Contiguous Sequence Research Articles

Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

This protocol uses quantitative polymerase chain reaction (qPCR) to measure the number of DNA molecules containing a specific contiguous sequence in a sample of interest (e.g., genomic DNA or cDNA generated by reverse transcription). The sample is subjected to fluorescence-based PCR amplification and, theoretically, during each cycle, two new duplex DNA molecules are produced for each duplex DNA molecule present in the sample. The progress of the reaction during PCR is evaluated by measuring the fluorescence of dsDNA-dye complexes in real time. In the early cycles, DNA duplication is not detected because inadequate amounts of DNA are made. At a certain threshold cycle, DNA-dye complexes double each cycle for 8-10 cycles, until the DNA concentration becomes so high and the primer concentration so low that the reassociation of the product strands blocks efficient synthesis of new DNA and the reaction plateaus. There are two types of measurements: (1) the relative change of the target sequence compared to a reference sequence and (2) the determination of molecule number in the starting sample. The first requires a reference sequence, and the second requires a sample of the target sequence with known numbers of the molecules of sequence to generate a standard curve. By identifying the threshold cycle at which a sample first begins to accumulate DNA-dye complexes exponentially, an estimation of the numbers of starting molecules in the sample can be extrapolated.

Noise-induced speed up in repetitively firing neurons occurs far from spike threshold

Injecting white noise currents has been shown to increase mean firing rate in several types of neuron. This speed up is generally attributed to the fact that depolarizing currents on the tails of the noise distribution push cells past spike threshold sooner than they would have otherwise. As part of a larger effort to understand the dynamic properties of rat subthalamic (STN) neurons, we have performed perforated patch recordings of these tonically active neurons in brain slices, exposing them to contiguous sequences of brief current pulses drawn from a Gaussian distribution. Pulsed noise disrupted firing pattern with no significant change in rate over a wide range of noise amplitudes, so long as the pulse durations were brief (<2 ms). With longer pulse durations, neurons increased their firing rate with noise, with greater increases for pulses of larger magnitude and longer duration. However, individual neurons displayed varying degrees of noise-induced speed up. We used phase resetting methods to understand the noise-induced changes in the mean and variance of the recorded interspike interval distributions. For each cell, a phase response curves (PRC) was derived by using linear interpolation to estimate the phase of the neuron between spikes, followed by a linear regression to estimate the sensitivity of the cell to input perturbations at each phase. We then compared Monte Carlo simulations of a one-dimensional phase model to predict the response patterns of individual neurons. Surprisingly, simulation results produced a good match to experimental mean and variances even at noise levels leading to autocorrelation histograms with no visible periodicity (CVs ~ 0.4). We then investigated simulations results in detail to understand the dynamic mechanism that underlies the noise-induced speed up in these neurons. Plots of the mean phase as a function of time revealed that the noise-induced phase advance occurred early in an interspike interval, well before trajectories approach the time of the next spike. This phase advance can be understood as ‘noise-induced drift’ in the Stratanovich interpretation of stochastic differential equations, with possible supralinear contributions for pulses of finite duration. Current research uses artificially constructed PRCs to examine how the increasing spread of the phase distribution during each intervals leads to an asymmetric sampling of the noise-induced drift, preventing the early noise-induced speed up from being canceled by an expected noise-induced slowing as the PRC approaches zero near the time of the next spike.

ChemInform Abstract: Synthesis and Properties of Long [n]Cumulenes (n ≥ 5)

AbstractReview: 177 refs.

Clustering of Short Read Sequences for de novo Transcriptome Assembly

Given the importance of transcriptome analysis in various biological studies and considering thevast amount of whole transcriptome sequencing data, it seems necessary to develop analgorithm to assemble transcriptome data. In this study we propose an algorithm fortranscriptome assembly in the absence of a reference genome. First, the contiguous sequencesare generated using de Bruijn graph with different k-mer lengths. Then, the eclectic mixtures ofsequences are gathered in order to form the final sequences. Lastly, the contiguous sequencesare clustered and the isoform groups are provided. This proposed algorithm is capable ofgenerating long contiguous sequences and accurately clustering them into isoform groups.Toevaluate our algorithm, we applied it to a simulated RNA-seq dataset of rat transcriptome and areal RNA-seq experiment of the loricaria gr. cataphracta transcriptome. The correctness of theassembled contigs was more than 95%, and our algorithm was able to reconstruct over 70% ofthe transcripts at more than 80% of the transcripts’ lengths. This study demonstrates thatapplying a sophisticated merging method improves transcriptome assembly. The source code isavailable upon request by contacting the corresponding author by email.

