Reducing false positive incidental findings with ensemble genotyping and logistic regression-based variant filtering methods

PubMed Central

Hwang, Kyu-Baek; Lee, In-Hee; Park, Jin-Ho; Hambuch, Tina; Choi, Yongjoon; Kim, MinHyeok; Lee, Kyungjoon; Song, Taemin; Neu, Matthew B.; Gupta, Neha; Kohane, Isaac S.; Green, Robert C.; Kong, Sek Won

2014-01-01

As whole genome sequencing (WGS) uncovers variants associated with rare and common diseases, an immediate challenge is to minimize false positive findings due to sequencing and variant calling errors. False positives can be reduced by combining results from orthogonal sequencing methods, but costly. Here we present variant filtering approaches using logistic regression (LR) and ensemble genotyping to minimize false positives without sacrificing sensitivity. We evaluated the methods using paired WGS datasets of an extended family prepared using two sequencing platforms and a validated set of variants in NA12878. Using LR or ensemble genotyping based filtering, false negative rates were significantly reduced by 1.1- to 17.8-fold at the same levels of false discovery rates (5.4% for heterozygous and 4.5% for homozygous SNVs; 30.0% for heterozygous and 18.7% for homozygous insertions; 25.2% for heterozygous and 16.6% for homozygous deletions) compared to the filtering based on genotype quality scores. Moreover, ensemble genotyping excluded > 98% (105,080 of 107,167) of false positives while retaining > 95% (897 of 937) of true positives in de novo mutation (DNM) discovery, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and ensemble genotyping would be essential to minimize false positive DNM candidates. PMID:24829188

A comparative evaluation between real time Roche COBas TAQMAN 48 HCV and bDNA Bayer Versant HCV 3.0.

PubMed

Giraldi, Cristina; Noto, Alessandra; Tenuta, Robert; Greco, Francesca; Perugini, Daniela; Spadafora, Mario; Bianco, Anna Maria Lo; Savino, Olga; Natale, Alfonso

2006-10-01

The HCV virus is a common human pathogen made of a single stranded RNA genome with 9600nt. This work compared two different commercial methods used for HCV viral load, the bDNA Bayer Versant HCV 3.0 and the RealTime Roche COBAS TaqMan 48 HCV. We compared the reproducibility and linearity of the two methods. Seventy-five plasma samples with genotypes 1 to 4, which represent the population (45% genotype 1; 24% genotype 2; 13% genotype 3; 18% genotype 4) were directly processed with the Versanto method based upon signal amplification; the same samples were first extracted (COBAS Ampliprep - TNAI) and then amplified using RealTime PCR (COBAS TaqMan 48). The results obtained indicate the same performance for both methods if they have genotype 1, but in samples with genotypes 2, 3 and 4 the RealTime PCR Roche method gave an underestimation in respect to the Bayer bDNA assay.

Significant variation between SNP-based HLA imputations in diverse populations: the last mile is the hardest.

PubMed

Pappas, D J; Lizee, A; Paunic, V; Beutner, K R; Motyer, A; Vukcevic, D; Leslie, S; Biesiada, J; Meller, J; Taylor, K D; Zheng, X; Zhao, L P; Gourraud, P-A; Hollenbach, J A; Mack, S J; Maiers, M

2018-05-22

Four single nucleotide polymorphism (SNP)-based human leukocyte antigen (HLA) imputation methods (e-HLA, HIBAG, HLA*IMP:02 and MAGPrediction) were trained using 1000 Genomes SNP and HLA genotypes and assessed for their ability to accurately impute molecular HLA-A, -B, -C and -DRB1 genotypes in the Human Genome Diversity Project cell panel. Imputation concordance was high (>89%) across all methods for both HLA-A and HLA-C, but HLA-B and HLA-DRB1 proved generally difficult to impute. Overall, <27.8% of subjects were correctly imputed for all HLA loci by any method. Concordance across all loci was not enhanced via the application of confidence thresholds; reliance on confidence scores across methods only led to noticeable improvement (+3.2%) for HLA-DRB1. As the HLA complex is highly relevant to the study of human health and disease, a standardized assessment of SNP-based HLA imputation methods is crucial for advancing genomic research. Considerable room remains for the improvement of HLA-B and especially HLA-DRB1 imputation methods, and no imputation method is as accurate as molecular genotyping. The application of large, ancestrally diverse HLA and SNP reference data sets and multiple imputation methods has the potential to make SNP-based HLA imputation methods a tractable option for determining HLA genotypes.

Efficient genotype compression and analysis of large genetic variation datasets

PubMed Central

Layer, Ryan M.; Kindlon, Neil; Karczewski, Konrad J.; Quinlan, Aaron R.

2015-01-01

Genotype Query Tools (GQT) is a new indexing strategy that expedites analyses of genome variation datasets in VCF format based on sample genotypes, phenotypes and relationships. GQT’s compressed genotype index minimizes decompression for analysis, and performance relative to existing methods improves with cohort size. We show substantial (up to 443 fold) performance gains over existing methods and demonstrate GQT’s utility for exploring massive datasets involving thousands to millions of genomes. PMID:26550772

Rare variant association analysis in case-parents studies by allowing for missing parental genotypes.

PubMed

Li, Yumei; Xiang, Yang; Xu, Chao; Shen, Hui; Deng, Hongwen

2018-01-15

The development of next-generation sequencing technologies has facilitated the identification of rare variants. Family-based design is commonly used to effectively control for population admixture and substructure, which is more prominent for rare variants. Case-parents studies, as typical strategies in family-based design, are widely used in rare variant-disease association analysis. Current methods in case-parents studies are based on complete case-parents data; however, parental genotypes may be missing in case-parents trios, and removing these data may lead to a loss in statistical power. The present study focuses on testing for rare variant-disease association in case-parents study by allowing for missing parental genotypes. In this report, we extended the collapsing method for rare variant association analysis in case-parents studies to allow for missing parental genotypes, and investigated the performance of two methods by using the difference of genotypes between affected offspring and their corresponding "complements" in case-parent trios and TDT framework. Using simulations, we showed that, compared with the methods just only using complete case-parents data, the proposed strategy allowing for missing parental genotypes, or even adding unrelated affected individuals, can greatly improve the statistical power and meanwhile is not affected by population stratification. We conclude that adding case-parents data with missing parental genotypes to complete case-parents data set can greatly improve the power of our strategy for rare variant-disease association.

Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

PubMed

Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

2015-12-01

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Using phyto-recurrent selection to choose Populus genotypes for phytoremediation of landfill leachate

Treesearch

Jill A. Zalesny; Ronald S., Jr. Zalesny; Adam H. Wiese; Richard B. Hall

2006-01-01

Information about the response of Populus genotypes to landfill leachate irrigation is needed, along with efficient methods for choosing genotypes based on leachate composition. We irrigated poplar clones during three cycles of phyto-recurrent selection to test whether genotypes responded differently to leachate and water, and to test whether our...

Choosing tree genotypes for phytoremediation of landfill leachate using phyto-recurrent selection

Treesearch

Jill A. Zalesny; Ronald S., Jr. Zalesny; Adam H. Wiese; Richard B. Hall

2007-01-01

Information about the response of poplar (Populus spp.) genotypes to landfill leachate irrigation is needed, along with efficient methods for choosing genotypes based on leachate composition. Poplar clones were irrigated during three cycles of phyto-recurrent selection to test whether genotypes responded differently to leachate and water, and to test...

Relationship of some upland rice genotype after gamma irradiation

NASA Astrophysics Data System (ADS)

Suliartini, N. W. S.; Wijayanto, T.; Madiki, A.; Boer, D.; Muhidin; Juniawan

2018-02-01

The objective of the research was to group local upland rice genotypes after being treated with gamma irradiation. The research materials were upland rice genotypes resulted from mutation of the second generation and two parents: Pae Loilo (K3D0) and Pae Pongasi (K2D0) Cultivars. The research was conducted at the Indonesian Sweetener and Fiber Crops Research Institute, Malang Regency, and used the augmented design method. Research data were analyzed with R Program. Eight hundred and seventy one genotypes were selected with the selection criteria were based on yields on the average parents added 1.5 standard deviation. Based on the selection, eighty genotypes were analyzed with cluster analyses. Nine observation variables were used to develop cluster dendrogram using average linked method. Genetic distance was measured by euclidean distance. The results of cluster dendrogram showed that tested genotypes were divided into eight groups. Group 1, 2, 7, and 8 each had one genotype, group 3 and 6 each had two genotypes, group 4 had 25 genotypes, and group 5 had 51 genotypes. Check genotypes formed a separate group. Group 6 had the highest yield per plant of 126.11 gram, followed by groups 5 and 4 of 97.63 and 94.08 gram, respectively.

[Detection and application of PIS genetic deficiency gene in dairy goat].

PubMed

Yang, Bo; Jia, Li-Li; Zhao, De-Chao; Meng, Li-Yun; Liu, Xue-Feng; Zhang, Yan-Jun; Zhang, Wen-Guang; Li, Jin-Quan

2012-07-01

The purpose of this study was to develop a molecular method for detecting polled intersex syndrome (PIS) genetic deficiency gene in dairy goat. Three pairs of primers, PIS-, PIS+, and NEI were designed based on PIS gene sequence (AF404302) to identify the PIS genetic deficiency genotype. For the normal phenotype, the fragments of 141 and 300 bp were obtained for the genotype PIS-PIS-, and 141, 449, and 300 bp for the genotype PIS-PIS+. For the PIS goat with the genotype PIS+PIS+, 449 and 300 bp were obtained. Two hundred and twenty-four dairy goats in one population were tested based on this method. The results showed that there were 150 PIS-PIS+, 70 PIS -PIS-, and 4 PIS+PIS+. The genotype frequency of PIS-PIS+ was 66.9%, and the gene frequency of PIS+ was 35.3% in the population. Therefore, the frequency of PIS offspring was over 12%. This study developed a method to detect PIS genetic deficiency dairy goat. The method could identify buck genotype accurately to avoid the occurrence of PIS genetic deficiency. The ease and accuracy show a strong potential of the method for use in marker assisted selection of dairy goats and healthy development of dairy goat industry.

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

PubMed Central

Dudley, Dawn M.; Chin, Emily N.; Bimber, Benjamin N.; Sanabani, Sabri S.; Tarosso, Leandro F.; Costa, Priscilla R.; Sauer, Mariana M.; Kallas, Esper G.; O.’Connor, David H.

2012-01-01

Background Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance. PMID:22574170

Selection of common bean genotypes for the Cerrado/Pantanal ecotone via mixed models and multivariate analysis.

PubMed

Corrêa, A M; Pereira, M I S; de Abreu, H K A; Sharon, T; de Melo, C L P; Ito, M A; Teodoro, P E; Bhering, L L

2016-10-17

The common bean, Phaseolus vulgaris, is predominantly grown on small farms and lacks accurate genotype recommendations for specific micro-regions in Brazil. This contributes to a low national average yield. The aim of this study was to use the methods of the harmonic mean of the relative performance of genetic values (HMRPGV) and the centroid, for selecting common bean genotypes with high yield, adaptability, and stability for the Cerrado/Pantanal ecotone region in Brazil. We evaluated 11 common bean genotypes in three trials carried out in the dry season in Aquidauana in 2013, 2014, and 2015. A likelihood ratio test detected a significant interaction between genotype x year, contributing 54% to the total phenotypic variation in grain yield. The three genotypes selected by the joint analysis of genotypic values in all years (Carioca Precoce, BRS Notável, and CNFC 15875) were the same as those recommended by the HMRPGV method. Using the centroid method, genotypes BRS Notável and CNFC 15875 were considered ideal genotypes based on their high stability to unfavorable environments and high responsiveness to environmental improvement. We identified a high association between the methods of adaptability and stability used in this study. However, the use of centroid method provided a more accurate and precise recommendation of the behavior of the evaluated genotypes.

Genotype calling from next-generation sequencing data using haplotype information of reads

PubMed Central

Zhi, Degui; Wu, Jihua; Liu, Nianjun; Zhang, Kui

2012-01-01

Motivation: Low coverage sequencing provides an economic strategy for whole genome sequencing. When sequencing a set of individuals, genotype calling can be challenging due to low sequencing coverage. Linkage disequilibrium (LD) based refinement of genotyping calling is essential to improve the accuracy. Current LD-based methods use read counts or genotype likelihoods at individual potential polymorphic sites (PPSs). Reads that span multiple PPSs (jumping reads) can provide additional haplotype information overlooked by current methods. Results: In this article, we introduce a new Hidden Markov Model (HMM)-based method that can take into account jumping reads information across adjacent PPSs and implement it in the HapSeq program. Our method extends the HMM in Thunder and explicitly models jumping reads information as emission probabilities conditional on the states of adjacent PPSs. Our simulation results show that, compared to Thunder, HapSeq reduces the genotyping error rate by 30%, from 0.86% to 0.60%. The results from the 1000 Genomes Project show that HapSeq reduces the genotyping error rate by 12 and 9%, from 2.24% and 2.76% to 1.97% and 2.50% for individuals with European and African ancestry, respectively. We expect our program can improve genotyping qualities of the large number of ongoing and planned whole genome sequencing projects. Contact: dzhi@ms.soph.uab.edu; kzhang@ms.soph.uab.edu Availability: The software package HapSeq and its manual can be found and downloaded at www.ssg.uab.edu/hapseq/. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22285565

LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms

PubMed Central

Money, Daniel; Gardner, Kyle; Migicovsky, Zoë; Schwaninger, Heidi; Zhong, Gan-Yuan; Myles, Sean

2015-01-01

Obtaining genome-wide genotype data from a set of individuals is the first step in many genomic studies, including genome-wide association and genomic selection. All genotyping methods suffer from some level of missing data, and genotype imputation can be used to fill in the missing data and improve the power of downstream analyses. Model organisms like human and cattle benefit from high-quality reference genomes and panels of reference genotypes that aid in imputation accuracy. In nonmodel organisms, however, genetic and physical maps often are either of poor quality or are completely absent, and there are no panels of reference genotypes available. There is therefore a need for imputation methods designed specifically for nonmodel organisms in which genomic resources are poorly developed and marker order is unreliable or unknown. Here we introduce LinkImpute, a software package based on a k-nearest neighbor genotype imputation method, LD-kNNi, which is designed for unordered markers. No physical or genetic maps are required, and it is designed to work on unphased genotype data from heterozygous species. It exploits the fact that markers useful for imputation often are not physically close to the missing genotype but rather distributed throughout the genome. Using genotyping-by-sequencing data from diverse and heterozygous accessions of apples, grapes, and maize, we compare LD-kNNi with several genotype imputation methods and show that LD-kNNi is fast, comparable in accuracy to the best-existing methods, and exhibits the least bias in allele frequency estimates. PMID:26377960

Advantages of continuous genotype values over genotype classes for GWAS in higher polyploids: a comparative study in hexaploid chrysanthemum.

PubMed

Grandke, Fabian; Singh, Priyanka; Heuven, Henri C M; de Haan, Jorn R; Metzler, Dirk

2016-08-24

Association studies are an essential part of modern plant breeding, but are limited for polyploid crops. The increased number of possible genotype classes complicates the differentiation between them. Available methods are limited with respect to the ploidy level or data producing technologies. While genotype classification is an established noise reduction step in diploids, it gains complexity with increasing ploidy levels. Eventually, the errors produced by misclassifications exceed the benefits of genotype classes. Alternatively, continuous genotype values can be used for association analysis in higher polyploids. We associated continuous genotypes to three different traits and compared the results to the output of the genotype caller SuperMASSA. Linear, Bayesian and partial least squares regression were applied, to determine if the use of continuous genotypes is limited to a specific method. A disease, a flowering and a growth trait with h (2) of 0.51, 0.78 and 0.91 were associated with a hexaploid chrysanthemum genotypes. The data set consisted of 55,825 probes and 228 samples. We were able to detect associating probes using continuous genotypes for multiple traits, using different regression methods. The identified probe sets were overlapping, but not identical between the methods. Baysian regression was the most restrictive method, resulting in ten probes for one trait and none for the others. Linear and partial least squares regression led to numerous associating probes. Association based on genotype classes resulted in similar values, but missed several significant probes. A simulation study was used to successfully validate the number of associating markers. Association of various phenotypic traits with continuous genotypes is successful with both uni- and multivariate regression methods. Genotype calling does not improve the association and shows no advantages in this study. Instead, use of continuous genotypes simplifies the analysis, saves computational time and results more potential markers.

Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data

USDA-ARS?s Scientific Manuscript database

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...

Usefulness of the HMRPGV method for simultaneous selection of upland cotton genotypes with greater fiber length and high yield stability.

PubMed

Farias, F J C; Carvalho, L P; Silva Filho, J L; Teodoro, P E

2016-08-19

The harmonic mean of the relative performance of genotypic predicted value (HMRPGV) method has been used to measure the genotypic stability and adaptability of various crops. However, its use in cotton is still restricted. This study aimed to use mixed models to select cotton genotypes that simultaneously result in longer fiber length, higher fiber yield, and phenotypic stability in both of these traits. Eight trials with 16 cotton genotypes were conducted in the 2008/2009 harvest in Mato Grosso State. The experimental design was randomized complete blocks with four replicates of each of the 16 genotypes. In each trial, we evaluated fiber yield and fiber length. The genetic parameters were estimated using the restricted maximum likelihood/best linear unbiased predictor method. Joint selection considering, simultaneously, fiber length, fiber yield, stability, and adaptability is possible with the HMRPGV method. Our results suggested that genotypes CNPA MT 04 2080 and BRS CEDRO may be grown in environments similar to those tested here and may be predicted to result in greater fiber length, fiber yield, adaptability, and phenotypic stability. These genotypes may constitute a promising population base in breeding programs aimed at increasing these trait values.

A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus.

PubMed

Pedersen, M S; Fahnøe, U; Hansen, T A; Pedersen, A G; Jenssen, H; Bukh, J; Schønning, K

2018-06-01

The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

PubMed Central

OKINO, Cintia Hiromi; MONTASSIER, Maria de Fátima Silva; de OLIVEIRA, Andressa Peres; MONTASSIER, Helio José

2018-01-01

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates. PMID:29491226

LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms.

PubMed

Money, Daniel; Gardner, Kyle; Migicovsky, Zoë; Schwaninger, Heidi; Zhong, Gan-Yuan; Myles, Sean

2015-09-15

Obtaining genome-wide genotype data from a set of individuals is the first step in many genomic studies, including genome-wide association and genomic selection. All genotyping methods suffer from some level of missing data, and genotype imputation can be used to fill in the missing data and improve the power of downstream analyses. Model organisms like human and cattle benefit from high-quality reference genomes and panels of reference genotypes that aid in imputation accuracy. In nonmodel organisms, however, genetic and physical maps often are either of poor quality or are completely absent, and there are no panels of reference genotypes available. There is therefore a need for imputation methods designed specifically for nonmodel organisms in which genomic resources are poorly developed and marker order is unreliable or unknown. Here we introduce LinkImpute, a software package based on a k-nearest neighbor genotype imputation method, LD-kNNi, which is designed for unordered markers. No physical or genetic maps are required, and it is designed to work on unphased genotype data from heterozygous species. It exploits the fact that markers useful for imputation often are not physically close to the missing genotype but rather distributed throughout the genome. Using genotyping-by-sequencing data from diverse and heterozygous accessions of apples, grapes, and maize, we compare LD-kNNi with several genotype imputation methods and show that LD-kNNi is fast, comparable in accuracy to the best-existing methods, and exhibits the least bias in allele frequency estimates. Copyright © 2015 Money et al.

Comparison of cytology, HPV DNA testing and HPV 16/18 genotyping alone or combined targeting to the more balanced methodology for cervical cancer screening.

PubMed

Chatzistamatiou, Kimon; Moysiadis, Theodoros; Moschaki, Viktoria; Panteleris, Nikolaos; Agorastos, Theodoros

2016-07-01

The objective of the present study was to identify the most effective cervical cancer screening algorithm incorporating different combinations of cytology, HPV testing and genotyping. Women 25-55years old recruited for the "HERMES" (HEllenic Real life Multicentric cErvical Screening) study were screened in terms of cytology and high-risk (hr) HPV testing with HPV 16/18 genotyping. Women positive for cytology or/and hrHPV were referred for colposcopy, biopsy and treatment. Ten screening algorithms based on different combinations of cytology, HPV testing and HPV 16/18 genotyping were investigated in terms of diagnostic accuracy. Three clusters of algorithms were formed according to the balance between effectiveness and harm caused by screening. The cluster showing the best balance included two algorithms based on co-testing and two based on HPV primary screening with HPV 16/18 genotyping. Among these, hrHPV testing with HPV 16/18 genotyping and reflex cytology (atypical squamous cells of undetermined significance - ASCUS threshold) presented the optimal combination of sensitivity (82.9%) and specificity relative to cytology alone (0.99) with 1.26 false positive rate relative to cytology alone. HPV testing with HPV 16/18 genotyping, referring HPV 16/18 positive women directly to colposcopy, and hrHPV (non 16/18) positive women to reflex cytology (ASCUS threshold), as a triage method to colposcopy, reflects the best equilibrium between screening effectiveness and harm. Algorithms, based on cytology as initial screening method, on co-testing or HPV primary without genotyping, and on HPV primary with genotyping but without cytology triage, are not supported according to the present analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Typing SNP based on the near-infrared spectroscopy and artificial neural network

NASA Astrophysics Data System (ADS)

Ren, Li; Wang, Wei-Peng; Gao, Yu-Zhen; Yu, Xiao-Wei; Xie, Hong-Ping

2009-07-01

Based on the near-infrared spectra (NIRS) of the measured samples as the discriminant variables of their genotypes, the genotype discriminant model of SNP has been established by using back-propagation artificial neural network (BP-ANN). Taking a SNP (857G > A) of N-acetyltransferase 2 (NAT2) as an example, DNA fragments containing the SNP site were amplified by the PCR method based on a pair of primers to obtain the three-genotype (GG, AA, and GA) modeling samples. The NIRS-s of the amplified samples were directly measured in transmission by using quartz cell. Based on the sample spectra measured, the two BP-ANN-s were combined to obtain the stronger ability of the three-genotype classification. One of them was established to compress the measured NIRS variables by using the resilient back-propagation algorithm, and another network established by Levenberg-Marquardt algorithm according to the compressed NIRS-s was used as the discriminant model of the three-genotype classification. For the established model, the root mean square error for the training and the prediction sample sets were 0.0135 and 0.0132, respectively. Certainly, this model could rightly predict the three genotypes (i.e. the accuracy of prediction samples was up to100%) and had a good robust for the prediction of unknown samples. Since the three genotypes of SNP could be directly determined by using the NIRS-s without any preprocessing for the analyzed samples after PCR, this method is simple, rapid and low-cost.

Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation

PubMed Central

2013-01-01

Background Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species. Results Genotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison. Conclusions GBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species. PMID:23324311

Using ancestry matching to combine family-based and unrelated samples for genome-wide association studies‡

PubMed Central

Crossett, Andrew; Kent, Brian P.; Klei, Lambertus; Ringquist, Steven; Trucco, Massimo; Roeder, Kathryn; Devlin, Bernie

2015-01-01

We propose a method to analyze family-based samples together with unrelated cases and controls. The method builds on the idea of matched case–control analysis using conditional logistic regression (CLR). For each trio within the family, a case (the proband) and matched pseudo-controls are constructed, based upon the transmitted and untransmitted alleles. Unrelated controls, matched by genetic ancestry, supplement the sample of pseudo-controls; likewise unrelated cases are also paired with genetically matched controls. Within each matched stratum, the case genotype is contrasted with control pseudo-control genotypes via CLR, using a method we call matched-CLR (mCLR). Eigenanalysis of numerous SNP genotypes provides a tool for mapping genetic ancestry. The result of such an analysis can be thought of as a multidimensional map, or eigenmap, in which the relative genetic similarities and differences amongst individuals is encoded in the map. Once constructed, new individuals can be projected onto the ancestry map based on their genotypes. Successful differentiation of individuals of distinct ancestry depends on having a diverse, yet representative sample from which to construct the ancestry map. Once samples are well-matched, mCLR yields comparable power to competing methods while ensuring excellent control over Type I error. PMID:20862653

Methods for discovering and validating relationships among genotyped animals

USDA-ARS?s Scientific Manuscript database

Genomic selection based on single-nucleotide polymorphisms (SNPs) has led to the collection of genotypes for over 2.2 million animals by the Council on Dairy Cattle Breeding in the United States. To assure that a genotype is assigned to the correct animal and that the animal’s pedigree is correct, t...

Dynamic variable selection in SNP genotype autocalling from APEX microarray data.

PubMed

Podder, Mohua; Welch, William J; Zamar, Ruben H; Tebbutt, Scott J

2006-11-30

Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide--adenine (A), thymine (T), cytosine (C) or guanine (G)--is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA) using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU) of St. Paul's Hospital (plus one negative PCR control sample). Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our model-based genotype calling algorithm captures the redundancy in the system considering all the underlying probe features of a particular SNP, automatically down-weighting any 'bad data' corresponding to image artifacts on the microarray slide or failure of a specific chemistry. In this regard, our method is able to automatically select the probes which work well and reduce the effect of other so-called bad performing probes in a sample-specific manner, for any number of SNPs.

VALIDATION OF MICROSATELLITE MARKERS FOR USE IN GENOTYPING POLYCLONAL PLASMODIUM FALCIPARUM INFECTIONS

PubMed Central

GREENHOUSE, BRYAN; MYRICK, ALISSA; DOKOMAJILAR, CHRISTIAN; WOO, JONATHAN M.; CARLSON, ELAINE J.; ROSENTHAL, PHILIP J.; DORSEY, GRANT

2006-01-01

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas. PMID:17123974

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China.

