa, M; Irie, R; Matsui, H; Nishikawa, T; Nakamura, Y; Yamaguchi, D; Peng, Z Z; Yamamoto, T; Nagai, K; Hayashi, K; Otsuki, T; Sugiyama, T; Ota, T; Suzuki, Y; Sugano, S; Isogai, T; Masuho, Y

2001-01-01

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

Bioinformatics Pipelines for Targeted Resequencing and Whole-Exome Sequencing of Human and Mouse Genomes: A Virtual Appliance Approach for Instant Deployment

PubMed Central

Saeed, Isaam; Wong, Stephen Q.; Mar, Victoria; Goode, David L.; Caramia, Franco; Doig, Ken; Ryland, Georgina L.; Thompson, Ella R.; Hunter, Sally M.; Halgamuge, Saman K.; Ellul, Jason; Dobrovic, Alexander; Campbell, Ian G.; Papenfuss, Anthony T.; McArthur, Grant A.; Tothill, Richard W.

2014-01-01

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/. PMID:24752294

Development of a monoclonal anitbody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor

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

Dussossoy, D.; Carayon, P.; Feraut, D.

1996-05-01

Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes form PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realizedmore » with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab. 34 refs., 7 figs.« less

Characterization of a novel chicken muscle disorder through differential gene expression and pathway analysis using RNA-sequencing.

PubMed

Mutryn, Marie F; Brannick, Erin M; Fu, Weixuan; Lee, William R; Abasht, Behnam

2015-05-21

Improvements in poultry production within the past 50 years have led to increased muscle yield and growth rate, which may be contributing to an increased rate and development of new muscle disorders in chickens. Previously reported muscle disorders and conditions are generally associated with poor meat quality traits and have a significant negative economic impact on the poultry industry. Recently, a novel myopathy phenotype has emerged which is characterized by palpably "hard" or tough breast muscle. The objective of this study is to identify the underlying biological mechanisms that contribute to this emerging muscle disorder colloquially referred to as "Wooden Breast", through the use of RNA-sequencing technology. We constructed cDNA libraries from five affected and six unaffected breast muscle samples from a line of commercial broiler chickens. After paired-end sequencing of samples using the Illumina Hiseq platform, we used Tophat to align the resulting sequence reads to the chicken reference genome and then used Cufflinks to find significant changes in gene transcript expression between each group. By comparing our gene list to previously published histology findings on this disorder and using Ingenuity Pathways Analysis (IPA®), we aim to develop a characteristic gene expression profile for this novel disorder through analyzing genes, gene families, and predicted biological pathways. Over 1500 genes were differentially expressed between affected and unaffected birds. There was an average of approximately 98 million reads per sample, across all samples. Results from the IPA analysis suggested "Diseases and Disorders" such as connective tissue disorders, "Molecular and Cellular Functions" such as cellular assembly and organization, cellular function and maintenance, and cellular movement, "Physiological System Development and Function" such as tissue development, and embryonic development, and "Top Canonical Pathways" such as, coagulation system, axonal guidance signaling, and acute phase response signaling, are associated with the Wooden Breast disease. There is convincing evidence by RNA-seq analysis to support localized hypoxia, oxidative stress, increased intracellular calcium, as well as the possible presence of muscle fiber-type switching, as key features of Wooden Breast Disease, which are supported by reported microscopic lesions of the disease.

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

PubMed Central

Variath, Murali Tottekkad; Joshi, Gopal; Bali, Sapinder; Agarwal, Manu; Kumar, Amar; Jagannath, Arun; Goel, Shailendra

2015-01-01

Background Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing. Principal Findings Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower. Conclusion Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection. PMID:26287743

A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.)

PubMed Central

2009-01-01

Background Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers. Results A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (≤1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with ≥ 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries. Conclusion Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species. PMID:19912666

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes. PMID:29250096

MetaSeq: privacy preserving meta-analysis of sequencing-based association studies.

