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Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs

NUCLEIC ACIDS RESEARCH 2017年第15期45卷 8731-8744页

作者： Fehlmann, Tobias Backes, Christina Kahraman, Mustafa Haas, Jan Ludwig, Nicole Posch, Andreas E. Wuerstle, Maximilian L. Huebenthal, Matthias Franke, Andre Meder, Benjamin Meese, Eckart Keller, Andreas Saarland Univ Chair Clin Bioinformat Saarbrucken Germany Hummingbird Diagnost GmbH Heidelberg Germany Univ Hosp Heidelberg Dept Internal Med 3 Heidelberg Germany German Ctr Cardiovasc Res DZHK Heidelberg Germany Klaus Tschira Inst Integrat Computat Cardiol Heidelberg Germany Saarland Univ Dept Human Genet Homburg Germany Ares Genet GmbH Vienna Austria Siemens Healthcare GmbH Strategy & Innovat Erlangen Germany Christian Albrechts Univ Kiel Inst Clin Mol Biol Kiel Germany

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive webbased tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-touse tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them.

关键词： Internet Computational Biology/*methods High-Throughput Nucleotide Sequencing/*methods MicroRNAs/*analysis Sequence analysis RNA/*methods Computational Biology/statistics & numerical data Data Interpretation Statistical High-Throughput Nucleotide Sequencing/statistics & numerical data Humans MicroRNAs/genetics microarray analysis/methods Sequence analysis RNA/statistics & numerical data Transcriptome Validation Studies as Topic

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Method validation of the biochip array technology for synthetic cannabinoids detection in urine

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BIOanalysis 2014年第21期6卷 2919-2930页

作者： Castaneto, Marisol S. Desrosiers, Nathalie A. Ellefsen, Kayla Anizan, Sebastien Martin, Thomas M. Klette, Kevin L. Huestis, Marilyn A. NIDA NIH Baltimore MD 21224 USA Univ Maryland Program Toxicol Baltimore MD 21201 USA Off Secretary Def Operat Readiness & Safety Washington DC USA

Background: Synthetic cannabinoids (SC) are widely-abused cannabimimetic drugs that do not screen positive in traditional cannabinoids immunoassays, making detection difficult. methods & results: The first commercially-available immunoassay for urinary SC was validated. Limits of detection (5-20 mu g/L), imprecision (< 13.1% intra-, < 37.7% inter-assay), and cross-reactivity profiles of 22 SC and 37 metabolites were obtained. A large negative bias (-80.8 to -28.0%) was observed. Sensitivity (98.3%), specificity (48.1%) and efficiency (53.9%) were determined from screening 20,017 urine specimens and confirming 1432 presumptive positive and 1069 selected negative specimens by LC-MS/MS. Cutoff optimization improved performance to 87.6% sensitivity, 85.2% specificity, and 85.4% efficiency. Conclusion: This high-throughput urine SC assay has good sensitivity and improved specificity and efficiency at modified cutoff concentrations.

关键词： Calibration Cannabinoids/metabolism Cannabinoids/standards Cannabinoids/urine Cross Reactions Humans Hydrogen-Ion Concentration Immunoassay/methods Immunoassay/methods Immunoassay/standards Immunoassay/standards Limit of Detection microarray analysis/methods microarray analysis/standards Substance Abuse Detection

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The Use of Protein-DNA, Chromatin Immunoprecipitation, and Transcriptome Arrays to Describe Transcriptional Circuits in the Dehydrated Male Rat Hypothalamus

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ENDOCRINOLOGY 2014年第11期155卷 4380-4390页

作者： Qiu, Jing Kleineidam, Anna Gouraud, Sabine Yao, Song Tieng Greenwood, Mingkwan Hoe, See Ziau Hindmarch, Charles Murphy, David Univ Bristol Sch Clin Sci Bristol BS1 3NY Avon England Univ Malaya Fac Med Dept Physiol Kuala Lumpur 50603 Malaysia

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

关键词： Animals Chromatin Immunoprecipitation Dehydration/genetics Dehydration/metabolism DNA/metabolism DNA-Binding Proteins/metabolism Gene Expression Profiling Gene Expression Regulation Gene Regulatory Networks Hypothalamus/chemistry Hypothalamus/metabolism Male microarray analysis/methods Promoter Regions, Genetic Rats Rats, Sprague-Dawley Transcription Factors/metabolism Transcriptome