PubMed

Sun, Mingjun; Jing, Zhigang; Di, Dongdong; Yan, Hao; Zhang, Zhicheng; Xu, Quangang; Zhang, Xiyue; Wang, Xun; Ni, Bo; Sun, Xiangxiang; Yan, Chengxu; Yang, Zhen; Tian, Lili; Li, Jinping; Fan, Weixing

2017-01-01

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2) was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2) and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs) were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS)-single-nucleotide polymorphism (SNP)-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

Effect of genotyped cows in the reference population on the genomic evaluation of Holstein cattle.

PubMed

Uemoto, Y; Osawa, T; Saburi, J

2017-03-01

This study evaluated the dependence of reliability and prediction bias on the prediction method, the contribution of including animals (bulls or cows), and the genetic relatedness, when including genotyped cows in the progeny-tested bull reference population. We performed genomic evaluation using a Japanese Holstein population, and assessed the accuracy of genomic enhanced breeding value (GEBV) for three production traits and 13 linear conformation traits. A total of 4564 animals for production traits and 4172 animals for conformation traits were genotyped using Illumina BovineSNP50 array. Single- and multi-step methods were compared for predicting GEBV in genotyped bull-only and genotyped bull-cow reference populations. No large differences in realized reliability and regression coefficient were found between the two reference populations; however, a slight difference was found between the two methods for production traits. The accuracy of GEBV determined by single-step method increased slightly when genotyped cows were included in the bull reference population, but decreased slightly by multi-step method. A validation study was used to evaluate the accuracy of GEBV when 800 additional genotyped bulls (POPbull) or cows (POPcow) were included in the base reference population composed of 2000 genotyped bulls. The realized reliabilities of POPbull were higher than those of POPcow for all traits. For the gain of realized reliability over the base reference population, the average ratios of POPbull gain to POPcow gain for production traits and conformation traits were 2.6 and 7.2, respectively, and the ratios depended on heritabilities of the traits. For regression coefficient, no large differences were found between the results for POPbull and POPcow. Another validation study was performed to investigate the effect of genetic relatedness between cows and bulls in the reference and test populations. The effect of genetic relationship among bulls in the reference population was also assessed. The results showed that it is important to account for relatedness among bulls in the reference population. Our studies indicate that the prediction method, the contribution ratio of including animals, and genetic relatedness could affect the prediction accuracy in genomic evaluation of Holstein cattle, when including genotyped cows in the reference population.

Evidence for two koi herpesvirus (KHV) genotypes in South Korea.

PubMed

Kim, Hyoung Jun; Kwon, Se Ryun

2013-06-13

The geographic distribution of koi herpesvirus (KHV) has recently been analyzed by polymerase chain reaction (PCR, based on the alleles of 3 domains) and sequence analysis using 3 regions of KHV genomic DNA (SphI-5, 9/5, and the thymidine kinase gene). In this study, samples from 6 carp showing symptoms of KHV infection in 2008 were examined for the presence of KHV by using PCR and cell culture isolation methods. KHV was detected in 2 (Pyeongtaek and Buan) of the samples. Sequence analysis revealed that the genotype of the KHV PT-08 isolate was Asia genotype variant 1 (A1), and the genotype of the KHV BA-08 isolate was European genotype variant 4 (E4). In addition, PCR patterns and sequence analysis based on the alleles of 3 domains of an alternate KHV classification system confirmed that the genotype of the KHV PT-08 isolate was CyHV3-J, and the genotype of the KHV BA-08 isolate was CyHV3-third genotype. To our knowledge, this is the first study to demonstrate the presence of 2 genotypes of KHV (genotype A1/CyHV3-J; genotype E4/CyHV3-third genotype) in South Korea.

Haplotype-Based Genotyping in Polyploids.

PubMed

Clevenger, Josh P; Korani, Walid; Ozias-Akins, Peggy; Jackson, Scott

2018-01-01

Accurate identification of polymorphisms from sequence data is crucial to unlocking the potential of high throughput sequencing for genomics. Single nucleotide polymorphisms (SNPs) are difficult to accurately identify in polyploid crops due to the duplicative nature of polyploid genomes leading to low confidence in the true alignment of short reads. Implementing a haplotype-based method in contrasting subgenome-specific sequences leads to higher accuracy of SNP identification in polyploids. To test this method, a large-scale 48K SNP array (Axiom Arachis2) was developed for Arachis hypogaea (peanut), an allotetraploid, in which 1,674 haplotype-based SNPs were included. Results of the array show that 74% of the haplotype-based SNP markers could be validated, which is considerably higher than previous methods used for peanut. The haplotype method has been implemented in a standalone program, HAPLOSWEEP, which takes as input bam files and a vcf file and identifies haplotype-based markers. Haplotype discovery can be made within single reads or span paired reads, and can leverage long read technology by targeting any length of haplotype. Haplotype-based genotyping is applicable in all allopolyploid genomes and provides confidence in marker identification and in silico-based genotyping for polyploid genomics.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

PubMed Central

Suyama, Yoshihisa; Matsuki, Yu

2015-01-01

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities. PMID:26593239

Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci.

PubMed

Løbersli, Inger; Haugum, Kjersti; Lindstedt, Bjørn-Arne

2012-01-01

Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains). Copyright © 2011 Elsevier B.V. All rights reserved.

TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing

PubMed Central

Sekizuka, Tsuyoshi; Yamashita, Akifumi; Murase, Yoshiro; Iwamoto, Tomotada; Mitarai, Satoshi; Kato, Seiya; Kuroda, Makoto

2015-01-01

Whole-genome sequencing (WGS) with next-generation DNA sequencing (NGS) is an increasingly accessible and affordable method for genotyping hundreds of Mycobacterium tuberculosis (Mtb) isolates, leading to more effective epidemiological studies involving single nucleotide variations (SNVs) in core genomic sequences based on molecular evolution. We developed an all-in-one web-based tool for genotyping Mtb, referred to as the Total Genotyping Solution for TB (TGS-TB), to facilitate multiple genotyping platforms using NGS for spoligotyping and the detection of phylogenies with core genomic SNVs, IS6110 insertion sites, and 43 customized loci for variable number tandem repeat (VNTR) through a user-friendly, simple click interface. This methodology is implemented with a KvarQ script to predict MTBC lineages/sublineages and potential antimicrobial resistance. Seven Mtb isolates (JP01 to JP07) in this study showing the same VNTR profile were accurately discriminated through median-joining network analysis using SNVs unique to those isolates. An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs. The results of in silico analyses using TGS-TB are consistent with those obtained using conventional molecular genotyping methods, suggesting that NGS short reads could provide multiple genotypes to discriminate multiple strains of Mtb, although longer NGS reads (≥300-mer) will be required for full genotyping on the TGS-TB web site. Most available short reads (~100-mer) can be utilized to discriminate the isolates based on the core genome phylogeny. TGS-TB provides a more accurate and discriminative strain typing for clinical and epidemiological investigations; NGS strain typing offers a total genotyping solution for Mtb outbreak and surveillance. TGS-TB web site: https://gph.niid.go.jp/tgs-tb/. PMID:26565975

PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

PubMed Central

Singh, Om P; Bali, Prerna; Hemingway, Janet; Subbarao, Sarala K; Dash, Aditya P; Adak, Tridibes

2009-01-01

Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at kdr locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped. PMID:19594947

Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

PubMed

Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

2018-01-01

Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

A rapid and reliable PCR method for genotyping the ABO blood group. II: A2 and O2 alleles.

PubMed

O'Keefe, D S; Dobrovic, A

1996-01-01

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O1 and O2) at the molecular level, and most current ABO genotyping methods only take into account the O1 allele. Determining the presence of the O2 allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A1 and A2 alleles, even though 10% of all white persons are blood group A2. We have developed a method for genotyping the ABO locus that takes the O2 and A2 alleles into account. Typing for A2 and O2 by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results.

Identification of polymorphic inversions from genotypes

PubMed Central

2012-01-01

Background Polymorphic inversions are a source of genetic variability with a direct impact on recombination frequencies. Given the difficulty of their experimental study, computational methods have been developed to infer their existence in a large number of individuals using genome-wide data of nucleotide variation. Methods based on haplotype tagging of known inversions attempt to classify individuals as having a normal or inverted allele. Other methods that measure differences between linkage disequilibrium attempt to identify regions with inversions but unable to classify subjects accurately, an essential requirement for association studies. Results We present a novel method to both identify polymorphic inversions from genome-wide genotype data and classify individuals as containing a normal or inverted allele. Our method, a generalization of a published method for haplotype data [1], utilizes linkage between groups of SNPs to partition a set of individuals into normal and inverted subpopulations. We employ a sliding window scan to identify regions likely to have an inversion, and accumulation of evidence from neighboring SNPs is used to accurately determine the inversion status of each subject. Further, our approach detects inversions directly from genotype data, thus increasing its usability to current genome-wide association studies (GWAS). Conclusions We demonstrate the accuracy of our method to detect inversions and classify individuals on principled-simulated genotypes, produced by the evolution of an inversion event within a coalescent model [2]. We applied our method to real genotype data from HapMap Phase III to characterize the inversion status of two known inversions within the regions 17q21 and 8p23 across 1184 individuals. Finally, we scan the full genomes of the European Origin (CEU) and Yoruba (YRI) HapMap samples. We find population-based evidence for 9 out of 15 well-established autosomic inversions, and for 52 regions previously predicted by independent experimental methods in ten (9+1) individuals [3,4]. We provide efficient implementations of both genotype and haplotype methods as a unified R package inveRsion. PMID:22321652

Shared Mycobacterium avium Genotypes Observed among Unlinked Clinical and Environmental Isolates

PubMed Central

Weigel, Kris M.; Yakrus, Mitchell A.; Becker, Annie L.; Chen, Hui-Ling; Fridley, Gina; Sikora, Arthur; Speake, Cate; Hilborn, Elizabeth D.; Pfaller, Stacy

2013-01-01

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event. PMID:23851084

High-throughput, image-based screening of pooled genetic variant libraries

PubMed Central

Emanuel, George; Moffitt, Jeffrey R.; Zhuang, Xiaowei

2018-01-01

Image-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants. PMID:29083401

Precision medicine: does ethnicity information complement genotype-based prescribing decisions?

PubMed Central

Shah, Rashmi R.; Gaedigk, Andrea

2017-01-01

Inter-ethnic differences in drug response are all too well known. These are underpinned by a number of factors, including pharmacogenetic differences across various ethnic populations. Precision medicine relies on genotype-based prescribing decisions with the aim of maximizing efficacy and mitigating the risks. When there is no access to genotyping tests, ethnicity is frequently regarded as a proxy of the patient’s probable genotype on the basis of overall population-based frequency of genetic variations in the ethnic group the patient belongs to, with some variations being ethnicity-specific. However, ever-increasing transcontinental migration of populations and the resulting admixing of populations have undermined the utility of self-identified ethnicity in predicting the genetic ancestry, and therefore the genotype, of the patient. An example of the relevance of genetic ancestry of a patient is the inadequate performance of European-derived pharmacogenetic dosing algorithms of warfarin in African Americans, Brazilians and Caribbean Hispanics. Consequently, genotyping a patient potentially requires testing for all known clinically actionable variants that the patient may harbour, and new variants that are likely to be identified using state-of the art next-generation sequencing-based methods. Furthermore, self-identified ethnicity is associated with a number of ethnicity-related attributes and non-genetic factors that potentially influence the risk of phenoconversion (genotype–phenotype discordance), which may adversely impact the success of genotype-based prescribing decisions. Therefore, while genotype-based prescribing decisions are important in implementing precision medicine, ethnicity should not be disregarded. PMID:29318005

Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data

PubMed Central

2016-01-01

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordable, datasets derived from HTS methods suffer from sequencing error, alignment errors, and missing data, all of which introduce noise and uncertainty to variant discovery and genotype calling. Under such circumstances, meaningful analysis of the data is difficult. Our primary interest lies in the issue of how one can accurately infer or impute missing genotypes in HTS-derived datasets. Many of the existing genotype imputation algorithms and software packages were primarily developed by and optimized for the human genetics community, a field where a complete and accurate reference genome has been constructed and SNP arrays have, in large part, been the common genotyping platform. We set out to answer two questions: 1) can we use existing imputation methods developed by the human genetics community to impute missing genotypes in datasets derived from non-human species and 2) are these methods, which were developed and optimized to impute ascertained variants, amenable for imputation of missing genotypes at HTS-derived variants? We selected Beagle v.4, a widely used algorithm within the human genetics community with reportedly high accuracy, to serve as our imputation contender. We performed a series of cross-validation experiments, using GBS data collected from the species Manihot esculenta by the Next Generation (NEXTGEN) Cassava Breeding Project. NEXTGEN currently imputes missing genotypes in their datasets using a LASSO-penalized, linear regression method (denoted ‘glmnet’). We selected glmnet to serve as a benchmark imputation method for this reason. We obtained estimates of imputation accuracy by masking a subset of observed genotypes, imputing, and calculating the sample Pearson correlation between observed and imputed genotype dosages at the site and individual level; computation time served as a second metric for comparison. We then set out to examine factors affecting imputation accuracy, such as levels of missing data, read depth, minor allele frequency (MAF), and reference panel composition. PMID:27537694

Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data.

PubMed

Chan, Ariel W; Hamblin, Martha T; Jannink, Jean-Luc

2016-01-01

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordable, datasets derived from HTS methods suffer from sequencing error, alignment errors, and missing data, all of which introduce noise and uncertainty to variant discovery and genotype calling. Under such circumstances, meaningful analysis of the data is difficult. Our primary interest lies in the issue of how one can accurately infer or impute missing genotypes in HTS-derived datasets. Many of the existing genotype imputation algorithms and software packages were primarily developed by and optimized for the human genetics community, a field where a complete and accurate reference genome has been constructed and SNP arrays have, in large part, been the common genotyping platform. We set out to answer two questions: 1) can we use existing imputation methods developed by the human genetics community to impute missing genotypes in datasets derived from non-human species and 2) are these methods, which were developed and optimized to impute ascertained variants, amenable for imputation of missing genotypes at HTS-derived variants? We selected Beagle v.4, a widely used algorithm within the human genetics community with reportedly high accuracy, to serve as our imputation contender. We performed a series of cross-validation experiments, using GBS data collected from the species Manihot esculenta by the Next Generation (NEXTGEN) Cassava Breeding Project. NEXTGEN currently imputes missing genotypes in their datasets using a LASSO-penalized, linear regression method (denoted 'glmnet'). We selected glmnet to serve as a benchmark imputation method for this reason. We obtained estimates of imputation accuracy by masking a subset of observed genotypes, imputing, and calculating the sample Pearson correlation between observed and imputed genotype dosages at the site and individual level; computation time served as a second metric for comparison. We then set out to examine factors affecting imputation accuracy, such as levels of missing data, read depth, minor allele frequency (MAF), and reference panel composition.

Sequence-Based Genotyping for Marker Discovery and Co-Dominant Scoring in Germplasm and Populations

PubMed Central

Truong, Hoa T.; Ramos, A. Marcos; Yalcin, Feyruz; de Ruiter, Marjo; van der Poel, Hein J. A.; Huvenaars, Koen H. J.; Hogers, René C. J.; van Enckevort, Leonora. J. G.; Janssen, Antoine; van Orsouw, Nathalie J.; van Eijk, Michiel J. T.

2012-01-01

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike. PMID:22662172

A method of genotyping by pedigree-based training-set for identification of QTLs associated with cucumber fruit size

USDA-ARS?s Scientific Manuscript database

Large sets of genomic data are becoming available for cucumber (Cucumis sativus), yet there is no tool for whole genome genotyping. Creation of saturated genetic maps depends on development of good markers. The present cucumber genetic maps are based on several hundreds of markers. However they are ...

A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

PubMed

Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

2014-01-01

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

A fast indirect method to compute functions of genomic relationships concerning genotyped and ungenotyped individuals, for diversity management.

PubMed

Colleau, Jean-Jacques; Palhière, Isabelle; Rodríguez-Ramilo, Silvia T; Legarra, Andres

2017-12-01

Pedigree-based management of genetic diversity in populations, e.g., using optimal contributions, involves computation of the [Formula: see text] type yielding elements (relationships) or functions (usually averages) of relationship matrices. For pedigree-based relationships [Formula: see text], a very efficient method exists. When all the individuals of interest are genotyped, genomic management can be addressed using the genomic relationship matrix [Formula: see text]; however, to date, the computational problem of efficiently computing [Formula: see text] has not been well studied. When some individuals of interest are not genotyped, genomic management should consider the relationship matrix [Formula: see text] that combines genotyped and ungenotyped individuals; however, direct computation of [Formula: see text] is computationally very demanding, because construction of a possibly huge matrix is required. Our work presents efficient ways of computing [Formula: see text] and [Formula: see text], with applications on real data from dairy sheep and dairy goat breeding schemes. For genomic relationships, an efficient indirect computation with quadratic instead of cubic cost is [Formula: see text], where Z is a matrix relating animals to genotypes. For the relationship matrix [Formula: see text], we propose an indirect method based on the difference between vectors [Formula: see text], which involves computation of [Formula: see text] and of products such as [Formula: see text] and [Formula: see text], where [Formula: see text] is a working vector derived from [Formula: see text]. The latter computation is the most demanding but can be done using sparse Cholesky decompositions of matrix [Formula: see text], which allows handling very large genomic and pedigree data files. Studies based on simulations reported in the literature show that the trends of average relationships in [Formula: see text] and [Formula: see text] differ as genomic selection proceeds. When selection is based on genomic relationships but management is based on pedigree data, the true genetic diversity is overestimated. However, our tests on real data from sheep and goat obtained before genomic selection started do not show this. We present efficient methods to compute elements and statistics of the genomic relationships [Formula: see text] and of matrix [Formula: see text] that combines ungenotyped and genotyped individuals. These methods should be useful to monitor and handle genomic diversity.

Stability and adaptability of popcorn genotypes in the State of Rio de Janeiro, Brazil.

PubMed

Pena, G F; do Amaral, A T; Gonçalves, L S A; Candido, L S; Vittorazzi, C; Ribeiro, R M; Freitas, S P

2012-08-31

This study aimed to obtain estimates of stability and adaptability of phase launched materials and materials recommended in the country, for the northern and northwestern regions of Rio de Janeiro State, Brazil, and made a comparative analysis of different methods to evaluate stability and adaptability of grain yield and popping expansion. To this end, 10 genotypes were evaluated (UNB2U-C3, UNB2U-C4, BRS Angela, Viçosa, Beija-Flor, IAC 112, IAC 125, Zélia, Jade, and UFVM2 Barão de Viçosa) in five environments. The Yates and Cochran method revealed that genotypes UFV2M Barão de Viçosa, BRS Angela and UNB2U-C3 were the most stable for grain yield. This method also indicated superiority of genotypes UNB2U-C3, UNB2U-C4, BRS Angela, Viçosa, IAC 125, and Zélia for popping expansion. The Plaisted and Peterson and Wricke methods demonstrated that genotypes Zélia and UNB2U-C4 were the most productive and stable. These methods indicated genotypes UNB2U-C3 and BRS Angela as the most stable for popping expansion. The Kang and Phan ranking system uses methods based on analysis of variance and classified population UNB2U-C4 as the genotype with the highest stability of grain production and confirmed cultivar BRS Angela as the most stable for popping expansion. Genotypes IAC 112 and UNB2U-C4 were the most stable and adapted for grain yield, according to the Lin and Binns method. The P(i) statistics also ranked UNB2U-C3 and UNB2U-C4 as the genotypes with the best predictability and capacity for popping expansion.

Multi-task Gaussian process for imputing missing data in multi-trait and multi-environment trials.

PubMed

Hori, Tomoaki; Montcho, David; Agbangla, Clement; Ebana, Kaworu; Futakuchi, Koichi; Iwata, Hiroyoshi

2016-11-01

A method based on a multi-task Gaussian process using self-measuring similarity gave increased accuracy for imputing missing phenotypic data in multi-trait and multi-environment trials. Multi-environmental trial (MET) data often encounter the problem of missing data. Accurate imputation of missing data makes subsequent analysis more effective and the results easier to understand. Moreover, accurate imputation may help to reduce the cost of phenotyping for thinned-out lines tested in METs. METs are generally performed for multiple traits that are correlated to each other. Correlation among traits can be useful information for imputation, but single-trait-based methods cannot utilize information shared by traits that are correlated. In this paper, we propose imputation methods based on a multi-task Gaussian process (MTGP) using self-measuring similarity kernels reflecting relationships among traits, genotypes, and environments. This framework allows us to use genetic correlation among multi-trait multi-environment data and also to combine MET data and marker genotype data. We compared the accuracy of three MTGP methods and iterative regularized PCA using rice MET data. Two scenarios for the generation of missing data at various missing rates were considered. The MTGP performed a better imputation accuracy than regularized PCA, especially at high missing rates. Under the 'uniform' scenario, in which missing data arise randomly, inclusion of marker genotype data in the imputation increased the imputation accuracy at high missing rates. Under the 'fiber' scenario, in which missing data arise in all traits for some combinations between genotypes and environments, the inclusion of marker genotype data decreased the imputation accuracy for most traits while increasing the accuracy in a few traits remarkably. The proposed methods will be useful for solving the missing data problem in MET data.

Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry.

PubMed

Kim, Sobin; Ulz, Michael E; Nguyen, Tuan; Li, Chi-Ming; Sato, Takaaki; Tycko, Benjamin; Ju, Jingyue

2004-05-01

A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.

Rapid Genotyping of Single Nucleotide Polymorphisms Influencing Warfarin Drug Response by Surface-Enhanced Laser Desorption and Ionization Time-of-Flight (SELDI-TOF) Mass Spectrometry

PubMed Central

Yang, Shangbin; Xu, LiHui; Wu, Haifeng M.

2010-01-01

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy. PMID:20075209

Rapid genotyping of single nucleotide polymorphisms influencing warfarin drug response by surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectrometry.

PubMed

Yang, Shangbin; Xu, LiHui; Wu, Haifeng M

2010-03-01

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy.

Improving genetic evaluation of litter size and piglet mortality for both genotyped and nongenotyped individuals using a single-step method.

PubMed

Guo, X; Christensen, O F; Ostersen, T; Wang, Y; Lund, M S; Su, G

2015-02-01

A single-step method allows genetic evaluation using information of phenotypes, pedigree, and markers from genotyped and nongenotyped individuals simultaneously. This paper compared genomic predictions obtained from a single-step BLUP (SSBLUP) method, a genomic BLUP (GBLUP) method, a selection index blending (SELIND) method, and a traditional pedigree-based method (BLUP) for total number of piglets born (TNB), litter size at d 5 after birth (LS5), and mortality rate before d 5 (Mort; including stillbirth) in Danish Landrace and Yorkshire pigs. Data sets of 778,095 litters from 309,362 Landrace sows and 472,001 litters from 190,760 Yorkshire sows were used for the analysis. There were 332,795 Landrace and 207,255 Yorkshire animals in the pedigree data, among which 3,445 Landrace pigs (1,366 boars and 2,079 sows) and 3,372 Yorkshire pigs (1,241 boars and 2,131 sows) were genotyped with the Illumina PorcineSNP60 BeadChip. The results showed that the 3 methods with marker information (SSBLUP, GBLUP, and SELIND) produced more accurate predictions for genotyped animals than the pedigree-based method. For genotyped animals, the average of reliabilities for all traits in both breeds using traditional BLUP was 0.091, which increased to 0.171 w+hen using GBLUP and to 0.179 when using SELIND and further increased to 0.209 when using SSBLUP. Furthermore, the average reliability of EBV for nongenotyped animals was increased from 0.091 for traditional BLUP to 0.105 for the SSBLUP. The results indicate that the SSBLUP is a good approach to practical genomic prediction of litter size and piglet mortality in Danish Landrace and Yorkshire populations.

TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces.

PubMed

Hansen, Heidi; Ben-David, Merav; McDonald, David B

2008-03-01

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method. © 2007 The Authors.

Development of an ELA-DRA gene typing method based on pyrosequencing technology.

PubMed

Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G

2008-11-01

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

Blood group genotyping: from patient to high-throughput donor screening.

PubMed

Veldhuisen, B; van der Schoot, C E; de Haas, M

2009-10-01

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

PubMed

Roy, Avijit; Ananthakrishnan, G; Hartung, John S; Brlansky, R H

2010-10-01

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Genome-wide comparisons of phylogenetic similarities between partial genomic regions and the full-length genome in Hepatitis E virus genotyping.

PubMed

Wang, Shuai; Wei, Wei; Luo, Xuenong; Cai, Xuepeng

2014-01-01

Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV) have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3'-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.

Impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott RealTime HCV Genotype II assay for hepatitis C genotyping.

PubMed

Sridhar, Siddharth; Yip, Cyril C Y; Chan, Jasper F W; To, Kelvin K W; Cheng, Vincent C C; Yuen, Kwok-Yung

2018-05-01

The Abbott RealTime HCV Genotype II assay (Abbott-RT-HCV assay) is a real-time PCR based genotyping method for hepatitis C virus (HCV). This study measured the impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott-RT-HCV assay. 517 samples were genotyped using the Abbott-RT-HCV assay over a one-year period, 34 (6.6%) were identified as HCV genotype 1 without further subtype designation raising the possibility of inaccurate genotyping. These samples were subjected to confirmatory sequencing. 27 of these 34 (79%) samples were genotype 1b while five (15%) were genotype 6. One HCV isolate was an inter-genotypic 1a/4o recombinant. This is a novel natural HCV recombinant that has never been reported. Inter-genotypic recombination and probe cross-reactivity can affect the accuracy of the Abbott-RT-HCV assay, both of which have significant implications on antiviral regimen choice. Confirmatory sequencing of ambiguous results is crucial for accurate genotyping. Copyright © 2018 Elsevier Inc. All rights reserved.