Application of the Codon-based Scoring Method in Motif Detection

A RNA or DNA sequence motif is a short sequence found within a particular nucleic acid sequence families. Most amino acid and nucleic acid sequences have some level of functional or structural similarities. These similarities are mostly represented by short, contiguous sequences called motif. Motif discovery is an important aspect of molecular biology. This is because the knowledge of these sequences helps determine their structural properties, signal sites and/or ligand-binding sites. In most cases, depending on the function of the motif, these contiguous regions can be highly conserved with an homology of nearly 100%. Several algorithms have been proposed for the discovery of motifs. In this paper, the codon-based scoring method is employed to detect motifs and with their invariants. The result obtained shows the reliability and robustness of the method as motifs are discovered irrespective of their length and position in a sequence.

Non-random DNA fragmentation in next-generation sequencing

Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.

General tests of independence based on empirical processes indexed by functions

The present paper is mainly concerned with the statistical tests of the independence problem between random vectors. We develop an approach based on general empirical processes indexed by a particular class of functions. We prove two abstract approximation theorems that include some existing results as particular cases. Finally, we characterize the limiting behavior of the Möbius transformation of empirical processes indexed by functions under contiguous sequences of alternatives.

Development and characterization of genomic SSR markers in Cynodon transvaalensis Burtt-Davy

Simple sequence repeat (SSR) markers are a major molecular tool for genetic and genomic research that have been extensively developed and used in major crops. However, few are available in African bermudagrass (Cynodon transvaalensis Burtt-Davy), an economically important warm-season turfgrass species. African bermudagrass is mainly used for hybridizations with common bermudagrass [C. dactylon var. dactylon (L.) Pers.] in the development of superior interspecific hybrid turfgrass cultivars. Accordingly, the major objective of this study was to develop and characterize a large set of SSR markers. Genomic DNA of C. transvaalensis '4200TN 24-2' from an Oklahoma State University (OSU) turf nursery was extracted for construction of four SSR genomic libraries enriched with [CA](n), [GA](n), [AAG](n), and [AAT](n) as core repeat motifs. A total of 3,064 clones were sequenced at the OSU core facility. The sequences were categorized into singletons and contiguous sequences to exclude redundancy. From the two sequence categories, 1,795 SSR loci were identified. After excluding duplicate SSRs by comparison with previously developed SSR markers using a nucleotide basic local alignment tool, 1,426 unique primer pairs (PPs) were designed. Out of the 1,426 designed PPs, 981 (68.8 %) amplified alleles of the expected size in the donor DNA. Polymorphisms of the SSR PPs tested in eight C. transvaalensis plants were 93 % polymorphic with 544 markers effective in all genotypes. Inheritance of the SSRs was examined in six F(1) progeny of African parents 'T577' × 'Uganda', indicating 917 markers amplified heritable alleles. The SSR markers developed in the study are the first large set of co-dominant markers in African bermudagrass and should be highly valuable for molecular and traditional breeding research.

The Past, Present, and Future of Human Centromere Genomics

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function.