PubMed

Singh, Angad Pal; Zafer, Samreen; Pe'er, Itsik

2013-01-01

Human genetics recently transitioned from GWAS to studies based on NGS data. For GWAS, small effects dictated large sample sizes, typically made possible through meta-analysis by exchanging summary statistics across consortia. NGS studies groupwise-test for association of multiple potentially-causal alleles along each gene. They are subject to similar power constraints and therefore likely to resort to meta-analysis as well. The problem arises when considering privacy of the genetic information during the data-exchange process. Many scoring schemes for NGS association rely on the frequency of each variant thus requiring the exchange of identity of the sequenced variant. As such variants are often rare, potentially revealing the identity of their carriers and jeopardizing privacy. We have thus developed MetaSeq, a protocol for meta-analysis of genome-wide sequencing data by multiple collaborating parties, scoring association for rare variants pooled per gene across all parties. We tackle the challenge of tallying frequency counts of rare, sequenced alleles, for metaanalysis of sequencing data without disclosing the allele identity and counts, thereby protecting sample identity. This apparent paradoxical exchange of information is achieved through cryptographic means. The key idea is that parties encrypt identity of genes and variants. When they transfer information about frequency counts in cases and controls, the exchanged data does not convey the identity of a mutation and therefore does not expose carrier identity. The exchange relies on a 3rd party, trusted to follow the protocol although not trusted to learn about the raw data. We show applicability of this method to publicly available exome-sequencing data from multiple studies, simulating phenotypic information for powerful meta-analysis. The MetaSeq software is publicly available as open source.

History and current status of wheat miRNAs using next-generation sequencing and their roles in development and stress.

PubMed

Budak, Hikmet; Khan, Zaeema; Kantar, Melda

2015-05-01

As small molecules that aid in posttranscriptional silencing, microRNA (miRNA) discovery and characterization have vastly benefited from the recent development and widespread application of next-generation sequencing (NGS) technologies. Several miRNAs were identified through sequencing of constructed small RNA libraries, whereas others were predicted by in silico methods using the recently accumulating sequence data. NGS was a major breakthrough in efforts to sequence and dissect the genomes of plants, including bread wheat and its progenitors, which have large, repetitive and complex genomes. Availability of survey sequences of wheat whole genome and its individual chromosomes enabled researchers to predict and assess wheat miRNAs both in the subgenomic and whole genome levels. Moreover, small RNA construction and sequencing-based studies identified several putative development- and stress-related wheat miRNAs, revealing their differential expression patterns in specific developmental stages and/or in response to stress conditions. With the vast amount of wheat miRNAs identified in recent years, we are approaching to an overall knowledge on the wheat miRNA repertoire. In the following years, more comprehensive research in relation to miRNA conservation or divergence across wheat and its close relatives or progenitors should be performed. Results may serve valuable in understanding both the significant roles of species-specific miRNAs and also provide us information in relation to the dynamics between miRNAs and evolution in wheat. Furthermore, putative development- or stress-related miRNAs identified should be subjected to further functional analysis, which may be valuable in efforts to develop wheat with better resistance and/or yield. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi.

PubMed

Song, Zhangyong; Yin, Youping; Jiang, Shasha; Liu, Juanjuan; Chen, Huan; Wang, Zhongkang

2013-06-19

Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia.

Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

PubMed

Zhang, Peter G Y; Yeung, Joanna; Gupta, Ishita; Ramirez, Miguel; Ha, Thomas; Swanson, Douglas J; Nagao-Sato, Sayaka; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; de Hoon, Michiel; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Goldowitz, Dan

2018-06-01

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Preparing the next generation of genomicists: a laboratory-style course in medical genomics.

PubMed

Linderman, Michael D; Bashir, Ali; Diaz, George A; Kasarskis, Andrew; Sanderson, Saskia C; Zinberg, Randi E; Mahajan, Milind; Shah, Hardik; Suckiel, Sabrina; Zweig, Micol; Schadt, Eric E

2015-08-12

The growing gap between the demand for genome sequencing and the supply of trained genomics professionals is creating an acute need to develop more effective genomics education. In response we developed "Practical Analysis of Your Personal Genome", a novel laboratory-style medical genomics course in which students have the opportunity to obtain and analyze their own whole genome. This report describes our motivations for and the content of a "practical" genomics course that incorporates personal genome sequencing and the lessons we learned during the first three iterations of this course.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Insights from Human/Mouse genome comparisons

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

Pennacchio, Len A.

2003-03-30

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

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

PubMed Central

Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2016-01-01

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

PubMed

Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

2011-01-01

cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

SeqFIRE: a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments.

PubMed

Ajawatanawong, Pravech; Atkinson, Gemma C; Watson-Haigh, Nathan S; Mackenzie, Bryony; Baldauf, Sandra L

2012-07-01

Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.

