Complementary DNA sequencing (cDNA): an eff ective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM)is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobenthos diff ers inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobenthos,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at fi ve stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA(cDNA)high-throughput sequencing of the V4 region of 18S rRNA gene.Results showed that the diversity of benthic ciliates detected by DNA(303 OTUs),and the cDNA(611 OTUs)sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly diff erent ciliate communities inside and outside of the YSCWM,but without statistical signifi cance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all)of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly infl uenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have signifi cant eff ects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

DNA Sequencing by Capillary Electrophoresis Using Quasi-inter-penetrating Network Formed by Polyacrylamide and Poly(N-hydroxymethylacrylamide)

Quasi-interpenetrating network formed by polyacrylamide and poly （N-hydroxymethylacrylamide） was designed, synthesized, and tested for DNA sequencing by capillary electrophoresis. The performance of quasi-IPN on DNA sequencing was determined by the acrylamide to N-hydroxymethylacrylamide molar ratio and sequencing temperature.

Genome Sequencing Using Graph Theory Approach

Genome sequencing is the process of determining in which order the nitrogenous bases also known as nucleotides within a DNA molecule are arranged. Every organism’s genome consists of a unique sequence of nucleotides. These nucleotides bases provide the phenotypes and genotypes of a cell. In mathematics, Graph theory is the study of mathematical objects known as graphs which are made of vertices (or nodes) connected by either directed edges or indirect edges. Determining the sequence in which these nucleotides are bonded can help scientists and researchers to compare DNA between organisms, which can help show how the organisms are related. In this research, we study how graph theory plays a vital part in genome sequencing and different types of graphs used during DNA sequencing. We are going to propose several ways graph theory is used to sequence the genome. We are as well, going to explore how the graphs like Hamiltonian graph, Euler graph, and de Bruijn graphs are used to sequence the genome and advantages and disadvantages associated with each graph.

Chaos game representation(CGR)-walk model for DNA sequences

Chaos game representation （CGR） is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features： one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA （p, d, q） model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA （p, d, q） model.

Comparison of Human Papillomavirus Detection and Genotyping with Four Different Prime Sets by PCR-Sequencing

Objective To assess and compare the Human Papillomavirus（HPV） detection efficiency and the potential clinical utility of PCR sequencing‐based technology.Methods Four HPV consensus primer sets（GP5＋/6＋,MGP,MY09/11,and PGMY09/11） were used in order to amplify a broad spectrum of HPV types for HPV infection in 325 cervical samples and the PCR products were sequenced afterwards for the HPV genotyping.Results The HPV‐positive rate was 75.4%,of which 35.5% harbored more than one HPV genotype.A total of 36 different genotypes was found,with HPV 16（24.1%） being the most prevalent,followed by HPV 58（13.3%） and HPV 52（9.6%）.There were substantial to almost perfect agreements between different primer sets regarding HPV detection efficiency,with the kappa value varying from 0.751 to 0.925,MGP,and PGMY09/11 were the most effective in detecting multiple infections（P0.001）.With each of the primer sets,a board range of HPV types could be identified,though there were several differences for a few genotypes.Conclusion The substantial agreement between PCR‐sequencing and HC2 for the detection of high‐risk HPV（kappa=0.761） indicated that PCR‐sequencing is also suitable for routine HPV screening.

A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

In this paper, Adomian decomposition method （ADM） with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear （PWL） hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks.

Molecular Taxonomy of Conogethes punctiferalis and Conogethes pinicolalis(Lepidoptera: Crambidae) Based on Mitochondrial DNA Sequences

Conogethes punctiferalis（Guenée）（Lepidoptera： Crambidae） was originally considered as one species with fruit-feeding type（FFT） and pinaceae-feeding type（PFT）, but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood（ML） parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalisand C. punctiferalis are significantly different.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

DNA Sequence Classification by Convolutional Neural Network

In recent years, a deep learning model called convolutional neural network with an ability of extracting features of high-level abstraction from minimum preprocessing data has been widely used. In this research, we proposed a new approach in classifying DNA sequences using the convolutional neural network while considering these sequences as text data. We used one-hot vectors to represent sequences as input to the model;therefore, it conserves the essential position information of each nucleotide in sequences. Using 12 DNA sequence datasets, we evaluated our proposed model and achieved significant improvements in all of these datasets. This result has shown a potential of using convolutional neural network for DNA sequence to solve other sequence problems in bioinformatics.

