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.

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.

Nanopore-based Fourth-generation DNA Sequencing Technology

Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein.Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale.In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

p21^(WAF1/CIP1) gene DNA sequencing and its expression in human osteosarcoma

Background Mutation and expression change of p21 WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21 WAF1/CIP1 gene in human osteosarcoma,p21 WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21 WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21 WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry,respectively. Results The occurrence of P21 protein in osteosarcoma was 17.78% (8/45),and p21 WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21 WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21 WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma,there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples’ DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy,the expression of p21 WAF1/CIP1 mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21 WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21 WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21 WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients,which can provide a basis for further research.

Single-molecule DNA sequencing using two-dimensionalTi2C(OH)2 MXene nanopores: A first-principles investigation

Nanopore-based devices have provided exciting opportunities to develop affordable label-free DNA sequencing platforms.Over a decade ago,graphene has been proposed as a two-dimensional(2D)nanopore membrane in order to achieve single-base resolution.However,it was experimentally revealed that clogging of the graphene nanopore can occur due to the hydrophobic nature of graphene,thus hindering the translocation of DNA.To overcome this problem,the exploration of alternative 2D materials has gained considerable interest over the last decade.Here we show that a Ti_(2)C-based MXene nanopore functionalized by hydroxyl groups(–OH)exhibits transverse conductance properties that allow for the distinction between all four naturally occurring DNA bases.We have used a combination of density functional theory and non-equilibrium Green’s function method to sample over multiple orientations of the nucleotides in the nanopore,as generated from molecular dynamics simulations.The conductance variation resulting from sweeping an applied gate voltage demonstrates that the Ti_(2)C-based MXene nanopore possesses high potential to rapidly and reliably sequence DNA.Our findings open the door to further theoretical and experimental explorations of MXene nanopores as a promising 2D material for nanopore-based DNA sensing.

Corrigendum to Nanopore-based Fourth- generation DNA Sequencing Technology＇ [GPB 144 （2015）- GPB 13]1 （4-16）]

The authors regret that there was an incorrect citation in the Table 1 of the article ＂Nanopore-based Fourth-generation DNA Sequencing Technology＂, which was published in the journal Genomics, Proteomics ＆ Bioinformatics, Issue 1, 2015. In the table, the image in the 4th row originally appeared in the article ＂Nanopore-based sequence-specific detection of duplex DNA for geno- mic profiling＂ published by Nano Letters in 2010. Therefore, Ref. [8], which was associated with the image in the current Table l, should be replaced by the reference： Singer A, Wanunu M, Morrison W, Kuhn H, Frank-Kamenetskii M, Meller A. Nanopore- based sequence specific detection of duplex DNA for genomic profiling. Nano Lett 2010; 10：738-42.＂ The copy right permission of this article has been obtained. The authors would like to apologize for any inconvenience caused.

DNA sequencing using nanopores and kinetic proofreading

We discuss the feasibility of using a nanopore sandwich device to implement the principle of kinetic proofreading to discriminate incorrect hybridizing oligonucleotides on a target DNA or RNA.We propose a method of sequencing DNA or RNA using this approach.The design parameters for such a DNA sequencer are estimated from the Hopfield・Ninio theory of kinetic proofreading and Schrodinger's first-passage-time distribution function.

Corrigendum to ‘Nanopore-based Fourth-generation DNA Sequencing Technology' [GPB 144(2015)–GPB 13/1(4–16)]

The authors regret that there were typos in the online version of Figure 4 published in Issue 1,2015.In the figure legend boxes of panels F and G,‘‘Ni’’should be corrected to‘‘N’’for the labeling of the green curves.The figure in the printed version has been corrected.The correct Figure 4 is shown below.The authors would like to apologize for any inconvenience caused.

Error correction coding paves an avenue to ultra-high accuracyof DNA sequencing

With the support from the National Natural Science Foundation of China,Prof.Huang Yanyi(黄岩谊)led a team at Peking University to demonstrate a novel approach,which combined fluorogenic sequencingby-synthesis(SBS)chemistry with an information theory-based error-correction coding scheme to

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.

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.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

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.

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.

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.

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.

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.

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.

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.