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Generation of a chemical gradient across an array of 256 cell cultures in a single chip

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ANALYST 2013年第19期138卷 5566-5571页

作者： Somaweera, Himali Ibragimov, Akif Pappas, Dimitri Texas Tech Univ Dept Chem & Biochem Lubbock TX 79409 USA Texas Tech Univ Dept Math & Stat Lubbock TX 79409 USA Texas Tech Univ Ctr Chem Biol Lubbock TX 79409 USA

A microfluidic diffusion diluter to create stable chemical gradients across an array of cell cultures was demonstrated. The device enabled concentration based studies to be conducted at 256 different concentrations across individual, low shear cell cultures. A gradient of staurosporine on cells stained with Mitotracker Deep Red (MTDR) showed a concentration-based effect on cell apoptosis across the cell culture array.

关键词： Cell Culture Techniques/instrumentation Cell Culture Techniques/instrumentation Cell Culture Techniques/methods Cell Culture Techniques/methods Cell Survival/physiology Humans microarray analysis/instrumentation microarray analysis/methods Microfluidic Analytical Techniques/instrumentation Microfluidic Analytical Techniques/methods

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Multiplex Genome Engineering Using CRISPR/Cas Systems

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SCIENCE 2013年第6121期339卷 819-823页

作者： Cong, Le Ran, F. Ann Cox, David Lin, Shuailiang Barretto, Robert Habib, Naomi Hsu, Patrick D. Wu, Xuebing Jiang, Wenyan Marraffini, Luciano A. Zhang, Feng Broad Inst MIT & Harvard Cambridge Ctr 7 Cambridge MA 02142 USA MIT Dept Biol Engn Dept Brain & Cognit Sci McGovern Inst Brain Res Cambridge MA 02139 USA Harvard Univ Sch Med Program Biol & Biomed Sci Boston MA 02115 USA Harvard Univ Sch Med Harvard MIT Hlth Sci & Technol Boston MA 02115 USA Harvard Univ Dept Mol & Cellular Biol Cambridge MA 02138 USA Tsinghua Univ Sch Life Sci Beijing 100084 Peoples R China Columbia Univ Coll Phys & Surg Dept Biochem & Mol Biophys New York NY 10032 USA MIT Computat & Syst Biol Grad Program Cambridge MA 02139 USA MIT Koch Inst Integrat Canc Res Cambridge MA 02139 USA Rockefeller Univ Bacteriol Lab New York NY 10065 USA

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

关键词： Animals Base Sequence Caspase 9/chemistry Caspase 9/genetics DNA/chemistry DNA/genetics DNA Cleavage Genetic Engineering/methods Genetic Loci Genome/genetics Humans Inverted Repeat Sequences/genetics Mice microarray analysis/methods Molecular Sequence Data Mutagenesis Recombinational DNA Repair RNA/chemistry RNA/genetics Streptococcus pyogenes/enzymology Streptococcus pyogenes/enzymology Streptococcus pyogenes/genetics Streptococcus pyogenes/genetics

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Characterizing the Surface Quality and Droplet Interface Shape for microarray Plates

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LANGMUIR 2012年第26期28卷 9961-9966页

作者： Schertzer, M. J. Ahamed, M. J. Ben-Mrad, R. Lea, P. Sullivan, P. E. Univ Toronto Dept Mech & Ind Engn Toronto ON M5S 3G8 Canada SQI Diagnost Toronto ON M9W 1A4 Canada Univ Calif Irvine Irvine CA 92697 USA

The variation in the surface quality of microarray plates was examined by measuring the contact angles of 480 droplets on five microarray plates. It was found that the measured contact angle did not accurately predict the droplet shape for moderate Bond numbers (similar to 0.5 <= N-B <= 5). By defining an apparent contact angle using the ratio of the contact radius to the height, the variance in the predicted interface shape decreased by greater than a factor of 3 for both local and globally averaged characteristics. The error in the predicted droplet height was also reduced by 3 orders of magnitude.

