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1
Steinke, Dirk, Miguel Vences, Walter Salzburger, and Axel Meyer. "TaxI: a software tool for DNA barcoding using distance methods." Philosophical Transactions of the Royal Society B: Biological Sciences 360, no. 1462 (2005): 1975–80. http://dx.doi.org/10.1098/rstb.2005.1729.
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DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding.
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McBride, L. J., S. M. Koepf, R. A. Gibbs, et al. "Automated DNA sequencing methods involving polymerase chain reaction." Clinical Chemistry 35, no. 11 (1989): 2196–201. http://dx.doi.org/10.1093/clinchem/35.11.2196.
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Abstract Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.
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WILSON, R., and L. HOOD. "High-throughput fluorescent DNA sequence analysis: Methods and automation." Methods 3, no. 1 (1991): 48–54. http://dx.doi.org/10.1016/s1046-2023(05)80163-x.
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Matsuo, Kouki. "Evaluation of methods for plant genomic DNA sequence analysis without DNA and PCR product purification." Plant Science 312 (November 2021): 111023. http://dx.doi.org/10.1016/j.plantsci.2021.111023.
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Mendizabal-Ruiz, Gerardo, Israel Román-Godínez, Sulema Torres-Ramos, Ricardo A. Salido-Ruiz, Hugo Vélez-Pérez, and J. Alejandro Morales. "Genomic signal processing for DNA sequence clustering." PeerJ 6 (January 24, 2018): e4264. http://dx.doi.org/10.7717/peerj.4264.
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Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.
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Gibson, Neil J. "The use of real-time PCR methods in DNA sequence variation analysis." Clinica Chimica Acta 363, no. 1-2 (2006): 32–47. http://dx.doi.org/10.1016/j.cccn.2005.06.022.
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Qi, Xingqin, Edgar Fuller, Qin Wu, and Cun-Quan Zhang. "Numerical Characterization of DNA Sequence Based on Dinucleotides." Scientific World Journal 2012 (2012): 1–6. http://dx.doi.org/10.1100/2012/104269.
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Sequence comparison is a primary technique for the analysis of DNA sequences. In order to make quantitative comparisons, one devises mathematical descriptors that capture the essence of the base composition and distribution of the sequence. Alignment methods and graphical techniques (where each sequence is represented by a curve in high-dimension Euclidean space) have been used popularly for a long time. In this contribution we will introduce a new nongraphical and nonalignment approach based on the frequencies of the dinucleotideXYin DNA sequences. The most important feature of this method is that it not only identifies adjacentXYpairs but also nonadjacentXYones whereXandYare separated by some number of nucleotides. This methodology preserves information in DNA sequence that is ignored by other methods. We test our method on the coding regions of exon-1 ofβ–globin for 11 species, and the utility of this new method is demonstrated.
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Stern, David B., Eduardo Castro Nallar, Jason Rathod, and Keith A. Crandall. "DNA Barcoding analysis of seafood accuracy in Washington, D.C. restaurants." PeerJ 5 (April 25, 2017): e3234. http://dx.doi.org/10.7717/peerj.3234.
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In Washington D.C., recent legislation authorizes citizens to test if products are properly represented and, if they are not, to bring a lawsuit for the benefit of the general public. Recent studies revealing the widespread phenomenon of seafood substitution across the United States make it a fertile area for consumer protection testing. DNA barcoding provides an accurate and cost-effective way to perform these tests, especially when tissue alone is available making species identification based on morphology impossible. In this study, we sequenced the 5′ barcoding region of the Cytochrome Oxidase I gene for 12 samples of vertebrate and invertebrate food items across six restaurants in Washington, D.C. and used multiple analytical methods to make identifications. These samples included several ambiguous menu listings, sequences with little genetic variation among closely related species and one sequence with no available reference sequence. Despite these challenges, we were able to make identifications for all samples and found that 33% were potentially mislabeled. While we found a high degree of mislabeling, the errors involved closely related species and we did not identify egregious substitutions as have been found in other cities. This study highlights the efficacy of DNA barcoding and robust analyses in identifying seafood items for consumer protection.
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Lu, Yue, Long Zhao, Zhao Li, and Xiangjun Dong. "Genetic Similarity Analysis Based on Positive and Negative Sequence Patterns of DNA." Symmetry 12, no. 12 (2020): 2090. http://dx.doi.org/10.3390/sym12122090.
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Similarity analysis of DNA sequences can clarify the homology between sequences and predict the structure of, and relationship between, them. At the same time, the frequent patterns of biological sequences explain not only the genetic characteristics of the organism, but they also serve as relevant markers for certain events of biological sequences. However, most of the aforementioned biological sequence similarity analysis methods are targeted at the entire sequential pattern, which ignores the missing gene fragment that may induce potential disease. The similarity analysis of such sequences containing a missing gene item is a blank. Consequently, some sequences with missing bases are ignored or not effectively analyzed. Thus, this paper presents a new method for DNA sequence similarity analysis. Using this method, we first mined not only positive sequential patterns, but also sequential patterns that were missing some of the base terms (collectively referred to as negative sequential patterns). Subsequently, we used these frequent patterns for similarity analysis on a two-dimensional plane. Several experiments were conducted in order to verify the effectiveness of this algorithm. The experimental results demonstrated that the algorithm can obtain various results through the selection of frequent sequential patterns and that accuracy and time efficiency was improved.
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Lipsky, Robert H., Chiara M. Mazzanti, Joseph G. Rudolph, et al. "DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms." Clinical Chemistry 47, no. 4 (2001): 635–44. http://dx.doi.org/10.1093/clinchem/47.4.635.
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Abstract Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution. Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
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Benabou, Sanae, Cyril Ruckebusch, Michel Sliwa, et al. "Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods." Nucleic Acids Research 47, no. 13 (2019): 6590–605. http://dx.doi.org/10.1093/nar/gkz522.
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Abstract Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson–Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.
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Nelson, Adin, Ivelise Rijo, Zhigang Zhang, Andrew D. Zelenetz, and Ariela Noy. "Feasiblity and Implication of Bidirectional Sequencing Using a Multiplex Framework 2 Region Primer for Somatic Mutation Analysis of the Immunoglobulin (IgH) Heavy Chain in Chronic Lymphocytic Leukemia (CLL)." Blood 110, no. 11 (2007): 4706. http://dx.doi.org/10.1182/blood.v110.11.4706.4706.
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Abstract Background: A somatic mutation rate of >2% of the immunoglobulin (IgH) heavy chain gene, in comparison to germline DNA, is a positive prognostic indicator in chronic lymphocytic leukemia (CLL). Genomic DNA is amplified using the Biomed-2 multiplex PCR primer set, sequenced in the reverse direction only using a 3′ JH primer, and compared to the germline sequence in VBase, a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences. It is unknown whether forward sequencing using the multiplex 5′ Biomed-2 primers in a single reaction is feasible or whether doing so improves the accuracy of the mutational analysis. Methods: DNA was extracted from peripheral blood or bone marrow mononuclear cells from patients with CLL using the QIAGEN QIAspin DNA mini-kit. The FR2 region of the IgH gene was amplified using tube B of the Biomed-2 multiplex PCR primer set, separated in 2% agarose gel, excised, and prepared for sequencing using the QIAGEN QIAquick gel extraction kit. Bidirectional DNA sequencing was performed with the full set of multiplex 5′ FR2 primers combined in one reaction for the forward sequence and in a second reaction with the Biomed-2 3′ JH primer for the reverse sequence. A consensus sequence was obtained and mutation rates were calculated based on the consensus sequence and the reverse sequence separately. Results: The FR2 region was amplified from 30 CLL patients. 20 samples produced bidirectional consensus DNA sequences, 2 sequenced only with multiplex FR2 primers, 6 sequenced only with a JH primer, and 2 produced no sequences. Compared with unidirectional JH sequencing, bidirectional sequencing did not reassign any patient in the germline IgH group to the mutated category. In 15 patients, the uni- and bi-directional sequencing were identical. In 4 patients, a single basepair difference was noted. A random permutation test yields a two-sided p-value as 12.5%, indicating that no significant difference was detected between the uni- and bi-directional sequencing. Multiplex bidirectional sequencing did provide sequence information within the CDR3 region at the 3′ terminus of the amplimer which is partially truncated when using the JH primer alone. Conclusion: Bidirectional sequencing does not provide a somatic IgH mutation rate that differs significantly from that obtained from the reverse sequence alone and does not likely influence the IgH mutational prognostic assignment. Nonetheless, the bidirectional consensus sequence adds bases in the CDR3 region which can be used for research applications such as the generation of patient-specific primers for PCR. It is also noteworthy that multiplex PCR using the Biomed-2 primers is feasible.
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Abbas, Ali Hadi, Haider Abas AL saegh, and Furkan Sabbar ALaraji. "Sequence diversity and evolution of infectious bursal disease virus in Iraq." F1000Research 10 (April 16, 2021): 293. http://dx.doi.org/10.12688/f1000research.28421.1.
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Background: Infectious Bursal Disease (IBD) is a highly infectious disease which causes huge economic losses to the poultry industry due to the direct impact of the illness and indirect consequences such as decreasing the general immunity of the flock, leaving it naive to other diseases. In Iraq, IBD is highly prevalent despite vaccination programs, yet studies on sequence diversity of the causative virus are still rare. Methods: A sample from Bursa of Fabricius from an IBD outbreak in a flock in the city of Najaf in Iraq was smeared on an FTA card. Amplicons of targeted regions in VP1 and VP2 genes were generated and sequenced. Sequences were then compared with other local and global sequences downloaded from GenBank repositories. Sequence alignment and DNA sequence analyses were achieved using MUSCLE, UGENE and MEGAx software. The molecular clock and sequence evolutionary analyses were applied using MEGAx tools. Results: The strain sequenced in this study belongs to a very virulent Infectious Bursal Disease Virus (vvIBDV) as the DNA and phylogenetic analysis of VP1 and VP2 gene sequences showed a mutual clustering with similar sequences belonging to vvIBDV genogroup 3. Analyses of the hyper variable region of VP2 gene (hvVP2) of IBDV isolates from Iraq indicates a presence of sequence diversity. Interestingly, the two vaccine strains Ventri IBDV Plus and ABIC MB71 that showed the highest sequence similarity to the local isolates in the hvVP2 region are not used in vaccination routine against IBDV in Iraq. Conclusion: Sequences of vvIBDV in Iraq are diverse. Remarkably, some of the available vaccine strains show high sequence similarity with local strains in Iraq; however, they are not included in the routine vaccination programs. Analysis of more samples involving more geographical regions is needed to draw a detailed map of antigenic diversity of IBDV in Iraq.
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Abbas, Ali Hadi, Haider Abas AL saegh, and Furkan Sabbar ALaraji. "Sequence diversity and evolution of infectious bursal disease virus in Iraq." F1000Research 10 (September 2, 2021): 293. http://dx.doi.org/10.12688/f1000research.28421.2.