Use of Sequenom Sample ID Plus® SNP Genotyping in Identification of FFPE Tumor Samples

PubMed Central

Miller, Jessica K.; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M. K.; Pasternack, Danielle; Bristow, Robert G.; Fraser, Michael; Boutros, Paul C.; McPherson, John D.

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework. PMID:24551080

Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

PubMed

Miller, Jessica K; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M K; Pasternack, Danielle; Bristow, Robert G; Fraser, Michael; Boutros, Paul C; McPherson, John D

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice.

PubMed

Gerstner, Arpad; DeFord, James H; Papaconstantinou, John

2003-07-25

Ames dwarfism is caused by a homozygous single nucleotide mutation in the pituitary specific prop-1 gene, resulting in combined pituitary hormone deficiency, reduced growth and extended lifespan. Thus, these mice serve as an important model system for endocrinological, aging and longevity studies. Because the phenotype of wild type and heterozygous mice is undistinguishable, it is imperative for successful breeding to accurately genotype these animals. Here we report a novel, yet simple, approach for prop-1 genotyping using PCR-based allele-specific amplification (PCR-ASA). We also compare this method to other potential genotyping techniques, i.e. PCR-based restriction fragment length polymorphism analysis (PCR-RFLP) and fluorescence automated DNA sequencing. We demonstrate that the single-step PCR-ASA has several advantages over the classical PCR-RFLP because the procedure is simple, less expensive and rapid. To further increase the specificity and sensitivity of the PCR-ASA, we introduced a single-base mismatch at the 3' penultimate position of the mutant primer. Our results also reveal that the fluorescence automated DNA sequencing has limitations for detecting a single nucleotide polymorphism in the prop-1 gene, particularly in heterozygotes.

Application of IS1311 locus 2 PCR-REA assay for the specific detection of 'Bison type' Mycobacterium avium subspecies paratuberculosis isolates of Indian origin.

PubMed

Singh, Ajay Vir; Chauhan, Devendra Singh; Singh, Abhinendra; Singh, Pravin Kumar; Sohal, Jagdip Singh; Singh, Shoor Vir

2015-01-01

Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), 'Bison type' is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn's disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between 'Indian Bison type' and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to 'Bison type'. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as 'Bison type', 'Cattle type' and 'Sheep type', respectively. IS1311 L2 PCR-REA method showed different restriction profiles of 'Bison type' genotype as compared to non-Indian DNA samples. IS1311 L2 PCR-REA method successfully discriminated 'Indian Bison type' from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification.

PubMed

Park, Jung Hun; Jang, Hyowon; Jung, Yun Kyung; Jung, Ye Lim; Shin, Inkyung; Cho, Dae-Yeon; Park, Hyun Gyu

2017-05-15

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry. Copyright © 2016 Elsevier B.V. All rights reserved.

An efficient algorithm to compute marginal posterior genotype probabilities for every member of a pedigree with loops

PubMed Central

2009-01-01

Background Marginal posterior genotype probabilities need to be computed for genetic analyses such as geneticcounseling in humans and selective breeding in animal and plant species. Methods In this paper, we describe a peeling based, deterministic, exact algorithm to compute efficiently genotype probabilities for every member of a pedigree with loops without recourse to junction-tree methods from graph theory. The efficiency in computing the likelihood by peeling comes from storing intermediate results in multidimensional tables called cutsets. Computing marginal genotype probabilities for individual i requires recomputing the likelihood for each of the possible genotypes of individual i. This can be done efficiently by storing intermediate results in two types of cutsets called anterior and posterior cutsets and reusing these intermediate results to compute the likelihood. Examples A small example is used to illustrate the theoretical concepts discussed in this paper, and marginal genotype probabilities are computed at a monogenic disease locus for every member in a real cattle pedigree. PMID:19958551

Breeding value prediction for production traits in layer chickens using pedigree or genomic relationships in a reduced animal model.

PubMed

Wolc, Anna; Stricker, Chris; Arango, Jesus; Settar, Petek; Fulton, Janet E; O'Sullivan, Neil P; Preisinger, Rudolf; Habier, David; Fernando, Rohan; Garrick, Dorian J; Lamont, Susan J; Dekkers, Jack C M

2011-01-21

Genomic selection involves breeding value estimation of selection candidates based on high-density SNP genotypes. To quantify the potential benefit of genomic selection, accuracies of estimated breeding values (EBV) obtained with different methods using pedigree or high-density SNP genotypes were evaluated and compared in a commercial layer chicken breeding line. The following traits were analyzed: egg production, egg weight, egg color, shell strength, age at sexual maturity, body weight, albumen height, and yolk weight. Predictions appropriate for early or late selection were compared. A total of 2,708 birds were genotyped for 23,356 segregating SNP, including 1,563 females with records. Phenotypes on relatives without genotypes were incorporated in the analysis (in total 13,049 production records).The data were analyzed with a Reduced Animal Model using a relationship matrix based on pedigree data or on marker genotypes and with a Bayesian method using model averaging. Using a validation set that consisted of individuals from the generation following training, these methods were compared by correlating EBV with phenotypes corrected for fixed effects, selecting the top 30 individuals based on EBV and evaluating their mean phenotype, and by regressing phenotypes on EBV. Using high-density SNP genotypes increased accuracies of EBV up to two-fold for selection at an early age and by up to 88% for selection at a later age. Accuracy increases at an early age can be mostly attributed to improved estimates of parental EBV for shell quality and egg production, while for other egg quality traits it is mostly due to improved estimates of Mendelian sampling effects. A relatively small number of markers was sufficient to explain most of the genetic variation for egg weight and body weight.

Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009-2010.

PubMed

Shak, Emma B; France, Anne Marie; Cowan, Lauren; Starks, Angela M; Grant, Juliana

2015-01-01

Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009-December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups.

Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009–2010

PubMed Central

Shak, Emma B.; Cowan, Lauren; Starks, Angela M.; Grant, Juliana

2015-01-01

Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009–December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups. PMID:26556930

Development and evaluation of a sensitive enzyme-linked oligonucleotide-sorbent assay for detection of polymerase chain reaction-amplified hepatitis C virus of genotypes 1-6.

PubMed

Huang, Rong-Yuan; Chang, Hao-Teng; Lan, Chung-Yu; Pai, Tun-Wen; Wu, Chao-Nan; Ling, Chung-Mei; Chang, Margaret Dah-Tsyr

2008-08-01

A high-throughput polymerase chain reaction (PCR)-based enzyme-linked oligonucleotide-sorbent assay (ELOSA) was developed for use in the diagnostic testing of serum from patients who may be infected with different hepatitis C virus (HCV) genotypes. Twelve genotype-specific 5'-aminated DNA-coated probes were designed based on the variable 5'-untranslated region sequences of the HCV genotypes 1-6. Using 100 clinical serum samples, the performance of the PCR-ELOSA method was compared with Roche's COBAS Amplicor HCV Monitor V2.0 assay and the VERSANT HCV genotype assay (LiPA), and the overall agreement was 99% at the level of HCV genotypes with a detection range of 2.0 x 10(2) to 1.0 x 10(7)IU/ml for PCR-ELOSA. The PCR-ELOSA was more comprehensive as demonstrated by the fact that approximately 20% of the samples with different subtypes could be discriminated by this method but not by LiPA. In addition, the PCR-ELOSA system showed high accuracy (CV

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

2018-01-01

ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.

PubMed

Li, Ying; Jortani, Saeed A; Ramey-Hartung, Bronwyn; Hudson, Elizabeth; Lemieux, Bertrand; Kong, Huimin

2011-01-14

The response to the anticoagulant drug warfarin is greatly affected by genetic polymorphisms in the VKORC1 and CYP2C9 genes. Genotyping these polymorphisms has been shown to be important in reducing the time of the trial and error process for finding the maintenance dose of warfarin thus reducing the risk of adverse effects of the drug. We developed a real-time isothermal DNA amplification system for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. For each SNP, real-time isothermal Helicase Dependent Amplification (HDA) reactions were performed to amplify a DNA fragment containing the SNP. Amplicons were detected by fluorescently labeled allele specific probes during real-time HDA amplification. Fifty clinical samples were analyzed by the HDA-based method, generating a total of 150 results. Of these, 148 were consistent between the HDA-based assays and a reference method. The two samples with unresolved HDA-based test results were repeated and found to be consistent with the reference method. The HDA-based assays demonstrated a clinically acceptable performance for genotyping the VKORC1 -1639G>A SNP and two SNPs (430C>T and 1075A>C) for the CYP2C9 enzyme (CYP2C9*2 and CYP2C9*3), all of which are relevant in warfarin pharmacogenentics. Copyright © 2010 Elsevier B.V. All rights reserved.

High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

PubMed

Distefano, Gaetano; Caruso, Marco; La Malfa, Stefano; Gentile, Alessandra; Wu, Shu-Biao

2012-01-01

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.

Accurate continuous geographic assignment from low- to high-density SNP data.

PubMed

Guillot, Gilles; Jónsson, Hákon; Hinge, Antoine; Manchih, Nabil; Orlando, Ludovic

2016-04-01

Large-scale genotype datasets can help track the dispersal patterns of epidemiological outbreaks and predict the geographic origins of individuals. Such genetically-based geographic assignments also show a range of possible applications in forensics for profiling both victims and criminals, and in wildlife management, where poaching hotspot areas can be located. They, however, require fast and accurate statistical methods to handle the growing amount of genetic information made available from genotype arrays and next-generation sequencing technologies. We introduce a novel statistical method for geopositioning individuals of unknown origin from genotypes. Our method is based on a geostatistical model trained with a dataset of georeferenced genotypes. Statistical inference under this model can be implemented within the theoretical framework of Integrated Nested Laplace Approximation, which represents one of the major recent breakthroughs in statistics, as it does not require Monte Carlo simulations. We compare the performance of our method and an alternative method for geospatial inference, SPA in a simulation framework. We highlight the accuracy and limits of continuous spatial assignment methods at various scales by analyzing genotype datasets from a diversity of species, including Florida Scrub-jay birds Aphelocoma coerulescens, Arabidopsis thaliana and humans, representing 41-197,146 SNPs. Our method appears to be best suited for the analysis of medium-sized datasets (a few tens of thousands of loci), such as reduced-representation sequencing data that become increasingly available in ecology. http://www2.imm.dtu.dk/∼gigu/Spasiba/ gilles.b.guillot@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence.

PubMed

Drost, Derek R; Novaes, Evandro; Boaventura-Novaes, Carolina; Benedict, Catherine I; Brown, Ryan S; Yin, Tongming; Tuskan, Gerald A; Kirst, Matias

2009-06-01

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ-synthesized microarray probes. Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa. A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.

Hidden Markov Model-Based CNV Detection Algorithms for Illumina Genotyping Microarrays.

PubMed

Seiser, Eric L; Innocenti, Federico

2014-01-01

Somatic alterations in DNA copy number have been well studied in numerous malignancies, yet the role of germline DNA copy number variation in cancer is still emerging. Genotyping microarrays generate allele-specific signal intensities to determine genotype, but may also be used to infer DNA copy number using additional computational approaches. Numerous tools have been developed to analyze Illumina genotype microarray data for copy number variant (CNV) discovery, although commonly utilized algorithms freely available to the public employ approaches based upon the use of hidden Markov models (HMMs). QuantiSNP, PennCNV, and GenoCN utilize HMMs with six copy number states but vary in how transition and emission probabilities are calculated. Performance of these CNV detection algorithms has been shown to be variable between both genotyping platforms and data sets, although HMM approaches generally outperform other current methods. Low sensitivity is prevalent with HMM-based algorithms, suggesting the need for continued improvement in CNV detection methodologies.

Simultaneous Genotype Calling and Haplotype Phasing Improves Genotype Accuracy and Reduces False-Positive Associations for Genome-wide Association Studies

PubMed Central

Browning, Brian L.; Yu, Zhaoxia

2009-01-01

We present a novel method for simultaneous genotype calling and haplotype-phase inference. Our method employs the computationally efficient BEAGLE haplotype-frequency model, which can be applied to large-scale studies with millions of markers and thousands of samples. We compare genotype calls made with our method to genotype calls made with the BIRDSEED, CHIAMO, GenCall, and ILLUMINUS genotype-calling methods, using genotype data from the Illumina 550K and Affymetrix 500K arrays. We show that our method has higher genotype-call accuracy and yields fewer uncalled genotypes than competing methods. We perform single-marker analysis of data from the Wellcome Trust Case Control Consortium bipolar disorder and type 2 diabetes studies. For bipolar disorder, the genotype calls in the original study yield 25 markers with apparent false-positive association with bipolar disorder at a p < 10−7 significance level, whereas genotype calls made with our method yield no associated markers at this significance threshold. Conversely, for markers with replicated association with type 2 diabetes, there is good concordance between genotype calls used in the original study and calls made by our method. Results from single-marker and haplotypic analysis of our method's genotype calls for the bipolar disorder study indicate that our method is highly effective at eliminating genotyping artifacts that cause false-positive associations in genome-wide association studies. Our new genotype-calling methods are implemented in the BEAGLE and BEAGLECALL software packages. PMID:19931040

An outbreak of penicillin resistant Streptococcus pneumoniae investigated by a polymerase chain reaction based genotyping method.

PubMed Central

Gillespie, S H; McHugh, T D; Hughes, J E; Dickens, A; Kyi, M S; Kelsey, M

1997-01-01

AIMS: To characterise the genotypes of penicillin resistant Streptococcus pneumoniae infecting patients in a care of the elderly ward and to study its transmission in a hospital environment. METHODS: Isolates of S pneumoniae were cultured from specimens obtained from patients who had been admitted to a care of the elderly ward where an outbreak had occurred. Penicillin resistant S pneumoniae were also obtained from a series of surveillance throat swabs taken from patients in the same ward. In addition, all penicillin resistant S pneumoniae isolated from specimens submitted for culture at the time of the outbreak were included. Four sensitive strains isolated from a routine microbiology laboratory were included as controls. A simple polymerase chain reaction (PCR) based genotyping method for the penicillin binding protein (PBP) genes 1a, 2x, and 2b was used to characterise the genotypes. RESULTS: Nine patients were infected with serotype 9 S pneumoniae. Four of these patients died; two deaths were directly attributable to the infection. Tested against a battery of haemolytic streptococci and other organisms found in the respiratory tract, only two false positive reactions for PBP 2x were found among S mitis. The method demonstrated that the outbreak strain had altered PBP 1a, 2b, and 2x genes, a pattern clearly distinguishable from other penicillin resistant strains isolated at the same time. CONCLUSIONS: This method is simple to perform and would enable many laboratories to characterise the genotype of penicillin resistant S pneumoniae and investigate transmission in their hospitals. Images PMID:9462268

A novel dNTP-limited PCR and HRM assay to detect Williams-Beuren syndrome.

PubMed

Zhang, Lichen; Zhang, Xiaoqing; You, Guoling; Yu, Yongguo; Fu, Qihua

2018-06-01

Williams-Beuren syndrome (WBS) is caused by a microdeletion of chromosome arm 7q11.23. A rapid and inexpensive genotyping method to detect microdeletion on 7q11.23 needs to be developed for the diagnosis of WBS. This study describes the development of a new type of molecular diagnosis method to detect microdeletion on 7q11.23 based upon high-resolution melting (HRM). Four genes on 7q11.23 were selected as the target genes for the deletion genotyping. dNTP-limited duplex PCR was used to amplify the reference gene, CFTR, and one of the four genes respectively on 7q11.23. An HRM assay was performed on the PCR products, and the height ratio of the negative derivative peaks between the target gene and reference gene was employed to analyze the copy number variation of the target region. A new genotyping method for detecting 7q11.23 deletion was developed based upon dNTP-limited PCR and HRM, which cost only 96 min. Samples from 15 WBS patients and 12 healthy individuals were genotyped by this method in a blinded fashion, and the sensitivity and specificity was 100% (95% CI, 0.80-1, and 95% CI, 0.75-1, respectively) which was proved by CytoScan HD array. The HRM assay we developed is an rapid, inexpensive, and highly accurate method for genotyping 7q11.23 deletion. It is potentially useful in the clinical diagnosis of WBS. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of case-only studies accounting for genotyping error.

PubMed

Cheng, K F

2007-03-01

The case-only design provides one approach to assess possible interactions between genetic and environmental factors. It has been shown that if these factors are conditionally independent, then a case-only analysis is not only valid but also very efficient. However, a drawback of the case-only approach is that its conclusions may be biased by genotyping errors. In this paper, our main aim is to propose a method for analysis of case-only studies when these errors occur. We show that the bias can be adjusted through the use of internal validation data, which are obtained by genotyping some sampled individuals twice. Our analysis is based on a simple and yet highly efficient conditional likelihood approach. Simulation studies considered in this paper confirm that the new method has acceptable performance under genotyping errors.

Genetic potential of black bean genotypes with predictable behaviors in multienvironment trials.

PubMed

Torga, P P; Melo, P G S; Pereira, H S; Faria, L C; Melo, L C

2016-10-24

The aim of this study was to evaluate the phenotypic stability and specific and broad adaptability of common black bean genotypes for the Central and Center-South regions of Brazil by using the Annicchiarico and AMMI (weighted average of absolute scores: WAAS, and weighted average of absolute scores and productivity: WAASP) methodologies. We carried out 69 trials, with 43 and 26 trials in the Central and Center-South regions, respectively. Thirteen genotypes were evaluated in a randomized block design with three replications, during the rainy, dry, and winter seasons in 2 years. To obtain estimates of specific adaptation, we analyzed the parameters for each method obtained in the two geographic regions separately. To estimate broad adaptation, we used the average of the parameters obtained from each region. The lines identified with high specific adaptation in each region were not the same based on the Annicchiarico and AMMI (WAAS) methodologies. It was not possible to identify the same genotypes with specific or broad stability by using these methods. By contrast, the Annicchiarico and AMMI (WAASP) methods presented very similar estimates of broad and specific adaptation. Based on these methods, the lines with more specific adaptation were CNFP 8000 and CNFP 7994, in the Central and Center-South regions, respectively, of which the CNFP 8000 line was more widely adapted.

Investigation of inversion polymorphisms in the human genome using principal components analysis.

PubMed

Ma, Jianzhong; Amos, Christopher I

2012-01-01

Despite the significant advances made over the last few years in mapping inversions with the advent of paired-end sequencing approaches, our understanding of the prevalence and spectrum of inversions in the human genome has lagged behind other types of structural variants, mainly due to the lack of a cost-efficient method applicable to large-scale samples. We propose a novel method based on principal components analysis (PCA) to characterize inversion polymorphisms using high-density SNP genotype data. Our method applies to non-recurrent inversions for which recombination between the inverted and non-inverted segments in inversion heterozygotes is suppressed due to the loss of unbalanced gametes. Inside such an inversion region, an effect similar to population substructure is thus created: two distinct "populations" of inversion homozygotes of different orientations and their 1:1 admixture, namely the inversion heterozygotes. This kind of substructure can be readily detected by performing PCA locally in the inversion regions. Using simulations, we demonstrated that the proposed method can be used to detect and genotype inversion polymorphisms using unphased genotype data. We applied our method to the phase III HapMap data and inferred the inversion genotypes of known inversion polymorphisms at 8p23.1 and 17q21.31. These inversion genotypes were validated by comparing with literature results and by checking Mendelian consistency using the family data whenever available. Based on the PCA-approach, we also performed a preliminary genome-wide scan for inversions using the HapMap data, which resulted in 2040 candidate inversions, 169 of which overlapped with previously reported inversions. Our method can be readily applied to the abundant SNP data, and is expected to play an important role in developing human genome maps of inversions and exploring associations between inversions and susceptibility of diseases.

Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes

PubMed Central

2013-01-01

Background Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system. Findings Shiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB. Conclusions We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly. PMID:23915543

SparRec: An effective matrix completion framework of missing data imputation for GWAS

NASA Astrophysics Data System (ADS)

Jiang, Bo; Ma, Shiqian; Causey, Jason; Qiao, Linbo; Hardin, Matthew Price; Bitts, Ian; Johnson, Daniel; Zhang, Shuzhong; Huang, Xiuzhen

2016-10-01

Genome-wide association studies present computational challenges for missing data imputation, while the advances of genotype technologies are generating datasets of large sample sizes with sample sets genotyped on multiple SNP chips. We present a new framework SparRec (Sparse Recovery) for imputation, with the following properties: (1) The optimization models of SparRec, based on low-rank and low number of co-clusters of matrices, are different from current statistics methods. While our low-rank matrix completion (LRMC) model is similar to Mendel-Impute, our matrix co-clustering factorization (MCCF) model is completely new. (2) SparRec, as other matrix completion methods, is flexible to be applied to missing data imputation for large meta-analysis with different cohorts genotyped on different sets of SNPs, even when there is no reference panel. This kind of meta-analysis is very challenging for current statistics based methods. (3) SparRec has consistent performance and achieves high recovery accuracy even when the missing data rate is as high as 90%. Compared with Mendel-Impute, our low-rank based method achieves similar accuracy and efficiency, while the co-clustering based method has advantages in running time. The testing results show that SparRec has significant advantages and competitive performance over other state-of-the-art existing statistics methods including Beagle and fastPhase.

Minimum number of measurements for evaluating Bertholletia excelsa.

PubMed

Baldoni, A B; Tonini, H; Tardin, F D; Botelho, S C C; Teodoro, P E

2017-09-27

Repeatability studies on fruit species are of great importance to identify the minimum number of measurements necessary to accurately select superior genotypes. This study aimed to identify the most efficient method to estimate the repeatability coefficient (r) and predict the minimum number of measurements needed for a more accurate evaluation of Brazil nut tree (Bertholletia excelsa) genotypes based on fruit yield. For this, we assessed the number of fruits and dry mass of seeds of 75 Brazil nut genotypes, from native forest, located in the municipality of Itaúba, MT, for 5 years. To better estimate r, four procedures were used: analysis of variance (ANOVA), principal component analysis based on the correlation matrix (CPCOR), principal component analysis based on the phenotypic variance and covariance matrix (CPCOV), and structural analysis based on the correlation matrix (mean r - AECOR). There was a significant effect of genotypes and measurements, which reveals the need to study the minimum number of measurements for selecting superior Brazil nut genotypes for a production increase. Estimates of r by ANOVA were lower than those observed with the principal component methodology and close to AECOR. The CPCOV methodology provided the highest estimate of r, which resulted in a lower number of measurements needed to identify superior Brazil nut genotypes for the number of fruits and dry mass of seeds. Based on this methodology, three measurements are necessary to predict the true value of the Brazil nut genotypes with a minimum accuracy of 85%.

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

PubMed

Riehm, Julia M; Projahn, Michaela; Vogler, Amy J; Rajerison, Minoaerisoa; Andersen, Genevieve; Hall, Carina M; Zimmermann, Thomas; Soanandrasana, Rahelinirina; Andrianaivoarimanana, Voahangy; Straubinger, Reinhard K; Nottingham, Roxanne; Keim, Paul; Wagner, David M; Scholz, Holger C

2015-06-01

Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

Setup of a Protocol of Molecular Diagnosis of β-Thalassemia Mutations in Tunisia using Denaturing High-Performance Liquid Chromatography (DHPLC).

PubMed

Sahli, Chaima Abdelhafidh; Ben Salem, Ikbel; Jouini, Latifa; Laouini, Naouel; Dabboubi, Rym; Hadj Fredj, Sondes; Siala, Hajer; Othmeni, Rym; Dakhlaoui, Boutheina; Fattoum, Slaheddine; Bibi, Amina; Messaoud, Taieb

2016-09-01

β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population. DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals. © 2016 Wiley Periodicals, Inc.

Similar predictions of etravirine sensitivity regardless of genotypic testing method used: comparison of available scoring systems.

PubMed

Vingerhoets, Johan; Nijs, Steven; Tambuyzer, Lotke; Hoogstoel, Annemie; Anderson, David; Picchio, Gaston

2012-01-01

The aims of this study were to compare various genotypic scoring systems commonly used to predict virological outcome to etravirine, and examine their concordance with etravirine phenotypic susceptibility. Six etravirine genotypic scoring systems were assessed: Tibotec 2010 (based on 20 mutations; TBT 20), Monogram, Stanford HIVdb, ANRS, Rega (based on 37, 30, 27 and 49 mutations, respectively) and virco(®)TYPE HIV-1 (predicted fold change based on genotype). Samples from treatment-experienced patients who participated in the DUET trials and with both genotypic and phenotypic data (n=403) were assessed using each scoring system. Results were retrospectively correlated with virological response in DUET. κ coefficients were calculated to estimate the degree of correlation between the different scoring systems. Correlation between the five scoring systems and the TBT 20 system was approximately 90%. Virological response by etravirine susceptibility was comparable regardless of which scoring system was utilized, with 70-74% of DUET patients determined as susceptible to etravirine by the different scoring systems achieving plasma viral load <50 HIV-1 RNA copies/ml. In samples classed as phenotypically susceptible to etravirine (fold change in 50% effective concentration ≤3), correlations with genotypic score were consistently high across scoring systems (≥70%). In general, the etravirine genotypic scoring systems produced similar results, and genotype-phenotype concordance was high. As such, phenotypic interpretations, and in their absence all genotypic scoring systems investigated, may be used to reliably predict the activity of etravirine.

A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms

PubMed Central

Blondal, Thorarinn; Waage, Benedikt G.; Smarason, Sigurdur V.; Jonsson, Frosti; Fjalldal, Sigridur B.; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V.