Flow structure through pool-riffle sequences and a conceptual model for their sustainability in gravel-bed rivers

Treesearch

D. Caamano; P. Goodwin; J. M. Buffington

2010-01-01

Detailed field measurements and simulations of three-dimensional flow structure were used to develop a conceptual model to explain the sustainability of self-formed pool-riffle sequences in gravel-bed rivers. The analysis was conducted at the Red River Wildlife Management Area in Idaho, USA, and enabled characterization of the flow structure through two consecutive...

Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

PubMed

Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur

2011-04-18

Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

Classifying next-generation sequencing data using a zero-inflated Poisson model.

PubMed

Zhou, Yan; Wan, Xiang; Zhang, Baoxue; Tong, Tiejun

2018-04-15

With the development of high-throughput techniques, RNA-sequencing (RNA-seq) is becoming increasingly popular as an alternative for gene expression analysis, such as RNAs profiling and classification. Identifying which type of diseases a new patient belongs to with RNA-seq data has been recognized as a vital problem in medical research. As RNA-seq data are discrete, statistical methods developed for classifying microarray data cannot be readily applied for RNA-seq data classification. Witten proposed a Poisson linear discriminant analysis (PLDA) to classify the RNA-seq data in 2011. Note, however, that the count datasets are frequently characterized by excess zeros in real RNA-seq or microRNA sequence data (i.e. when the sequence depth is not enough or small RNAs with the length of 18-30 nucleotides). Therefore, it is desired to develop a new model to analyze RNA-seq data with an excess of zeros. In this paper, we propose a Zero-Inflated Poisson Logistic Discriminant Analysis (ZIPLDA) for RNA-seq data with an excess of zeros. The new method assumes that the data are from a mixture of two distributions: one is a point mass at zero, and the other follows a Poisson distribution. We then consider a logistic relation between the probability of observing zeros and the mean of the genes and the sequencing depth in the model. Simulation studies show that the proposed method performs better than, or at least as well as, the existing methods in a wide range of settings. Two real datasets including a breast cancer RNA-seq dataset and a microRNA-seq dataset are also analyzed, and they coincide with the simulation results that our proposed method outperforms the existing competitors. The software is available at http://www.math.hkbu.edu.hk/∼tongt. xwan@comp.hkbu.edu.hk or tongt@hkbu.edu.hk. Supplementary data are available at Bioinformatics online.

Comprehensive analysis of genetic variations in strictly-defined Leber congenital amaurosis with whole-exome sequencing in Chinese.

PubMed

Wang, Shi-Yuan; Zhang, Qi; Zhang, Xiang; Zhao, Pei-Quan

2016-01-01

To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis (LCA) in Chinese. LCA subjects and their families were retrospectively collected from 2013 to 2015. Firstly, whole-exome sequencing was performed in patients who had underwent gene mutation screening with nothing found, and then homozygous sites was selected, candidate sites were annotated, and pathogenic analysis was conducted using softwares including Sorting Tolerant from Intolerant (SIFT), Polyphen-2, Mutation assessor, Condel, and Functional Analysis through Hidden Markov Models (FATHMM). Furthermore, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of pathogenic genes were performed followed by co-segregation analysis using Fisher exact Test. Sanger sequencing was used to validate single-nucleotide variations (SNVs). Expanded verification was performed in the rest patients. Totally 51 LCA families with 53 patients and 24 family members were recruited. A total of 104 SNVs (66 LCA-related genes and 15 co-segregated genes) were submitted for expand verification. The frequencies of homozygous mutation of KRT12 and CYP1A1 were simultaneously observed in 3 families. Enrichment analysis showed that the potential pathogenic genes were mainly enriched in functions related to cell adhesion, biological adhesion, retinoid metabolic process, and eye development biological adhesion. Additionally, WFS1 and STAU2 had the highest homozygous frequencies. LCA is a highly heterogeneous disease. Mutations in KRT12, CYP1A1, WFS1, and STAU2 may be involved in the development of LCA.

Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

PubMed Central

2012-01-01

Background For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. Results From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties. Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. Conclusions An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene expression studies strongly suggested that the difference in leaf Cd accumulation between the two specific tobacco cultivars is dependent solely on genetic factors and genetic variability rather than on the environment. PMID:23190529

Factor structure of paediatric timed motor examination and its relationship with IQ

PubMed Central

MARTIN, REBECCA; TIGERA, CASSIE; DENCKLA, MARTHA B; MAHONE, E MARK

2012-01-01

AIM Brain systems supporting higher cognitive and motor control develop in a parallel manner, dependent on functional integrity and maturation of related regions, suggesting neighbouring neural circuitry. Concurrent examination of motor and cognitive control can provide a window into neurological development. However, identification of performance-based measures that do not correlate with IQ has been a challenge. METHOD Timed motor performance from the Physical and Neurological Examination of Subtle Signs and IQ were analysed in 136 children aged 6 to 16 (mean age 10y 2.6mo, SD 2y 6.4mo; 98 female, 38male) attending an outpatient neuropsychology clinic and 136 right-handed comparison individuals aged 6 to 16 (mean age 10y 3.1mo, SD 2y 6.1mo; 98 female, 38male). Timed activities – three repetitive movements (toe tapping, hand patting, finger tapping) and three sequenced movements (heel–toe tap, hand pronate/supinate, finger sequencing) each performed on the right and left – were included in exploratory factor analyses. RESULTS Among comparison individuals, factor analysis yielded two factors – repetitive and sequenced movements – with the sequenced factor significantly predictive of Verbal IQ (VIQ) (ΔR2=0.018, p=0.019), but not the repetitive factor (ΔR2=0.004, p=0.39). Factor analysis within the clinical group yielded two similar factors (repetitive and sequenced), both significantly predictive of VIQ, (ΔR2=0.028, p=0.015; ΔR2=0.046, p=0.002 respectively). INTERPRETATION Among typical children, repetitive timed tasks may be independent of IQ; however, sequenced tasks share more variance, implying shared neural substrates. Among neurologically vulnerable populations, however, both sequenced and repetitive movements covary with IQ, suggesting that repetitive speed is more indicative of underlying neurological integrity. PMID:20412260

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions.

PubMed

El-Mogharbel, Nisrine; Wakefield, Matthew; Deakin, Janine E; Tsend-Ayush, Enkhjargal; Grützner, Frank; Alsop, Amber; Ezaz, Tariq; Marshall Graves, Jennifer A

2007-01-01

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.

Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

PubMed Central

2012-01-01

Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

VIPER: Visualization Pipeline for RNA-seq, a Snakemake workflow for efficient and complete RNA-seq analysis.

PubMed

Cornwell, MacIntosh; Vangala, Mahesh; Taing, Len; Herbert, Zachary; Köster, Johannes; Li, Bo; Sun, Hanfei; Li, Taiwen; Zhang, Jian; Qiu, Xintao; Pun, Matthew; Jeselsohn, Rinath; Brown, Myles; Liu, X Shirley; Long, Henry W

2018-04-12

RNA sequencing has become a ubiquitous technology used throughout life sciences as an effective method of measuring RNA abundance quantitatively in tissues and cells. The increase in use of RNA-seq technology has led to the continuous development of new tools for every step of analysis from alignment to downstream pathway analysis. However, effectively using these analysis tools in a scalable and reproducible way can be challenging, especially for non-experts. Using the workflow management system Snakemake we have developed a user friendly, fast, efficient, and comprehensive pipeline for RNA-seq analysis. VIPER (Visualization Pipeline for RNA-seq analysis) is an analysis workflow that combines some of the most popular tools to take RNA-seq analysis from raw sequencing data, through alignment and quality control, into downstream differential expression and pathway analysis. VIPER has been created in a modular fashion to allow for the rapid incorporation of new tools to expand the capabilities. This capacity has already been exploited to include very recently developed tools that explore immune infiltrate and T-cell CDR (Complementarity-Determining Regions) reconstruction abilities. The pipeline has been conveniently packaged such that minimal computational skills are required to download and install the dozens of software packages that VIPER uses. VIPER is a comprehensive solution that performs most standard RNA-seq analyses quickly and effectively with a built-in capacity for customization and expansion.

Systematic analysis of coding and noncoding DNA sequences using methods of statistical linguistics

NASA Technical Reports Server (NTRS)

Mantegna, R. N.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Peng, C. K.; Simons, M.; Stanley, H. E.