Navigator gated flow sensitive gradient echo sequence for quantification of aortic valve regurgitation

Background Exact quantification of aortic valve regurgitation is a challenge. Patients are typically asymptomatic and echocardiography is unreliable in certain conditions, particularly in eccentric regurgitation jets. Cardiac magnetic resonance (CMR) is a valuable alternative in this situation, but the use of conventional sequences without respiration correction is limited by motion artifacts and long acquisition times caused by necessary signal averaging. Novel navigator gated flow sensitive gradient echo sequence avoid motion artifacts caused by respiration and in addition allow for a higher temporal resolution eventually leading to more reliable results. Methods On 31 patients with varying degrees of aortic regurgitation we performed both standard (StdSeq) and navigator gated (NavSeq) flow sensitive gradient echo sequences. Primary parameter was the regurgitation fraction defined as the quotient of anterograde and retrograde flow over a cross section of the ascendent aorta just above the maximal excursion of the aortic leaflets. Since a gold standard for quantification of aortic regurgitation is missing, we correlated these values with the enddiastolic volume of the left ventricle (LVEDV) assessed by a contiguous stack short axis cine-SSFP sequences. This parameter is directly influenced by the regurgitant volume. For comparison we

Building classifier ensembles for B-cell epitope prediction.

Identification of B-cell epitopes in target antigens is a critical step in epitope-driven vaccine design, immunodiagnostic tests, and antibody production. B-cell epitopes could be linear, i.e., a contiguous amino acid sequence fragment of an antigen, or conformational, i.e., amino acids that are often not contiguous in the primary sequence but appear in close proximity within the folded 3D antigen structure. Numerous computational methods have been proposed for predicting both types of B-cell epitopes. However, the development of tools for reliably predicting B-cell epitopes remains a major challenge in immunoinformatics.Classifier ensembles a promising approach for combining a set of classifiers such that the overall performance of the resulting ensemble is better than the predictive performance of the best individual classifier. In this chapter, we show how to build a classifier ensemble for improved prediction of linear B-cell epitopes. The method can be easily adapted to build classifier ensembles for predicting conformational epitopes.

Optimal single 3T MR imaging sequence for HDR brachytherapy of cervical cancer.

PurposeThe superior image quality of 3 tesla (3T) magnetic resonance (MR) imaging in cervical cancer offers the potential to use a single image set for brachytherapy. This study aimed to determine a suitable single sequence for contouring tumour and organs at risk, applicator reconstruction, and treatment planning.Material and methodsA 3T (Skyra, Siemens Healthcare AG, Germany) MR imaging system with an 18 channel body matrix coil generated HDR cervical cancer brachytherapy planning images on 20 cases using plastic-based treatment applicators. Seven different T2-weighted Turbo Spin Echo (TSE) sequences including both 3D and contiguous 2D scans based on sagittal, axial (transverse), and oblique planes were analysed. Each image set was assessed for total scanning time and usefulness in tumour localization via inter- and intra-observer analysis of high-risk clinical target volume (HR CTV) contouring. Applicator reconstruction in the treatment planning system was also considered.ResultsThe intra-observer difference in HR CTV volumes between 2D and 3D axial-based image sets was low with an average difference of 3.1% for each observer. 2D and 3D sagittal image sets had the highest intra- and inter observer differences (over 15%). A 2D axial ‘double oblique’ sequence was found to produce the best intra- (average difference of 0.6%) and inter-observer (mean SD of 9.2%) consistency and greatest conformity (average 0.80).ConclusionsThere was little difference between 2D and 3D-based scanning sequences; however the increased scanning time of 3D sequences have potential to introduce greater patient motion artifacts. A contiguous 2D sequence based on an axial T2-weighted turbo-spin-echo (TSE) sequence orientated in all planes of the treatment applicator provided consistent tumour delineation whilst allowing applicator reconstruction and treatment planning.

Automatic Estimation of Multiple Motion Fields From Video Sequences Using a Region Matching Based Approach

Estimation of velocity fields from a video sequence is an important step towards activity classification in a surveillance system. It has been recently shown that multiple motion fields estimated from trajectories are an efficient tool to describe the movement of objects, allowing an automatic classification of activities in the scene. However, the trajectory detection in noisy environments is difficult, usually requiring some sort manual editing to complete or correct them. This paper proposes two novel contributions. First, an automatic method for building pedestrian trajectories in far-field surveillance scenarios is presented not requiring user intervention. This basically comprises the detection of multiple moving objects in a video sequence through the detection of the active regions, followed by the estimation of the velocity fields that is accomplished by performing region matching of the above regions at consecutive time instants. This leads to a sequence of centroids and corresponding velocity vectors, describing the local motions presented in the image. A motion correspondence algorithm is then applied to group the centroids in a contiguous sequence of frames into trajectories corresponding to each moving object. The second contribution is a method for automatically finding the trajectories from a library of previously computed ones. Experiments on extensive video sequences from university campuses show that motion fields can be reliably estimated from these automatically detected trajectories, leading to a fully automatic procedure for the estimation of multiple motion fields.