2003-01-01

A new MALDI–TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3′-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation

USDA-ARS?s Scientific Manuscript database

Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker d...

A video method to study Drosophila sleep.

PubMed

Zimmerman, John E; Raizen, David M; Maycock, Matthew H; Maislin, Greg; Pack, Allan I

2008-11-01

To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements. A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images. The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation. Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep.

Characterization of six human disease-associated inversion polymorphisms.

PubMed

Antonacci, Francesca; Kidd, Jeffrey M; Marques-Bonet, Tomas; Ventura, Mario; Siswara, Priscillia; Jiang, Zhaoshi; Eichler, Evan E

2009-07-15

The human genome is a highly dynamic structure that shows a wide range of genetic polymorphic variation. Unlike other types of structural variation, little is known about inversion variants within normal individuals because such events are typically balanced and are difficult to detect and analyze by standard molecular approaches. Using sequence-based, cytogenetic and genotyping approaches, we characterized six large inversion polymorphisms that map to regions associated with genomic disorders with complex segmental duplications mapping at the breakpoints. We developed a metaphase FISH-based assay to genotype inversions and analyzed the chromosomes of 27 individuals from three HapMap populations. In this subset, we find that these inversions are less frequent or absent in Asians when compared with European and Yoruban populations. Analyzing multiple individuals from outgroup species of great apes, we show that most of these large inversion polymorphisms are specific to the human lineage with two exceptions, 17q21.31 and 8p23 inversions, which are found to be similarly polymorphic in other great ape species and where the inverted allele represents the ancestral state. Investigating linkage disequilibrium relationships with genotyped SNPs, we provide evidence that most of these inversions appear to have arisen on at least two different haplotype backgrounds. In these cases, discovery and genotyping methods based on SNPs may be confounded and molecular cytogenetics remains the only method to genotype these inversions.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

PubMed

Gomgnimbou, Michel Kiréopori; Abadia, Edgar; Zhang, Jian; Refrégier, Guislaine; Panaiotov, Stefan; Bachiyska, Elizabeta; Sola, Christophe

2012-10-01

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Distribution of Diego blood group alleles and identification of four novel mutations on exon 19 of SLC4A1 gene in the Chinese Han population by polymerase chain reaction sequence-based typing.

PubMed

Xu, X G; He, J; He, Y M; Tao, S D; Ying, Y L; Zhu, F M; Lv, H J; Yan, L X

2011-04-01

The Diego blood group system plays an important role in transfusion medicine. Genotyping of DI1 and DI2 alleles is helpful for the investigation into haemolytic disease of the newborn (HDN) and for the development of rare blood group databases. Here, we set up a polymerase chain reaction sequence-based typing (PCR-SBT) method for genotyping of Diego blood group alleles. Specific primers for exon 19 of the solute carrier family 4, anion exchanger, member1 (SLC4A1) gene were designed, and our PCR-SBT method was established and optimized for Diego genotyping. A total of 1053 samples from the Chinese Han population and the family members of a rare proband with DI1/DI1 genotype were investigated by the PCR-SBT method. An allele-specific primer PCR (PCR-ASP) was used to verify the reliability of the PCR-SBT method. The frequencies of DI1 and DI2 alleles in the Chinese Han population were 0.0247 and 0.9753, respectively. Six new single nucleotide polymorphisms (SNPs) were found in the sequenced regions of the SLC4A1 gene, and four novel SNPs located in the exon 19, in which one SNP could cause an amino acid alteration of Ala858Ser on erythrocyte anion exchanger protein 1. The genotypes for Diego blood group were identical among 41 selected samples with PCR-ASP and PCR-SBT. The PCR-SBT method can be used in Diego genotyping as a substitute of serological technique when the antisera is lacking and was suitable for screening large numbers of donors in rare blood group databases. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem.

PubMed

Saracevic, Andrea; Simundic, Ana-Maria; Celap, Ivana; Luzanic, Valentina

2013-07-01

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.

Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

PubMed Central

Tinker, Nicholas A.; Bekele, Wubishet A.; Hattori, Jiro

2016-01-01

Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species. PMID:26818073

Theory‐based pharmacokinetics and pharmacodynamics of S‐ and R‐warfarin and effects on international normalized ratio: influence of body size, composition and genotype in cardiac surgery patients

PubMed Central

Xue, Ling; Holford, Nick; Ding, Xiao‐liang; Shen, Zhen‐ya; Huang, Chen‐rong; Zhang, Hua; Zhang, Jing‐jing; Guo, Zhe‐ning; Xie, Cheng; Zhou, Ling; Chen, Zhi‐yao; Liu, Lin‐sheng

2016-01-01

Aims The aims of this study are to apply a theory‐based mechanistic model to describe the pharmacokinetics (PK) and pharmacodynamics (PD) of S‐ and R‐warfarin. Methods Clinical data were obtained from 264 patients. Total concentrations for S‐ and R‐warfarin were measured by ultra‐high performance liquid tandem mass spectrometry. Genotypes were measured using pyrosequencing. A sequential population PK parameter with data method was used to describe the international normalized ratio (INR) time course. Data were analyzed with NONMEM. Model evaluation was based on parameter plausibility and prediction‐corrected visual predictive checks. Results Warfarin PK was described using a one‐compartment model. CYP2C9 *1/*3 genotype had reduced clearance for S‐warfarin, but increased clearance for R‐warfarin. The in vitro parameters for the relationship between prothrombin complex activity (PCA) and INR were markedly different (A = 0.560, B = 0.386) from the theory‐based values (A = 1, B = 0). There was a small difference between healthy subjects and patients. A sigmoid Emax PD model inhibiting PCA synthesis as a function of S‐warfarin concentration predicted INR. Small R‐warfarin effects was described by competitive antagonism of S‐warfarin inhibition. Patients with VKORC1 AA and CYP4F2 CC or CT genotypes had lower C50 for S‐warfarin. Conclusion A theory‐based PKPD model describes warfarin concentrations and clinical response. Expected PK and PD genotype effects were confirmed. The role of predicted fat free mass with theory‐based allometric scaling of PK parameters was identified. R‐warfarin had a minor effect compared with S‐warfarin on PCA synthesis. INR is predictable from 1/PCA in vivo. PMID:27763679

Ultra-low-density genotype panels for breed assignment of Angus and Hereford cattle.

PubMed

Judge, M M; Kelleher, M M; Kearney, J F; Sleator, R D; Berry, D P

2017-06-01

Angus and Hereford beef is marketed internationally for apparent superior meat quality attributes; DNA-based breed authenticity could be a useful instrument to ensure consumer confidence on premium meat products. The objective of this study was to develop an ultra-low-density genotype panel to accurately quantify the Angus and Hereford breed proportion in biological samples. Medium-density genotypes (13 306 single nucleotide polymorphisms (SNPs)) were available on 54 703 commercial and 4042 purebred animals. The breed proportion of the commercial animals was generated from the medium-density genotypes and this estimate was regarded as the gold-standard breed composition. Ten genotype panels (100 to 1000 SNPs) were developed from the medium-density genotypes; five methods were used to identify the most informative SNPs and these included the Delta statistic, the fixation (F st) statistic and an index of both. Breed assignment analyses were undertaken for each breed, panel density and SNP selection method separately with a programme to infer population structure using the entire 13 306 SNP panel (representing the gold-standard measure). Breed assignment was undertaken for all commercial animals (n=54 703), animals deemed to contain some proportion of Angus based on pedigree (n=5740) and animals deemed to contain some proportion of Hereford based on pedigree (n=5187). The predicted breed proportion of all animals from the lower density panels was then compared with the gold-standard breed prediction. Panel density, SNP selection method and breed all had a significant effect on the correlation of predicted and actual breed proportion. Regardless of breed, the Index method of SNP selection numerically (but not significantly) outperformed all other selection methods in accuracy (i.e. correlation and root mean square of prediction) when panel density was ⩾300 SNPs. The correlation between actual and predicted breed proportion increased as panel density increased. Using 300 SNPs (selected using the global index method), the correlation between predicted and actual breed proportion was 0.993 and 0.995 in the Angus and Hereford validation populations, respectively. When SNP panels optimised for breed prediction in one population were used to predict the breed proportion of a separate population, the correlation between predicted and actual breed proportion was 0.034 and 0.044 weaker in the Hereford and Angus populations, respectively (using the 300 SNP panel). It is necessary to include at least 300 to 400 SNPs (per breed) on genotype panels to accurately predict breed proportion from biological samples.

A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.

PubMed

Côté, Nathalie M L; Daligault, Julien; Pruvost, Mélanie; Bennett, E Andrew; Gorgé, Olivier; Guimaraes, Silvia; Capelli, Nicolas; Le Bailly, Matthieu; Geigl, Eva-Maria; Grange, Thierry

2016-01-01

Human gastrointestinal parasites are good indicators for hygienic conditions and health status of past and present individuals and communities. While microscopic analysis of eggs in sediments of archeological sites often allows their taxonomic identification, this method is rarely effective at the species level, and requires both the survival of intact eggs and their proper identification. Genotyping via PCR-based approaches has the potential to achieve a precise species-level taxonomic determination. However, so far it has mostly been applied to individual eggs isolated from archeological samples. To increase the throughput and taxonomic accuracy, as well as reduce costs of genotyping methods, we adapted a PCR-based approach coupled with next-generation sequencing to perform precise taxonomic identification of parasitic helminths directly from archeological sediments. Our study of twenty-five 100 to 7,200 year-old archeological samples proved this to be a powerful, reliable and efficient approach for species determination even in the absence of preserved eggs, either as a stand-alone method or as a complement to microscopic studies.

Approaches for geospatial processing of field-based high-throughput plant phenomics data from ground vehicle platforms

USDA-ARS?s Scientific Manuscript database

Understanding the genetic basis of complex plant traits requires connecting genotype to phenotype information, known as the “G2P question.” In the last three decades, genotyping methods have become highly developed. Much less innovation has occurred for measuring plant traits (phenotyping), particul...

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts

EPA Science Inventory

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels o...

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: genetic markers and characterization of cellulolytic potential.

PubMed

Koeck, Daniela E; Zverlov, Vladimir V; Liebl, Wolfgang; Schwarz, Wolfgang H

2014-07-01

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters. Copyright © 2014 Elsevier GmbH. All rights reserved.

Simultaneous selection for cowpea (Vigna unguiculata L.) genotypes with adaptability and yield stability using mixed models.

PubMed

Torres, F E; Teodoro, P E; Rodrigues, E V; Santos, A; Corrêa, A M; Ceccon, G

2016-04-29

The aim of this study was to select erect cowpea (Vigna unguiculata L.) genotypes simultaneously for high adaptability, stability, and yield grain in Mato Grosso do Sul, Brazil using mixed models. We conducted six trials of different cowpea genotypes in 2005 and 2006 in Aquidauana, Chapadão do Sul, Dourados, and Primavera do Leste. The experimental design was randomized complete blocks with four replications and 20 genotypes. Genetic parameters were estimated by restricted maximum likelihood/best linear unbiased prediction, and selection was based on the harmonic mean of the relative performance of genetic values method using three strategies: selection based on the predicted breeding value, having considered the performance mean of the genotypes in all environments (no interaction effect); the performance in each environment (with an interaction effect); and the simultaneous selection for grain yield, stability, and adaptability. The MNC99542F-5 and MNC99-537F-4 genotypes could be grown in various environments, as they exhibited high grain yield, adaptability, and stability. The average heritability of the genotypes was moderate to high and the selective accuracy was 82%, indicating an excellent potential for selection.

Selection of core animals in the Algorithm for Proven and Young using a simulation model.

PubMed

Bradford, H L; Pocrnić, I; Fragomeni, B O; Lourenco, D A L; Misztal, I

2017-12-01

The Algorithm for Proven and Young (APY) enables the implementation of single-step genomic BLUP (ssGBLUP) in large, genotyped populations by separating genotyped animals into core and non-core subsets and creating a computationally efficient inverse for the genomic relationship matrix (G). As APY became the choice for large-scale genomic evaluations in BLUP-based methods, a common question is how to choose the animals in the core subset. We compared several core definitions to answer this question. Simulations comprised a moderately heritable trait for 95,010 animals and 50,000 genotypes for animals across five generations. Genotypes consisted of 25,500 SNP distributed across 15 chromosomes. Genotyping errors and missing pedigree were also mimicked. Core animals were defined based on individual generations, equal representation across generations, and at random. For a sufficiently large core size, core definitions had the same accuracies and biases, even if the core animals had imperfect genotypes. When genotyped animals had unknown parents, accuracy and bias were significantly better (p ≤ .05) for random and across generation core definitions. © 2017 The Authors. Journal of Animal Breeding and Genetics Published by Blackwell Verlag GmbH.

Association of polymorphisms of exon 2 of the growth hormone gene with production performance in Huoyan goose.

PubMed

Zhang, Yang; Zhu, Zhen; Xu, Qi; Chen, Guohong

2014-01-07

Primers based on the cDNA sequence of the goose growth hormone (GH) gene in GenBank were designed to amplify exon 2 of the GH gene in Huoyan goose. A total of 552 individuals were brooded in one batch and raised in Liaoning and Jiangsu Provinces, China. Single nucleotide polymorphisms (SNPs) of exon 2 in the GH gene were detected by the polymerase chain reaction (single strand conformation polymorphism method). Homozygotes were subsequently cloned, sequenced and analyzed. Two SNP mutations were detected, and 10 genotypes (referred to as AA, BB, CC, DD, AB, AC, AD, BC, BD and CD) were obtained. Allele D was predominant, and the frequencies of the 10 genotypes fit the Hardy-Weinberg equilibrium in the male, female and whole populations according to the chi-square test. Based on SNP types, the 10 genotypes were combined into three main genotypes. Multiple comparisons were carried out between different genotypes and production traits when the geese were 10 weeks old. Some indices of production performance were significantly (p < 0.05) associated with the genotype. Particularly, geese with genotype AB or BB were highly productive. Thus, these genotypes may serve as selection markers for production traits in Huoyan geese.

Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label.

PubMed

Liu, Ke; Pan, Dun; Wen, Yanqin; Zhang, Honglu; Chao, Jie; Wang, Lihua; Song, Shiping; Fan, Chunhai; Shi, Yongyong

2018-02-01

The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high-efficiency hybridizable DNA origami-based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof-of-concept, five genotype B and six genotype C are detected in 11 HBV-infected patients' blood DNA samples of Han Chinese population in the single-blinded test. The AFM image-based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relatedness of Indian flax genotypes (Linum usitatissimum L.): an inter-simple sequence repeat (ISSR) primer assay.

PubMed

Rajwade, Ashwini V; Arora, Ritu S; Kadoo, Narendra Y; Harsulkar, Abhay M; Ghorpade, Prakash B; Gupta, Vidya S

2010-06-01

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03-0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard's similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.

Using Genotype Abundance to Improve Phylogenetic Inference

PubMed Central

Mesin, Luka; Victora, Gabriel D; Minin, Vladimir N; Matsen, Frederick A

2018-01-01

Abstract Modern biological techniques enable very dense genetic sampling of unfolding evolutionary histories, and thus frequently sample some genotypes multiple times. This motivates strategies to incorporate genotype abundance information in phylogenetic inference. In this article, we synthesize a stochastic process model with standard sequence-based phylogenetic optimality, and show that tree estimation is substantially improved by doing so. Our method is validated with extensive simulations and an experimental single-cell lineage tracing study of germinal center B cell receptor affinity maturation. PMID:29474671

Identity-by-Descent-Based Phasing and Imputation in Founder Populations Using Graphical Models

PubMed Central

Palin, Kimmo; Campbell, Harry; Wright, Alan F; Wilson, James F; Durbin, Richard

2011-01-01

Accurate knowledge of haplotypes, the combination of alleles co-residing on a single copy of a chromosome, enables powerful gene mapping and sequence imputation methods. Since humans are diploid, haplotypes must be derived from genotypes by a phasing process. In this study, we present a new computational model for haplotype phasing based on pairwise sharing of haplotypes inferred to be Identical-By-Descent (IBD). We apply the Bayesian network based model in a new phasing algorithm, called systematic long-range phasing (SLRP), that can capitalize on the close genetic relationships in isolated founder populations, and show with simulated and real genome-wide genotype data that SLRP substantially reduces the rate of phasing errors compared to previous phasing algorithms. Furthermore, the method accurately identifies regions of IBD, enabling linkage-like studies without pedigrees, and can be used to impute most genotypes with very low error rate. Genet. Epidemiol. 2011. © 2011 Wiley Periodicals, Inc.35:853-860, 2011 PMID:22006673

A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

PubMed

Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

2016-10-01

Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

QuASAR: quantitative allele-specific analysis of reads

PubMed Central

Harvey, Chris T.; Moyerbrailean, Gregory A.; Davis, Gordon O.; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

2015-01-01

Motivation: Expression quantitative trait loci (eQTL) studies have discovered thousands of genetic variants that regulate gene expression, enabling a better understanding of the functional role of non-coding sequences. However, eQTL studies are costly, requiring large sample sizes and genome-wide genotyping of each sample. In contrast, analysis of allele-specific expression (ASE) is becoming a popular approach to detect the effect of genetic variation on gene expression, even within a single individual. This is typically achieved by counting the number of RNA-seq reads matching each allele at heterozygous sites and testing the null hypothesis of a 1:1 allelic ratio. In principle, when genotype information is not readily available, it could be inferred from the RNA-seq reads directly. However, there are currently no existing methods that jointly infer genotypes and conduct ASE inference, while considering uncertainty in the genotype calls. Results: We present QuASAR, quantitative allele-specific analysis of reads, a novel statistical learning method for jointly detecting heterozygous genotypes and inferring ASE. The proposed ASE inference step takes into consideration the uncertainty in the genotype calls, while including parameters that model base-call errors in sequencing and allelic over-dispersion. We validated our method with experimental data for which high-quality genotypes are available. Results for an additional dataset with multiple replicates at different sequencing depths demonstrate that QuASAR is a powerful tool for ASE analysis when genotypes are not available. Availability and implementation: http://github.com/piquelab/QuASAR. Contact: fluca@wayne.edu or rpique@wayne.edu Supplementary information: Supplementary Material is available at Bioinformatics online. PMID:25480375

A novel environmental DNA approach to quantify the cryptic invasion of non-native genotypes.

PubMed

Uchii, Kimiko; Doi, Hideyuki; Minamoto, Toshifumi

2016-03-01

The invasion of non-native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non-native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA-based approach to quantitatively monitor the invasion of non-native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non-native DNA based on a single-nucleotide polymorphism using cycling probe technology in real-time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non-native DNA in the water was well correlated to the biomass ratio of native and non-native genotypes. This method provided quantitative estimates for the proportion of native and non-native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non-native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non-native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation. © 2015 John Wiley & Sons Ltd.

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.

PubMed

Galan, Maxime; Guivier, Emmanuel; Caraux, Gilles; Charbonnel, Nathalie; Cosson, Jean-François

2010-05-11

High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.

[Hepatitis B virus genotype E infection in Turkey: the detection of the first case].

PubMed

Sayan, Murat; Sanlıdağ, Tamer; Akçalı, Sinem; Arıkan, Ayşe

2014-10-01

Hepatitis B virus (HBV) infection is a global major health problem. Currently, 10 genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of the previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey. However, recent studies indicated that other HBV genotypes are also seen with an increasing rate. Although epidemiological and clinical information on genotype E infection is currently limited, it is known that genotype E infection is common in West and Central Africa. In this report, the first case of HBV genotype E infection in Turkey was presented. A 22-year-old Nigerian male employee who resided in Manisa for five years was admitted to Celal Bayar University Hospital Manisa, Turkey, for his routine check-up. Since HBsAg was found positive, other HBV markers were tested with a repeated serum sample. Laboratory findings were as follows; HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc (+), anti-HCV (-), anti-HIV (-), ALT: 44 U/L and AST: 45 U/L. HBV-DNA level was detected as 700 IU/ml by real-time PCR (Artus HBV QS RGQ Qiagen, Germany). HBV-DNA isolated from the serum sample of the patient was amplified by PCR and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis and Inno-LIPA HBV genotyping method (Innogenetics, Belgium) was performed to determine multiple HBV genotype infection. On the basis of those methods the genotype of the virus was identified as genotype E. The partial sequences of the HBV polymerase gene were loaded to the international DNA data bank (GenBank) for contribution to the global HBV surveillance. This report emphasized that besides genotype D the other HBV genotypes could be found in Turkey. Since the patient was an inactive HBsAg carrier before his residence in Turkey, this case was regarded as an imported HBV genotype E case. In conclusion, detection of different HBV genotypes, their epidemiology and molecular characteristics are important for both national and global HBV surveillance and better clinical approach.

Evaluation of the Epidemiological Relevance of Variable-Number Tandem-Repeat Genotyping of Mycobacterium bovis and Comparison of the Method with IS6110 Restriction Fragment Length Polymorphism Analysis and Spoligotyping†

PubMed Central

Allix, Caroline; Walravens, Karl; Saegerman, Claude; Godfroid, Jacques; Supply, Philip; Fauville-Dufaux, Maryse

2006-01-01

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, with isolates taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen. PMID:16757584

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

Genotype Imputation with Millions of Reference Samples

PubMed Central

Browning, Brian L.; Browning, Sharon R.

2016-01-01

We present a genotype imputation method that scales to millions of reference samples. The imputation method, based on the Li and Stephens model and implemented in Beagle v.4.1, is parallelized and memory efficient, making it well suited to multi-core computer processors. It achieves fast, accurate, and memory-efficient genotype imputation by restricting the probability model to markers that are genotyped in the target samples and by performing linear interpolation to impute ungenotyped variants. We compare Beagle v.4.1 with Impute2 and Minimac3 by using 1000 Genomes Project data, UK10K Project data, and simulated data. All three methods have similar accuracy but different memory requirements and different computation times. When imputing 10 Mb of sequence data from 50,000 reference samples, Beagle’s throughput was more than 100× greater than Impute2’s throughput on our computer servers. When imputing 10 Mb of sequence data from 200,000 reference samples in VCF format, Minimac3 consumed 26× more memory per computational thread and 15× more CPU time than Beagle. We demonstrate that Beagle v.4.1 scales to much larger reference panels by performing imputation from a simulated reference panel having 5 million samples and a mean marker density of one marker per four base pairs. PMID:26748515

Simultaneous Cocirculation of Both European Varicella-Zoster Virus Genotypes (E1 and E2) in Mexico City▿

PubMed Central

Rodríguez-Castillo, Araceli; Vaughan, Gilberto; Ramírez-González, José Ernesto; Escobar-Gutiérrez, Alejandro

2010-01-01

Full-length genome analysis of varicella-zoster virus (VZV) has shown that viral strains can be classified into seven different genotypes: European (E), Mosaic (M), and Japanese (J), and the E and M genotypes can be further subclassified into E1, E2, and M1 through 4, respectively. The distribution of the main VZV genotypes in Mexico was described earlier, demonstrating the predominance of E genotype, although other genotypes (M1 and M4) were also identified. However, no information regarding the circulation of either E genotype in the country is available. In the present study, we confirm the presence of both E1 and E2 genotypes in the country and explore the possibility of coinfection as the triggering factor for increased virulence among severe cases. A total of 61 different European VZV isolates collected in the Mexico City metropolitan area from 2005 to 2006 were typed by using a PCR method based on genotype-specific primer amplification. Fifty isolates belonged to the E1 genotype, and the eleven remaining samples were classified as E2 genotypes. No coinfection with both E genotypes was identified among these specimens. We provide here new information on the distribution of VZV genotypes circulating in Mexico City. PMID:20220168

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization

PubMed Central

Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael

2017-01-01

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution. PMID:28045981

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

PubMed

Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael; Ambur, Ole Herman

2017-01-01

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

ALCHEMY: a reliable method for automated SNP genotype calling for small batch sizes and highly homozygous populations

PubMed Central

Wright, Mark H.; Tung, Chih-Wei; Zhao, Keyan; Reynolds, Andy; McCouch, Susan R.; Bustamante, Carlos D.

2010-01-01

Motivation: The development of new high-throughput genotyping products requires a significant investment in testing and training samples to evaluate and optimize the product before it can be used reliably on new samples. One reason for this is current methods for automated calling of genotypes are based on clustering approaches which require a large number of samples to be analyzed simultaneously, or an extensive training dataset to seed clusters. In systems where inbred samples are of primary interest, current clustering approaches perform poorly due to the inability to clearly identify a heterozygote cluster. Results: As part of the development of two custom single nucleotide polymorphism genotyping products for Oryza sativa (domestic rice), we have developed a new genotype calling algorithm called ‘ALCHEMY’ based on statistical modeling of the raw intensity data rather than modelless clustering. A novel feature of the model is the ability to estimate and incorporate inbreeding information on a per sample basis allowing accurate genotyping of both inbred and heterozygous samples even when analyzed simultaneously. Since clustering is not used explicitly, ALCHEMY performs well on small sample sizes with accuracy exceeding 99% with as few as 18 samples. Availability: ALCHEMY is available for both commercial and academic use free of charge and distributed under the GNU General Public License at http://alchemy.sourceforge.net/ Contact: mhw6@cornell.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20926420

snpAD: An ancient DNA genotype caller.