Differential DNA methylation profiles of human B lymphocytes and Epstein-Barr virus-immortalized B lymphocytes

Objective： This study aimed to comprehensively assess Epstein-Barr virus（EBV）-induced methylation alterations of B cell across whole genome.Methods： We compared DNA methylation patterns of primary B cells and corresponding lymphoblastoid cell lines（LCLs） from eight participants. The genome-wide DNA methylation profiles were compared at over 850,000 genome-wide methylation sites.Results： DNA methylation analysis revealed 87,732 differentially methylated Cp G sites, representing approximately 12.41% of all sites in LCLs compared to primary B cells. The hypermethylated and hypomethylated Cp G sites were about 22.75% or 77.25%, respectively. Only 0.8% of hypomethylated sites and 4.5% of hypermethylated sites were located in Cp G islands, whereas 8.0% of hypomethylated sites and 16.3% of hypermethylated sites were located in shore（N_shore and S_shore）. Using principal component analysis of the DNA methylation profiles, primary B cells and LCLs could be accurately predicted. Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） analysis of differently methylated genes revealed that most of the top GO biological processes were related to cell activation and immune response, and some top enrichment pathways were related with activation and malignant transformation of human B cells.Conclusions： Our study demonstrated genome-wide DNA methylation variations between primary B cells and corresponding LCLs, which might yield new insight on the methylation mechanism of EBV-induced immortalization.

Active motif finder-a bio-tool based on mutational structures in DNA sequences

Active Motif Finder （AMF） is a novel algorithmic tool, designed based on mutations in DNA sequences. Tools available at present for finding motifs are based on matching a given motif in the query sequence. AMF describes a new algorithm that identifies the occurrences of patterns which possess all kinds of mutations like insertion, deletion and mismatch. The algorithm is mainly based on the Alignment Score Matrix （ASM） computation by com paring input motif with full length sequence. Much of the effort in bioinformatics is directed to identify these motifs in the sequences of newly discovered genes. The proposed bio-tool serves as an open resource for analysis and useful for studying polymorphisms in DNA sequences. AMF can be searched via a user-friendly interface. This tool is intended to serve the scientific community working in the areas of chemical and structural biology, and is freely available to all users, at http：//www.sastra.edu/scbt/amf/.

DNA diagnostics for reliable and universal identification of Helicobacter pylori

Reliable diagnostics are a major challenge for the detection and treatment of Helicobacter pylori(H.pylori)infection.Currently at the forefront are non-invasive urea breath test(UBT)and stool antigen test(SAT).Polymerase chain reaction(PCR)is not endorsed due to nonspecific primers and the threat of false-positives.The specificity of DNA amplification can be achieved by nested PCR(NPCR),which involves two rounds of PCR.If the primers are properly designed for the variable regions of the 16S rRNA gene,it is not difficult to develop an NPCR assay for the unambiguous identification of H.pylori.Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy,stool,and saliva samples,each from the same individuals,and compared with available H.pylori assays,namely histology,rapid urease test,SAT,and 13C-UBT.The assay was much more sensitive than simple PCR,and it was equally sensitive in biopsy samples as the 13CUBT test,which is considered the gold standard.In addition,it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of H.pylori-specific DNA.However,due to the threshold and lower abundance,the sensitivity was much lower in amplifications from stool or saliva.Reliable detection in saliva also complicates the ability of H.pylori to survive in the oral cavity aside from and independent of the stomach.The reason for the lower sensitivity in stool is DNA degradation;therefore,a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon.The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results.Surprisingly,this NPCR revealed the presence of H.pylori in twice the number of samples as SAT,indicating that many patients are misdiagnosed,not treated by antibiotics,and their problems are interpreted as chronic.Thus,it is unclear how to properly diagnose H.pylori in practice.In the first approach,SAT or UBT is sufficient.If samples are negative,the 148 bp amplicon NPCR assay should be performed.If problems persist,patients should not be considered negative,but due to threshold H.pylori abundance,they should be periodically tested.The advantage of NPCR over UBT is that it can be used universally,including questionable samples taken from patients with achlorhydria,receiving proton pump inhibitors,antibiotics,bismuth compound,intestinal metaplasia,or gastric ulcer bleeding.