Noncontiguous SCHEMA protein recombination.

SCHEMA is a method of designing protein recombination libraries that contain a large fraction of functional proteins with a high degree of mutational diversity. In the previous chapter, we illustrated the method for designing libraries by swapping contiguous sequence elements. Here, we introduce the NCR ("noncontiguous recombination") algorithm to identify optimal designs for swapping elements that are contiguous in the 3-D structure but not necessarily in the primary sequence. To exemplify the method, NCR is used to recombine three fungal cellobiohydrolases (CBH1s) to produce a library containing more than 500,000 novel chimeric sequences.

Potential impact on kidney infection: a whole-genome analysis of Leptospira santarosai serovar Shermani

Leptospira santarosai serovar Shermani is the most frequently encountered serovar, and it causes leptospirosis and tubulointerstitial nephritis in Taiwan. This study aims to complete the genome sequence of L. santarosai serovar Shermani and analyze the transcriptional responses of L. santarosai serovar Shermani to renal tubular cells. To assemble this highly repetitive genome, we combined reads that were generated from four next-generation sequencing platforms by using hybrid assembly approaches to finish two-chromosome contiguous sequences without gaps by validating the data with optical restriction maps and Sanger sequencing. Whole-genome comparison studies revealed a 28-kb region containing genes that encode transposases and hypothetical proteins in L. santarosai serovar Shermani, but this region is absent in other pathogenic Leptospira spp. We found that lipoprotein gene expression in both L. santarosai serovar Shermani and L. interrogans serovar Copenhageni were upregulated upon interaction with renal tubular cells, and LSS19962, a L. santarosai serovar Shermani-specific gene within a 28-kb region that encodes hypothetical proteins, was upregulated in L. santarosai serovar Shermani-infected renal tubular cells. Lipoprotein expression during leptospiral infection might facilitate the interactions of leptospires within kidneys. The availability of the whole-genome sequence of L. santarosai serovar Shermani would make it the first completed sequence of this species, and its comparison with that of other Leptospira spp. may provide invaluable information for further studies in leptospiral pathogenesis.

High potential source for biomass degradation enzyme discovery and environmental aspects revealed through metagenomics of Indian buffalo rumen.

The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837)

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite of salmonid fish severely affecting cultured salmon production in the North Atlantic. Salmon louse control on farms currently relies in part upon the use of veterinary drugs; however, as only a limited number of salmon delousing agents are available, there are concerns about development of drug resistance in salmon lice. The common anti-salmon louse drug SLICE® (Merck Animal Health) contains the avermectin emamectin benzoate (EMB). Members of the large gene superfamily of ABC (ATP-binding cassette) transporters have been identified as potential avermectin resistance factors in parasitic nematodes. In salmon lice, only three ABC transporters have been cloned and studied to date. We report here upon the isolation of four novel L. salmonis ABC transporters, and employ an inhibitor-based approach to assess roles of L. salmonis ABC transporters in the toxicology of EMB. To isolate salmon louse ABC transporters, publicly available L. salmonis expressed sequence tags (ESTs) were subjected to homology searches, and the retrieved ESTs assembled into contiguous sequences and annotated. Potential ABC drug transporters isolated by this approach included four multidrug resistance-associated proteins (MRPs). In addition, five ABC proteins likely having roles unrelated to drug resistance were obtained. Quantitative real time PCR (RT-qPCR) was used to analyse mRNA levels of the MRPs in L. salmonis strains differing in EMB susceptibility. In the absence of EMB, all studied MRPs showed similar transcript levels in the drug-susceptible strain S and the moderately EMB-resistant strain R. Moreover, mRNA expression of the four studied MRPs remained unaffected by exposure to EMB. Taken together, the results of RT-qPCR analyses did not provide evidence for roles of the studied MRPs as factors affecting EMB susceptibility. Further experiments used an inhibitor-based approach to investigate the roles of ABC transporters in EMB toxicity. In immotility bioassays, salmon lice of the two strains were exposed to EMB, provided alone or in combination with cyclosporin A or verapamil, which are known inhibitors of P-glycoprotein and MRPs. Cyclosporin A increased EMB toxicity to a similar degree with both strains, suggesting the biochemical factors affected by this inhibitor show similar levels in both strains. In contrast, verapamil increased EMB effects only in the R strain. This result could be indicative of an enhanced expression of hitherto unknown ABC transporters in the moderately EMB-resistant R strain. More research is required to identify the target of verapamil in salmon lice.

