385 research outputs found

    These are not the k-mers you are looking for: efficient online k-mer counting using a probabilistic data structure

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    K-mer abundance analysis is widely used for many purposes in nucleotide sequence analysis, including data preprocessing for de novo assembly, repeat detection, and sequencing coverage estimation. We present the khmer software package for fast and memory efficient online counting of k-mers in sequencing data sets. Unlike previous methods based on data structures such as hash tables, suffix arrays, and trie structures, khmer relies entirely on a simple probabilistic data structure, a Count-Min Sketch. The Count-Min Sketch permits online updating and retrieval of k-mer counts in memory which is necessary to support online k-mer analysis algorithms. On sparse data sets this data structure is considerably more memory efficient than any exact data structure. In exchange, the use of a Count-Min Sketch introduces a systematic overcount for k-mers; moreover, only the counts, and not the k-mers, are stored. Here we analyze the speed, the memory usage, and the miscount rate of khmer for generating k-mer frequency distributions and retrieving k-mer counts for individual k-mers. We also compare the performance of khmer to several other k-mer counting packages, including Tallymer, Jellyfish, BFCounter, DSK, KMC, Turtle and KAnalyze. Finally, we examine the effectiveness of profiling sequencing error, k-mer abundance trimming, and digital normalization of reads in the context of high khmer false positive rates. khmer is implemented in C++ wrapped in a Python interface, offers a tested and robust API, and is freely available under the BSD license at github.com/ged-lab/khmer

    Don't Thrash: How to Cache Your Hash on Flash

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    This paper presents new alternatives to the well-known Bloom filter data structure. The Bloom filter, a compact data structure supporting set insertion and membership queries, has found wide application in databases, storage systems, and networks. Because the Bloom filter performs frequent random reads and writes, it is used almost exclusively in RAM, limiting the size of the sets it can represent. This paper first describes the quotient filter, which supports the basic operations of the Bloom filter, achieving roughly comparable performance in terms of space and time, but with better data locality. Operations on the quotient filter require only a small number of contiguous accesses. The quotient filter has other advantages over the Bloom filter: it supports deletions, it can be dynamically resized, and two quotient filters can be efficiently merged. The paper then gives two data structures, the buffered quotient filter and the cascade filter, which exploit the quotient filter advantages and thus serve as SSD-optimized alternatives to the Bloom filter. The cascade filter has better asymptotic I/O performance than the buffered quotient filter, but the buffered quotient filter outperforms the cascade filter on small to medium data sets. Both data structures significantly outperform recently-proposed SSD-optimized Bloom filter variants, such as the elevator Bloom filter, buffered Bloom filter, and forest-structured Bloom filter. In experiments, the cascade filter and buffered quotient filter performed insertions 8.6-11 times faster than the fastest Bloom filter variant and performed lookups 0.94-2.56 times faster.Comment: VLDB201

    In Situ Diazotroph Population Dynamics Under Different Resource Ratios in the North Pacific Subtropical Gyre.

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    Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models

    Dinitrogen fixation in the unicellular diazotroph Crocosphaera watsonii

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    Crocosphaera watsonii is an abundant organism in the tropical and subtropical ocean and is considered to be an important contributor to the marine nitrogen cycle due to its ability to fix dinitrogen (N2). Light-dark cycles were used to determine the diel variation in N2 fixation and photosynthesis in the unicellular diazotrophic cyanobacterium C. watsonii WH8501. A first set of experiments showed that N2 fixation and photosynthesis were separated temporally with N2 fixation during the dark and photosynthesis during the light periods. Due to the storage of carbon reserves during the day and subsequent respiration at night, C. watsonii links its cellular nitrogen and carbon metabolism. N2 fixation and photosynthesis appear to be regulated and optimized by a circadian rhythm. Gene expression analysis demonstrated cyclic patterns of the major genes involved in nitrogen and carbon metabolism. However, the patterns of expression did not coincide with patterns of activity with the gene expression peaking several hours prior to the activity. In a second set of experiments, N2 fixation and photosynthesis were simultaneously measurable at population level in the respective dark period, i.e. an artificial light period instead of a dark period. Single-cell analysis using nanoSIMS revealed that the co-occurrence of N2 fixation and photosynthesis at population level was probably due to each individual cell’s capability of coping with the O2-sensitivity of the nitrogenase enzyme. In addition, single-cell rates showed large variability with the highest rate about six times higher than the mean rate during the normal dark N2 fixation. The experimental work with a pure culture of a diazotroph provided the opportunity for a re-assessment of the two commonly used methods for the measurement of N2 fixation in both field and laboratory studies, i.e. the 15N2-tracer addition method and the acetylene reduction assay