Low dimensional chaos in the AT and GC skew profiles of DNA sequences

This paper investigates the existence of low-dimensional deterministic chaos in the AT and GC skew profiles of DNA sequences. It has taken DNA sequences from eight organisms as samples. The skew profiles are analysed using continuous wavelet transform and then nonlinear time series methods. The invariant measures of correlation dimension and the largest Lyapunov exponent are calculated. It is demonstrated that the AT and GC skew profiles of these DNA sequences all exhibit low dimensional chaotic behaviour. It suggests that chaotic properties may be ubiquitous in the DNA sequences of all organisms.

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

LONG-RANGE CORRELATIONS IN DNA SEQUENCES USING TWO-DIMENSIONAL DNA WALKS

The characterization of long-range correlations and fractal properties of DNA sequences has proved to be adifficult though rewarding task mainly due to the mosaic character of DNA consisting of many patches of various lengthswith different nucleotide constitutions.In this paper we investigate statistical correlations among different positions in DNAsequences using the two-dimensional DNA walk.The root-mean-square fluctuation F(l)is described by a power law.Theautocorrelation function C(l),which is used to measure the linear dependence and periodicity,exists a power law ofC(l)-l^(-μ).We also calculate the mean-square distance<R^2(l)>along the DNA chain,and it may be expressed as<R^2(l)>-l^(?)with 2>γ>1.Our investigations can provide some insights into long-range correlations in DNA sequences.

Characteristics of alternating current hopping conductivity in DNA sequences

This paper presents a model to describe alternating current （AC） conductivity of DNA sequences, in which DNA is considered as a one-dimensional （1D） disordered system, and electrons transport via hopping between localized states. It finds that AC conductivity in DNA sequences increases as the frequency of the external electric field rises, and it takes the form of σac（ω） - ω2 ln^2（1/ω）. Also AC conductivity of DNA sequences increases with the increase of temperature, this phenomenon presents characteristics of weak temperature-dependence. Meanwhile, the AC conductivity in an offdiagonally correlated case is much larger than that in the uncorrelated case of the Anderson limit in low temperatures, which indicates that the off-diagonal correlations in DNA sequences have a great effect on the AC conductivity, while at high temperature the off-diagonal correlations no longer play a vital role in electric transport. In addition, the proportion of nucleotide pairs p also plays an important role in AC electron transport of DNA sequences. For p 〈 0.5, the conductivity of DNA sequence decreases with the increase of p, while for p ≥ 0.5, the conductivity increases with the increase of p.