PubMed

Prüfer, Kay

2018-06-21

The study of ancient genomes can elucidate the evolutionary past. However, analyses are complicated by base-modifications in ancient DNA molecules that result in errors in DNA sequences. These errors are particularly common near the ends of sequences and pose a challenge for genotype calling. I describe an iterative method that estimates genotype frequencies and errors along sequences to allow for accurate genotype calling from ancient sequences. The implementation of this method, called snpAD, performs well on high-coverage ancient data, as shown by simulations and by subsampling the data of a high-coverage Neandertal genome. Although estimates for low-coverage genomes are less accurate, I am able to derive approximate estimates of heterozygosity from several low-coverage Neandertals. These estimates show that low heterozygosity, compared to modern humans, was common among Neandertals. The C ++ code of snpAD is freely available at http://bioinf.eva.mpg.de/snpAD/. Supplementary data are available at Bioinformatics online.

Genotyping-by-sequencing for estimating relatedness in nonmodel organisms: Avoiding the trap of precise bias.

PubMed

Attard, Catherine R M; Beheregaray, Luciano B; Möller, Luciana M

2018-05-01

There has been remarkably little attention to using the high resolution provided by genotyping-by-sequencing (i.e., RADseq and similar methods) for assessing relatedness in wildlife populations. A major hurdle is the genotyping error, especially allelic dropout, often found in this type of data that could lead to downward-biased, yet precise, estimates of relatedness. Here, we assess the applicability of genotyping-by-sequencing for relatedness inferences given its relatively high genotyping error rate. Individuals of known relatedness were simulated under genotyping error, allelic dropout and missing data scenarios based on an empirical ddRAD data set, and their true relatedness was compared to that estimated by seven relatedness estimators. We found that an estimator chosen through such analyses can circumvent the influence of genotyping error, with the estimator of Ritland (Genetics Research, 67, 175) shown to be unaffected by allelic dropout and to be the most accurate when there is genotyping error. We also found that the choice of estimator should not rely solely on the strength of correlation between estimated and true relatedness as a strong correlation does not necessarily mean estimates are close to true relatedness. We also demonstrated how even a large SNP data set with genotyping error (allelic dropout or otherwise) or missing data still performs better than a perfectly genotyped microsatellite data set of tens of markers. The simulation-based approach used here can be easily implemented by others on their own genotyping-by-sequencing data sets to confirm the most appropriate and powerful estimator for their data. © 2017 John Wiley & Sons Ltd.

The coding region of the UFGT gene is a source of diagnostic SNP markers that allow single-locus DNA genotyping for the assessment of cultivar identity and ancestry in grapevine (Vitis vinifera L.)

PubMed Central

2013-01-01

Background Vitis vinifera L. is one of society’s most important agricultural crops with a broad genetic variability. The difficulty in recognizing grapevine genotypes based on ampelographic traits and secondary metabolites prompted the development of molecular markers suitable for achieving variety genetic identification. Findings Here, we propose a comparison between a multi-locus barcoding approach based on six chloroplast markers and a single-copy nuclear gene sequencing method using five coding regions combined with a character-based system with the aim of reconstructing cultivar-specific haplotypes and genotypes to be exploited for the molecular characterization of 157 V. vinifera accessions. The analysis of the chloroplast target regions proved the inadequacy of the DNA barcoding approach at the subspecies level, and hence further DNA genotyping analyses were targeted on the sequences of five nuclear single-copy genes amplified across all of the accessions. The sequencing of the coding region of the UFGT nuclear gene (UDP-glucose: flavonoid 3-0-glucosyltransferase, the key enzyme for the accumulation of anthocyanins in berry skins) enabled the discovery of discriminant SNPs (1/34 bp) and the reconstruction of 130 V. vinifera distinct genotypes. Most of the genotypes proved to be cultivar-specific, and only few genotypes were shared by more, although strictly related, cultivars. Conclusion On the whole, this technique was successful for inferring SNP-based genotypes of grapevine accessions suitable for assessing the genetic identity and ancestry of international cultivars and also useful for corroborating some hypotheses regarding the origin of local varieties, suggesting several issues of misidentification (synonymy/homonymy). PMID:24298902

Generalized disequilibrium test for association in qualitative traits incorporating imprinting effects based on extended pedigrees.

PubMed

Li, Jian-Long; Wang, Peng; Fung, Wing Kam; Zhou, Ji-Yuan

2017-10-16

For dichotomous traits, the generalized disequilibrium test with the moment estimate of the variance (GDT-ME) is a powerful family-based association method. Genomic imprinting is an important epigenetic phenomenon and currently, there has been increasing interest of incorporating imprinting to improve the test power of association analysis. However, GDT-ME does not take imprinting effects into account, and it has not been investigated whether it can be used for association analysis when the effects indeed exist. In this article, based on a novel decomposition of the genotype score according to the paternal or maternal source of the allele, we propose the generalized disequilibrium test with imprinting (GDTI) for complete pedigrees without any missing genotypes. Then, we extend GDTI and GDT-ME to accommodate incomplete pedigrees with some pedigrees having missing genotypes, by using a Monte Carlo (MC) sampling and estimation scheme to infer missing genotypes given available genotypes in each pedigree, denoted by MCGDTI and MCGDT-ME, respectively. The proposed GDTI and MCGDTI methods evaluate the differences of the paternal as well as maternal allele scores for all discordant relative pairs in a pedigree, including beyond first-degree relative pairs. Advantages of the proposed GDTI and MCGDTI test statistics over existing methods are demonstrated by simulation studies under various simulation settings and by application to the rheumatoid arthritis dataset. Simulation results show that the proposed tests control the size well under the null hypothesis of no association, and outperform the existing methods under various imprinting effect models. The existing GDT-ME and the proposed MCGDT-ME can be used to test for association even when imprinting effects exist. For the application to the rheumatoid arthritis data, compared to the existing methods, MCGDTI identifies more loci statistically significantly associated with the disease. Under complete and incomplete imprinting effect models, our proposed GDTI and MCGDTI methods, by considering the information on imprinting effects and all discordant relative pairs within each pedigree, outperform all the existing test statistics and MCGDTI can recapture much of the missing information. Therefore, MCGDTI is recommended in practice.

Pathogen profiling for disease management and surveillance.

PubMed

Sintchenko, Vitali; Iredell, Jonathan R; Gilbert, Gwendolyn L

2007-06-01

The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen profiling integrates microbial genomics data into communicable disease control by consolidating phenotypic identity-based methods with DNA microarrays, proteomics, metabolomics and sequence-based typing. Sharing data on pathogen profiles should facilitate our understanding of transmission patterns and the dynamics of epidemics.

efficient association study design via power-optimized tag SNP selection

PubMed Central

HAN, BUHM; KANG, HYUN MIN; SEO, MYEONG SEONG; ZAITLEN, NOAH; ESKIN, ELEAZAR

2008-01-01

Discovering statistical correlation between causal genetic variation and clinical traits through association studies is an important method for identifying the genetic basis of human diseases. Since fully resequencing a cohort is prohibitively costly, genetic association studies take advantage of local correlation structure (or linkage disequilibrium) between single nucleotide polymorphisms (SNPs) by selecting a subset of SNPs to be genotyped (tag SNPs). While many current association studies are performed using commercially available high-throughput genotyping products that define a set of tag SNPs, choosing tag SNPs remains an important problem for both custom follow-up studies as well as designing the high-throughput genotyping products themselves. The most widely used tag SNP selection method optimizes over the correlation between SNPs (r2). However, tag SNPs chosen based on an r2 criterion do not necessarily maximize the statistical power of an association study. We propose a study design framework that chooses SNPs to maximize power and efficiently measures the power through empirical simulation. Empirical results based on the HapMap data show that our method gains considerable power over a widely used r2-based method, or equivalently reduces the number of tag SNPs required to attain the desired power of a study. Our power-optimized 100k whole genome tag set provides equivalent power to the Affymetrix 500k chip for the CEU population. For the design of custom follow-up studies, our method provides up to twice the power increase using the same number of tag SNPs as r2-based methods. Our method is publicly available via web server at http://design.cs.ucla.edu. PMID:18702637

Biplot analysis of strawberry genotypes recommended for the State of Espírito Santo.

PubMed

Costa, A F; Teodoro, P E; Bhering, L L; Leal, N R; Tardin, F D; Daher, R F

2016-08-26

Most strawberry genotypes grown commercially in Brazil originate from breeding programs in the United States, and are therefore not adapted to the various soil and climatic conditions found in Brazil. Thus, quantifying the magnitude of genotype x environment (GE) interactions serves as a primary means for increasing average Brazilian strawberry yields, and helps provide specific recommendations for farmers on which genotypes meet high yield and phenotypic stability thresholds. The aim of this study was to use AMMI (additive main effects and multiplicative interaction) and GGE biplot (genotype main effects + genotype x environment interaction) analyses to identify high-yield, stable strawberry genotypes grown at three locations in Espírito Santo for two agricultural years. We evaluated seven strawberry genotypes (Dover, Camino Real, Ventana, Camarosa, Seascape, Diamante, and Aromas) at three locations (Domingos Martins, Iúna, and Muniz Freire) in agricultural years 2006 and 2007, totaling six study environments. Joint analysis of variance was calculated using yield data (t/ha), and AMMI and GGE biplot analysis was conducted following the detection of a significant genotypes x agricultural years x locations (G x A x L) interaction. During the two agricultural years, evaluated locations were allocated to different regions on biplot graphics using both methods, indicating distinctions among them. Based on the results obtained from the two methods used in this study to investigate the G x A x L interaction, we recommend growing the Camarosa genotype for production at the three locations assessed due to the high frequency of favorable alleles, which were expressed in all localities evaluated regardless of the agricultural year.

A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment

PubMed Central

Shiran, M R; Chowdry, J; Rostami-Hodjegan, A; Ellis, S W; Lennard, M S; Iqbal, M Z; Lagundoye, O; Seivewright, N; Tucker, G T

2003-01-01

Aims To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT). Methods Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma. Results Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity. Conclusions There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients. PMID:12895196

Evaluation of genetic divergence among clones of conilon coffee after scheduled cycle pruning.

PubMed

Dalcomo, J M; Vieira, H D; Ferreira, A; Lima, W L; Ferrão, R G; Fonseca, A F A; Ferrão, M A G; Partelli, F L

2015-11-30

Coffea canephora genotypes from the breeding program of Instituto Capixaba de Pesquisa e Extensão Rural were evaluated, and genetic diversity was estimated with the aim of future improvement strategies. From an initial group of 55 genotypes, 18 from the region of Castelo, ES, were selected, and three clones of the cultivars "Vitória" and "robusta tropical." Upon completion of the scheduled cycle pruning, 17 morphoagronomic traits were measured in the 22 genotypes selected. The principal components method was used to evaluate the contributions relative to the traits. The genetic dissimilarity matrix was obtained through Mahalanobis generalized distance, and genotypes were grouped using the hierarchical method based on the mean of the distances. The most promising clones of Avaliação Castelo were AC02, AC03, AC12, AC13, AC22, AC24, AC26, AC27, AC28, AC29, AC30, AC35, AC36, AC37, AC39, AC40, AC43, and AC46. These methods detected high genetic variability, grouping, by similarity, the genotypes in five groups. The trait that contributed the least to genetic divergence was the number of leaves in plagiotropic branches; however, this was not eliminated, because discarding it altered the groups. There are superior genotypes with potential for use in the next stages of the breeding program, aimed at both the composition of clonal variety and hybridizations.

CC Genotype Donors for the Interleukin-28B Single-Nucleotide Polymorphism are Associated with Better Outcomes in Hepatitis C after Liver Transplant

PubMed Central

Firpi, Roberto J.; Dong, Huijia; Clark, Virginia C.; Soldevila-Pico, Consuelo; Morelli, Giuseppe; Cabrera, Roniel; Norkina, Oxana; Shuster, Jonathan J.; Nelson, David R.; Liu, Chen

2012-01-01

Background/Aims Interleukin-28B (IL-28B) polymorphism is the strongest pretreatment predictor of viral clearance in the hepatitis C (HCV) population. Donor and recipient IL-28B genomic background may play an important role in post-transplant HCV recurrence. We sought to examine the role of IL-28B polymorphisms of donor and recipients in liver transplant patients with recurrent HCV and its impact on the response to interferon-based therapy. Methods The cohort study consisted of 135 adult liver transplant patients who received interferon-based therapy for recurrent HCV between 1996 and 2005 at the University of Florida. IL-28B single nucleotide polymorphism (rs. 12979860) was characterized using liver tissue from all donors and recipients. Results The CC genotype was observed in approximately 30% of donors and recipients. Sustained viral response (SVR) to HCV therapy was 100% if both recipient and donor were CC genotype, while the SVR was only 25% if neither donor nor recipient had a CC genotype. (Recipient, p=0.025, Donor, p<0.001). Recipients and donors with CC genotype had less fibrosis than recipients with genotypes CT and TT, but the difference was not statistically significant. IL-28B genotype did not seem to play a role in the overall survival in these patients. Conclusion In conclusion, recipient and donor CC genotype is associated with a better treatment response to interferon-based therapy after liver transplant. Our study suggests that using CC genotype donor livers for HCV patients may improve the overall clinical outcome after liver transplantation. PMID:23107586

Factor regression for interpreting genotype-environment interaction in bread-wheat trials.

PubMed

Baril, C P

1992-05-01

The French INRA wheat (Triticum aestivum L. em Thell.) breeding program is based on multilocation trials to produce high-yielding, adapted lines for a wide range of environments. Differential genotypic responses to variable environment conditions limit the accuracy of yield estimations. Factor regression was used to partition the genotype-environment (GE) interaction into four biologically interpretable terms. Yield data were analyzed from 34 wheat genotypes grown in four environments using 12 auxiliary agronomic traits as genotypic and environmental covariates. Most of the GE interaction (91%) was explained by the combination of only three traits: 1,000-kernel weight, lodging susceptibility and spike length. These traits are easily measured in breeding programs, therefore factor regression model can provide a convenient and useful prediction method of yield.

Effects of Using Personal Genotype Data on Student Learning and Attitudes in a Pharmacogenomics Course

PubMed Central

McDonough, Caitrin W.; Elsey, Amanda R.; Burkley, Benjamin; Cavallari, Larisa H.; Johnson, Julie A.

2016-01-01

Objective. To evaluate the impact of personal genotyping and a novel educational approach on student attitudes, knowledge, and beliefs regarding pharmacogenomics and genomic medicine. Methods. Two online elective courses (pharmacogenomics and genomic medicine) were offered to student pharmacists at the University of Florida using a flipped-classroom, patient-centered teaching approach. In the pharmacogenomics course, students could be genotyped and apply results to patient cases. Results. Thirty-four and 19 student pharmacists completed the pharmacogenomics and genomic medicine courses, respectively, and 100% of eligible students (n=34) underwent genotyping. Student knowledge improved after the courses. Seventy-four percent (n=25) of students reported better understanding of pharmacogenomics based on having undergone genotyping. Conclusions. Completion of a novel pharmacogenomics elective course sequence that incorporated personal genotyping and genomic medicine was associated with increased student pharmacist knowledge and improved clinical confidence with pharmacogenomics. PMID:27756930

Investigation of Inversion Polymorphisms in the Human Genome Using Principal Components Analysis

PubMed Central

Ma, Jianzhong; Amos, Christopher I.

2012-01-01

Despite the significant advances made over the last few years in mapping inversions with the advent of paired-end sequencing approaches, our understanding of the prevalence and spectrum of inversions in the human genome has lagged behind other types of structural variants, mainly due to the lack of a cost-efficient method applicable to large-scale samples. We propose a novel method based on principal components analysis (PCA) to characterize inversion polymorphisms using high-density SNP genotype data. Our method applies to non-recurrent inversions for which recombination between the inverted and non-inverted segments in inversion heterozygotes is suppressed due to the loss of unbalanced gametes. Inside such an inversion region, an effect similar to population substructure is thus created: two distinct “populations” of inversion homozygotes of different orientations and their 1∶1 admixture, namely the inversion heterozygotes. This kind of substructure can be readily detected by performing PCA locally in the inversion regions. Using simulations, we demonstrated that the proposed method can be used to detect and genotype inversion polymorphisms using unphased genotype data. We applied our method to the phase III HapMap data and inferred the inversion genotypes of known inversion polymorphisms at 8p23.1 and 17q21.31. These inversion genotypes were validated by comparing with literature results and by checking Mendelian consistency using the family data whenever available. Based on the PCA-approach, we also performed a preliminary genome-wide scan for inversions using the HapMap data, which resulted in 2040 candidate inversions, 169 of which overlapped with previously reported inversions. Our method can be readily applied to the abundant SNP data, and is expected to play an important role in developing human genome maps of inversions and exploring associations between inversions and susceptibility of diseases. PMID:22808122

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

PubMed Central

Hu, Guohong; Wang, Hui-Yun; Greenawalt, Danielle M.; Azaro, Marco A.; Luo, Minjie; Tereshchenko, Irina V.; Cui, Xiangfeng; Yang, Qifeng; Gao, Richeng; Shen, Li; Li, Honghua

2006-01-01

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of ∼160 000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300 000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from . PMID:16982644

Repetitive sequences based on genotyping of Candida albicans isolates obtained from Iranian patients with human immunodeficiency virus

PubMed Central

Tamai, Iradj Ashrafi; Salehi, Taghi Zahraei; Sharifzadeh, Aghil; Shokri, Hojjatollah; Khosravi, Ali Reza

2014-01-01

Objective(s): Candidiasis infection caused by Candida albicans has been known as a major problem in patients with immune disorders. The objective of this study was to genotype the C. albicans isolates obtained from oral cavity of patients with positive human immunodeficiency virus (HIV+) with or/and without oropharyngeal candidiasis (OPC). Materials and Methods: A total of 100 C. albicans isolates from Iranian HIV+patients were genotyped using specific PCR primers of the 25S rDNA and RPS genes. Results: The frequencies of genotypes A, B and C which were achieved using 25S rDNA , were 66, 24 and 10 percent, respectively. In addition, genotypes D and E were not found in this study. Each C. albicans genotype was further classified into four subtypes (types 2, 3, 2/3 and 3/4) by PCR amplification targeting RPS sequence. Conclusion: In general, genotype A3 constituted the majority of understudy clinical isolates obtained from oral cavity of Iranian HIV+ patients. PMID:25691923

Identification of genotypes of Influenza A virus in Malaysia

PubMed Central

MM, Rahman; KK, Wong; I, Isahak; ZZ, Rashid; H, Alfizah

2014-01-01

Objective: Influenza is considered as an emerging disease until today. The present study was undertaken to determine the prevalent genotypes of Influenza A virus in Malaysia. Methods: Influenza A virus was identified from respiratory specimens by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Phylogenetic analysis of the identified isolates was performed and genotypes were detected. Results: A total number of 505 throat swabs and nasopharyngeal aspirates were examined by rRT-PCR at Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in which 65(12.87%) were positive for influenza A. The identified isolates were successfully genotyped by phylogenetic analysis. The identified influenza A genotypes were: H1N1 (42), H3N2 (20) and H5N1 (3). Conclusion: The findings indicated that 3 genotypes were circulating in Malaysia during 2011 in which H1N1 was the predominant. Results added new genotype (H5N1) identification record in Malaysia that may be added in data base of WHO and CDC. PMID:25225528

QuASAR: quantitative allele-specific analysis of reads.

PubMed

Harvey, Chris T; Moyerbrailean, Gregory A; Davis, Gordon O; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

2015-04-15

Expression quantitative trait loci (eQTL) studies have discovered thousands of genetic variants that regulate gene expression, enabling a better understanding of the functional role of non-coding sequences. However, eQTL studies are costly, requiring large sample sizes and genome-wide genotyping of each sample. In contrast, analysis of allele-specific expression (ASE) is becoming a popular approach to detect the effect of genetic variation on gene expression, even within a single individual. This is typically achieved by counting the number of RNA-seq reads matching each allele at heterozygous sites and testing the null hypothesis of a 1:1 allelic ratio. In principle, when genotype information is not readily available, it could be inferred from the RNA-seq reads directly. However, there are currently no existing methods that jointly infer genotypes and conduct ASE inference, while considering uncertainty in the genotype calls. We present QuASAR, quantitative allele-specific analysis of reads, a novel statistical learning method for jointly detecting heterozygous genotypes and inferring ASE. The proposed ASE inference step takes into consideration the uncertainty in the genotype calls, while including parameters that model base-call errors in sequencing and allelic over-dispersion. We validated our method with experimental data for which high-quality genotypes are available. Results for an additional dataset with multiple replicates at different sequencing depths demonstrate that QuASAR is a powerful tool for ASE analysis when genotypes are not available. http://github.com/piquelab/QuASAR. fluca@wayne.edu or rpique@wayne.edu Supplementary Material is available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

In silico genomic analyses reveal three distinct lineages of Escherichia coli O157:H7, one of which is associated with hyper-virulence.

PubMed

Laing, Chad R; Buchanan, Cody; Taboada, Eduardo N; Zhang, Yongxiang; Karmali, Mohamed A; Thomas, James E; Gannon, Victor Pj

2009-06-29

Many approaches have been used to study the evolution, population structure and genetic diversity of Escherichia coli O157:H7; however, observations made with different genotyping systems are not easily relatable to each other. Three genetic lineages of E. coli O157:H7 designated I, II and I/II have been identified using octamer-based genome scanning and microarray comparative genomic hybridization (mCGH). Each lineage contains significant phenotypic differences, with lineage I strains being the most commonly associated with human infections. Similarly, a clade of hyper-virulent O157:H7 strains implicated in the 2006 spinach and lettuce outbreaks has been defined using single-nucleotide polymorphism (SNP) typing. In this study an in silico comparison of six different genotyping approaches was performed on 19 E. coli genome sequences from 17 O157:H7 strains and single O145:NM and K12 MG1655 strains to provide an overall picture of diversity of the E. coli O157:H7 population, and to compare genotyping methods for O157:H7 strains. In silico determination of lineage, Shiga-toxin bacteriophage integration site, comparative genomic fingerprint, mCGH profile, novel region distribution profile, SNP type and multi-locus variable number tandem repeat analysis type was performed and a supernetwork based on the combination of these methods was produced. This supernetwork showed three distinct clusters of strains that were O157:H7 lineage-specific, with the SNP-based hyper-virulent clade 8 synonymous with O157:H7 lineage I/II. Lineage I/II/clade 8 strains clustered closest on the supernetwork to E. coli K12 and E. coli O55:H7, O145:NM and sorbitol-fermenting O157 strains. The results of this study highlight the similarities in relationships derived from multi-locus genome sampling methods and suggest a "common genotyping language" may be devised for population genetics and epidemiological studies. Future genotyping methods should provide data that can be stored centrally and accessed locally in an easily transferable, informative and extensible format based on comparative genomic analyses.

Multiplex SNaPshot-a new simple and efficient CYP2D6 and ADRB1 genotyping method.

PubMed

Ben, Songtao; Cooper-DeHoff, Rhonda M; Flaten, Hanna K; Evero, Oghenero; Ferrara, Tracey M; Spritz, Richard A; Monte, Andrew A

2016-04-23

Reliable, inexpensive, high-throughput genotyping methods are required for clinical trials. Traditional assays require numerous enzyme digestions or are too expensive for large sample volumes. Our objective was to develop an inexpensive, efficient, and reliable assay for CYP2D6 and ADRB1 accounting for numerous polymorphisms including gene duplications. We utilized the multiplex SNaPshot® custom genotype method to genotype CYP2D6 and ADRB1. We compared the method to reference standards genotyped using the Taqman Copy Number Variant Assay followed by pyrosequencing quantification and determined assigned genotype concordance. We genotyped 119 subjects. Seven (5.9 %) were found to be CYP2D6 poor metabolizers (PMs), 18 (15.1 %) intermediate metabolizers (IMs), 89 (74.8 %) extensive metabolizers (EMs), and 5 (4.2 %) ultra-rapid metabolizers (UMs). We genotyped two variants in the β1-adrenoreceptor, rs1801253 (Gly389Arg) and rs1801252 (Ser49Gly). The Gly389Arg genotype is Gly/Gly 18 (15.1 %), Gly/Arg 58 (48.7 %), and Arg/Arg 43 (36.1 %). The Ser49Gly genotype is Ser/Ser 82 (68.9 %), Ser/Gly 32 (26.9), and Gly/Gly 5 (4.2 %). The multiplex SNaPshot method was concordant with genotypes in reference samples. The multiplex SNaPshot method allows for specific and accurate detection of CYP2D6 genotypes and ADRB1 genotypes and haplotypes. This platform is simple and efficient and suited for high throughput.

Frequency of uridine monophosphate synthase Gly(213)Ala polymorphism in Caucasian gastrointestinal cancer patients and healthy subjects, investigated by means of new, rapid genotyping assays.

PubMed

Gusella, Milena; Bertolaso, Laura; Bolzonella, Caterina; Pasini, Felice; Padrini, Roberto

2011-10-01

Uridine monophosphate synthase (UMPS) is a fundamental enzyme in pyrimidine synthesis. A single-nucleotide polymorphism, a G-C transversion at the 638th nucleotide, was demonstrated to increase UMPS activity and suggested to have clinical effects. The aims of this study were to set up simple genotyping methods and investigate the UMPS 638G>C polymorphism in the Caucasian population. Two hundred forty-one patients with gastrointestinal cancers and 189 healthy subjects were enrolled. Genomic DNA was extracted from peripheral blood. A polymerase chain reaction-restriction fragment length polymorphism (RFLP) method was implemented using a forward primer incorporating a mismatched base to produce an artificial restriction site and BsrI restriction enzyme digestion; a denaturing high performance liquid chromatography (DHPLC) method was developed to further speed up UMPS genotyping. A 153 bp UMPS gene fragment was successfully amplified and analyzed in all samples. RFLP and DHPLC results showed a 100% match and where confirmed by direct sequencing. UMPS genotype distribution was similar in patients with cancer and control subjects. Although no association was detected between UMPS variants and gastrointestinal cancer risk in Caucasians, polymerase chain reaction-RFLP with BsrI digestion and DHPLC set up at 59°C are reliable and cost-effective methods to genotype UMPS.

A forensic perspective on the genetic identification of grapevine (Vitis vinifera L.) varieties using STR markers.

PubMed

Santos, Sara; Oliveira, Manuela; Amorim, António; van Asch, Barbara

2014-11-01

The grapevine (Vitis vinifera subsp. vinifera) is one of the most important agricultural crops worldwide. A long interest in the historical origins of ancient and cultivated current grapevines, as well as the need to establish phylogenetic relationships and parentage, solve homonymies and synonymies, fingerprint cultivars and clones, and assess the authenticity of plants and wines has encouraged the development of genetic identification methods. STR analysis is currently the most commonly used method for these purposes. A large dataset of grapevines genotypes for many cultivars worldwide has been produced in the last decade using a common set of recommended dinucleotide nuclear STRs. This type of marker has been replaced by long core-repeat loci in standardized state-of-the-art human forensic genotyping. The first steps toward harmonized grapevine genotyping have already been taken to bring the genetic identification methods closer to human forensic STR standards by previous authors. In this context, we bring forward a set of basic suggestions that reinforce the need to (i) guarantee trueness-to-type of the sample; (ii) use the long core-repeat markers; (iii) verify the specificity and amplification consistency of PCR primers; (iv) sequence frequent alleles and use these standardized allele ladders; (v) consider mutation rates when evaluating results of STR-based parentage and pedigree analysis; (vi) genotype large and representative samples in order to obtain allele frequency databases; (vii) standardize genotype data by establishing allele nomenclature based on repeat number to facilitate information exchange and data compilation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Short-read, high-throughput sequencing technology for STR genotyping

PubMed Central

Bornman, Daniel M.; Hester, Mark E.; Schuetter, Jared M.; Kasoji, Manjula D.; Minard-Smith, Angela; Barden, Curt A.; Nelson, Scott C.; Godbold, Gene D.; Baker, Christine H.; Yang, Boyu; Walther, Jacquelyn E.; Tornes, Ivan E.; Yan, Pearlly S.; Rodriguez, Benjamin; Bundschuh, Ralf; Dickens, Michael L.; Young, Brian A.; Faith, Seth A.

2013-01-01

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples. PMID:25621315

Methylenetetrahydrofolate reductase C677T polymorphism predicts response and time to progression to gemcitabine-based chemotherapy for advanced non-small cell lung cancer in a Chinese Han population*

PubMed Central

Hong, Wei; Wang, Kai; Zhang, Yi-ping; Kou, Jun-yan; Hong, Dan; Su, Dan; Mao, Wei-min; Yu, Xin-min; Xie, Fa-jun; Wang, Xiao-jian

2013-01-01

Objective: The aim of this study was to evaluate the association between the methylenetetrahydrofolate reductase (MTHFR) C677T excision repair cross-complementation group 1 (ERCC1) genetic polymorphisms and the clinical efficacy of gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC). Methods: A total of 135 chemonaive patients with unresectable advanced NSCLC were treated with gemcitabine/platinum regimens. The polymorphisms of MTHFR C677T, ERCC1 C8092A, and ERCC1 C118T were genotyped using the TaqMan methods. Results: The overall response rate was 28.9%. Patients with MTHFR CC genotype had a higher rate of objective response than patients with variant genotype (TT or CT) (41.2% versus 19.1%, P=0.01). Median time to progression (TTP) of patients with MTHFR CC genotype was longer than that of patients with variant genotype (7.6 months versus 5.0 months, P=0.003). No significant associations were obtained between ERCC1 C118T and C8092A polymorphisms and both response and survival. Conclusions: Our data suggest the value of MTHFR C677T polymorphism as a possible predictive marker of response and TTP in advanced NSCLC patients treated with gemcitabine/platinum. PMID:23463763

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

PubMed

Ansart-Pirenne, H; Martin-Blanc, S; Le Pennec, P-Y; Rouger, P; Cartron, J-P; Tournamille, C

2007-02-01

The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

DISSCO: direct imputation of summary statistics allowing covariates

PubMed Central

Xu, Zheng; Duan, Qing; Yan, Song; Chen, Wei; Li, Mingyao; Lange, Ethan; Li, Yun

2015-01-01

Background: Imputation of individual level genotypes at untyped markers using an external reference panel of genotyped or sequenced individuals has become standard practice in genetic association studies. Direct imputation of summary statistics can also be valuable, for example in meta-analyses where individual level genotype data are not available. Two methods (DIST and ImpG-Summary/LD), that assume a multivariate Gaussian distribution for the association summary statistics, have been proposed for imputing association summary statistics. However, both methods assume that the correlations between association summary statistics are the same as the correlations between the corresponding genotypes. This assumption can be violated in the presence of confounding covariates. Methods: We analytically show that in the absence of covariates, correlation among association summary statistics is indeed the same as that among the corresponding genotypes, thus serving as a theoretical justification for the recently proposed methods. We continue to prove that in the presence of covariates, correlation among association summary statistics becomes the partial correlation of the corresponding genotypes controlling for covariates. We therefore develop direct imputation of summary statistics allowing covariates (DISSCO). Results: We consider two real-life scenarios where the correlation and partial correlation likely make practical difference: (i) association studies in admixed populations; (ii) association studies in presence of other confounding covariate(s). Application of DISSCO to real datasets under both scenarios shows at least comparable, if not better, performance compared with existing correlation-based methods, particularly for lower frequency variants. For example, DISSCO can reduce the absolute deviation from the truth by 3.9–15.2% for variants with minor allele frequency <5%. Availability and implementation: http://www.unc.edu/∼yunmli/DISSCO. Contact: yunli@med.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25810429

A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

PubMed

Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

2017-10-01

Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

Precise genotyping and recombination detection of Enterovirus

PubMed Central

2015-01-01

Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue.

PubMed

Strobl, Frederic; Ross, J Alexander; Stelzer, Ernst H K

2017-01-01

The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of T. castaneum adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.

Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia.

PubMed

Pan, Weida; Fu, Yeqi; Abdullahi, Auwalu Yusuf; Wang, Mingwei; Shi, Xianli; Yang, Fang; Yu, Xingang; Yan, Xinxin; Zhang, Pan; Hang, Jianxiong; Li, Guoqing

2017-04-01

To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5'-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10 -5 and 4.88 × 10 -5  ng/μL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.

Assessment of genetic diversity in lettuce (Lactuca sativa L.) germplasm using RAPD markers.

PubMed

Sharma, Shubhangi; Kumar, Pankaj; Gambhir, Geetika; Kumar, Ramesh; Srivastava, D K

2018-01-01

The importance of germplasm characterization is an important link between the conservation and utilization of plant genetic resources in various breeding programmes. In the present study, genetic variability and relationships among 25 Lactuca sativa L. genotypes were tested using random amplified polymorphic DNA (RAPD) molecular markers. A total of 45 random decamer oligonucleotide primers were examined to generate RAPD profiles, out of these reproducible patterns were obtained with 22 primers. A total of 87 amplicon were obtained, out of which all were polymorphic and 7 were unique bands. The level of polymorphism across genotypes was 100% as revealed by RAPD. Genetic similarity matrix, based on Jaccard's coefficients ranged from 13.7 to 84.10% indicating a wide genetic base. Dendrogram was constructed by unweighted pair group method with arithmetic averages method. RAPD technology could be useful for identification of different accessions as well as assessing the genetic similarity among different genotypes of lettuce. The study reveals the limited genetic base and the needs to diversify using new sources from the germplasm.

Identification of Species and Sources of Cryptosporidium Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool

PubMed Central

Xiao, Lihua; Alderisio, Kerri; Limor, Josef; Royer, Michael; Lal, Altaf A.

2000-01-01

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples. PMID:11097935

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Genotype Imputation with Millions of Reference Samples.

PubMed

Browning, Brian L; Browning, Sharon R

2016-01-07

We present a genotype imputation method that scales to millions of reference samples. The imputation method, based on the Li and Stephens model and implemented in Beagle v.4.1, is parallelized and memory efficient, making it well suited to multi-core computer processors. It achieves fast, accurate, and memory-efficient genotype imputation by restricting the probability model to markers that are genotyped in the target samples and by performing linear interpolation to impute ungenotyped variants. We compare Beagle v.4.1 with Impute2 and Minimac3 by using 1000 Genomes Project data, UK10K Project data, and simulated data. All three methods have similar accuracy but different memory requirements and different computation times. When imputing 10 Mb of sequence data from 50,000 reference samples, Beagle's throughput was more than 100× greater than Impute2's throughput on our computer servers. When imputing 10 Mb of sequence data from 200,000 reference samples in VCF format, Minimac3 consumed 26× more memory per computational thread and 15× more CPU time than Beagle. We demonstrate that Beagle v.4.1 scales to much larger reference panels by performing imputation from a simulated reference panel having 5 million samples and a mean marker density of one marker per four base pairs. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Comparison of Three Different Commercial Kits for the Human Papilloma Virus Genotyping.

PubMed

Lim, Yong Kwan; Choi, Jee-Hye; Park, Serah; Kweon, Oh Joo; Park, Ae Ja

2016-11-01

High-risk type human papilloma virus (HPV) is the most important cause of cervical cancer. Recently, real-time polymerase chain reaction and reverse blot hybridization assay-based HPV DNA genotyping kits are developed. So, we compared the performances of different three HPV genotyping kits using different analytical principles and methods. Two hundred positive and 100 negative cervical swab specimens were used. DNA was extracted and all samples were tested by the MolecuTech REBA HPV-ID, Anyplex II HPV28 Detection, and HPVDNAChip. Direct sequencing was performed as a reference method for confirming high-risk HPV genotypes 16, 18, 45, 52, and 58. Although high-level agreement results were observed in negative samples, three kits showed decreased interassay agreement as screening setting in positive samples. Comparing the genotyping results, three assays showed acceptable sensitivity and specificity for the detection of HPV 16 and 18. Otherwise, various sensitivities showed in the detection of HPV 45, 52, and 58. The three assays had dissimilar performance of HPV screening capacity and exhibited moderate level of concordance in HPV genotyping. These discrepant results were unavoidable due to difference in type-specific analytical sensitivity and lack of standardization; therefore, we suggested that the efforts to standardization of HPV genotyping kits and adjusting analytical sensitivity would be important for the best clinical performance. © 2016 Wiley Periodicals, Inc.

GStream: Improving SNP and CNV Coverage on Genome-Wide Association Studies

PubMed Central

Alonso, Arnald; Marsal, Sara; Tortosa, Raül; Canela-Xandri, Oriol; Julià, Antonio

2013-01-01

We present GStream, a method that combines genome-wide SNP and CNV genotyping in the Illumina microarray platform with unprecedented accuracy. This new method outperforms previous well-established SNP genotyping software. More importantly, the CNV calling algorithm of GStream dramatically improves the results obtained by previous state-of-the-art methods and yields an accuracy that is close to that obtained by purely CNV-oriented technologies like Comparative Genomic Hybridization (CGH). We demonstrate the superior performance of GStream using microarray data generated from HapMap samples. Using the reference CNV calls generated by the 1000 Genomes Project (1KGP) and well-known studies on whole genome CNV characterization based either on CGH or genotyping microarray technologies, we show that GStream can increase the number of reliably detected variants up to 25% compared to previously developed methods. Furthermore, the increased genome coverage provided by GStream allows the discovery of CNVs in close linkage disequilibrium with SNPs, previously associated with disease risk in published Genome-Wide Association Studies (GWAS). These results could provide important insights into the biological mechanism underlying the detected disease risk association. With GStream, large-scale GWAS will not only benefit from the combined genotyping of SNPs and CNVs at an unprecedented accuracy, but will also take advantage of the computational efficiency of the method. PMID:23844243

A Video Method to Study Drosophila Sleep

PubMed Central

Zimmerman, John E.; Raizen, David M.; Maycock, Matthew H.; Maislin, Greg; Pack, Allan I.

2008-01-01

Study Objectives: To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements. Design: A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images. Measurements and Results: The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation. Conclusions: Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep. Citation: Zimmerman JE; Raizen DM; Maycock MH; Maislin G; Pack AI. A video method to study drosophila sleep. SLEEP 2008;31(11):1587–1598. PMID:19014079

A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

PubMed

Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

2010-09-01

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

High prevalence of ACE DD genotype among north Indian end stage renal disease patients.

PubMed

Tripathi, Gaurav; Dharmani, Poonam; Khan, Faisal; Sharma, R K; Pandirikkal, Vinod; Agrawal, Suraksha

2006-10-17

The Renin-Angiotensin system (RAS) is a key regulator of both blood pressure and kidney functions and their interaction. In such a situation, genetic variability in the genes of different components of RAS is likely to contribute for its heterogeneous association in the renal disease patients. Angiotensin converting enzyme-1 (ACE-1) is an important component of RAS which determines the vasoactive peptide Angiotensin-II. In the present study, we have investigated 127 ESRD patients and 150 normal healthy controls from north India to deduce the association between ACE gene polymorphism and ESRD. The inclusion criteria for patients included a constantly elevated serum creatinine level above normal range (ranging from 3.4 to 15.8) and further the patients were recommended for renal transplantation. A total of 150 normal healthy controls were also genotyped for ACE I/D polymorphism. The criterion of defining control sample as normal was totally based on the absence of any kidney disease determined from the serum creatinin level. Genotyping of ACE I/D were assayed by polymerase chain reaction (PCR) based DNA amplification using specific flanking primers Based on the method described elsewhere. The difference of DD and II genotypes was found highly significant among the two groups (p = 0.025; OR = 3.524; 95% CI = 1.54-8.07). The combined genotype DD v/s ID+II comparison validated that DD genotype is a high risk genotype for ESRD (p = 0.001; OR = 5.74; 95% CI limit = 3.4-8.5). However, no correlation was obtained for different biochemical parameters of lipid profile and renal function among DD and non DD genotype. Interestingly, approximately 87% of the DD ESRD patients were found hypertensive in comparison to the 65% patients of non DD genotype Based on these observations we conclude that ACE DD genotype implicate a strong possible role in the hypertensive state and in renal damage among north Indians. The study will help in predetermining the timing, type and doses of anti-hypertensive therapy for ESRD patients.

Methods for MHC genotyping in non-model vertebrates.

PubMed

Babik, W

2010-03-01

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

No Evidence for Synergy Between Human Papillomavirus Genotypes for the Risk of High-Grade Squamous Intraepithelial Lesions in a Large Population-Based Study

PubMed Central

Wentzensen, Nicolas; Nason, Martha; Schiffman, Mark; Dodd, Lori; Hunt, William C.; Wheeler, Cosette M.

2014-01-01

Background. Multiple human papillomavirus (HPV) genotypes may be independently or synergistically associated with risk of high-grade squamous intraepithelial lesions (HSILs). We evaluated the risk of HSIL in women concomitantly infected with multiple HPV genotypes. Methods. A population-based stratified sample of 59 664 cervical cytology specimens from women residing in New Mexico were evaluated for cytologic abnormalities and HPV genotypes. We calculated the risk of HSIL in women infected with a single HPV genotype and the risk in those infected with multiple HPV genotypes. Results. The highest risk of HSIL was observed for HPV-16 (0.036), followed by HPV-33 (0.028), HPV-58 (0.024), and HPV-18 (0.022). For most types, we observed a greater risk of HSIL in women infected with multiple carcinogenic HPV types. In contrast, the risk of HSIL was similar in women infected with HPV-16 and other types, compared with women infected with HPV-16 only. We observed an increased but plateauing risk of HSIL in women infected with multiple types, compared with those infected with a single type, with risk ratios of 1.5 (95% confidence interval [CI], 1.2–1.8), 1.7 (95% CI, 1.3–2.4), and 1.4 (95% CI, 0.83–2.5) for women infected with 2, 3, and ≥4 genotypes, respectively. Conclusions. In the largest population-based study of HPV genotypes and cytologic outcomes so far, we did not see more than additive effects of HPV types on the risk of HSIL in women infected with multiple types. PMID:24179110

The Discriminatory Value of CYP2D6 Genotyping in Predicting the Dextromethorphan/Dextrorphan Phenotype in Women with Breast Cancer

PubMed Central

Trojan, Andreas; Vergopoulos, Athanasios; Breitenstein, Urs; Seifert, Burkhardt; Rageth, Christoph; Joechle, Wolfgang

2012-01-01

Background The growth inhibitory effect of tamoxifen is used for the treatment of breast cancer. Tamoxifen efficacy is mediated by its biotransformation, predominantly via the cytochrome P450 2D6 (CYP2D6) isoenzyme, to the active metabolite endoxifen. We investigated the relationship of CYP2D6 genotypes to the metabolism of dextromethorphan (DM), which is frequently used as a surrogate marker for the formation of endoxifen. Methods The CYP2D6 genotype was determined by polymerase chain reaction (PCR) in previously untreated patients with hormone receptor-positive invasive breast cancer considered to receive antihormonal therapy. The DM/dextrorphan (DX) urinary excretion ratios were obtained in a subset of patients by high-pressure liquid chromatography (HPLC)-mediated urine analysis after intake of 25 mg DM. The relationships of genotype and corresponding phenotype were statistically analyzed for association. Results From 151 patients predicted based on their genotype data for the ‘traditional’ CYP2D6 phenotype classes poor, intermediate, extensive and ultrarapid, 83 patients were examined for their DM/DX urinary ratios. The genotype-based poor metabolizer status correlated with the DM/DX ratios, whereas the intermediate, extensive and ultrarapid genotypes could not be distinguished based on their phenotype. Citalopram intake did not significantly influence the phenotype. Conclusions The DM metabolism can be reliably used to assess the CYP2D6 enzyme activity. The correlation with the genotype can be incomplete and the metabolic ratios do not seem to be compromised by citalopram. DM phenotyping may provide a standardized tool to better assess the CYP2D6 metabolic capacity. PMID:22553469

First detection of multiple knockdown resistance (kdr)-like mutations in voltage-gated sodium channel using three new genotyping methods in Anopheles sinensis from Guangxi Province, China.

PubMed

Tan, Wei L; Li, Chun X; Wang, Zhong M; Liu, Mei D; Dong, Yan D; Feng, Xiang Y; Wu, Zhi M; Guo, Xiao X; Xing, Dan; Zhang, Ying M; Wang, Zhong C; Zhao, Tong Y

2012-09-01

To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.

Bayesian GGE biplot models applied to maize multi-environments trials.

PubMed

de Oliveira, L A; da Silva, C P; Nuvunga, J J; da Silva, A Q; Balestre, M

2016-06-17

The additive main effects and multiplicative interaction (AMMI) and the genotype main effects and genotype x environment interaction (GGE) models stand out among the linear-bilinear models used in genotype x environment interaction studies. Despite the advantages of their use to describe genotype x environment (AMMI) or genotype and genotype x environment (GGE) interactions, these methods have known limitations that are inherent to fixed effects models, including difficulty in treating variance heterogeneity and missing data. Traditional biplots include no measure of uncertainty regarding the principal components. The present study aimed to apply the Bayesian approach to GGE biplot models and assess the implications for selecting stable and adapted genotypes. Our results demonstrated that the Bayesian approach applied to GGE models with non-informative priors was consistent with the traditional GGE biplot analysis, although the credible region incorporated into the biplot enabled distinguishing, based on probability, the performance of genotypes, and their relationships with the environments in the biplot. Those regions also enabled the identification of groups of genotypes and environments with similar effects in terms of adaptability and stability. The relative position of genotypes and environments in biplots is highly affected by the experimental accuracy. Thus, incorporation of uncertainty in biplots is a key tool for breeders to make decisions regarding stability selection and adaptability and the definition of mega-environments.

HRM and SNaPshot as alternative forensic SNP genotyping methods.

PubMed

Mehta, Bhavik; Daniel, Runa; McNevin, Dennis

2017-09-01

Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

Next-generation genotype imputation service and methods.

PubMed

Das, Sayantan; Forer, Lukas; Schönherr, Sebastian; Sidore, Carlo; Locke, Adam E; Kwong, Alan; Vrieze, Scott I; Chew, Emily Y; Levy, Shawn; McGue, Matt; Schlessinger, David; Stambolian, Dwight; Loh, Po-Ru; Iacono, William G; Swaroop, Anand; Scott, Laura J; Cucca, Francesco; Kronenberg, Florian; Boehnke, Michael; Abecasis, Gonçalo R; Fuchsberger, Christian

2016-10-01

Genotype imputation is a key component of genetic association studies, where it increases power, facilitates meta-analysis, and aids interpretation of signals. Genotype imputation is computationally demanding and, with current tools, typically requires access to a high-performance computing cluster and to a reference panel of sequenced genomes. Here we describe improvements to imputation machinery that reduce computational requirements by more than an order of magnitude with no loss of accuracy in comparison to standard imputation tools. We also describe a new web-based service for imputation that facilitates access to new reference panels and greatly improves user experience and productivity.

Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR.

PubMed

Nilyanimit, Pornjarim; Chansaenroj, Jira; Poomipak, Witthaya; Praianantathavorn, Kesmanee; Payungporn, Sunchai; Poovorawan, Yong

2018-03-01

Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations. © The Korean Society for Laboratory Medicine

Minimum number of measurements for evaluating soursop (Annona muricata L.) yield.

PubMed

Sánchez, C F B; Teodoro, P E; Londoño, S; Silva, L A; Peixoto, L A; Bhering, L L

2017-05-31

Repeatability studies on fruit species are of great importance to identify the minimum number of measurements necessary to accurately select superior genotypes. This study aimed to identify the most efficient method to estimate the repeatability coefficient (r) and predict the minimum number of measurements needed for a more accurate evaluation of soursop (Annona muricata L.) genotypes based on fruit yield. Sixteen measurements of fruit yield from 71 soursop genotypes were carried out between 2000 and 2016. In order to estimate r with the best accuracy, four procedures were used: analysis of variance, principal component analysis based on the correlation matrix, principal component analysis based on the phenotypic variance and covariance matrix, and structural analysis based on the correlation matrix. The minimum number of measurements needed to predict the actual value of individuals was estimated. Principal component analysis using the phenotypic variance and covariance matrix provided the most accurate estimates of both r and the number of measurements required for accurate evaluation of fruit yield in soursop. Our results indicate that selection of soursop genotypes with high fruit yield can be performed based on the third and fourth measurements in the early years and/or based on the eighth and ninth measurements at more advanced stages.

Identification and Correction of Sample Mix-Ups in Expression Genetic Data: A Case Study

PubMed Central

Broman, Karl W.; Keller, Mark P.; Broman, Aimee Teo; Kendziorski, Christina; Yandell, Brian S.; Sen, Śaunak; Attie, Alan D.

2015-01-01

In a mouse intercross with more than 500 animals and genome-wide gene expression data on six tissues, we identified a high proportion (18%) of sample mix-ups in the genotype data. Local expression quantitative trait loci (eQTL; genetic loci influencing gene expression) with extremely large effect were used to form a classifier to predict an individual’s eQTL genotype based on expression data alone. By considering multiple eQTL and their related transcripts, we identified numerous individuals whose predicted eQTL genotypes (based on their expression data) did not match their observed genotypes, and then went on to identify other individuals whose genotypes did match the predicted eQTL genotypes. The concordance of predictions across six tissues indicated that the problem was due to mix-ups in the genotypes (although we further identified a small number of sample mix-ups in each of the six panels of gene expression microarrays). Consideration of the plate positions of the DNA samples indicated a number of off-by-one and off-by-two errors, likely the result of pipetting errors. Such sample mix-ups can be a problem in any genetic study, but eQTL data allow us to identify, and even correct, such problems. Our methods have been implemented in an R package, R/lineup. PMID:26290572

Identification and Correction of Sample Mix-Ups in Expression Genetic Data: A Case Study.

PubMed

Broman, Karl W; Keller, Mark P; Broman, Aimee Teo; Kendziorski, Christina; Yandell, Brian S; Sen, Śaunak; Attie, Alan D

2015-08-19

In a mouse intercross with more than 500 animals and genome-wide gene expression data on six tissues, we identified a high proportion (18%) of sample mix-ups in the genotype data. Local expression quantitative trait loci (eQTL; genetic loci influencing gene expression) with extremely large effect were used to form a classifier to predict an individual's eQTL genotype based on expression data alone. By considering multiple eQTL and their related transcripts, we identified numerous individuals whose predicted eQTL genotypes (based on their expression data) did not match their observed genotypes, and then went on to identify other individuals whose genotypes did match the predicted eQTL genotypes. The concordance of predictions across six tissues indicated that the problem was due to mix-ups in the genotypes (although we further identified a small number of sample mix-ups in each of the six panels of gene expression microarrays). Consideration of the plate positions of the DNA samples indicated a number of off-by-one and off-by-two errors, likely the result of pipetting errors. Such sample mix-ups can be a problem in any genetic study, but eQTL data allow us to identify, and even correct, such problems. Our methods have been implemented in an R package, R/lineup. Copyright © 2015 Broman et al.

An automated genotyping tool for enteroviruses and noroviruses.

PubMed

Kroneman, A; Vennema, H; Deforche, K; v d Avoort, H; Peñaranda, S; Oberste, M S; Vinjé, J; Koopmans, M

2011-06-01

Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons. Copyright © 2011 Elsevier B.V. All rights reserved.

SNPs at 3'-UTR of the bovine CDIPT gene associated with Qinchuan cattle meat quality traits.

PubMed

Fu, C Z; Wang, H; Mei, C G; Wang, J L; Jiang, B J; Ma, X H; Wang, H B; Cheng, G; Zan, L S

2013-03-13

The CDIPT is crucial to the fatty acid metabolic pathway, intracellular signal transduction and energy metabolism in eukaryotic cells. We detected three SNPs at 3'-untranslated regions (UTR), named 3'-UTR_108 A > G, 3'-UTR_448 G > A and 3'-UTR_477 C > G, of the CDIPT gene in 618 Qinchuan cattle using PCR-RFLP and DNA sequencing methods. At each of the three SNPs, we found three genotypes named as follows: AA, AB, BB (3'-UTR_108 A > G), CC, CD, DD (3'-UTR_448 G > A) and EE, EF, FF (3'-UTR_477 C > G.). Based on association analysis of these SNPs with ultrasound measurement traits, individuals of genotype BB had a significantly larger loin muscle area than genotype AA. Individuals of genotype CC had significantly thicker back fat than individuals of genotype DD. Individuals of genotype EE also had significantly thicker back fat than did individuals of genotype FF. We conclude that these SNPs of the CDIPT gene could be used as molecular markers for selecting and breeding beef cattle with superior body traits, depending on breeding goals.

Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India.

PubMed

Chandramuki, Akepati; Khanna, Neelam; Shashkina, Elena; Kurepina, Natalia; Mathema, Barun; Kreiswirth, Barry N; Venkataswamy, Manjunatha M

2017-01-01

Specific genotypes of Mycobacterium tuberculosis (MTB) have been reported to cause outbreaks of pulmonary tuberculosis (TB) in geographical areas that are endemic to TB. However, since there is little epidemiological evidence on the association of particular genotypes that cause tuberculous meningitis (TBM), we sought to investigate the association of specific MTB strains with infection of the central nervous system (CNS). We carried out a genetic characterisation of 89 MTB isolates from TBM patients at a Southern Indian tertiary neurocare centre and compared the genotypes with strains of pulmonary TB isolated from Indian immigrants in New York City. We applied the standard methods of genotyping of MTB, namely, IS6110-based restriction fragment length polymorphism and spoligotyping for strain identification, along with principal genetic grouping and single-nucleotide polymorphism cluster analysis. The analysis revealed a high-level of diversity amongst the strain population. The genotypes of the isolates from TBM patients paralleled the pulmonary TB strain population recovered from the Indian immigrants in NYC. We conclude that there is no apparent association between genotypes of MTB and propensity to infect CNS tissue.

Integrating environmental covariates and crop modeling into the genomic selection framework to predict genotype by environment interactions.

PubMed

Heslot, Nicolas; Akdemir, Deniz; Sorrells, Mark E; Jannink, Jean-Luc

2014-02-01

Development of models to predict genotype by environment interactions, in unobserved environments, using environmental covariates, a crop model and genomic selection. Application to a large winter wheat dataset. Genotype by environment interaction (G*E) is one of the key issues when analyzing phenotypes. The use of environment data to model G*E has long been a subject of interest but is limited by the same problems as those addressed by genomic selection methods: a large number of correlated predictors each explaining a small amount of the total variance. In addition, non-linear responses of genotypes to stresses are expected to further complicate the analysis. Using a crop model to derive stress covariates from daily weather data for predicted crop development stages, we propose an extension of the factorial regression model to genomic selection. This model is further extended to the marker level, enabling the modeling of quantitative trait loci (QTL) by environment interaction (Q*E), on a genome-wide scale. A newly developed ensemble method, soft rule fit, was used to improve this model and capture non-linear responses of QTL to stresses. The method is tested using a large winter wheat dataset, representative of the type of data available in a large-scale commercial breeding program. Accuracy in predicting genotype performance in unobserved environments for which weather data were available increased by 11.1% on average and the variability in prediction accuracy decreased by 10.8%. By leveraging agronomic knowledge and the large historical datasets generated by breeding programs, this new model provides insight into the genetic architecture of genotype by environment interactions and could predict genotype performance based on past and future weather scenarios.

Prevalence and genotyping ofToxoplasma gondii among Saudi pregnant women in Saudi Arabia.

PubMed

Alghamdi, Jawahir; Elamin, Maha Hussein; Alhabib, Samia

2016-11-01

Introduction: Toxoplasma gondii ( T. gondii ) is an intracellular protozoan that can infect all mammals, who serve as intermediate host. It causes congenital, neurological, eyes complications and mild or asymptomatic infections in humans. Purpose of this study: To investigate not only the prevalence of T. gondii , but also to find out its genotyping using multiple sequential molecular methods to predict exactly the precise genotyping of T. gondii among Saudi pregnant women. Methods: A cross-sectional study was conducted using multi-stage methods. Initial stage involved enrolment of 250 Saudi pregnant women from multi-centre healthcare and community based settings in the capital of Saudi Arabia Riyadh. The second stage was embracement of the laboratory investigation that included Enzyme immunoassay (ELISA), DNA extraction, PCR, nested-PCR assay, and genotyping of the seropositive cases. Results: 203 women agreed to take part in our study with a response rate of 81.2% (203/250). Using ELISA, we found that the prevalence of Toxoplasma gondii IgG and IgM antibodies was 32.5% and 6.4%, respectively. We found that 29 samples (80.6%) were of genotype II; however 7 samples (19.4%) were of genotype III. Conclusion: Defining the population structure of T. gondii from Saudi Arabia has important implications for transmission, immunogenicity, pathogenesis, and in planning preventive strategies. Relationship between such variation in structure and disease manifestation in pregnant women is still difficult to assess due to the role of host immune status and genetic background on the control of infection, and of other parasitic features such as the infecting dose or parasite stage. Our finding of the genotyping of T. gondii might facilitate and inform future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission among Saudi women.

Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

PubMed

Stachler, Matthew; Jia, Yonghui; Sharaf, Nematullah; Wade, Jacqueline; Longtine, Janina; Garcia, Elizabeth; Sholl, Lynette M

2015-01-01

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

DISSCO: direct imputation of summary statistics allowing covariates.

PubMed

Xu, Zheng; Duan, Qing; Yan, Song; Chen, Wei; Li, Mingyao; Lange, Ethan; Li, Yun

2015-08-01

Imputation of individual level genotypes at untyped markers using an external reference panel of genotyped or sequenced individuals has become standard practice in genetic association studies. Direct imputation of summary statistics can also be valuable, for example in meta-analyses where individual level genotype data are not available. Two methods (DIST and ImpG-Summary/LD), that assume a multivariate Gaussian distribution for the association summary statistics, have been proposed for imputing association summary statistics. However, both methods assume that the correlations between association summary statistics are the same as the correlations between the corresponding genotypes. This assumption can be violated in the presence of confounding covariates. We analytically show that in the absence of covariates, correlation among association summary statistics is indeed the same as that among the corresponding genotypes, thus serving as a theoretical justification for the recently proposed methods. We continue to prove that in the presence of covariates, correlation among association summary statistics becomes the partial correlation of the corresponding genotypes controlling for covariates. We therefore develop direct imputation of summary statistics allowing covariates (DISSCO). We consider two real-life scenarios where the correlation and partial correlation likely make practical difference: (i) association studies in admixed populations; (ii) association studies in presence of other confounding covariate(s). Application of DISSCO to real datasets under both scenarios shows at least comparable, if not better, performance compared with existing correlation-based methods, particularly for lower frequency variants. For example, DISSCO can reduce the absolute deviation from the truth by 3.9-15.2% for variants with minor allele frequency <5%. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Evidence for an African cluster of human head and body lice with variable colors and interbreeding of lice between continents.

PubMed

Veracx, Aurélie; Boutellis, Amina; Merhej, Vicky; Diatta, Georges; Raoult, Didier

2012-01-01

Human head lice and body lice have been classified based on phenotypic characteristics, including geographical source, ecotype (preferred egg laying site hair or clothes), shape and color. More recently, genotypic studies have been based on mitochondrial genes, nuclear genes and intergenic spacers. Mitochondrial genetic analysis reclassified lice into three genotypes (A, B and C). However, no previous study has attempted to correlate both genotypic and phenotypic data. Lice were collected in four African countries: Senegal, Burundi, Rwanda and Ethiopia and were photographed to compare their colors. The Multi-Spacer-Typing (MST) method was used to genotype lice belonging to the worldwide Clade A, allowing a comparison of phenotypic and genotypic data. No congruence between louse color and genotype has been identified. Phylogenetic analysis of the spacer PM2, performed including lice from other sources, showed the existence of an African cluster of human lice. However, the analysis of other spacers suggested that lice from different areas are interbreeding. We identified two geotypes of Clade A head and body lice including one that is specifically African, that can be either black or grey and can live on the head or in clothing. We also hypothesized that lice from different areas are interbreeding.

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

PubMed

Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

2016-02-24

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Digital camera and smartphone as detectors in paper-based chemiluminometric genotyping of single nucleotide polymorphisms.

PubMed

Spyrou, Elena M; Kalogianni, Despina P; Tragoulias, Sotirios S; Ioannou, Penelope C; Christopoulos, Theodore K

2016-10-01

Chemi(bio)luminometric assays have contributed greatly to various areas of nucleic acid analysis due to their simplicity and detectability. In this work, we present the development of chemiluminometric genotyping methods in which (a) detection is performed by using either a conventional digital camera (at ambient temperature) or a smartphone and (b) a lateral flow assay configuration is employed for even higher simplicity and suitability for point of care or field testing. The genotyping of the C677T single nucleotide polymorphism (SNP) of methylenetetrahydropholate reductase (MTHFR) gene is chosen as a model. The interrogated DNA sequence is amplified by polymerase chain reaction (PCR) followed by a primer extension reaction. The reaction products are captured through hybridization on the sensing areas (spots) of the strip. Streptavidin-horseradish peroxidase conjugate is used as a reporter along with a chemiluminogenic substrate. Detection of the emerging chemiluminescence from the sensing areas of the strip is achieved by digital camera or smartphone. For this purpose, we constructed a 3D-printed smartphone attachment that houses inexpensive lenses and converts the smartphone into a portable chemiluminescence imager. The device enables spatial discrimination of the two alleles of a SNP in a single shot by imaging of the strip, thus avoiding the need of dual labeling. The method was applied successfully to genotyping of real clinical samples. Graphical abstract Paper-based genotyping assays using digital camera and smartphone as detectors.

Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates.

PubMed

Curtin, C; Kennedy, E; Henschke, P A

2012-07-01

The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. A published microplate-based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Molecular mapping and breeding with microsatellite markers.

PubMed

Lightfoot, David A; Iqbal, Muhammad J

2013-01-01

In genetics databases for crop plant species across the world, there are thousands of mapped loci that underlie quantitative traits, oligogenic traits, and simple traits recognized by association mapping in populations. The number of loci will increase as new phenotypes are measured in more diverse genotypes and genetic maps based on saturating numbers of markers are developed. A period of locus reevaluation will decrease the number of important loci as those underlying mega-environmental effects are recognized. A second wave of reevaluation of loci will follow from developmental series analysis, especially for harvest traits like seed yield and composition. Breeding methods to properly use the accurate maps of QTL are being developed. New methods to map, fine map, and isolate the genes underlying the loci will be critical to future advances in crop biotechnology. Microsatellite markers are the most useful tool for breeders. They are codominant, abundant in all genomes, highly polymorphic so useful in many populations, and both economical and technically easy to use. The selective genotyping approaches, including genotype ranking (indexing) based on partial phenotype data combined with favorable allele data and bulked segregation event (segregant) analysis (BSA), will be increasingly important uses for microsatellites. Examples of the methods for developing and using microsatellites derived from genomic sequences are presented for monogenic, oligogenic, and polygenic traits. Examples of successful mapping, fine mapping, and gene isolation are given. When combined with high-throughput methods for genotyping and a genome sequence, the use of association mapping with microsatellite markers will provide critical advances in the analysis of crop traits.

Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

PubMed Central

Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R.; Kale, Mohan M.

2016-01-01

Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae). There are four serotypes of Dengue Virus (DENV-1 to DENV-4), each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis revealed that the worldwide population of DENV-2 strains is subdivided into fifteen lineages. The population structure of DENV-2 is spatiotemporal and is shaped by episodic positive selection and recombination. Intra-genotype diversity was observed in four genotypes (Asian/American, Asian I, cosmopolitan and sylvatic). Episodic positive selection on envelope and non-structural genes translates into antigenic diversity and appears to be responsible for emergence of strains/lineages in DENV-2 genotypes. Understanding of the genotype diversity and emerging lineages will be useful to design strategies for epidemiological surveillance and vaccine design. PMID:27635316

Genotypic analysis of human immunodeficiency virus type 1 env V3 loop sequences: bioinformatics prediction of coreceptor usage among 28 infected mother-infant pairs in a drug-naive population.

PubMed

Duri, Kerina; Soko, White; Gumbo, Felicity; Kristiansen, Knut; Mapingure, Munyaradzi; Stray-Pedersen, Babill; Muller, Fredrik

2011-04-01

We sought to predict virus coreceptor utilization using a simple bioinformatics method based on genotypic analysis of human immunodeficiency virus types 1 (HIV-1) env V3 loop sequences of 28 infected but drug-naive women during pregnancy and their infected infants and to better understand coreceptor usage in vertical transmission dynamics. The HIV-1 env V3 loop was sequenced from plasma samples and analyzed for viral coreceptor usage and subtype in a cohort of HIV-1-infected pregnant women. Predicted maternal frequencies of the X4, R5X4, and R5 genotypes were 7%, 11%, and 82%, respectively. Antenatal plasma viral load was higher, with a mean log(10) (SD) of 4.8 (1.6) and 3.6 (1.2) for women with the X4 and R5 genotypes, respectively, p = 0.078. Amino acid substitution from the conserved V3 loop crown motif GPGQ to GPGR and lymphadenopathy were associated with the X4 genotype, p = 0.031 and 0.043, respectively. The maternal viral coreceptor genotype was generally preserved in vertical transmission and was predictive of the newborn's viral genotype. Infants born to mothers with X4 genotypes were more likely to have lower birth weights relative to those born to mothers with the R5 genotype, with a mean weight (SD) of 2870 (±332) and 3069 (±300) g, respectively. These data show that at least in HIV-1 subtype C, R5 coreceptor usage is the most predominant genotype, which is generally preserved following vertical transmission and is associated with the V3 GPGQ crown motif. Therefore, antiretroviral-naive pregnant women and their infants can benefit from ARV combination therapies that include R5 entry inhibitors following prediction of their coreceptor genotype using simple bioinformatics methods.

A Partial Least Square Approach for Modeling Gene-gene and Gene-environment Interactions When Multiple Markers Are Genotyped

PubMed Central

Wang, Tao; Ho, Gloria; Ye, Kenny; Strickler, Howard; Elston, Robert C.

2008-01-01

Genetic association studies achieve an unprecedented level of resolution in mapping disease genes by genotyping dense SNPs in a gene region. Meanwhile, these studies require new powerful statistical tools that can optimally handle a large amount of information provided by genotype data. A question that arises is how to model interactions between two genes. Simply modeling all possible interactions between the SNPs in two gene regions is not desirable because a greatly increased number of degrees of freedom can be involved in the test statistic. We introduce an approach to reduce the genotype dimension in modeling interactions. The genotype compression of this approach is built upon the information on both the trait and the cross-locus gametic disequilibrium between SNPs in two interacting genes, in such a way as to parsimoniously model the interactions without loss of useful information in the process of dimension reduction. As a result, it improves power to detect association in the presence of gene-gene interactions. This approach can be similarly applied for modeling gene-environment interactions. We compare this method with other approaches: the corresponding test without modeling any interaction, that based on a saturated interaction model, that based on principal component analysis, and that based on Tukey’s 1-df model. Our simulations suggest that this new approach has superior power to that of the other methods. In an application to endometrial cancer case-control data from the Women’s Health Initiative (WHI), this approach detected AKT1 and AKT2 as being significantly associated with endometrial cancer susceptibility by taking into account their interactions with BMI. PMID:18615621

A partial least-square approach for modeling gene-gene and gene-environment interactions when multiple markers are genotyped.

PubMed

Wang, Tao; Ho, Gloria; Ye, Kenny; Strickler, Howard; Elston, Robert C

2009-01-01

Genetic association studies achieve an unprecedented level of resolution in mapping disease genes by genotyping dense single nucleotype polymorphisms (SNPs) in a gene region. Meanwhile, these studies require new powerful statistical tools that can optimally handle a large amount of information provided by genotype data. A question that arises is how to model interactions between two genes. Simply modeling all possible interactions between the SNPs in two gene regions is not desirable because a greatly increased number of degrees of freedom can be involved in the test statistic. We introduce an approach to reduce the genotype dimension in modeling interactions. The genotype compression of this approach is built upon the information on both the trait and the cross-locus gametic disequilibrium between SNPs in two interacting genes, in such a way as to parsimoniously model the interactions without loss of useful information in the process of dimension reduction. As a result, it improves power to detect association in the presence of gene-gene interactions. This approach can be similarly applied for modeling gene-environment interactions. We compare this method with other approaches, the corresponding test without modeling any interaction, that based on a saturated interaction model, that based on principal component analysis, and that based on Tukey's one-degree-of-freedom model. Our simulations suggest that this new approach has superior power to that of the other methods. In an application to endometrial cancer case-control data from the Women's Health Initiative, this approach detected AKT1 and AKT2 as being significantly associated with endometrial cancer susceptibility by taking into account their interactions with body mass index.

Rapid diagnosis of common deletional α-thalassemia in the Chinese population by qPCR based on identical primer homologous fragments.

PubMed

Long, Ju

2016-05-01

In China, -(SEA), -α(3.7) and -α(4.2) are common deletional α-thalassemia alleles. Gap-PCR is the currently used detection method for these alleles, whose disadvantages include time-consuming procedure and increased potential for PCR product contamination. Therefore, this detection method needs to be improved. Based on identical-primer homologous fragments, a qPCR system was developed for deletional α-thalassemia genotyping, which was composed of a group of quantitatively-related primers and their corresponding probes plus two groups of qualitatively-related primers and their corresponding probes. In order to verify the accuracy of the qPCR system, known genotype samples and random samples are employed. The standard curve result demonstrated that designed primers and probes all yielded good amplification efficiency. In the tests of known genotype samples and random samples, sample detection results were consistent with verification results. In detecting αα, -(SEA), -α(3.7) and -α(4.2) alleles, deletional α-thalassemia alleles are accurately detected by this method. In addition, this method is provided with a wider detection range, greater speed and reduced PCR product contamination risk when compared with current common gap-PCR detection reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Lactobacillus from Algerian Goat’S Milk Based on Phenotypic, 16S rDNA Sequencing and their Technological Properties

PubMed Central

Marroki, Ahmed; Zúñiga, Manuel; Kihal, Mabrouk; Pérez- Martínez, Gaspar

2011-01-01

Nineteen strains of Lactobacillus isolated from goat’s milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. PMID:24031617

Joint genotype- and ancestry-based genome-wide association studies in admixed populations.

PubMed

Szulc, Piotr; Bogdan, Malgorzata; Frommlet, Florian; Tang, Hua

2017-09-01

In genome-wide association studies (GWAS) genetic loci that influence complex traits are localized by inspecting associations between genotypes of genetic markers and the values of the trait of interest. On the other hand, admixture mapping, which is performed in case of populations consisting of a recent mix of two ancestral groups, relies on the ancestry information at each locus (locus-specific ancestry). Recently it has been proposed to jointly model genotype and locus-specific ancestry within the framework of single marker tests. Here, we extend this approach for population-based GWAS in the direction of multimarker models. A modified version of the Bayesian information criterion is developed for building a multilocus model that accounts for the differential correlation structure due to linkage disequilibrium (LD) and admixture LD. Simulation studies and a real data example illustrate the advantages of this new approach compared to single-marker analysis or modern model selection strategies based on separately analyzing genotype and ancestry data, as well as to single-marker analysis combining genotypic and ancestry information. Depending on the signal strength, our procedure automatically chooses whether genotypic or locus-specific ancestry markers are added to the model. This results in a good compromise between the power to detect causal mutations and the precision of their localization. The proposed method has been implemented in R and is available at http://www.math.uni.wroc.pl/~mbogdan/admixtures/. © 2017 WILEY PERIODICALS, INC.

Genotyping of polyomavirus BK by Real Time PCR for VP1 gene.

PubMed

Gambarino, Stefano; Costa, Cristina; Astegiano, Sara; Piasentin, Elsa Alessio; Segoloni, Giuseppe P; Cavallo, Rossana; Bergallo, Massimiliano

2011-10-01

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I-IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and "hot spots" of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.

High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

PubMed

Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

2013-01-01

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. Copyright © 2012 Wiley Periodicals, Inc.

The transmission/disequilibrium test and parental-genotype reconstruction: the reconstruction-combined transmission/ disequilibrium test.

PubMed Central

Knapp, M

1999-01-01

Spielman and Ewens recently proposed a method for testing a marker for linkage with a disease, which combines data from families with and without information on parental genotypes. For some families without parental-genotype information, it may be possible to reconstruct missing parental genotypes from the genotypes of their offspring. The treatment of such a reconstructed family as if parental genotypes have been typed, however, can introduce bias. In the present study, a new method is presented that employs parental-genotype reconstruction and corrects for the biases resulting from reconstruction. The results of an application of this method to a real data set and of a simulation study suggest that this approach may increase the power to detect linkage. PMID:10053021

Genetic divergence of physiological-quality traits of seeds in a population of peppers.

PubMed

Pessoa, A M S; Barroso, P A; do Rêgo, E R; Medeiros, G D A; Bruno, R L A; do Rêgo, M M

2015-10-16

Brazil has a great diversity of Capsicum peppers that can be used in breeding programs. The objective of this study was to evaluate genetic variation in traits related to the physiological quality of seeds of Capsicum annuum L. in a segregating F2 population and its parents. A total of 250 seeds produced by selfing in the F1 generation resulting from crosses between UFPB 77.3 and UFPB 76 were used, with 100 seeds of both parents used as additional controls, totaling 252 genotypes. The seeds were germinated in gerboxes containing substrate blotting paper moistened with distilled water. Germination and the following vigor tests were evaluated: first count, germination velocity index, and root and shoot lengths. Data were subjected to analysis of variance, and means were compared by Scott and Knott's method at 1% probability. Tocher's clustering based on Mahalanobis distance and canonical variable analysis with graphic dispersion of genotypes were performed, and genetic parameters were estimated. All variables were found to be significant by the F test (P ≤ 0.01) and showed high heritability and a CVg/CVe ratio higher than 1.0, indicating genetic differences among genotypes. Parents (genotypes 1 and 2) formed distinct groups in all clustering methods. Genotypes 3, 104, 153, and 232 were found to be the most divergent according to Tocher's clustering method, and this was mainly due to early germination, which was observed on day 14, and would therefore be selected. Understanding the phenotypic variability among these 252 genotypes will serve as a basis for continuing the breeding program within this family.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

PubMed

Merker, Matthias; Kohl, Thomas A; Niemann, Stefan; Supply, Philip

2017-01-01

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

PubMed

Shirasu, Naoto; Kuroki, Masahide

2014-01-01

We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

Quantitative analysis of low-density SNP data for parentage assignment and estimation of family contributions to pooled samples.

PubMed

Henshall, John M; Dierens, Leanne; Sellars, Melony J

2014-09-02

While much attention has focused on the development of high-density single nucleotide polymorphism (SNP) assays, the costs of developing and running low-density assays have fallen dramatically. This makes it feasible to develop and apply SNP assays for agricultural species beyond the major livestock species. Although low-cost low-density assays may not have the accuracy of the high-density assays widely used in human and livestock species, we show that when combined with statistical analysis approaches that use quantitative instead of discrete genotypes, their utility may be improved. The data used in this study are from a 63-SNP marker Sequenom® iPLEX Platinum panel for the Black Tiger shrimp, for which high-density SNP assays are not currently available. For quantitative genotypes that could be estimated, in 5% of cases the most likely genotype for an individual at a SNP had a probability of less than 0.99. Matrix formulations of maximum likelihood equations for parentage assignment were developed for the quantitative genotypes and also for discrete genotypes perturbed by an assumed error term. Assignment rates that were based on maximum likelihood with quantitative genotypes were similar to those based on maximum likelihood with perturbed genotypes but, for more than 50% of cases, the two methods resulted in individuals being assigned to different families. Treating genotypes as quantitative values allows the same analysis framework to be used for pooled samples of DNA from multiple individuals. Resulting correlations between allele frequency estimates from pooled DNA and individual samples were consistently greater than 0.90, and as high as 0.97 for some pools. Estimates of family contributions to the pools based on quantitative genotypes in pooled DNA had a correlation of 0.85 with estimates of contributions from DNA-derived pedigree. Even with low numbers of SNPs of variable quality, parentage testing and family assignment from pooled samples are sufficiently accurate to provide useful information for a breeding program. Treating genotypes as quantitative values is an alternative to perturbing genotypes using an assumed error distribution, but can produce very different results. An understanding of the distribution of the error is required for SNP genotyping platforms.

Exome sequencing of a multigenerational human pedigree.

PubMed

Hedges, Dale J; Hedges, Dale; Burges, Dan; Powell, Eric; Almonte, Cherylyn; Huang, Jia; Young, Stuart; Boese, Benjamin; Schmidt, Mike; Pericak-Vance, Margaret A; Martin, Eden; Zhang, Xinmin; Harkins, Timothy T; Züchner, Stephan

2009-12-14

Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.

Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.

PubMed

Cowan, Lauren S; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

Using Propidium Monoazide as an Enrichment Step to Remove Extraneous DMA and Non-Viable Organisms for Cryptosporidium Genotyping

EPA Science Inventory

Cryptosporidium is an important protozoan parasite that continues to cause waterborne disease outbreaks worldwide. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in s...

Improving accuracy of genomic prediction in Brangus cattle by adding animals with imputed low-density SNP genotypes.