关键词： microarray analysis/methods Surface Properties

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Molecular blood typing augments serologic testing and allows for enhanced matching of red blood cells for transfusion in patients with sickle cell disease

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TRANSFUSION 2012年第2期52卷 381-388页

作者： Wilkinson, Katie Harris, Samantha Gaur, Prashant Haile, Askale Armour, Rosalind Teramura, Gayle Delaney, Meghan Puget Sound Blood Ctr Seattle WA 98104 USA Puget Sound Inst Pathol Seattle WA USA

BACKGROUND: Sickle cell disease (SCD) patients have dissimilar red blood cell (RBC) phenotypes compared to the primarily Caucasian blood donor base due, in part, to underlying complex Rh and silenced Duffy expression. Gene arraybased technology offers high-throughput antigen typing of blood donors and can identify patients with altered genotypes. The purpose of the study was to ascertain if RBC components drawn from predominantly Caucasian donors could provide highly antigen-matched products for molecularly typed SCD patients. STUDY DESIGN AND methods: SCD patients were genotyped by a molecular array (HEA Beadchip, BioArray Solutions). The extended antigen phenotype (C, c, E, e, K, k, Jka, Jkb, Fya, Fyb, S, s) was used to query the inventory using different matching algorithms;the resulting number of products was recorded. RESULTS: A mean of 96.2 RBC products was available for each patient at basic-level, 34 at mid-level, and 16.3 at high-level stringency. The number of negative antigens correlated negatively with the number of available products. The Duffy silencing mutation in the promoter region (67T> C) (GATA) was found in 96.5% of patients. Allowing Fy(b+) products for patients with GATA increased the number of available products by up to 180%, although it does not ensure prevention of Duffy antibodies in all patients. CONCLUSIONS: This feasibility study provides evidence that centers with primarily Caucasian donors may be able to provide highly antigen-matched products. Knowledge of the GATA status expands the inventory of antigen-matched products. Further work is needed to determine the most clinically appropriate match level for SCD patients.

关键词： Adult Algorithms Anemia, Sickle Cell/blood Anemia, Sickle Cell/epidemiology Anemia, Sickle Cell/immunology Anemia, Sickle Cell/therapy Blood Donors Blood Group Incompatibility/epidemiology Blood Group Incompatibility/prevention & control Blood Grouping and Crossmatching/methods Blood Grouping and Crossmatching/methods Erythrocyte Transfusion Humans microarray analysis/methods Middle Aged Molecular Typing/methods Prevalence Serologic Tests/statistics & numerical data Serologic Tests/statistics & numerical data Serologic Tests/utilization Serologic Tests/utilization Up-Regulation Young Adult

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Signal enhancement of a micro-arrayed polydiacetylene (PDA) biosensor using gold nanoparticles

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ANALYST 2012年第5期137卷 1241-1246页

作者： Won, Sang Ho Sim, Sang Jun Korea Univ Dept Chem & Biol Engn Seoul 136713 South Korea Sungkyunkwan Univ Sch Chem Engn Suwon 440746 South Korea

Polydiacetylene (PDA) liposomes possess unique properties that allow liposomes to change color and emit fluorescence in response to stimuli such as temperature, antibody-antigen interaction, pH, mechanical stress, and organic solvent. They have been studied extensively as signal transducers in biosensor applications. Here, we describe an antibody-based biosensor using PDA liposomes for detection of human immunoglobulin E (hIgE). Target hIgE chemically bound to hIgE monoclonal antibodies immobilized on PDA liposomes and the fluorescent signals were slightly increased depending on the target protein concentration. As the primary response, the hIgE could be detected to below 10 ng mL(-1). However, fluorescent signals were dramatically increased depending on the target protein concentration when gold nanoparticle-conjugated polyclonal antibody probes were added on the PDA liposomes after the primary immune reaction. A PDA liposome biosensor could detect the hIgE as low as 0.1 ng mL(-1) and the sensitivity was increased up to one hundred times higher than the primary response. As a result, we confirmed that gold nanoparticle-conjugated polyclonal antibody probes efficiently enhanced the fluorescent signal of the PDA liposome biosensor chip. This strategy can be useful to detect proteins of ultra-low concentration.