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Background: Infectious Bursal Disease (IBD) is a highly infectious disease which causes huge economic losses to the poultry industry due to the direct impact of the illness and indirect consequences such as decreasing the general immunity of the flock, leaving it naive to other diseases. In Iraq, IBD is highly prevalent despite vaccination programs, yet studies on sequence diversity of the causative virus are still rare. Methods: A sample from Bursa of Fabricius from an IBD outbreak in a flock in the city of Najaf in Iraq was smeared on an FTA card. Amplicons of targeted regions in VP1 and VP2 genes were generated and sequenced. Sequences were then compared with other local and global sequences downloaded from GenBank repositories. Sequence alignment and DNA sequence analyses were achieved using MUSCLE, UGENE and MEGAx software. The molecular clock and sequence evolutionary analyses were applied using MEGAx tools. Results: The strain sequenced in this study belongs to a very virulent Infectious Bursal Disease Virus (vvIBDV) as the DNA and phylogenetic analysis of VP1 and VP2 gene sequences showed a mutual clustering with similar sequences belonging to vvIBDV genogroup 3. Analyses of the hyper variable region of VP2 gene (hvVP2) of IBDV isolates from Iraq indicates a presence of sequence diversity. Interestingly, the two vaccine strains Ventri IBDV Plus and ABIC MB71 that showed the highest sequence similarity to the local isolates in the hvVP2 region are not used in vaccination routine against IBDV in Iraq. Conclusion: Sequences of vvIBDV in Iraq are diverse. Remarkably, some of the available vaccine strains show high sequence similarity with local strains in Iraq; however, they are not included in the routine vaccination programs. Analysis of more samples involving more geographical regions is needed to draw a detailed map of antigenic diversity of IBDV in Iraq.
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Tang, Yi-Wei, Alexander Von Graevenitz, Michael G. Waddington, et al. "Identification of Coryneform Bacterial Isolates by Ribosomal DNA Sequence Analysis." Journal of Clinical Microbiology 38, no. 4 (2000): 1676–78. http://dx.doi.org/10.1128/jcm.38.4.1676-1678.2000.
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Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%)Corynebacterium isolates and 5 of 6 (83.3%)Corynebacterium-related isolates, respectively. Within theCorynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significantCorynebacterium diphtheriae (4 of 4) andCorynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter,Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.
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House, Brent L., Michael W. Mortimer, and Michael L. Kahn. "New Recombination Methods for Sinorhizobium meliloti Genetics." Applied and Environmental Microbiology 70, no. 5 (2004): 2806–15. http://dx.doi.org/10.1128/aem.70.5.2806-2815.2004.
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ABSTRACT The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.
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Christoffersen, M. L., M. E. Araújo, and M. A. M. Moreira. "A molecular method for a qualitative analysis of potentially coding sequences of DNA." Brazilian Journal of Biology 64, no. 3a (2004): 383–98. http://dx.doi.org/10.1590/s1519-69842004000300003.
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Total sequence phylogenies have low information content. Ordinary misconceptions are that character quality can be ignored and that relying on computer algorithms is enough. Despite widespread preference for a posteriori methods of character evaluation, a priori methods are necessary to produce transformation series that are independent of tree topologies. We propose a stepwise qualitative method for analyzing protein sequences. Informative codons are selected, alternative amino acid transformation series are analyzed, and most parsimonious transformations are hypothesized. We conduct four phylogenetic analyses of philodryanine snakes. The tree based on all nucleotides produces least resolution. Trees based on the exclusion of third positions, on an asymmetric step matrix, and on our protocol, produce similar results. Our method eliminates noise by hypothesizing explicit transformation series for each informative protein-coding amino acid. This approaches qualitative methods for morphological data, in which only characters successfully interpreted in a phylogenetic context are used in cladistic analyses. The method allows utilizing character information contained in the original sequence alignment and, therefore, has higher resolution in inferring a phylogenetic tree than some traditional methods (such as distance methods).
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Gomes, A. R., S. Vinga, M. Zavolan, and H. de Lencastre. "Analysis of the Genetic Variability of Virulence-Related Loci in Epidemic Clones of Methicillin-Resistant Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 49, no. 1 (2005): 366–79. http://dx.doi.org/10.1128/aac.49.1.366-379.2005.
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ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) isolates have previously been classified into major epidemic clonal types by pulsed-field gel electrophoresis in combination with multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec typing. We aimed to investigate whether genetic variability in potentially polymorphic domains of virulence-related factors could provide another level of differentiation in a diverse collection of epidemic MRSA clones. The target regions of strains representative of epidemic clones and genetically related methicillin-susceptible S. aureus isolates from the 1960s that were sequenced included the R domains of clfA and clfB; the D, W, and M regions of fnbA and fnbB; and three regions in the agr operon. Sequence variation ranged from very conserved regions, such as those for RNAIII and the agr interpromoter region, to the highly polymorphic R regions of the clf genes. The sequences of the clf R domains could be grouped into six major sequence types on the basis of the sequences in their 3′ regions. Six sequence types were also observed for the fnb sequences at the amino acid level. From an evolutionary point of view, it was interesting that a small DNA stretch at the 3′ clf R-domain sequence and the fnb sequences agreed with the results of MLST for this set of strains. In particular, clfB R-domain sequences, which had a high discriminatory capacity and with which the types distinguished were congruent with those obtained by other molecular typing methods, have potential for use for the typing of S. aureus. Clone- and strain-specific sequence motifs in the clf and fnb genes may represent useful additions to a typing methodology with a DNA array.
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Macas, Jiří, Pavel Neumann, Petr Novák, and Jiming Jiang. "Global sequence characterization of rice centromeric satellite based on oligomer frequency analysis in large-scale sequencing data." Bioinformatics 26, no. 17 (2010): 2101–8. http://dx.doi.org/10.1093/bioinformatics/btq343.
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Abstract Motivation: Satellite DNA makes up significant portion of many eukaryotic genomes, yet it is relatively poorly characterized even in extensively sequenced species. This is, in part, due to methodological limitations of traditional methods of satellite repeat analysis, which are based on multiple alignments of monomer sequences. Therefore, we employed an alternative, alignment-free, approach utilizing k-mer frequency statistics, which is in principle more suitable for analyzing large sets of satellite repeat data, including sequence reads from next generation sequencing technologies. Results: k-mer frequency spectra were determined for two sets of rice centromeric satellite CentO sequences, including 454 reads from ChIP-sequencing of CENH3-bound DNA (7.6 Mb) and the whole genome Sanger sequencing reads (5.8 Mb). k-mer frequencies were used to identify the most conserved sequence regions and to reconstruct consensus sequences of complete monomers. Reconstructed consensus sequences as well as the assessment of overall divergence of k-mer spectra revealed high similarity of the two datasets, suggesting that CentO sequences associated with functional centromeres (CENH3-bound) do not significantly differ from the total population of CentO, which includes both centromeric and pericentromeric repeat arrays. On the other hand, considerable differences were revealed when these methods were used for comparison of CentO populations between individual chromosomes of the rice genome assembly, demonstrating preferential sequence homogenization of the clusters within the same chromosome. k-mer frequencies were also successfully used to identify and characterize smRNAs derived from CentO repeats. Contact: macas@umbr.cas.cz Supplementary information: Supplementary data are available at Bioinformatics online.
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Sardaraz, Muhammad, Muhammad Tahir, and Ataul Aziz Ikram. "Advances in high throughput DNA sequence data compression." Journal of Bioinformatics and Computational Biology 14, no. 03 (2016): 1630002. http://dx.doi.org/10.1142/s0219720016300021.
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Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted.
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Volobuev, AN N., ES S. Petrov, and NP P. Romanchuk. "BIOPHYSICAL BASES OF GENOME ORGANIZATION." Science and Innovations in Medicine 2, no. 4 (2017): 13–17. http://dx.doi.org/10.35693/2500-1388-2017-0-4-13-17.
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Aim - the analysis of nucleotide sequences of DNA molecules and the bases of the information storage with the help of DNA. Material and methods - the study is based on Markov chain theory and Bayesian Information Criterion. Results. Principles of genetic code construction were investigated. Specific nucleotide sequences were analyzed using Markov chain theory; the method of sequencing nucleotide sequences was described. Conclusion. A nucleotide sequence has certain restrictions associated with complementarity of the bases along DNA chain. These restrictions at the level of triplet sequence can be eliminated by degeneracy of the genetic code.
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Zhang, Shao Xuan, Xin Rui Liu, Bo Chuan Wang, Yun Hui Ling, De Jun Sun, and Guang Zhu Lin. "Comparison of the ITS Sequences of 5 Common Potentilla Species in Jilin Province of China." Advanced Materials Research 554-556 (July 2012): 1690–93. http://dx.doi.org/10.4028/www.scientific.net/amr.554-556.1690.
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To find the differences in the internal transcribed spacer(ITS) sequences and provide scientific data for the authentication of Potentilla chinensis and its related species, we extracted the genome DNA from the leaves of 5 common Potetilla species in Jilin Province, amplified the ITS region using ITS universal primers of angiosperm, and sequenced the purified PCR products directly. Polymorphism of ITS sequences was found within P. chinensis and the sequence data suggested that our samples of this species might be related to hybridization. Other 4 species showed intraspecies-stability in ITS sequence. The ITS sequences of these 5 Potentilla species are significantly different. So ITS sequence analysis and other methods derived from it can be used in authentication of Potentilla.
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Tan, Chengjie, Shanshan Li, and Ping Zhu. "4D Graphical representation research of DNA sequences." International Journal of Biomathematics 08, no. 01 (2015): 1550004. http://dx.doi.org/10.1142/s1793524515500047.
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Graphical representation of DNA sequences is a key component in studying biological problems. In order to gain new insights in DNA sequences, this paper combined the digitized methods of single-base, base pairs and coding in triplet bases with the times of base appearing, and then a novel 4D graphical representation method of DNA sequences was put forward. It was a one-to-one correspondence of the arbitrary DNA sequence and 4D graphical representation, that avoided causing non-unique 4D graphical representation and overlapping lines. The method could reflect the biological information features of DNA sequence more comprehensively and effectively without any losses. Based on the 4D graphical representation, we used the geometric center of 4D graphical representation as eigenvalue of DNA sequences analyses, which kept the original features of the data, and then established the Euclidean distances and included angles between vectors' terminal point for similarity analyses of the first extron of the beta-globulin gene among 11 species. Finally, we established the graph of systematic hierarchical cluster analysis of 11 species to observe more easily the relationship between species. A positive outcome was reached, and the results were in accord with biological taxonomy, which also supported the rationality and effectiveness of the novel 4D graphical representation.
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Lastuti, Nunuk Dyah Retno, Ali Rohman, Didik Handiyatno, Dony Chrismanto, and Kurnia Desiandura. "Sequence analysis of the cytochrome c oxidase subunit 1 gene of Sarcoptes scabiei isolated from goats and rabbits in East Java, Indonesia." July-2019 12, no. 7 (2019): 959–64. http://dx.doi.org/10.14202/vetworld.2019.959-964.
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Aim: This study aimed to sequence the Cytochrome c oxidase (COX-1) gene sequence from mitochondrial DNA of Sarcoptes scabiei isolated from Lamongan goats and Mojokerto rabbits, align it with DNA isolated from Zi'gong rabbit (GenBank accession No. EU256389.1), and produce a phylogenetic analysis of S. scabiei COX-1 gene. Materials and Methods: S. scabiei mites were obtained from goats and rabbits, and DNA was extracted using QIAamp DNA Mini Kit. The forward and reverse primer sequences were designed based on the DNA sequence of an S. scabiei COX-1 gene isolated from the Zi'gong rabbit (5'-TCTTAGGGGCTGGATTTAGTATG-3' and 5'-AGTTCCTCTACCAGTTCCAC-3', respectively). To confirm sequencing output, the sequence resulting from the reverse primer was inverted and aligned to the sequence from the forward primer using Clone Manager Professional Version 9 for Windows (Scientific & Educational Software; http://www.scied.com). This alignment was subsequently used to build a phylogenetic tree, using the Neighbor- Joining method, in the MEGA6 program (https://www.megasoftware.net/). Results: Polymerase chain reaction (PCR) products from S. scabiei isolates from Lamongan goats and Mojokerto rabbits produced bands of around 290 bp with 2% agarose gel electrophoresis. Comparing the DNA sequences of the S. scabiei COX-1 gene with those isolated from Lamongan goats and Mojokerto rabbits showed 99% homology. Conclusion: PCR products of the S. scabiei COX-1 gene isolated from Lamongan goats and Mojokerto rabbits were around 290 bp long. The sequences had more than 99% homology. The sequences of the COX-1 gene of S. scabiei from Lamongan goats and Mojokerto rabbits were relatively close to the sequence of the gene in S. scabiei obtained from various hosts according to National Center for Biotechnology Information data.
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Dvorská, L., M. Bartoš, G. Martin, W. Erler, and I. Pavlík. "Strategies for differentiation, identification and typing of medically important species of mycobacteria by molecular methods." Veterinární Medicína 46, No. 11–12 (2001): 309–28. http://dx.doi.org/10.17221/7890-vetmed.
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Molecular biology methods offer new opportunities to differentiate, identify and type bacterial species and strains. These methods use the variability of nucleic sequences of genes such as 16S rDNA,&nbsp;beta subunit RNA-ase (rpoB), gyrase (gyrB), rDNA internal transcribed spacer and other genes. The aim of this paper is to provide comprehensive information about the methods available to differentiate and identify species of mycobacteria at the DNA sequence level. The methods discussed in the review include PCR, PCR-REA, sequencing analysis, spoligotyping and DNA fingerprinting. These methods have been applied to both the &ldquo;universal&rdquo; part of the genome and to specific mycobacterial genes.
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Buldyrev, S. V., N. V. Dokholyan, A. L. Goldberger, et al. "Analysis of DNA sequences using methods of statistical physics." Physica A: Statistical Mechanics and its Applications 249, no. 1-4 (1998): 430–38. http://dx.doi.org/10.1016/s0378-4371(97)00503-7.
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Suvorova, Yulia M., Maria A. Korotkova, and Eugene V. Korotkov. "Comparative analysis of periodicity search methods in DNA sequences." Computational Biology and Chemistry 53 (December 2014): 43–48. http://dx.doi.org/10.1016/j.compbiolchem.2014.08.008.
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Liu, Bin. "BioSeq-Analysis: a platform for DNA, RNA and protein sequence analysis based on machine learning approaches." Briefings in Bioinformatics 20, no. 4 (2017): 1280–94. http://dx.doi.org/10.1093/bib/bbx165.
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AbstractWith the avalanche of biological sequences generated in the post-genomic age, one of the most challenging problems is how to computationally analyze their structures and functions. Machine learning techniques are playing key roles in this field. Typically, predictors based on machine learning techniques contain three main steps: feature extraction, predictor construction and performance evaluation. Although several Web servers and stand-alone tools have been developed to facilitate the biological sequence analysis, they only focus on individual step. In this regard, in this study a powerful Web server called BioSeq-Analysis (http://bioinformatics.hitsz.edu.cn/BioSeq-Analysis/) has been proposed to automatically complete the three main steps for constructing a predictor. The user only needs to upload the benchmark data set. BioSeq-Analysis can generate the optimized predictor based on the benchmark data set, and the performance measures can be reported as well. Furthermore, to maximize user’s convenience, its stand-alone program was also released, which can be downloaded from http://bioinformatics.hitsz.edu.cn/BioSeq-Analysis/download/, and can be directly run on Windows, Linux and UNIX. Applied to three sequence analysis tasks, experimental results showed that the predictors generated by BioSeq-Analysis even outperformed some state-of-the-art methods. It is anticipated that BioSeq-Analysis will become a useful tool for biological sequence analysis.
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Brody, Thomas, Amarendra Yavatkar, Alexander Kuzin, and Ward F. Odenwald. "Ultraconserved Non-coding DNA Within Diptera and Hymenoptera." G3 Genes|Genomes|Genetics 10, no. 9 (2020): 3015–24. http://dx.doi.org/10.1534/g3.120.401502.
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Abstract This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved sequence elements in these phylogenetic groups. We compared non-coding sequences found within and flanking Drosophila developmental genes to homologous sequences in Ceratitis capitata and Musca domestica. Many of the conserved sequence blocks (CSBs) that constitute Drosophila cis-regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca. Also conserved is the position but not necessarily the orientation of many of these ultraconserved CSBs (uCSBs) with respect to flanking genes. Using the mosquito EvoPrint algorithm, we have also identified uCSBs shared among distantly related mosquito species. Side by side comparison of bee and ant EvoPrints of selected developmental genes identify uCSBs shared between these two Hymenoptera, as well as less conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in these dipterans and Hymenoptera will lead to a greater understanding of their evolutionary origin and function of their conserved non-coding sequences and aid in discovery of core elements of enhancers. This study applies the phylogenetic footprinting program EvoPrinter to detection of ultraconserved non-coding sequence elements in Diptera, including flies and mosquitos, and Hymenoptera, including ants and bees. EvoPrinter outputs an interspecies comparison as a single sequence in terms of the input reference sequence. Ultraconserved sequences flanking known developmental genes were detected in Ceratitis and Musca when compared with Drosophila species, in Aedes and Culex when compared with Anopheles, and between ants and bees. Our methods are useful in detecting and understanding the core evolutionarily hardened sequences required for gene regulation.
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Starke, Ingo C., Wilfried Vahjen, Robert Pieper, and Jürgen Zentek. "The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons." Molecular Biology International 2014 (July 10, 2014): 1–10. http://dx.doi.org/10.1155/2014/548683.
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In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.
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Inbamalar, T. M., and R. Sivakumar. "Improved Algorithm for Analysis of DNA Sequences Using Multiresolution Transformation." Scientific World Journal 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/786497.
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Bioinformatics and genomic signal processing use computational techniques to solve various biological problems. They aim to study the information allied with genetic materials such as the deoxyribonucleic acid (DNA), the ribonucleic acid (RNA), and the proteins. Fast and precise identification of the protein coding regions in DNA sequence is one of the most important tasks in analysis. Existing digital signal processing (DSP) methods provide less accurate and computationally complex solution with greater background noise. Hence, improvements in accuracy, computational complexity, and reduction in background noise are essential in identification of the protein coding regions in the DNA sequences. In this paper, a new DSP based method is introduced to detect the protein coding regions in DNA sequences. Here, the DNA sequences are converted into numeric sequences using electron ion interaction potential (EIIP) representation. Then discrete wavelet transformation is taken. Absolute value of the energy is found followed by proper threshold. The test is conducted using the data bases available in the National Centre for Biotechnology Information (NCBI) site. The comparative analysis is done and it ensures the efficiency of the proposed system.
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Gernandt, David S., Garth Holman, Christopher Campbell, et al. "Phylogenetics of extant and fossil Pinaceae: methods for increasing topological stability." Botany 94, no. 9 (2016): 863–84. http://dx.doi.org/10.1139/cjb-2016-0064.
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Relationships of living and fossil Pinaceae were inferred using parsimony and Bayesian inference of morphological characters and plastid and nuclear DNA sequences. When considering extant taxa only, adding molecular to morphological characters resulted in markedly increased resolution and branch support compared with analysis of morphology alone. Including 45 fossil taxa resulted in drastically decreased resolution in morphology-based consensus trees. We evaluated the effect on branch support and resolution of including DNA sequences, deleting fossils lacking information for cone scale apices and seeds, using reduced consensus methods, and using implied weighting, and found that the greatest improvements were found by including DNA sequences and using implied weighting. The tree topologies from parsimony and Bayesian inference confirm previous findings that the fossil genus Pseudoaraucaria and a few species of Pityostrobus from the Lower Cretaceous are related to abietoid genera, and that other species of Pityostrobus are pinoid and closely related to Pinus. Focusing phylogenetic analyses on the most complete fossil cones, specifically those that are anatomically preserved and include both cone scale apices and seeds, and taking into account homoplasy, resulted in the clearest hypotheses for the timing and sequence of diversification in the family.
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Rajaei, Pedram, Khadijeh Hoda Jahanian, Amin Beheshti, Shahab S. Band, Abdollah Dehzangi, and Hamid Alinejad-Rokny. "VIRMOTIF: A User-Friendly Tool for Viral Sequence Analysis." Genes 12, no. 2 (2021): 186. http://dx.doi.org/10.3390/genes12020186.
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Bioinformatics and computational biology have significantly contributed to the generation of vast and important knowledge that can lead to great improvements and advancements in biology and its related fields. Over the past three decades, a wide range of tools and methods have been developed and proposed to enhance performance, diagnosis, and throughput while maintaining feasibility and convenience for users. Here, we propose a new user-friendly comprehensive tool called VIRMOTIF to analyze DNA sequences. VIRMOTIF brings different tools together as one package so that users can perform their analysis as a whole and in one place. VIRMOTIF is able to complete different tasks, including computing the number or probability of motifs appearing in DNA sequences, visualizing data using the matplotlib and heatmap libraries, and clustering data using four different methods, namely K-means, PCA, Mean Shift, and ClusterMap. VIRMOTIF is the only tool with the ability to analyze genomic motifs based on their frequency and representation (D-ratio) in a virus genome.
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CWIKLINSKI, K., F. N. J. KOOYMAN, D. C. K. VAN DOORN, J. B. MATTHEWS, and J. E. HODGKINSON. "New insights into sequence variation in the IGS region of 21 cyathostomin species and the implication for molecular identification." Parasitology 139, no. 8 (2012): 1063–73. http://dx.doi.org/10.1017/s0031182012000467.
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SUMMARYCyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3–62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means†.
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Phillips, Ruth B., Susan A. Manley, and Thomas J. Daniels. "Systematics of the Salmonid Genus Salvelinus Inferred from Ribosomal DNA Sequences." Canadian Journal of Fisheries and Aquatic Sciences 51, S1 (1994): 198–204. http://dx.doi.org/10.1139/f94-305.
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DNA sequences of the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA were determined for six species of the genus Salvelinus: S. alpinus (Arctic char), S. malma (Dolly Varden), S. confluentus (bull trout), S. leucomaenis (Japanese char), S. fontinalis (brook trout), and S. namaycush (lake trout), and for Hucho perryi (huchen). The ITS2 sequence data (approximately 375 base pairs (bp)) were combined with previously determined sequence data for the ITS1 (approximately 575 bp), giving a total of 981 bp of aligned sequence for each species. Phylogenetic analysis of the aligned sequences was done using maximum parsimony, maximum likelihood, and distance matrix methods with H. perryi as an outgroup. The results were consistent with previous work based on comparisons of morphologies, allozymes, and karyotypes. Comparison of these results with those based on mitochondrial DNA sequences suggests that hybridization may have occurred between S. confluentus and S. alpinus or S. malma.
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Blackwood, Christopher B., Adam Oaks, and Jeffrey S. Buyer. "Phylum- and Class-Specific PCR Primers for General Microbial Community Analysis." Applied and Environmental Microbiology 71, no. 10 (2005): 6193–98. http://dx.doi.org/10.1128/aem.71.10.6193-6198.2005.
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ABSTRACT Amplification of a particular DNA fragment from a mixture of organisms by PCR is a common first step in methods of examining microbial community structure. The use of group-specific primers in community DNA profiling applications can provide enhanced sensitivity and phylogenetic detail compared to domain-specific primers. Other uses for group-specific primers include quantitative PCR and library screening. The purpose of the present study was to develop several primer sets targeting commonly occurring and important groups. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes and for parts of both the 18S ribosomal sequence and the internal transcribed spacer region of Basidiomycota were examined. Primers were tested by comparison to sequences in the ARB 2003 database, and chosen primers were further tested by cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to be placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.
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Dewhirst, Floyd E., Zeli Shen, Michael S. Scimeca, et al. "Discordant 16S and 23S rRNA Gene Phylogenies for the Genus Helicobacter: Implications for Phylogenetic Inference and Systematics." Journal of Bacteriology 187, no. 17 (2005): 6106–18. http://dx.doi.org/10.1128/jb.187.17.6106-6118.2005.
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ABSTRACT Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31′ and 27′. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.
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Zhang, Peng, Bertrand Boisson, Peter D. Stenson, et al. "SeqTailor: a user-friendly webserver for the extraction of DNA or protein sequences from next-generation sequencing data." Nucleic Acids Research 47, W1 (2019): W623—W631. http://dx.doi.org/10.1093/nar/gkz326.
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Abstract Human whole-genome-sequencing reveals about 4 000 000 genomic variants per individual. These data are mostly stored as VCF-format files. Although many variant analysis methods accept VCF as input, many other tools require DNA or protein sequences, particularly for splicing prediction, sequence alignment, phylogenetic analysis, and structure prediction. However, there is no existing webserver capable of extracting DNA/protein sequences for genomic variants from VCF files in a user-friendly and efficient manner. We developed the SeqTailor webserver to bridge this gap, by enabling rapid extraction of (i) DNA sequences around genomic variants, with customizable window sizes and options to annotate the splice sites closest to the variants and to consider the neighboring variants within the window; and (ii) protein sequences encoded by the DNA sequences around genomic variants, with built-in SnpEff annotator and customizable window sizes. SeqTailor supports 11 species, including: human (GRCh37/GRCh38), chimpanzee, mouse, rat, cow, chicken, lizard, zebrafish, fruitfly, Arabidopsis and rice. Standalone programs are provided for command-line-based needs. SeqTailor streamlines the sequence extraction process, and accelerates the analysis of genomic variants with software requiring DNA/protein sequences. It will facilitate the study of genomic variation, by increasing the feasibility of sequence-based analysis and prediction. The SeqTailor webserver is freely available at http://shiva.rockefeller.edu/SeqTailor/.
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van Belkum, Alex, Stewart Scherer, Loek van Alphen, and Henri Verbrugh. "Short-Sequence DNA Repeats in Prokaryotic Genomes." Microbiology and Molecular Biology Reviews 62, no. 2 (1998): 275–93. http://dx.doi.org/10.1128/mmbr.62.2.275-293.1998.
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SUMMARY Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10−4 per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.
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Jenkins, Bethany D., Grieg F. Steward, Steven M. Short, Bess B. Ward, and Jonathan P. Zehr. "Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray." Applied and Environmental Microbiology 70, no. 3 (2004): 1767–76. http://dx.doi.org/10.1128/aem.70.3.1767-1776.2004.
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ABSTRACT Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.
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Igarashi, Yoji, Daisuke Mori, Susumu Mitsuyama, et al. "A Preliminary Metagenome Analysis Based on a Combination of Protein Domains." Proteomes 7, no. 2 (2019): 19. http://dx.doi.org/10.3390/proteomes7020019.
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Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions.
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NYEO, SU-LONG, and I.-CHING YANG. "CODON DISTRIBUTIONS IN DNA SEQUENCE OF ESCHERICHIA COLI." Journal of Biological Systems 10, no. 01 (2002): 47–60. http://dx.doi.org/10.1142/s0218339002000299.
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The distributions of codons in the DNA sequence of Escherichia coli K-12 are studied by using several statistical methods of analysis. Codons corresponding to the amino acids leucine, alanine and isoleucine are considered. The pair distributions of the codons as a function of the pair separation are evaluated and are seen to decay exponentially. The exponential decay constants have a linear relation with the numbers of the codons, indicating that the codons are randomly distributed in the sequence. The pair correlation and power spectral methods also show similar statistical behavior of codons in the sequence, with the exception that there appear very small peaks about the frequency f=0.286 in the power spectra of the amino acids leucine, alanine and isoleucine. Such a frequency reflects a periodicity of about 3.5 amino acids and a general helical structure of the proteins of the bacterium.
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Datta, Sibnarayan, Reji Gopalakrishnan, Soumya Chatterjee, and Vijay Veer. "Phylogenetic Characterization of a Novel Insect-Specific Flavivirus Detected in a Culex Pool, Collected from Assam, India." Intervirology 58, no. 3 (2015): 149–54. http://dx.doi.org/10.1159/000381901.
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Objective: We report the phylogenetic characterization of a unique flavivirus sequence detected in a wild Culex tritaeniorhynchus mosquito pool, collected from the northeast Indian state of Assam. Methods: DNA and RNA were extracted from field-collected mosquito pools. Extracts were subjected to PCR and reverse transcriptase PCR amplification using universal and type-specific primers for direct detection of flavivirus-specific viral nucleic acids. An amplified flavivirus nonstructural protein 5 (NS5) genetic region was sequenced and BLAST searched, and phylogenetic analyses performed with reference sequences retrieved from GenBank. Results: Phylogenetic analyses revealed the sequence to be related to insect-specific flaviviruses (ISFs) of the genus Flavivirus, family Flaviviridae. Despite being related to the Palm Creek virus (PCV; an ISF very recently reported from Northern Australia), the present sequence (provisionally named Assam virus) was found to be highly divergent from PCV and other ISF sequences available in GenBank. The partial NS5 sequence analysis demonstrated low nucleotide sequence identity (66-77%) with known ISFs reported from other parts of the globe. Conclusion: Findings of this study suggest the presence of a candidate novel ISF - the first to be reported from India.
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Xu, Liu, and Masahide Seki. "Recent advances in the detection of base modifications using the Nanopore sequencer." Journal of Human Genetics 65, no. 1 (2019): 25–33. http://dx.doi.org/10.1038/s10038-019-0679-0.
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Abstract DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.
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Enaigbe, Andrew A., Obhioze Augustine Akpoka, and Stanley Odaro Imade. "ASSESSMENT OF 16S rRNA SEQUENCE AND HETEROTROPHIC PLATE COUNT (HPC) METHODS OF IDENTIFYING BACTERIA FROM DRINKING WATER SYSTEMS IN BENIN CITY METROPOLIS." Bacterial Empire 2, no. 3 (2019): 63. http://dx.doi.org/10.36547/be.2019.2.3.63-69.
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A variety of simple culture-based tests which are proposed to recover a wide range of microorganisms from water are collectively known as heterotrophic plate count (HPC) and used as an indirect indicator to give information about water quality. The aim of this study was to assess the heterotrophic plate count (HPC) culture-based dependent and 16S rRNA independent techniques of identifying bacteria. The HPC was conducted by incubating a filtered sample of water on R2A agar plates and enumerating the number of resultant bacterial colonies that grow on each plate. The molecular analysis was performed by extracting the deoxyribonucleic acid (DNA) from bacterial isolate and polymerase chain reaction (PCR) was done to obtain the amplicons (PCR products). Purified PCR products were sequenced by ABI V3.1 Big dye kit and the analysis of sequence was conducted by the basic local alignment search tool (BLAST) to identify their closest relatives. A total number of 17 isolates of Pseudomonas, Bacillus and Proteus were phenotypically identified, while the nucleotide sequence revealed the presence of 59 diverse strains with distribution (percentage) of Pseudomonas (25) 42.2 %, Bacillus 17 (28.8 %) and Proteus (17) 28.8 %. The investigated isolates when compared from gene data-base recorded genetic relatedness with similarity index of 61 to 100 %. The 16S rRNA gene sequences from DNA extractions showed microbial consortia in drinking water to comprise of a broad array of bacterial diversity, including those of critical concern to human health such as Pseudomonas and Bacillus. Finally, there was no significant correlation between the HPC test and 16S rRNA sequence analysis.
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Vuddhakul, Varaporn, Toshihiro Nakai, Chiho Matsumoto, et al. "Analysis of gyrB and toxRGene Sequences of Vibrio hollisae and Development ofgyrB- and toxR-Targeted PCR Methods for Isolation of V. hollisae from the Environment and Its Identification." Applied and Environmental Microbiology 66, no. 8 (2000): 3506–14. http://dx.doi.org/10.1128/aem.66.8.3506-3514.2000.
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ABSTRACT Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio(toxR). A portion of the gyrB sequence ofV. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the correspondingVibrio parahaemolyticus gyrB sequence. The toxRgene of V. hollisae was cloned utilizing a htpGgene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from otherVibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 102 CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37°C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the abovehtpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification ofV. hollisae.
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Shah, Mohammad Monir, Hirotoshi Iihara, Makiko Noda, et al. "dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus." International Journal of Systematic and Evolutionary Microbiology 57, no. 1 (2007): 25–30. http://dx.doi.org/10.1099/ijs.0.64205-0.
Full textAbstract:
In the last few years, many attempts have been made to use conserved gene sequences for identification and for phylogenetic studies of Staphylococcus species. In an effort to identify a more reliable approach, a dnaJ gene sequence-based database was created. In this study, an approximately 883 bp portion of the dnaJ gene sequence from 45 staphylococcal type strains was compared with 16S rRNA and other conserved gene (hsp60, sodA and rpoB) sequences available in public databases. Nucleotide sequence comparisons revealed that the staphylococcal dnaJ gene showed higher discrimination (mean similarity 77.6 %) than the 16S rRNA (mean similarity 97.4 %), rpoB (mean similarity 86 %), hsp60 (mean similarity 82 %) and sodA (mean similarity 81.5 %) genes. Analysis of the dnaJ gene sequence from 20 Staphylococcus isolates representing two clinically important species showed <1 % sequence divergence. Phylogenetic data obtained from the dnaJ gene sequence were in general agreement with those of 16S rRNA gene sequence analysis and DNA–DNA reassociation studies. In conclusion, the dnaJ gene sequence-based assay is an effective alternative to currently used methods, including 16S rRNA gene sequencing, for identification and taxonomical analysis of Staphylococcus species.
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Pitkäranta, M., T. Meklin, A. Hyvärinen, et al. "Analysis of Fungal Flora in Indoor Dust by Ribosomal DNA Sequence Analysis, Quantitative PCR, and Culture." Applied and Environmental Microbiology 74, no. 1 (2007): 233–44. http://dx.doi.org/10.1128/aem.00692-07.
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ABSTRACT In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.
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Zhang, Shao Xuan, Xin Rui Liu, Yun Hui Ling, Bo Chuan Wang, De Jun Sun, and Guang Zhu Lin. "Comparison of Chloroplast TrnL-F Sequences of 5 Common Potentilla Species in Jilin Province of China." Advanced Materials Research 554-556 (July 2012): 1730–33. http://dx.doi.org/10.4028/www.scientific.net/amr.554-556.1730.
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To find the differences in the trnL-F sequences and provide scientific data for the authentication of Potentilla chinensis and its related species, we extracted the genome DNA from the leaves of 5 common Potetilla species in Jilin Province, amplified the trnL-F region using universal primers of angiosperm, and sequenced the purified PCR products directly. We found that the trnL-F sequences of different species of Potentilla are significantly different. So trn-L sequence analysis and other methods derived from it can be used in authentication of Potentilla.
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Spampinato, Claudia, and Darío Leonardi. "Molecular Fingerprints to IdentifyCandidaSpecies." BioMed Research International 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/923742.
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A wide range of molecular techniques have been developed for genotypingCandidaspecies. Among them, multilocus sequence typing (MLST) and microsatellite length polymorphisms (MLP) analysis have recently emerged. MLST relies on DNA sequences of internal regions of various independent housekeeping genes, while MLP identifies microsatellite instability. Both methods generate unambiguous and highly reproducible data. Here, we review the results achieved by using these two techniques and also provide a brief overview of a new method based on high-resolution DNA melting (HRM). This method identifies sequence differences by subtle deviations in sample melting profiles in the presence of saturating fluorescent DNA binding dyes.