    Uudsete meetodite arendamine ligandide s idumisomaduste uurimiseks melanokortiini 4 retseptorile

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    VĂ€itekirja elektrooniline versioon ei sisalda publikatsioone.KĂ€esolev doktoritöö „Uudsete meetodite arendamine ligandide sidumisomaduste uurimiseks melanokortiini 4 retseptorile“ kĂ€sitleb uuringutega, mille ĂŒheks eesmĂ€rgiks oli fluorestsentsil pĂ”hinevate katsesĂŒsteemide vĂ€ljatöötamine, mida saaks kasutada uute melanokortiini retseptorite spetsiifiliste ligandide avastamiseks. Melanokortiini retseptorid osalevad mitmete oluliste fĂŒsioloogiliste funktsioonide regulatsioonil nagu pigmentatsioon, seksuaal- ja toitumiskĂ€itumine, energiatasakaalu reguleerimine, valu ja keha temperatuuri reguleerimine, immuunsed ja pĂ”letikuvastased reaktsioonid, jne. Seega on melanokortiini retseptoritele spetsiifilised ligandid perspektiivsed ravimikandidaadid selliste haiguste ravimiseks nagu rasvumine ja anoreksia, melanoom, erektsiooni ja seksuaalsuse hĂ€ired, aga ka Ă€revushĂ€ired ning depressioon. Uute tĂ”husate ravimite leidmine sĂ”ltub suurel mÀÀral ka meie teadmistest retseptor-ligandide vastasmĂ”ju mehhanisme kohta ning oskusest kasutada neid teadmisi uudsete efektiivsete raviainete disainimiseks. Lisaks sellele, oleneb tihti ka meetodist, mis on antud juhul meie „silmadeks“ selles katsesĂŒsteemis, millist toimeaine mĂ”ju me ĂŒldse nĂ€eme ja kui hĂ€sti me seda detekteerida ning iseloomustada suudame. Siin töös arendatud uudsed fluorestsentsil pĂ”hinevad kastesĂŒsteemid vĂ”imaldavad loobuda radioaktiivsete ĂŒhendite kasutamisest ning jĂ€lgida retseptor-ligandi vastasmĂ”jusid reaalajas. See vĂ”imaldab saada tĂ€iendavat informatsiooni melanokortiinse sĂŒsteemi funktsioneerimise kohta, aga ka luua automatiseeritud katsesĂŒsteem uute aktiivsete ĂŒhendite leidmiseks.Current PhD thesis “Development of assay systems for characterisation of ligand binding properties to melanocortin 4 receptors” describes our progress of melanocortin receptor studies connected with development of novel assay systems that would facilitate the discovery of novel receptor specific ligands. Melanocortin receptors are involved in regulation of wide variety of physiological functions like pigmentation, sexual behaviour, regulation of energy balance and feeding behaviours, temperature control and pain sense, inflammatory and immune responses and others. Thus, ligands for these receptors have a remarkable therapeutic potential for treatment of several human disorders like obesity and anorexia, melanoma, erectile dysfunction and sexual motivation, as well as anxiety and depression. Success in discovery of new drugs, in many aspects depends from our ability to understand the mechanisms of receptor-ligand interactions and use this to design novel, receptor subtype selective, potent and metabolically suitable drugs. Besides that, assay properties may play a very important role in interpretation of results, as the ability to see the effect of the drug depends on the “eyes” of the assay through which it is monitored. Fluorescence anisotropy-based assay systems we developed avoid the use of radioactive ligands and allow on-line monitoring of receptor-ligand interactions that would improve general understanding of the melanocortin system

    To investigate the physiological role of arcuate nucleus cocaine- and amphetamine- regulated transcript in energy homeostasis