关键词： Biosensing Techniques/methods Gold/chemistry Humans Immunoglobulin E/analysis Liposomes/chemistry Metal Nanoparticles/chemistry microarray analysis/methods Particle Size Polyacetylenes/chemistry Polymers/chemistry Spectrometry, Fluorescence

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DNAzyme-based fluorescent microarray for highly selective and sensitive detection of lead(II)

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ANALYST 2012年第1期137卷 70-72页

作者： Liu, Meiying Lou, Xinhui Du, Juan Guan, Ming Wang, Jing Ding, Xiaofan Zhao, Jianlong Capital Normal Univ Dept Chem Beijing 100084 Peoples R China Capital Normal Univ Dept Chem Beijing 100084 Peoples R China Chinese Acad Sci Grad Sch Beijing 100049 Peoples R China Chinese Acad Sci Shanghai Inst Microsyst & Informat Technol State Key Lab Transducer Technol Shanghai 200050 Peoples R China

A facile microarray-based fluorescent sensor for the detection of lead (II) was developed based on the catalytic cleavages of the substrates by a DNAzyme upon its binding to Pb2+. The release of the fluorophore labelled substrates resulted in the decrease of fluorescence intensity. The sensor had a quantifiable detection range from 1 nM to 1 mu M and a selectivity of >20 fold for Pb2+ over other metal ions.

关键词： Biosensing Techniques/methods Catalytic Domain Cations, Divalent DNA, Catalytic/chemistry DNA, Catalytic/metabolism Fluorescent Dyes Lead/analysis Lead/chemistry Lead/metabolism microarray analysis/methods Sensitivity and Specificity Spectrometry, Fluorescence/methods

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Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia

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BLOOD 2012年第10期119卷 2376-2384页

作者： Kuramitsu, Madoka Sato-Otsubo, Aiko Morio, Tomohiro Takagi, Masatoshi Toki, Tsutomu Terui, Kiminori Wang, RuNan Kanno, Hitoshi Ohga, Shouichi Ohara, Akira Kojima, Seiji Kitoh, Toshiyuki Goi, Kumiko Kudo, Kazuko Matsubayashi, Tadashi Mizue, Nobuo Ozeki, Michio Masumi, Atsuko Momose, Haruka Takizawa, Kazuya Mizukami, Takuo Yamaguchi, Kazunari Ogawa, Seishi Ito, Etsuro Hamaguchi, Isao Natl Inst Infect Dis Dept Safety Res Blood & Biol Prod Tokyo 2080011 Japan Univ Tokyo Grad Sch Sci Canc Genom Project Tokyo 113 Japan Tokyo Med & Dent Univ Dept Pediat & Dev Biol Grad Sch Med Bunkyo Ku Tokyo Japan Hirosaki Univ Dept Pediat Grad Sch Med Hirosaki Aomori 0368562 Japan Tokyo Womens Med Univ Dept Transfus Med & Cell Proc Tokyo Japan Kyushu Univ Dept Pediat Grad Sch Med Sci Fukuoka 812 Japan Toho Univ Dept Pediat 1 Sch Med Tokyo Japan Nagoya Univ Dept Pediat Grad Sch Med Nagoya Aichi 4648601 Japan Shiga Med Ctr Children Dept Hematol Oncol Shiga Japan Univ Yamanashi Dept Pediat Sch Med Yamanashi Japan Shizuoka Childrens Hosp Div Hematol & Oncol Shizuoka Japan Seirei Hamamatsu Gen Hosp Dept Pediat Shizuoka Japan Kushiro City Gen Hosp Dept Pediat Kushiro Hokkaido Japan Gifu Univ Dept Pediat Grad Sch Med Gifu Japan

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes. (Blood. 2012;119(10):2376-2384)

关键词： Anemia, Diamond-Blackfan/ethnology Anemia, Diamond-Blackfan/genetics Anemia, Diamond-Blackfan/pathology Asian Continental Ancestry Group/genetics Child, Preschool DNA Mutational analysis/methods Female Gene Deletion Genetic Association Studies Humans Infant Infant, Newborn Japan Japan Male microarray analysis/methods Polymerase Chain Reaction Polymorphism, Single Nucleotide Reproducibility of Results Ribosomal Proteins/genetics

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