Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

Background: Cryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains. Results: The majority of the putative species-specific genes examined were in fact common to C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes. Conclusions: Most of the genes identified as being species specific during bioinformatics in Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia

A new multi locus variable number of tandem repeat analysis scheme for epidemiological surveillance of Xanthomonas vasicola pv. musacearum, the plant pathogen causing bacterial wilt on banana and enset

Xanthomonas vasicola pv. musacearum (Xvm) which causes Xanthomonas wilt (XW) on banana (Musa accuminata x balbisiana) and enset (Ensete ventricosum), is closely related to the species Xanthomonas vasicola that contains the pathovars vasculorum (Xvv) and holcicola (Xvh), respectively pathogenic to sugarcane and sorghum. Xvm is considered a monomorphic bacterium whose intra-pathovar diversity remains poorly understood. With the sudden emergence of Xvm within east and central Africa coupled with the unknown origin of one of the two sublineages suggested for Xvm, attention has shifted to adapting technologies that focus on identifying the origin and distribution of the genetic diversity within this pathogen. Although microbiological and conventional molecular diagnostics have been useful in pathogen identification. Recent advances have ushered in an era of genomic epidemiology that aids in characterizing monomorphic pathogens. To unravel the origin and pathways of the recent emergence of XW in Eastern and Central Africa, there was a need for a genotyping tool adapted for molecular epidemiology. Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA) is able to resolve the evolutionary patterns and invasion routes of a pathogen. In this study, we identified microsatellite loci from nine published Xvm genome sequences. Of the 36 detected microsatellite loci, 21 were selected for primer design and 19 determined to be highly typeable, specific, reproducible and polymorphic with two- to four- alleles per locus on a sub-collection. The 19 markers were multiplexed and applied to genotype 335 Xvm strains isolated from seven countries over several years. The microsatellite markers grouped the Xvm collection into three clusters; with two similar to the SNP-based sublineages 1 and 2 and a new cluster 3, revealing an unknown diversity in Ethiopia. Five of the 19 markers had alleles present in both Xvm and Xanthomonas vasicola pathovars holcicola and vasculorum, supporting the phylogenetic closeliness of these three pathovars. Thank to the public availability of the haplotypes on the MLVABank database, this highly reliable and polymorphic genotyping tool can be further used in a transnational surveillance network to monitor the spread and evolution of XW throughout Africa.. It will inform and guide management of Xvm both in banana-based and enset-based cropping systems. Due to the suitability of MLVA-19 markers for population genetic analyses, this genotyping tool will also be used in future microevolution studies

V-MitoSNP: visualization of human mitochondrial SNPs

BACKGROUND: Mitochondrial single nucleotide polymorphisms (mtSNPs) constitute important data when trying to shed some light on human diseases and cancers. Unfortunately, providing relevant mtSNP genotyping information in mtDNA databases in a neatly organized and transparent visual manner still remains a challenge. Amongst the many methods reported for SNP genotyping, determining the restriction fragment length polymorphisms (RFLPs) is still one of the most convenient and cost-saving methods. In this study, we prepared the visualization of the mtDNA genome in a way, which integrates the RFLP genotyping information with mitochondria related cancers and diseases in a user-friendly, intuitive and interactive manner. The inherent problem associated with mtDNA sequences in BLAST of the NCBI database was also solved. DESCRIPTION: V-MitoSNP provides complete mtSNP information for four different kinds of inputs: (1) color-coded visual input by selecting genes of interest on the genome graph, (2) keyword search by locus, disease and mtSNP rs# ID, (3) visualized input of nucleotide range by clicking the selected region of the mtDNA sequence, and (4) sequences mtBLAST. The V-MitoSNP output provides 500 bp (base pairs) flanking sequences for each SNP coupled with the RFLP enzyme and the corresponding natural or mismatched primer sets. The output format enables users to see the SNP genotype pattern of the RFLP by virtual electrophoresis of each mtSNP. The rate of successful design of enzymes and primers for RFLPs in all mtSNPs was 99.1%. The RFLP information was validated by actual agarose electrophoresis and showed successful results for all mtSNPs tested. The mtBLAST function in V-MitoSNP provides the gene information within the input sequence rather than providing the complete mitochondrial chromosome as in the NCBI BLAST database. All mtSNPs with rs number entries in NCBI are integrated in the corresponding SNP in V-MitoSNP. CONCLUSION: V-MitoSNP is a web-based software platform that provides a user-friendly and interactive interface for mtSNP information, especially with regard to RFLP genotyping. Visual input and output coupled with integrated mtSNP information from MITOMAP and NCBI make V-MitoSNP an ideal and complete visualization interface for human mtSNPs association studies

CSGM Designer: a platform for designing cross-species intron-spanning genic markers linked with genome information of legumes.

BackgroundGenetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. An important class of genetic markers is those based on orthologous genes, because they can guide hypotheses about conserved gene function, a situation that is well documented for a number of agronomic traits. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species.ResultsHere we report an efficient platform for the design of cross-species gene-derived markers in legumes. The automated platform, named CSGM Designer (URL: http://tgil.donga.ac.kr/CSGMdesigner), facilitates rapid and systematic design of cross-species genic markers. The underlying database is composed of genome data from five legume species whose genomes are substantially characterized. Use of CSGM is enhanced by graphical displays of query results, which we describe as "circular viewer" and "search-within-results" functions. CSGM provides a virtual PCR representation (eHT-PCR) that predicts the specificity of each primer pair simultaneously in multiple genomes. CSGM Designer output was experimentally validated for the amplification of orthologous genes using 16 genotypes representing 12 crop and model legume species, distributed among the galegoid and phaseoloid clades. Successful cross-species amplification was obtained for 85.3% of PCR primer combinations.ConclusionCSGM Designer spans the divide between well-characterized crop and model legume species and their less well-characterized relatives. The outcome is PCR primers that target highly conserved genes for polymorphism discovery, enabling functional inferences and ultimately facilitating trait-associated molecular breeding

Molecular characterization and exclusion of porcine GUSB as a candidate gene for congenital hernia inguinalis/scrotalis

BACKGROUND: Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine β-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region. RESULTS: A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs. CONCLUSION: On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia

Ghrelin alleles and use of the same for genetically typing animals

Disclosed herein are genetic markers for animal growth, appetite and fatness, methods for identifying such markers, and methods of screening animals to determine those more likely to produce desired growth, appetite and fatness and preferably selecting those animals for future breeding purposes. The markers are based upon the presence or absence of certain polymorphisms in the Ghrelin gene

Genetic determinants of response to aspirin and warfarin and development of silicon nanowire based genotyping

PhD ThesisChronic diseases such as cardiovascular diseases and colorectal cancer are the leading cause of mortality worldwide. Commonly used drugs such as aspirin and warfarin are shown to effective at reducing the risk of chronic diseases but have a narrow therapeutic window and are associated with adverse drug reactions, particularly, hemorrhage. Identification of pharmacogenetic markers such as single nucleotide polymorphisms (SNPs) that could help deliver personalized dose could help improve the risk-benefit ratio. Furthermore, development of a rapid point of care genotyping device consisting of a pharmacogenetic SNP panel for aspirin and warfarin could help implement personalized medicine in the clinical setting. Analysis of candidate SNPs in aspirin’s pharmacokinetic and pharmacodynamic pathways was carried out to explain variation in aspirin’s colorectal chemopreventive efficacy using two large population based case-control datasets. Associations and interactions were tested using logistic regression models and meta-analysis of the 2 datasets. A novel sitespecific association for rs1799853 (OR=0.73, 95% CI=0.60-0.90, P=0.003) and rs1105879 (OR=1.16, 95% CI=1.02-1.32, P=0.03) with colon cancer risk was observed. Furthermore, stratification by aspirin use showed increased risk of colorectal cancer in aspirin users but not in non-users carrying variant allele of the SNPs rs4936367 and rs7112513 in PAFAH1B2 gene and rs2070959 and rs1105879 in UGT1A6 gene (Pinteraction<0.05 for all). These results provide insight into aspirin’s differential chemopreventive efficacy and the neoplastic transformation of cells in colon and rectum. Utility of clinically validated pharmacogenetic dosing algorithms consisting of three warfarin dose associated SNPs from the European population needs to tested in the Gujarati Indians, an Indian sub-population. Dose prediction accuracy of the algorithms was compared between Gujarati Indian and European population. Mean squared difference of both pharmacogenetic algorithms was higher in Gujarati Indian compared to European population (Klein et al 2009, 216.3 v/s 160.7, P=0.05; Gage et al 2008, 170.6 v/s 143.2, P=0.07). Poor prediction accuracy could be explained by the presence of study subjects requiring dose for target INR range 2.5-3.5 and low frequency of the VKORC1 rs9923231 variant, which is the most important genetic determinant of warfarin dosing in Europeans. Therefore, the SNP panel and dosing algorithms developed from European populations cannot be assumed to have utility in the Gujarati Indian population. Finally, to help develop a rapid, point-of care, silicon nanowire (SiNW) based SNP genotyping device, a panel of isothermal melting probes were designed to genotype three warfarin dose associated SNPs. Testing of hybridization and washing conditions to have optimal hybridization kinetics between the probe and target DNA and high target sequence specificity was carried out using custom designed microarray platform. Accurate genotype calls for all 3 SNPs in 2 anonymised samples using empirically optimized hybridization and washing conditions was carried out successfully. Current work highlighted associations between probe characteristics and hybridization parameters, which would be useful in designing and testing probes on the SiNW platform. Identification, validation and testing of clinical utility of population specific pharmacogenetic markers along with development and deployment of ultra-rapid point of care genotyping technologies could help deliver personalized risk-benefit ratio for aspirin and warfarin

Steps and Tools for PCR-Based Technique Design

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for technique standardization

Single nucleotide polymorphisms associated with the human electroencephalogram during desflurane anaesthesia

General anaesthesia is an induced state that enables a person to endure surgical procedures without pain or recollection. There is substantial individual variability in the response to anaesthesia and in order to avoid adverse effects caused by either under- or over-sedation, anaesthetic drug administration must be tailored to suit the individual patient. This requires a means to monitor the depth of general anaesthesia. The electroencephalogram (EEG) records the electrical activity of the brain and enables effects of anaesthetic drugs on brain functioning to be monitored. Quantitative EEG monitors, such as the Bispectral (BIS) index monitor, process the raw EEG and provide a numerical output that is often used to measure the depth of general anaesthesia during surgery. Due to a number of factors including clinical conditions and genetic variability in the EEG, the BIS value can at times be misleading. To identify genetic variations associated with EEG phenotypes relevant to anaesthesia monitoring, an association analysis was performed for 34 single nucleotide polymorphisms (SNPs) in a sample of 125 surgical patients undergoing general, gynaecological or orthopaedic surgery. During surgery, patients were anaesthetised with the volatile anaesthetic agent desflurane, the depth of anaesthesia was measured using BIS monitoring and the raw was also EEG recorded. SNP genotyping was performed for 13 SNPs in five candidate genes; SGIP1, GABRA2, CACNA1G, HCN1 and HCN2, using the polymerase chain reaction and restriction fragment length polymorphism analysis. An additional 21 SNPs in 15 genes involved in various inflammatory and other immune-related pathways were genotyped by Sequenom MassARRAY at the Australian Genome Research Facility. Six SNPs in five different genes were found to be associated with spindle amplitude (SGIP1, GABRA2, HCN1, IL1B and MYD88), and a further five SNPs were associated with either delta frequency (IL10), or end tidal desflurane concentration (ETDC) (CACNA1G, CRP, MYD88 and TGFB1). The strongest associations were identified for a single SNP located in the 3' UTR of MYD88 (rs6853). The rs6853 A/G genotype was associated with higher median spindle amplitude (p = 0.0040) and spindle amplitude relative to ETDC (p = 0.0006), and lower EDTC (p = 0.0095) than the A/A genotype. No rs6853 G/G homozygotes were identified in the study sample. MYD88 acts as an adaptor protein in the interleukin-1 receptor and toll-like receptor signalling pathways. Within the brain, cytokines are thought to act as neuronal modulators and influence neurotransmitter signalling and ion channel activity. The association of MYD88 with spindle amplitude, in conjunction with IL1B, suggests that cytokines may influence the EEG during general anaesthesia. Thus cytokine mediated regulation of neuronal activity is speculated to underlie the reported associations. All reported effect sizes were small (0.77- to 1.55-fold) and associated genes had four distinct types of function; ion channels, neurotransmitter signalling, endocytosis, and cytokine signalling. This suggests that numerous genes in different pathways, each with a small, possibly additive effect, are involved in regulating the EEG during general anaesthesia