1995-01-01

We compare the statistical properties of coding and noncoding regions in eukaryotic and viral DNA sequences by adapting two tests developed for the analysis of natural languages and symbolic sequences. The data set comprises all 30 sequences of length above 50 000 base pairs in GenBank Release No. 81.0, as well as the recently published sequences of C. elegans chromosome III (2.2 Mbp) and yeast chromosome XI (661 Kbp). We find that for the three chromosomes we studied the statistical properties of noncoding regions appear to be closer to those observed in natural languages than those of coding regions. In particular, (i) a n-tuple Zipf analysis of noncoding regions reveals a regime close to power-law behavior while the coding regions show logarithmic behavior over a wide interval, while (ii) an n-gram entropy measurement shows that the noncoding regions have a lower n-gram entropy (and hence a larger "n-gram redundancy") than the coding regions. In contrast to the three chromosomes, we find that for vertebrates such as primates and rodents and for viral DNA, the difference between the statistical properties of coding and noncoding regions is not pronounced and therefore the results of the analyses of the investigated sequences are less conclusive. After noting the intrinsic limitations of the n-gram redundancy analysis, we also briefly discuss the failure of the zeroth- and first-order Markovian models or simple nucleotide repeats to account fully for these "linguistic" features of DNA. Finally, we emphasize that our results by no means prove the existence of a "language" in noncoding DNA.

An Imaging And Graphics Workstation For Image Sequence Analysis

NASA Astrophysics Data System (ADS)

Mostafavi, Hassan

1990-01-01

This paper describes an application-specific engineering workstation designed and developed to analyze imagery sequences from a variety of sources. The system combines the software and hardware environment of the modern graphic-oriented workstations with the digital image acquisition, processing and display techniques. The objective is to achieve automation and high throughput for many data reduction tasks involving metric studies of image sequences. The applications of such an automated data reduction tool include analysis of the trajectory and attitude of aircraft, missile, stores and other flying objects in various flight regimes including launch and separation as well as regular flight maneuvers. The workstation can also be used in an on-line or off-line mode to study three-dimensional motion of aircraft models in simulated flight conditions such as wind tunnels. The system's key features are: 1) Acquisition and storage of image sequences by digitizing real-time video or frames from a film strip; 2) computer-controlled movie loop playback, slow motion and freeze frame display combined with digital image sharpening, noise reduction, contrast enhancement and interactive image magnification; 3) multiple leading edge tracking in addition to object centroids at up to 60 fields per second from both live input video or a stored image sequence; 4) automatic and manual field-of-view and spatial calibration; 5) image sequence data base generation and management, including the measurement data products; 6) off-line analysis software for trajectory plotting and statistical analysis; 7) model-based estimation and tracking of object attitude angles; and 8) interface to a variety of video players and film transport sub-systems.

Microbes, metagenomes and marine mammals: enabling the next generation of scientist to enter the genomic era

PubMed Central

2013-01-01

Background The revolution in DNA sequencing technology continues unabated, and is affecting all aspects of the biological and medical sciences. The training and recruitment of the next generation of researchers who are able to use and exploit the new technology is severely lacking and potentially negatively influencing research and development efforts to advance genome biology. Here we present a cross-disciplinary course that provides undergraduate students with practical experience in running a next generation sequencing instrument through to the analysis and annotation of the generated DNA sequences. Results Many labs across world are installing next generation sequencing technology and we show that the undergraduate students produce quality sequence data and were excited to participate in cutting edge research. The students conducted the work flow from DNA extraction, library preparation, running the sequencing instrument, to the extraction and analysis of the data. They sequenced microbes, metagenomes, and a marine mammal, the Californian sea lion, Zalophus californianus. The students met sequencing quality controls, had no detectable contamination in the targeted DNA sequences, provided publication quality data, and became part of an international collaboration to investigate carcinomas in carnivores. Conclusions Students learned important skills for their future education and career opportunities, and a perceived increase in students’ ability to conduct independent scientific research was measured. DNA sequencing is rapidly expanding in the life sciences. Teaching undergraduates to use the latest technology to sequence genomic DNA ensures they are ready to meet the challenges of the genomic era and allows them to participate in annotating the tree of life. PMID:24007365

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

PubMed

Kadumuri, Rajashekar Varma; Vadrevu, Ramakrishna

2017-10-01

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

SINEBase: a database and tool for SINE analysis.

PubMed

Vassetzky, Nikita S; Kramerov, Dmitri A

2013-01-01

SINEBase (http://sines.eimb.ru) integrates the revisited body of knowledge about short interspersed elements (SINEs). A set of formal definitions concerning SINEs was introduced. All available sequence data were screened through these definitions and the genetic elements misidentified as SINEs were discarded. As a result, 175 SINE families have been recognized in animals, flowering plants and green algae. These families were classified by the modular structure of their nucleotide sequences and the frequencies of different patterns were evaluated. These data formed the basis for the database of SINEs. The SINEBase website can be used in two ways: first, to explore the database of SINE families, and second, to analyse candidate SINE sequences using specifically developed tools. This article presents an overview of the database and the process of SINE identification and analysis.