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    Cocaine- and amphetamine- regulated transcript (CART) was originally identified as a mRNA transcript upregulated in rats in response to administration of cocaine and amphetamine. CART is widely expressed in the central nervous system (CNS) with high levels of expression in hypothalamic nuclei such as the arcuate nucleus (ARC). CART was initially thought to act as an anorectic peptide since it is coexpressed with the anorectic neuropeptide pro-opiomelanocortin (POMC) in the ARC. In addition, intracerebroventricular (ICV) administration of CART (55-102) peptide inhibits feeding and administration of anti-CART antibody results in stimulation of feeding. However, subsequent studies have suggested CART may also act as an orexigen since injection of CART (55-102) specifically into the ARC and ventromedial nucleus (VMN) of the hypothalamus results in a significant increase in food intake. These data suggest CART acts through both anorectic and orexigenic circuits. Given the importance of the hypothalamus in the regulation of energy homeostasis, and the role of the ARC in integrating peripheral signals, it is essential to elucidate the role of ARC derived CART. In order to elucidate CART’s true physiological role in the ARC I used a combination of genetic approaches. I generated a recombinant Adeno-associated virus (rAAV) expressing CART antisense (CART-AS) and a transgenic mouse model which utilises the POMC promoter to drive expression of CART-AS. In the transgenic CART-AS model mice exhibited a significantly higher body weight relative to control animals, no significant difference in food intake was observed. In addition, mice expressing the CART-AS transgene demonstrated a reduction in uncoupling protein-1 (UCP- 1) mRNA expression in brown adipose tissue (BAT) which is suggestive of decreased thermogenesis. This may explain the observed increase in body weight in the transgenic mice. Bilateral intra-ARC injections of rAAV-CART-AS resulted in a significant increase in cumulative food intake and body weight gain compared to control animals. There was no significant difference in activity or metabolism levels. The data presented in my thesis provides an important contribution to understanding the role of CART within the ARC. The results from my genetic studies appear to suggest that ARC derived CART has an anorectic role

    Arkansas Bulletin of Water Research - Issue 2018

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    The Arkansas Bulletin of Water Research is a publication of the Arkansas Water Resources Center (AWRC). This bulletin is produced in an effort to share water research relevant to Arkansas water stakeholders in an easily searchable and aesthetically engaging way. This is the second publication of the bulletin and will be published annually. The submission of a paper to this bulletin is appropriate for topics at all related to water resources, by anyone conducting water research or investigations. This includes but is not limited to university researchers, consulting firms, watershed groups, and other agencies. Prospective authors should read the “Introduction to the Arkanasas Bulletin of Water Research” contained within this publication and should refer to the AWRC website for additional infromation. https://arkansas-water-center.uark.edu

    The role of unicellular cyanobacteria in nitrogen fixation and assimilation in subtropical marine waters

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    Biological N2 fixation constitutes the major source of nitrogen in open ocean systems, regulating the marine nitrogen inventory and primary productivity. Symbiotic relationships between phytoplankton and N2 fixing microorganisms (diazotrophs) have been suggested to play a significant role in the ecology and biogeochemistry in these oceanic regions. The widely distributed, uncultured N2 fixing cyanobacterium UCYN A was suggested to live in symbiosis since it has unprecedented genome reduction, including the lack of genes encoding for oxygen evolving photosystem II and the tricarboxylic acid cycle. This thesis aims to study carbon and nitrogen metabolism on field populations of UCYN A using molecular biology, as well as mass spectrometry tools to visualize metabolic activity on a single cell scale. The development of a 16S rRNA oligonucleotide probe specifically targeting UCYN A cells and its successful application on environmental samples (Manuscript I and II) revealed a symbiotic partnership with a unicellular prymnesiophyte. We demonstrated a nutrient transfer in carbon and nitrogen compounds between these two partner cells, providing an explanation how these diazotrophs thrive in open ocean systems. Further, UCYN A can also associate with globally abundant calcifying prymnesiophyte members, e.g. Braarudosphaera bigelowii, indicating that this symbiosis might impact the efficiency of the biological carbon pump. In manuscript III, we provided quantitative information on the cellular abundance and distribution of UCYN A cells in the North Atlantic Ocean and identified the eukaryotic partner cell as Haptophyta (including prymnesiophyte) via double Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD FISH). The UCYN A Haptophyta association was the dominant form (87.0±6.1%) over free living UCYN A cells. Interestingly, we also detected UCYN A cells living in association with unknown eukaryotes and non calcifying Haptophyta cells, raising questions about the host specificity. During a follow up study (Manuscript IV), we conducted various nutrient amendment experiments (including iron, phosphorus, ammonium nitrate and Saharan Dust) in order to examine physiological interactions between individual UCYN A and Haptophyta cells. Single cell measurements using nanometer scale secondary ion mass spectrometry (nanoSIMS) revealed a tight physiological coupling in the transfer of carbon (R2 = 0.6232; n = 44) and nitrogen (R2 = 0.9659; n = 44) between host and symbiont. N2 fixation was mainly stimulated when iron rich Saharan Dust was added, emphasizing on aeolian dust deposition in seawater as a major parameter in constraining N2 fixation of UCYN A. Moreover, when fixed nitrogen species (ammonium and nitrate) were added, a third unknown microbial partner cell was observed within individual UCYN A Haptophyta associations, but their menaing is unclear. Based on this thesis work we revealed how UCYN A cells thrive in the environment and established a culture independent technique to assess the in situ activity in respect to CO2 and N2 fixation of this ecological relevant group of microorganisms. Furthermore, this unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N2 fixation