Employing heat maps to mine associations in structured routine care data

Mining the electronic medical record (EMR) has the potential to deliver new medical knowledge about causal effects, which are hidden in statistical associations between different patient attributes. It is our goal to detect such causal mechanisms within current research projects which include e.g. the detection of determinants of imminent ICU readmission. An iterative statistical approach to examine each set of considered attribute pairs delivers potential answers but is difficult to interpret. Therefore, we aimed to improve the interpretation of the resulting matrices by the use of heat maps. We propose strategies to adapt heat maps for the search for associations and causal effects within routine EMR data. Heat maps visualize tabulated metric datasets as grid-like choropleth maps, and thus present measures of association between numerous attribute pairs clearly arranged. Basic assumptions about plausible exposures and outcomes are used to allocate distinct attribute sets to both matrix dimensions. The image then avoids certain redundant graphical elements and provides a clearer picture of the supposed associations. Specific color schemes have been chosen to incorporate preexisting information about similarities between attributes. The use of measures of association as a clustering input has been taken as a trigger to apply transformations which ensure that distance metrics always assume finite values and treat positive and negative associations in the same way. To evaluate the general capability of the approach, we conducted analyses of simulated datasets and assessed diagnostic and procedural codes in a large routine care dataset. Simulation results demonstrate that the proposed clustering procedure rearranges attributes similar to simulated statistical associations. Thus, heat maps are an excellent tool to indicate whether associations concern the same attributes or different ones, and whether affected attribute sets conform to any preexisting relationship between attributes. The dendrograms help in deciding if contiguous sequences of attributes effectively correspond to homogeneous attribute associations. The exemplary analysis of a routine care dataset revealed patterns of associations that follow plausible medical constellations for several diseases and the associated medical procedures and activities. Cases with breast cancer (ICD C50), for example, appeared to be associated with radiation therapy (8-52). In cross check, approximately 60 percent of the attribute pairs in this dataset showed a strong negative association, which can be explained by diseases treated in a medical specialty which routinely does not perform the respective procedures in these cases. The corresponding diagram clearly reflects these relationships in the shape of coherent subareas. We could demonstrate that heat maps of measures of association are effective for the visualization of patterns in routine care EMRs. The adjustable method for the assignment of attributes to image dimensions permits a balance between the display of ample information and a favorable level of graphical complexity. The scope of the search can be adapted by the use of pre-existing assumptions about plausible effects to select exposure and outcome attributes. Thus, the proposed method promises to simplify the detection of undiscovered causal effects within routine EMR data.

A new computing method for extracting contiguous phraseological sequences from academic text corpora

This study aims to develop a new computing method for extracting contiguous phraseological sequences (PSs) of various lengths from academic text corpora by measuring internal associations of n-grams. We construct a new normalizing algorithm of probability-weighted average for refining the MI measure and enhancing precision in extracting PSs from corpora. This computing method is applied to the data in a medium-sized text corpus of academic English. Results indicate that the resultant new MI measure can provide statistics which better reveal internal associations within an n-gram, regardless of size. Lexico-grammatical sequences extracted with this method are more complete and less arbitrary in terms of grammar and semantics. The method can be applied to treating a variety of linguistic phenomenon, ranging from well-established phrases to likely phrasal entities, thus having potentially practical applications in corpus-based studies of phraseology and natural language processing.

De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes

Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion.