To evaluate the contribution of non-synonymous-coding variants of known familial and genome-wide association studies (GWAS)-linked genes for Parkinson's disease (PD) to PD risk in the East Asian population, we sequenced all the coding exons of 39 PD-related disease genes and evaluated the accumulation of rare non-synonymous-coding variants in 375 early-onset PD cases and 399 controls. We also genotyped 782 non-synonymous-coding variants of these genes in 710 late-onset PD cases and 9046 population controls. Significant enrichment of LRRK2 variants was observed in both early- and late-onset PD (odds ratio = 1.58; 95% confidence interval = 1.29-1.93; P = 8.05 × 10(-6)). Moderate enrichment was also observed in FGF20, MCCC1, GBA and ITGA8. Half of the rare variants anticipated to cause loss of function of these genes were present in healthy controls. Overall, non-synonymous-coding variants of known familial and GWAS-linked genes appear to make a limited contribution to PD risk, suggesting that clinical sequencing of these genes will provide limited information for risk prediction and molecular diagnosis.
A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.
The commercial freshwater crayfish Cherax quadricarinatus complete mitochondrial genome was recovered from partial genome sequencing using the MiSeq Personal Sequencer. The mitogenome has 15,869 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The base composition of C. quadricarinatus is 32.16% for T, 23.39% for C, 33.26% for A, and 11.19% for G, with an AT bias of 65.42%.
The mitogenome of the black yabby, Geocharax gracilis, was sequenced using the MiSeq Personal Sequencer. It has 15,924 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 23 transfer RNAs, and a non-coding AT-rich region. The base composition of G. gracilis mitogenome is 32.18% for T, 22.32% for C, 34.83% for A, and 10.68% for G, with an AT bias of 67.01%. The mitogenome gene order is typical for that of parastacid crayfish with the exception of some minor rearrangements involving tRNA genes.
The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96 % identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (1990) and Burma (1976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (1974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94 % identity to the French Guyana strain (1989) in genotype V while isolate DS212/110306 had 96 % identity to the Nauru Island strain (1974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country.
Oil palm is the second largest source of edible oil which contributes to approximately 20% of the world's production of oils and fats. In order to understand the molecular biology involved in in vitro propagation, flowering, efficient utilization of nitrogen sources and root diseases, we have initiated an expressed sequence tag (EST) analysis on oil palm.
Previously, direct-proportional length-based DNA computing (DPLB-DNAC) for solving weighted graph problems has been reported. The proposed DPLB-DNAC has been successfully applied to solve the shortest path problem, which is an instance of weighted graph problems. The design and development of DPLB-DNAC is important in order to extend the capability of DNA computing for solving numerical optimization problem. According to DPLB-DNAC, after the initial pool generation, the initial solution is subjected to amplification by polymerase chain reaction and, finally, the output of the computation is visualized by gel electrophoresis. In this paper, however, we give more attention to the initial pool generation of DPLB-DNAC. For this purpose, two kinds of initial pool generation methods, which are generally used for solving weighted graph problems, are evaluated. Those methods are hybridization-ligation and parallel overlap assembly (POA). It is found that for DPLB-DNAC, POA is better than that of the hybridization-ligation method, in terms of population size, generation time, material usage, and efficiency, as supported by the results of actual experiments.
Cancer starts when cells in a part of the body start to grow out of control. In fact cells become cancer cells because of DNA damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to study the cancer genes, DNA walk plots of genomes of patients with lung cancer were generated using a program written in MATLAB language. The data so obtained was checked for fractal property by computing the fractal dimension using a program written in MATLAB. Also, the correlation of damaged DNA was studied using the Hurst exponent measure. We have found that the damaged DNA sequences are exhibiting higher degree of fractality and less correlation compared with normal DNA sequences. So we confirmed this method can be used for early detection of lung cancer. The method introduced in this research not only is useful for diagnosis of lung cancer but also can be applied for detection and growth analysis of different types of cancers.
Matched MeSH terms: Sequence Analysis, DNA/statistics & numerical data
Perspicuous assessments of taxonomic boundaries and discovery of cryptic taxa are of paramount importance in interpreting ecological and evolutionary phenomena among black flies (Simuliidae) and combating associated vector-borne diseases. Simulium tani Takaoka & Davies is the largest and perhaps the most taxonomically challenging species complex of black flies in the Oriental Region. We use a DNA sequence-based method to delineate currently recognized chromosomal and morphological taxa in the S. tani complex on the Southeast Asian mainland and Taiwan, while elucidating their phylogenetic relationships. A molecular approach using multiple genes, coupled with morphological and chromosomal data, supported recognition of cytoform K and morphoform 'b' as valid species; indicated that S. xuandei, cytoform L, and morphoform 'a' contain possible cryptic species; and suggested that cytoform B is in the early stages of reproductive isolation whereas lineage sorting is incomplete in cytoforms A, C, and G.
Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future.
he Chinese tiger frog Hoplobatrachus rugulosus is widely distributed in southern China, Malaysia, Myanmar, Thailand, and Vietnam. It is listed in Appendix II of CITES as the only Class II nationally-protected frog in China. The bred tiger frog known as the Thailand tiger frog, is also identified as H. rugulosus. Our analysis of the Cyt b gene showed high genetic divergence (13.8%) between wild and bred samples of tiger frog. Unexpected genetic divergence of the complete mt genome (14.0%) was also observed between wild and bred samples of tiger frog. Yet, the nuclear genes (NCX1, Rag1, Rhod, Tyr) showed little divergence between them. Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H. rugulosus may be a cryptic species complex. Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis, and H. rugulosus (BT) as a sister clade to H. rugulosus (WT). We suggest that we should prevent Thailand tiger frogs (bred type) from escaping into wild environments lest they produce hybrids with Chinese tiger frogs (wild type).
In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.
Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
Nile tilapia (Oreochromis niloticus) is a globally significant aquaculture species rapidly gaining status as a farmed commodity. In West Africa, wild Nile tilapia genetic resources are abundant yet knowledge of fine-scale population structure and patterns of natural genetic variation are limited. Coinciding with this is a burgeoning growth in tilapia aquaculture in Ghana and other countries within the region underpinned by locally available genetic resources. Using 192 single nucleotide polymorphism (SNP) markers this study conducted a genetic survey of Nile tilapia throughout West Africa, sampling 23 wild populations across eight countries (Benin, Burkina Faso, Côte d'Ivoire, Ghana, Togo, Mali, Gambia and Senegal), representing the major catchments of the Volta, Niger, Senegal and Gambia River basins. A pattern of isolation-by-distance and significant spatial genetic structure was identified throughout West Africa (Global FST = 0.144), which largely corresponds to major river basins and, to a lesser extent, sub-basins. Two populations from the Gambia River (Kudang and Walekounda), one from the western Niger River (Lake Sélingué) and one from the upper Red Volta River (Kongoussi) showed markedly lower levels of diversity and high genetic differentiation compared to all other populations, suggesting genetically isolated populations occurring across the region. Genetic structure within the Volta Basin did not always follow the pattern expected for sub-river basins. This study identifies clear genetic structuring and differentiation amongst West African Nile tilapia populations, which concur with broad patterns found in previous studies. In addition, we provide new evidence for fine-scale genetic structuring within the Volta Basin and previously unidentified genetic differences of populations in Gambia. The 192 SNP marker suite used in this study is a useful tool for differentiating tilapia populations and we recommend incorporating this marker suite into future population screening of O. niloticus. Our results form the basis of a solid platform for future research on wild tilapia genetic resources in West Africa, and the identification of potentially valuable germplasm for use in ongoing breeding programs for aquaculture.
In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome-scale studies to characterize both model and non-model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome-wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site-associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms-enabling the exploration of diverse evolutionary and conservation questions.
Plasmodium knowlesi is an important causative agent of malaria in humans of Southeast Asia. Macaques are natural hosts for this parasite, but little is conclusively known about its patterns of transmission within and between these hosts. Here, we apply a comprehensive phylogenetic approach to test for patterns of cryptic population genetic structure between P. knowlesi isolated from humans and long-tailed macaques from the state of Sarawak in Malaysian Borneo. Our approach differs from previous investigations through our exhaustive use of archival 18S Small Subunit rRNA (18S) gene sequences from Plasmodium and Hepatocystis species, our inclusion of insertion and deletion information during phylogenetic inference, and our application of Bayesian phylogenetic inference to this problem. We report distinct clades of P. knowlesi that predominantly contained sequences from either human or macaque hosts for paralogous A-type and S-type 18S gene loci. We report significant partitioning of sequence distances between host species across both types of loci, and confirmed that sequences of the same locus type showed significantly biased assortment into different clades depending on their host species. Our results support the zoonotic potential of Plasmodium knowlesi, but also suggest that humans may be preferentially infected with certain strains of this parasite. Broadly, such patterns could arise through preferential zoonotic transmission of some parasite lineages or a disposition of parasites to transmit within, rather than between, human and macaque hosts. Available data are insufficient to address these hypotheses. Our results suggest that the epidemiology of P. knowlesi may be more complicated than previously assumed, and highlight the need for renewed and more vigorous explorations of transmission patterns in the fifth human malarial parasite.
The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome.
Natural hybridization has been considered a source of taxonomic complexity in Cryptocoryne. A combined study of DNA sequencing data from the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnK-matK region of chloroplast DNA was used to identify the parents of Cryptocoryne putative hybrids from Peninsular Malaysia. Based on the intermediate morphology and sympatric distribution area, the plants were tentatively identified as the hybrid Cryptocoryne ×purpurea nothovar. purpurea. The plants were pollen sterile and had long been considered as hybrids, supposedly between two related and co-existing species, C. cordata var. cordata and C. griffithii. The status of C. ×purpurea nothovar. purpurea was independently confirmed by the presence of an additive ITS sequence pattern from these two parental species in hybrid individuals. An analysis of the chloroplast trnK-matK sequences showed that the hybridization is bidirectional with the putative hybrids sharing identical sequences from C. cordata var. cordata and C. griffithii, indicating that both putative parental species had been the maternal parent in different accessions.