SINEBase: a database and tool for SINE analysis

PubMed Central

Vassetzky, Nikita S.; Kramerov, Dmitri A.

2013-01-01

SINEBase (http://sines.eimb.ru) integrates the revisited body of knowledge about short interspersed elements (SINEs). A set of formal definitions concerning SINEs was introduced. All available sequence data were screened through these definitions and the genetic elements misidentified as SINEs were discarded. As a result, 175 SINE families have been recognized in animals, flowering plants and green algae. These families were classified by the modular structure of their nucleotide sequences and the frequencies of different patterns were evaluated. These data formed the basis for the database of SINEs. The SINEBase website can be used in two ways: first, to explore the database of SINE families, and second, to analyse candidate SINE sequences using specifically developed tools. This article presents an overview of the database and the process of SINE identification and analysis. PMID:23203982

Algorithm to find distant repeats in a single protein sequence

PubMed Central

Banerjee, Nirjhar; Sarani, Rangarajan; Ranjani, Chellamuthu Vasuki; Sowmiya, Govindaraj; Michael, Daliah; Balakrishnan, Narayanasamy; Sekar, Kanagaraj

2008-01-01

Distant repeats in protein sequence play an important role in various aspects of protein analysis. A keen analysis of the distant repeats would enable to establish a firm relation of the repeats with respect to their function and three-dimensional structure during the evolutionary process. Further, it enlightens the diversity of duplication during the evolution. To this end, an algorithm has been developed to find all distant repeats in a protein sequence. The scores from Point Accepted Mutation (PAM) matrix has been deployed for the identification of amino acid substitutions while detecting the distant repeats. Due to the biological importance of distant repeats, the proposed algorithm will be of importance to structural biologists, molecular biologists, biochemists and researchers involved in phylogenetic and evolutionary studies. PMID:19052663

RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

PubMed Central

Munger, Steven C.; Raghupathy, Narayanan; Choi, Kwangbom; Simons, Allen K.; Gatti, Daniel M.; Hinerfeld, Douglas A.; Svenson, Karen L.; Keller, Mark P.; Attie, Alan D.; Hibbs, Matthew A.; Graber, Joel H.; Chesler, Elissa J.; Churchill, Gary A.

2014-01-01

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations. PMID:25236449

Analysis and Prediction of Exon Skipping Events from RNA-Seq with Sequence Information Using Rotation Forest.

PubMed

Du, Xiuquan; Hu, Changlin; Yao, Yu; Sun, Shiwei; Zhang, Yanping

2017-12-12

In bioinformatics, exon skipping (ES) event prediction is an essential part of alternative splicing (AS) event analysis. Although many methods have been developed to predict ES events, a solution has yet to be found. In this study, given the limitations of machine learning algorithms with RNA-Seq data or genome sequences, a new feature, called RS (RNA-seq and sequence) features, was constructed. These features include RNA-Seq features derived from the RNA-Seq data and sequence features derived from genome sequences. We propose a novel Rotation Forest classifier to predict ES events with the RS features (RotaF-RSES). To validate the efficacy of RotaF-RSES, a dataset from two human tissues was used, and RotaF-RSES achieved an accuracy of 98.4%, a specificity of 99.2%, a sensitivity of 94.1%, and an area under the curve (AUC) of 98.6%. When compared to the other available methods, the results indicate that RotaF-RSES is efficient and can predict ES events with RS features.

Predicting PDZ domain mediated protein interactions from structure

PubMed Central

2013-01-01

Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on training–testing domain sequence similarity. Using both predictors, we defined a functional map of human PDZ domain biology and predict novel PDZ domain function. Users may access our structure-based and previous sequence-based predictors at http://webservice.baderlab.org/domains/POW. PMID:23336252

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

PubMed

de la Bastide, Paul Y; Leung, Wai Lam; Hintz, William E

2015-01-01

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Transcriptomic analysis of flower development in tea (Camellia sinensis (L.)).