    The role of unicellular cyanobacteria in nitrogen fixation and assimilation in subtropical marine waters

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    Biological N2 fixation constitutes the major source of nitrogen in open ocean systems, regulating the marine nitrogen inventory and primary productivity. Symbiotic relationships between phytoplankton and N2 fixing microorganisms (diazotrophs) have been suggested to play a significant role in the ecology and biogeochemistry in these oceanic regions. The widely distributed, uncultured N2 fixing cyanobacterium UCYN–A was suggested to live in symbiosis since it has unprecedented genome reduction, including the lack of genes encoding for oxygen–evolving photosystem II and the tricarboxylic acid cycle. This thesis aims to study carbon and nitrogen metabolism on field populations of UCYN–A using molecular biology, as well as mass spectrometry tools to visualize metabolic activity on a single cell scale. The development of a 16S rRNA oligonucleotide probe specifically targeting UCYN– A cells and its successful application on environmental samples (Manuscript I and II) revealed a symbiotic partnership with a unicellular prymnesiophyte. We demonstrated a nutrient transfer in carbon and nitrogen compounds between these two partner cells, providing an explanation how these diazotrophs thrive in open ocean systems. Further, UCYN–A can also associate with globally abundant calcifying prymnesiophyte members, e.g. Braarudosphaera bigelowii, indicating that this symbiosis might impact the efficiency of the biological carbon pump. In manuscript III, we provided quantitative information on the cellular abundance and distribution of UCYN–A cells in the North Atlantic Ocean and identified the eukaryotic partner cell as Haptophyta (including prymnesiophyte) via double Catalyzed Reporter Deposition–Fluorescence In Situ Hybridization (CARD–FISH). The UCYN–A–Haptophyta association was the dominant form (87.0±6.1%) over free–living UCYN–A cells. Interestingly, we also detected UCYN–A cells living in association with unknown eukaryotes and non–calcifying Haptophyta cells, raising questions about the host specificity. During a follow up study (Manuscript IV), we conducted various nutrient amendment experiments (including iron, phosphorus, ammonium–nitrate and Saharan Dust) in order to examine physiological interactions between individual UCYN–A and Haptophyta cells. Single cell measurements using nanometer scale secondary ion mass spectrometry (nanoSIMS) revealed a tight physiological coupling in the transfer of carbon (R2 = 0.6232; n = 44) and nitrogen (R2 = 0.9659; n = 44) between host and symbiont. N2 fixation was mainly stimulated when iron–rich Saharan Dust was added, emphasizing on aeolian dust deposition in seawater as a major parameter in constraining N2 fixation of UCYN–A. Moreover, when fixed nitrogen species (ammonium and nitrate) were added, a third unknown microbial partner II cell was observed within individual UCYN–A–Haptophyta associations, but their menaing is unclear. Based on this thesis work we revealed how UCYN–A cells thrive in the environment and established a culture–independent technique to assess the in situ activity in respect to CO2 and N2 fixation of this ecological relevant group of microorganisms. Furthermore, this unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N2 fixation
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