An Optimized Neural Network with Bat Algorithm for DNA Sequence Classification

Recently, many researchers have used nature inspired metaheuristicalgorithms due to their ability to perform optimally on complex problems. Tosolve problems in a simple way, in the recent era bat algorithm has becomefamous due to its high tendency towards convergence to the global optimummost of the time. But, still the standard bat with random walk has a problemof getting stuck in local minima. In order to solve this problem, this researchproposed bat algorithm with levy flight random walk. Then, the proposedBat with Levy flight algorithm is further hybridized with three differentvariants of ANN. The proposed BatLFBP is applied to the problem ofinsulin DNA sequence classification of healthy homosapien. For classificationperformance, the proposed models such as Bat levy flight Artificial NeuralNetwork (BatLFANN) and Bat levy Flight Back Propagation (BatLFBP) arecompared with the other state-of-the-art algorithms like Bat Artificial NeuralNetwork (BatANN), Bat back propagation (BatBP), Bat Gaussian distribution Artificial Neural Network (BatGDANN). And Bat Gaussian distributionback propagation (BatGDBP), in-terms of means squared error (MSE) andaccuracy. From the perspective of simulations results, it is show that theproposed BatLFANN achieved 99.88153% accuracy with MSE of 0.001185,and BatLFBP achieved 99.834185 accuracy with MSE of 0.001658 on WL5.While on WL10 the proposed BatLFANN achieved 99.89899% accuracy withMSE of 0.00101, and BatLFBP achieved 99.84473% accuracy with MSE of0.004553. Similarly, on WL15 the proposed BatLFANN achieved 99.82853%accuracy with MSE of 0.001715, and BatLFBP achieved 99.3262% accuracywith MSE of 0.006738 which achieve better accuracy as compared to the otherhybrid models.

The Potential of DNA Barcode-Based Delineation Using Seven Putative Candidate Loci of the Plastid Region in Inferring Molecular Diversity of Cowpea at Sub-Species Level

The novelty and suitability of the mitochondrial gene CO1 in DNA barcoding as a reliable identification tool in animal species are undisputed. This is attributed to its standardized sequencing segment of the mitochondrial cytochrome c oxidase-1 gene (CO1) which has the necessary universality and variability making it a generally acceptable barcode region. CO1 is a haploid single locus that is uniparentally-inherited. Protein-coding regions are present in high-copy numbers making it an ideal barcode. The mitochondrial oxidase subunit I (COI) gene is a robust barcode with a suitable threshold for delineating animals and is not subject to drastic length variation, frequent mononucleotide repeats or microinversions. However, a low nucleotide substitution rate of plant mitochondrial genome [mtDNA] precludes the use of CO1 as a universal plant DNA barcode and makes the search for alternative barcode regions necessary. Currently, there exists no universal barcode for plants. The plastid region reveals leading candidate loci as appropriate DNA barcodes yet to be explored in biodiversity studies in Kenya. Four of these plastid regions are portions of coding genes (matK, rbcL, rpoB, and rpoC1), and three noncoding spacers (atpF-atpH, trnH-psbA, and psbK-psbL) which emerge as ideal candidate DNA loci. While different research groups propose various combinations of these loci, there exists no consensus;the lack thereof impedes progress in getting a suitable universal DNA barcode. Little research has attempted to investigate and document the applicability and extend of effectiveness of different DNA regions as barcodes to delineate cowpea at subspecies level. In this study we sought to test feasibility of the seven putative candidate DNA loci singly and in combination in order to establish a suitable single and multi-locus barcode regions that can have universal application in delineating diverse phylogeographic groups of closely related Kenyan cowpea variants. In this study, our focus was based on genetic parameters including analyses of intra- and infra-specific genetic divergence based on intra- and infra-specific K2P distances;calculation of Wilcoxon signed rank tests of intra-specific divergence among loci and coalescence analyses to delineate independent genetic clusters. Knowledge of DNA candidate loci that are informative will reveal the suitability of DNA barcoding as a tool in biodiversity studies. Results of this study indicate that: matK, trnH-psbA, psbK-psbL, and rbcL are good barcodes for delineating intra and infraspecific distances at single loci level. However, among the combinations, matK + trnH-psbA, rpoB + atpF-atpH + matK are the best barcodes in delineating cowpea subvariants. rbcL gene can be a suitable barcode marker at single locus level, but overall, multi locus approach appears more informative than single locus approach. The present study hopes to immensely contribute to the scanty body of knowledge on the novelty of DNA barcoding in cataloguing closely related cowpea variants at molecular level and hopes to open up future research on genomics and the possibility of use of conserved regions within DNA in inferring phylogenetic relationships among Kenyan cowpea variants.