PubMed

Liu, Feng; Wang, Yu; Ding, Zhaotang; Zhao, Lei; Xiao, Jun; Wang, Linjun; Ding, Shibo

2017-10-05

Flowering is a critical and complicated process in plant development, involving interactions of numerous endogenous and environmental factors, but little is known about the complex network regulating flower development in tea plants. In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptomic analysis assembles gene-related information involved in reproductive growth of C. sinensis. Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction were enriched among the DEGs. Furthermore, 207 flowering-associated unigenes were identified from our database. Some transcription factors, such as WRKY, ERF, bHLH, MYB and MADS-box were shown to be up-regulated in floral transition, which might play the role of progression of flowering. Furthermore, 14 genes were selected for confirmation of expression levels using quantitative real-time PCR (qRT-PCR). The comprehensive transcriptomic analysis presents fundamental information on the genes and pathways which are involved in flower development in C. sinensis. Our data also provided a useful database for further research of tea and other species of plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptome analysis of pecan seeds at different developing stages and identification of key genes involved in lipid metabolism

PubMed Central

Shah, Faheem Afzal; Wang, Qiaojian; Wang, Zhaocheng; Wu, Lifang

2018-01-01

Pecan is an economically important nut crop tree due to its unique texture and flavor properties. The pecan seed is rich of unsaturated fatty acid and protein. However, little is known about the molecular mechanisms of the biosynthesis of fatty acids in the developing seeds. In this study, transcriptome sequencing of the developing seeds was performed using Illumina sequencing technology. Pecan seed embryos at different developmental stages were collected and sequenced. The transcriptomes of pecan seeds at two key developing stages (PA, the initial stage and PS, the fast oil accumulation stage) were also compared. A total of 82,155 unigenes, with an average length of 1,198 bp from seven independent libraries were generated. After functional annotations, we detected approximately 55,854 CDS, among which, 2,807 were Transcription Factor (TF) coding unigenes. Further, there were 13,325 unigenes that showed a 2-fold or greater expression difference between the two groups of libraries (two developmental stages). After transcriptome analysis, we identified abundant unigenes that could be involved in fatty acid biosynthesis, degradation and some other aspects of seed development in pecan. This study presents a comprehensive dataset of transcriptomic changes during the seed development of pecan. It provides insights in understanding the molecular mechanisms responsible for fatty acid biosynthesis in the seed development. The identification of functional genes will also be useful for the molecular breeding work of pecan. PMID:29694395

Transcriptome analysis of pecan seeds at different developing stages and identification of key genes involved in lipid metabolism.

PubMed

Xu, Zheng; Ni, Jun; Shah, Faheem Afzal; Wang, Qiaojian; Wang, Zhaocheng; Wu, Lifang; Fu, Songling

2018-01-01

Pecan is an economically important nut crop tree due to its unique texture and flavor properties. The pecan seed is rich of unsaturated fatty acid and protein. However, little is known about the molecular mechanisms of the biosynthesis of fatty acids in the developing seeds. In this study, transcriptome sequencing of the developing seeds was performed using Illumina sequencing technology. Pecan seed embryos at different developmental stages were collected and sequenced. The transcriptomes of pecan seeds at two key developing stages (PA, the initial stage and PS, the fast oil accumulation stage) were also compared. A total of 82,155 unigenes, with an average length of 1,198 bp from seven independent libraries were generated. After functional annotations, we detected approximately 55,854 CDS, among which, 2,807 were Transcription Factor (TF) coding unigenes. Further, there were 13,325 unigenes that showed a 2-fold or greater expression difference between the two groups of libraries (two developmental stages). After transcriptome analysis, we identified abundant unigenes that could be involved in fatty acid biosynthesis, degradation and some other aspects of seed development in pecan. This study presents a comprehensive dataset of transcriptomic changes during the seed development of pecan. It provides insights in understanding the molecular mechanisms responsible for fatty acid biosynthesis in the seed development. The identification of functional genes will also be useful for the molecular breeding work of pecan.

A Novel Cylindrical Representation for Characterizing Intrinsic Properties of Protein Sequences.

PubMed

Yu, Jia-Feng; Dou, Xiang-Hua; Wang, Hong-Bo; Sun, Xiao; Zhao, Hui-Ying; Wang, Ji-Hua

2015-06-22

The composition and sequence order of amino acid residues are the two most important characteristics to describe a protein sequence. Graphical representations facilitate visualization of biological sequences and produce biologically useful numerical descriptors. In this paper, we propose a novel cylindrical representation by placing the 20 amino acid residue types in a circle and sequence positions along the z axis. This representation allows visualization of the composition and sequence order of amino acids at the same time. Ten numerical descriptors and one weighted numerical descriptor have been developed to quantitatively describe intrinsic properties of protein sequences on the basis of the cylindrical model. Their applications to similarity/dissimilarity analysis of nine ND5 proteins indicated that these numerical descriptors are more effective than several classical numerical matrices. Thus, the cylindrical representation obtained here provides a new useful tool for visualizing and charactering protein sequences. An online server is available at http://biophy.dzu.edu.cn:8080/CNumD/input.jsp .

Enabling large-scale next-generation sequence assembly with Blacklight

PubMed Central

Couger, M. Brian; Pipes, Lenore; Squina, Fabio; Prade, Rolf; Siepel, Adam; Palermo, Robert; Katze, Michael G.; Mason, Christopher E.; Blood, Philip D.

2014-01-01

Summary A variety of extremely challenging biological sequence analyses were conducted on the XSEDE large shared memory resource Blacklight, using current bioinformatics tools and encompassing a wide range of scientific applications. These include genomic sequence assembly, very large metagenomic sequence assembly, transcriptome assembly, and sequencing error correction. The data sets used in these analyses included uncategorized fungal species, reference microbial data, very large soil and human gut microbiome sequence data, and primate transcriptomes, composed of both short-read and long-read sequence data. A new parallel command execution program was developed on the Blacklight resource to handle some of these analyses. These results, initially reported previously at XSEDE13 and expanded here, represent significant advances for their respective scientific communities. The breadth and depth of the results achieved demonstrate the ease of use, versatility, and unique capabilities of the Blacklight XSEDE resource for scientific analysis of genomic and transcriptomic sequence data, and the power of these resources, together with XSEDE support, in meeting the most challenging scientific problems. PMID:25294974

A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance.

PubMed

Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Cisneros, Jose Luis Bellod; Jurtz, Vanessa; Larsen, Mette Voldby; Hasman, Henrik; Aarestrup, Frank Møller; Lund, Ole

2016-01-01

Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available.

Machine learning applications in genetics and genomics.

PubMed

Libbrecht, Maxwell W; Noble, William Stafford

2015-06-01

The field of machine learning, which aims to develop computer algorithms that improve with experience, holds promise to enable computers to assist humans in the analysis of large, complex data sets. Here, we provide an overview of machine learning applications for the analysis of genome sequencing data sets, including the annotation of sequence elements and epigenetic, proteomic or metabolomic data. We present considerations and recurrent challenges in the application of supervised, semi-supervised and unsupervised machine learning methods, as well as of generative and discriminative modelling approaches. We provide general guidelines to assist in the selection of these machine learning methods and their practical application for the analysis of genetic and genomic data sets.

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.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

PubMed

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-07-01

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

PubMed

Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

2016-11-01

High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region.

PubMed Central

Ashburner, M; Misra, S; Roote, J; Lewis, S E; Blazej, R; Davis, T; Doyle, C; Galle, R; George, R; Harris, N; Hartzell, G; Harvey, D; Hong, L; Houston, K; Hoskins, R; Johnson, G; Martin, C; Moshrefi, A; Palazzolo, M; Reese, M G; Spradling, A; Tsang, G; Wan, K; Whitelaw, K; Celniker, S

1999-01-01

A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized "Adh region." A computational analysis of the sequence predicts 218 protein-coding genes, 11 tRNAs, and 17 transposable element sequences. At least 38 of the protein-coding genes are arranged in clusters of from 2 to 6 closely related genes, suggesting extensive tandem duplication. The gene density is one protein-coding gene every 13 kb; the transposable element density is one element every 171 kb. Of 73 genes in this region identified by genetic analysis, 49 have been located on the sequence; P-element insertions have been mapped to 43 genes. Ninety-five (44%) of the known and predicted genes match a Drosophila EST, and 144 (66%) have clear similarities to proteins in other organisms. Genes known to have mutant phenotypes are more likely to be represented in cDNA libraries, and far more likely to have products similar to proteins of other organisms, than are genes with no known mutant phenotype. Over 650 chromosome aberration breakpoints map to this chromosome region, and their nonrandom distribution on the genetic map reflects variation in gene spacing on the DNA. This is the first large-scale analysis of the genome of D. melanogaster at the sequence level. In addition to the direct results obtained, this analysis has allowed us to develop and test methods that will be needed to interpret the complete sequence of the genome of this species.Before beginning a Hunt, it is wise to ask someone what you are looking for before you begin looking for it. Milne 1926 PMID:10471707