PubMed

Lopes, F B; Wu, X-L; Li, H; Xu, J; Perkins, T; Genho, J; Ferretti, R; Tait, R G; Bauck, S; Rosa, G J M

2018-02-01

Reliable genomic prediction of breeding values for quantitative traits requires the availability of sufficient number of animals with genotypes and phenotypes in the training set. As of 31 October 2016, there were 3,797 Brangus animals with genotypes and phenotypes. These Brangus animals were genotyped using different commercial SNP chips. Of them, the largest group consisted of 1,535 animals genotyped by the GGP-LDV4 SNP chip. The remaining 2,262 genotypes were imputed to the SNP content of the GGP-LDV4 chip, so that the number of animals available for training the genomic prediction models was more than doubled. The present study showed that the pooling of animals with both original or imputed 40K SNP genotypes substantially increased genomic prediction accuracies on the ten traits. By supplementing imputed genotypes, the relative gains in genomic prediction accuracies on estimated breeding values (EBV) were from 12.60% to 31.27%, and the relative gain in genomic prediction accuracies on de-regressed EBV was slightly small (i.e. 0.87%-18.75%). The present study also compared the performance of five genomic prediction models and two cross-validation methods. The five genomic models predicted EBV and de-regressed EBV of the ten traits similarly well. Of the two cross-validation methods, leave-one-out cross-validation maximized the number of animals at the stage of training for genomic prediction. Genomic prediction accuracy (GPA) on the ten quantitative traits was validated in 1,106 newly genotyped Brangus animals based on the SNP effects estimated in the previous set of 3,797 Brangus animals, and they were slightly lower than GPA in the original data. The present study was the first to leverage currently available genotype and phenotype resources in order to harness genomic prediction in Brangus beef cattle. © 2018 Blackwell Verlag GmbH.

Potential use of random amplified polymorphic DNA (RAPD) technique to study the genetic diversity in Indian mustard (Brassica juncea) and its relationship to heterosis.

PubMed

Jain, A; Bhatia, S; Banga, S S; Prakash, S; Lakshmikumaran, M

1994-04-01

RAPD assays were performed, using 34 arbitrary decamer oligonucleotide primers and six combinations of two primers, to detect inherent variations and genetic relationships among 12 Indian and 11 exotic B. juncea genotypes. Of 595 amplification products identified, 500 of them were polymorphic across all genotypes. A low level of genetic variability was detected among the Indian genotypes, while considerable polymorphism was present among the exotic ones. Based on the pair-wise comparisons of amplification products the genetic similarity was calculated using Jaccard's similarity coefficients and a dendrogram was constructed using an unweighted pair group method was arithmetical averages (UPGMA). On the basis of this analysis the genotypes were clustered into two groups, A and B. Group A comprised only exotic genotypes, whereas all the Indian genotypes and four of the exotic genotypes were clustered in group B. Almost similar genotypic rankings could also be established by computing as few as 200 amplification products. In general, a high per cent of heterosis was recorded in crosses involving Indian x exotic genotypes. On the other hand, when crosses were made amongst Indian or exotic genotypes, about 80% of them exhibited negative heterosis. Results from this study indicate that, despite the lack of direct correlation between the genetic distance and the degree of heterosis, genetic diversity forms a very useful guide not only for investigating the relationships among Brassica genotypes but also in the selection of parents for heterotic hybrid combinations.

Haplotype estimation using sequencing reads.

PubMed

Delaneau, Olivier; Howie, Bryan; Cox, Anthony J; Zagury, Jean-François; Marchini, Jonathan

2013-10-03

High-throughput sequencing technologies produce short sequence reads that can contain phase information if they span two or more heterozygote genotypes. This information is not routinely used by current methods that infer haplotypes from genotype data. We have extended the SHAPEIT2 method to use phase-informative sequencing reads to improve phasing accuracy. Our model incorporates the read information in a probabilistic model through base quality scores within each read. The method is primarily designed for high-coverage sequence data or data sets that already have genotypes called. One important application is phasing of single samples sequenced at high coverage for use in medical sequencing and studies of rare diseases. Our method can also use existing panels of reference haplotypes. We tested the method by using a mother-father-child trio sequenced at high-coverage by Illumina together with the low-coverage sequence data from the 1000 Genomes Project (1000GP). We found that use of phase-informative reads increases the mean distance between switch errors by 22% from 274.4 kb to 328.6 kb. We also used male chromosome X haplotypes from the 1000GP samples to simulate sequencing reads with varying insert size, read length, and base error rate. When using short 100 bp paired-end reads, we found that using mixtures of insert sizes produced the best results. When using longer reads with high error rates (5-20 kb read with 4%-15% error per base), phasing performance was substantially improved. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Optimized pH method for DNA elution from buccal cells collected in Whatman FTA cards.

PubMed

Lema, Carolina; Kohl-White, Kendra; Lewis, Laurie R; Dao, Dat D

2006-01-01

DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.

Identification of soybean genotypes with high stability for the Brazilian macro-region 402 via biplot analysis.

PubMed

Junior, E U Ramos; Brogin, R L; Godinho, V P C; Botelho, F J E; Tardin, F D; Teodoro, P E

2017-09-27

Biplot analysis has often been used to recommend genotypes from different crops in the presence of the genotype x environment interaction (GxE). The objective of this study was to verify the association between the AMMI and GGE biplot methods and to select soybean genotypes that simultaneously meet high grain yield and stability to the environments belonging to the Edaphoclimatic Region 402, from Soybean Cultivation Region 4 (Mid-West), which comprises the Center North and West of Mato Grosso, and the southern region of Rondônia. Grain yield of 12 soybean genotypes was evaluated in seven competition trials of soybean cultivars in the 2014/2015 harvest. Significant GxE interaction revealed the need to use methods for recommending genotypes with adaptability and yield stability. The methods were complementary regarding the recommendation of the best genotypes. The AMMI analysis recommended MG/BR46 (Conquista) (G10) widely for all environments evaluated, whereas the BRY23-55012 (G9) and BRAS11-0149 (G2) were the most indicated genotypes by the GGE biplot method. However, the methods were concordant as to Porto Velho (PV1) environment that contributed least to the GxE interaction.

DISTMIX: direct imputation of summary statistics for unmeasured SNPs from mixed ethnicity cohorts.

PubMed

Lee, Donghyung; Bigdeli, T Bernard; Williamson, Vernell S; Vladimirov, Vladimir I; Riley, Brien P; Fanous, Ayman H; Bacanu, Silviu-Alin

2015-10-01

To increase the signal resolution for large-scale meta-analyses of genome-wide association studies, genotypes at unmeasured single nucleotide polymorphisms (SNPs) are commonly imputed using large multi-ethnic reference panels. However, the ever increasing size and ethnic diversity of both reference panels and cohorts makes genotype imputation computationally challenging for moderately sized computer clusters. Moreover, genotype imputation requires subject-level genetic data, which unlike summary statistics provided by virtually all studies, is not publicly available. While there are much less demanding methods which avoid the genotype imputation step by directly imputing SNP statistics, e.g. Directly Imputing summary STatistics (DIST) proposed by our group, their implicit assumptions make them applicable only to ethnically homogeneous cohorts. To decrease computational and access requirements for the analysis of cosmopolitan cohorts, we propose DISTMIX, which extends DIST capabilities to the analysis of mixed ethnicity cohorts. The method uses a relevant reference panel to directly impute unmeasured SNP statistics based only on statistics at measured SNPs and estimated/user-specified ethnic proportions. Simulations show that the proposed method adequately controls the Type I error rates. The 1000 Genomes panel imputation of summary statistics from the ethnically diverse Psychiatric Genetic Consortium Schizophrenia Phase 2 suggests that, when compared to genotype imputation methods, DISTMIX offers comparable imputation accuracy for only a fraction of computational resources. DISTMIX software, its reference population data, and usage examples are publicly available at http://code.google.com/p/distmix. dlee4@vcu.edu Supplementary Data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Genetic Diversity Analysis of Highly Incomplete SNP Genotype Data with Imputations: An Empirical Assessment

PubMed Central

Fu, Yong-Bi

2014-01-01

Genotyping by sequencing (GBS) recently has emerged as a promising genomic approach for assessing genetic diversity on a genome-wide scale. However, concerns are not lacking about the uniquely large unbalance in GBS genotype data. Although some genotype imputation has been proposed to infer missing observations, little is known about the reliability of a genetic diversity analysis of GBS data, with up to 90% of observations missing. Here we performed an empirical assessment of accuracy in genetic diversity analysis of highly incomplete single nucleotide polymorphism genotypes with imputations. Three large single-nucleotide polymorphism genotype data sets for corn, wheat, and rice were acquired, and missing data with up to 90% of missing observations were randomly generated and then imputed for missing genotypes with three map-independent imputation methods. Estimating heterozygosity and inbreeding coefficient from original, missing, and imputed data revealed variable patterns of bias from assessed levels of missingness and genotype imputation, but the estimation biases were smaller for missing data without genotype imputation. The estimates of genetic differentiation were rather robust up to 90% of missing observations but became substantially biased when missing genotypes were imputed. The estimates of topology accuracy for four representative samples of interested groups generally were reduced with increased levels of missing genotypes. Probabilistic principal component analysis based imputation performed better in terms of topology accuracy than those analyses of missing data without genotype imputation. These findings are not only significant for understanding the reliability of the genetic diversity analysis with respect to large missing data and genotype imputation but also are instructive for performing a proper genetic diversity analysis of highly incomplete GBS or other genotype data. PMID:24626289

Malaria in pregnant Cameroonian women: the effect of age and gravidity on submicroscopic and mixed-species infections and multiple parasite genotypes.

PubMed

Walker-Abbey, Annie; Djokam, Rosine R T; Eno, Anna; Leke, Rose F G; Titanji, Vincent P K; Fogako, Josephine; Sama, Grace; Thuita, Lucy H; Beardslee, Eliza; Snounou, Georges; Zhou, Ainong; Taylor, Diane Wallace

2005-03-01

Polymerase chain reaction (PCR)-based methods were used to investigate malaria in pregnant women residing in Yaounde, Cameroon. Microscopy and species-specific PCR-based diagnosis show that at delivery 82.4% of the women were infected with Plasmodium falciparum (27.5% blood-smear positive and 54.9% submicroscopic infections). The prevalence of P. malariae and P. ovale was 7.6% and 2.5%, respectively, with 9.4% infected with more than one species. Based on genotyping of the merozoite surface protein 1 (msp-1) and msp-2 alleles, the mean number of genetically different P. falciparum parasites in peripheral blood was 3.4 (range = 1-9) and 3.5 (range 1-8) in the placenta. Plasmodium falciparum detected by microscopy and PCR as well as mixed-species infections were significantly higher in women < or = 20 years old and paucigravidae, but maternal anemia was associated only with microscopic detection of parasites. Neither submicroscopic infections nor number of parasite genotypes decreased significantly with age or gravidity. Thus, pregnancy-associated immunity helps reduce malaria to submicroscopic levels, but does not reduce the number of circulating parasite genotypes.

Bayesian Multi-Trait Analysis Reveals a Useful Tool to Increase Oil Concentration and to Decrease Toxicity in Jatropha curcas L.

PubMed Central

Silva Junqueira, Vinícius; de Azevedo Peixoto, Leonardo; Galvêas Laviola, Bruno; Lopes Bhering, Leonardo; Mendonça, Simone; Agostini Costa, Tania da Silveira; Antoniassi, Rosemar

2016-01-01

The biggest challenge for jatropha breeding is to identify superior genotypes that present high seed yield and seed oil content with reduced toxicity levels. Therefore, the objective of this study was to estimate genetic parameters for three important traits (weight of 100 seed, oil seed content, and phorbol ester concentration), and to select superior genotypes to be used as progenitors in jatropha breeding. Additionally, the genotypic values and the genetic parameters estimated under the Bayesian multi-trait approach were used to evaluate different selection indices scenarios of 179 half-sib families. Three different scenarios and economic weights were considered. It was possible to simultaneously reduce toxicity and increase seed oil content and weight of 100 seed by using index selection based on genotypic value estimated by the Bayesian multi-trait approach. Indeed, we identified two families that present these characteristics by evaluating genetic diversity using the Ward clustering method, which suggested nine homogenous clusters. Future researches must integrate the Bayesian multi-trait methods with realized relationship matrix, aiming to build accurate selection indices models. PMID:27281340

Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.).

PubMed

Kamel, Katarzyna A; Kroc, Magdalena; Święcicki, Wojciech

2015-01-01

Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lupinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.

Optimization of Benzoisothiazole dioxide inhibitory activity of the NS5B polymerase of HCV genotype 4 using ligand-steered homological modeling, reaction-driven scaffold-hopping and Enovo workflow.

PubMed

Mahmoud, Amr Hamed; Mohamed Abouzid, Khaled Abouzid; El Ella, Dalal Abd El Rahman Abou; Hamid Ismail, Mohamed Abdel

2011-01-01

Infection caused by hepatitis C virus (HCV) is a significant world health problem for which novel therapies are in urgent demand. The virus is highly prevalent in the Middle East and Africa particularly Egypt with more than 90% of infections due to genotype 4. Nonstructural (NS5B) viral proteins have emerged as an attractive target for HCV antivirals discovery. A potent class of inhibitors having benzisothiazole dioxide scaffold has been identified on this target, however they were mainly active on genotype 1 while exhibiting much lowered activity on other genotypes due to the high degree of mutation of its binding site. Based on this fact, we employed a novel strategy to optimize this class on genotype 4. This strategy depends on using a refined ligand-steered homological model of this genotype to study the mutation binding energies of the binding site amino acid residues, the essential features for interaction and provide a structure-based pharmacophore model that can aid optimization. This model was applied on a focused library which was generated using a reaction-driven scaffold-hopping strategy. The hits retrieved were subjected to Enovo pipeline pilot optimization workflow that employs R-group enumeration, core-constrained protein docking using modified CDOCKER and finally ranking of poses using an accurate molecular mechanics generalized Born with surface area method.

S-genotype identification based on allele-specific PCR in Japanese pear

PubMed Central

Nashima, Kenji; Terakami, Shingo; Nishio, Sogo; Kunihisa, Miyuki; Nishitani, Chikako; Saito, Toshihiro; Yamamoto, Toshiya

2015-01-01

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs. PMID:26175617

First insights into species and genotypes of Echinococcus in South Africa.

PubMed

Mogoye, Benjamin K; Menezes, Colin N; Wong, Michelle L; Stacey, Sarah; von Delft, Dirk; Wahlers, Kerstin; Wassermann, Marion; Romig, Thomas; Kern, Peter; Grobusch, Martin P; Frean, John

2013-09-23

Cystic echinococcosis is a serious and neglected parasitic zoonosis that is regarded as an emerging disease world-wide. Effective control of the disease is based on understanding the variability of Echinococcus granulosus (sensu lato), as genotypic characteristics may influence lifecycle patterns, development rate, and transmission. No molecular epidemiological research has previously been conducted to shed light on genotypes responsible for the disease in South Africa. To identify strains circulating in the country, parasite material was collected from patients between August 2010 and September 2012 and analyzed by PCR/RFLP methods. A total of 32 samples was characterized as E. granulosus sensu stricto (G1-G3) (81%), E. canadensis (G6/7) (16%) and E. ortleppi (G5) (3%). Furthermore, two co-amplifying G6/7 genotypes were confirmed as G7 by sequencing. This is the first report on genotyping of Echinococcus species in South Africa, and, to the best of our knowledge, the first report of the G5 and G7 genotypes from humans in Africa. Copyright © 2013 Elsevier B.V. All rights reserved.

Hepatitis C virus therapy with peg-interferon and ribavirin in Myanmar: A resource-constrained country

PubMed Central

Hlaing, Naomi Khaing Than; Banerjee, Debolina; Mitrani, Robert; Arker, Soe Htet; Win, Kyaw San; Tun, Nyan Lin; Thant, Zaw; Win, Khin Maung; Reddy, K Rajender

2016-01-01

AIM To investigate peg-interferon (peg-IFN) and ribavirin (RBV) therapy in Myanmar and to predict sustained virologic response (SVR). METHODS This single-center, open-label, study was conducted in Myanmar between 2009 and 2014. A total of 288 patients infected with HCV genotypes 1, 2, 3 and 6 were treated with peg-IFN alpha-2a (180 μg/wk) or alpha-2b (50 to 100 μg as a weight-based dose) and RBV as a weight-based dose (15 mg/kg/d). Treatment duration was 48 wk for genotypes 1 and 6, 24 wk for genotype 2, and 24 or 48 wk for genotype 3 based on rapid virologic response (RVR). Those co-infected with hepatitis B received 48 wk of therapy. RESULTS Overall, SVR was achieved for 82% of patients and the therapy was well tolerated. All patients achieved SVR at equivalent rates regardless of HCV genotype (P = 0.314). Low fibrosis scores (P < 0.001), high baseline albumin levels (P = 0.028) and low baseline viral loads (P = 0.029) all independently predicted SVR. On the other hand, IL-28B TT and CC genotypes were not found to significantly predict SVR (P = 0.634; P = 0.618). Among those who completed treatment, the occurrence of RVR showed a > 96% positive predictive value for achieving SVR. Treatment duration did not significantly impact the likelihood of achieving SVR for patients infected with genotype 3 HCV (P = 0.371). The most common adverse events were fatigue (71%) and poor appetite (60%). Among patients with genotype 3 HCV, more patients in the 48-wk treatment group required erythropoietin than in the 24-wk treatment group (61.1% vs 49.2%). CONCLUSION SVR rates were high with peg-IFN and RBV therapy in Myanmar. Fibrosis scores, baseline albumin, HCV RNA levels and RVR independently predicted SVR. PMID:27920482

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

PubMed

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz; Strapagiel, Dominik

2017-11-03

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

PubMed

Valverde, Estefanía J; Cano, Irene; Castro, Dolores; Paley, Richard K; Borrego, Juan J

2017-03-01

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

Association of ACE, FABP2 and GST genes polymorphism with essential hypertension risk among a North Indian population.

PubMed

Abbas, Shania; Raza, Syed Tasleem; Chandra, Anu; Rizvi, Saliha; Ahmed, Faisal; Eba, Ale; Mahdi, Farzana

2015-01-01

Hypertension has a multi-factorial background based on genetic and environmental interactive factors. ACE, FABP2 and GST genes have been suggested to be involved in the development of hypertension. However, the results have been inconsistent. The present study was carried out to investigate the association of ACE (rs4646994), FABP2 (rs1799883) and GST (GSTM1 null or positive genotype and GSTT1 null or positive genotype) genes polymorphism with essential HTN cases and controls. This study includes 138 essential hypertension (HTN) patients and 116 age-, sex- and ethnicity-matched control subjects. GST (GSTM1 null or positive genotype and GSTT1 null or positive genotype) genes polymorphisms were evaluated by multiplex PCR, ACE (rs4646994) gene polymorphisms by PCR and FABP2 (rs1799883) gene polymorphisms by PCR-RFLP method. Significant differences were obtained in the frequencies of ACE DD, II genotype (p = 0.006, 0.003), GSTT1 null, GSTM1 positive genotype (p = 0.048, 0.010) and FABP2 Ala54/Ala54 genotype (p = 0.049) between essential HTN cases and controls. It is concluded that ACE (rs 4646994), FABP2 (rs1799883) and GST (GSTM1 null or positive genotype and GSTT1 null or positive genotype) genes polymorphism are associated with HTN. Further investigation with a larger sample size may be required to validate this study.

HLA genotyping by next-generation sequencing of complementary DNA.

PubMed

Segawa, Hidenobu; Kukita, Yoji; Kato, Kikuya

2017-11-28

Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA. Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database. The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology. The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Modeling and E-M estimation of haplotype-specific relative risks from genotype data for a case-control study of unrelated individuals.

PubMed

Stram, Daniel O; Leigh Pearce, Celeste; Bretsky, Phillip; Freedman, Matthew; Hirschhorn, Joel N; Altshuler, David; Kolonel, Laurence N; Henderson, Brian E; Thomas, Duncan C

2003-01-01

The US National Cancer Institute has recently sponsored the formation of a Cohort Consortium (http://2002.cancer.gov/scpgenes.htm) to facilitate the pooling of data on very large numbers of people, concerning the effects of genes and environment on cancer incidence. One likely goal of these efforts will be generate a large population-based case-control series for which a number of candidate genes will be investigated using SNP haplotype as well as genotype analysis. The goal of this paper is to outline the issues involved in choosing a method of estimating haplotype-specific risk estimates for such data that is technically appropriate and yet attractive to epidemiologists who are already comfortable with odds ratios and logistic regression. Our interest is to develop and evaluate extensions of methods, based on haplotype imputation, that have been recently described (Schaid et al., Am J Hum Genet, 2002, and Zaykin et al., Hum Hered, 2002) as providing score tests of the null hypothesis of no effect of SNP haplotypes upon risk, which may be used for more complex tasks, such as providing confidence intervals, and tests of equivalence of haplotype-specific risks in two or more separate populations. In order to do so we (1) develop a cohort approach towards odds ratio analysis by expanding the E-M algorithm to provide maximum likelihood estimates of haplotype-specific odds ratios as well as genotype frequencies; (2) show how to correct the cohort approach, to give essentially unbiased estimates for population-based or nested case-control studies by incorporating the probability of selection as a case or control into the likelihood, based on a simplified model of case and control selection, and (3) finally, in an example data set (CYP17 and breast cancer, from the Multiethnic Cohort Study) we compare likelihood-based confidence interval estimates from the two methods with each other, and with the use of the single-imputation approach of Zaykin et al. applied under both null and alternative hypotheses. We conclude that so long as haplotypes are well predicted by SNP genotypes (we use the Rh2 criteria of Stram et al. [1]) the differences between the three methods are very small and in particular that the single imputation method may be expected to work extremely well. Copyright 2003 S. Karger AG, Basel

Theory-based pharmacokinetics and pharmacodynamics of S- and R-warfarin and effects on international normalized ratio: influence of body size, composition and genotype in cardiac surgery patients.

PubMed

Xue, Ling; Holford, Nick; Ding, Xiao-Liang; Shen, Zhen-Ya; Huang, Chen-Rong; Zhang, Hua; Zhang, Jing-Jing; Guo, Zhe-Ning; Xie, Cheng; Zhou, Ling; Chen, Zhi-Yao; Liu, Lin-Sheng; Miao, Li-Yan

2017-04-01

The aims of this study are to apply a theory-based mechanistic model to describe the pharmacokinetics (PK) and pharmacodynamics (PD) of S- and R-warfarin. Clinical data were obtained from 264 patients. Total concentrations for S- and R-warfarin were measured by ultra-high performance liquid tandem mass spectrometry. Genotypes were measured using pyrosequencing. A sequential population PK parameter with data method was used to describe the international normalized ratio (INR) time course. Data were analyzed with NONMEM. Model evaluation was based on parameter plausibility and prediction-corrected visual predictive checks. Warfarin PK was described using a one-compartment model. CYP2C9 *1/*3 genotype had reduced clearance for S-warfarin, but increased clearance for R-warfarin. The in vitro parameters for the relationship between prothrombin complex activity (PCA) and INR were markedly different (A = 0.560, B = 0.386) from the theory-based values (A = 1, B = 0). There was a small difference between healthy subjects and patients. A sigmoid E max PD model inhibiting PCA synthesis as a function of S-warfarin concentration predicted INR. Small R-warfarin effects was described by competitive antagonism of S-warfarin inhibition. Patients with VKORC1 AA and CYP4F2 CC or CT genotypes had lower C50 for S-warfarin. A theory-based PKPD model describes warfarin concentrations and clinical response. Expected PK and PD genotype effects were confirmed. The role of predicted fat free mass with theory-based allometric scaling of PK parameters was identified. R-warfarin had a minor effect compared with S-warfarin on PCA synthesis. INR is predictable from 1/PCA in vivo. © 2016 The British Pharmacological Society.

Genome-wide association tests of inversions with application to psoriasis

PubMed Central

Ma, Jianzhong; Xiong, Momiao; You, Ming; Lozano, Guillermina; Amos, Christopher I.

2014-01-01

Although inversions have occasionally been found to be associated with disease susceptibility through interrupting a gene or its regulatory region, or by increasing the risk for deleterious secondary rearrangements, no association study has been specifically conducted for risks associated with inversions, mainly because existing approaches to detecting and genotyping inversions do not readily scale to a large number of samples. Based on our recently proposed approach to identifying and genotyping inversions using principal components analysis (PCA), we herein develop a method of detecting association between inversions and disease in a genome-wide fashion. Our method uses genotype data for single nucleotide polymorphisms (SNPs), and is thus cost-efficient and computationally fast. For an inversion polymorphism, local PCA around the inversion region is performed to infer the inversion genotypes of all samples. For many inversions, we found that some of the SNPs inside an inversion region are fixed in the two lineages of different orientations and thus can serve as surrogate markers. Our method can be applied to case-control and quantitative trait association studies to identify inversions that may interrupt a gene or the connection between a gene and its regulatory agents. Our method also offers a new venue to identify inversions that are responsible for disease-causing secondary rearrangements. We illustrated our proposed approach to case-control data for psoriasis and identified novel associations with a few inversion polymorphisms. PMID:24623382

Genetic divergence in the common bean (Phaseolus vulgaris L.) in the Cerrado-Pantanal ecotone.

PubMed

da Silva, F A; Corrêa, A M; Teodoro, P E; Lopes, K V; Corrêa, C C G

2017-03-30

Evaluating genetic diversity among genotypes is important for providing parameters for the identification of superior genotypes, because the choice of parents that form segregating populations is crucial. Our objectives were to i) evaluate agronomic performance; ii) compare clustering methods; iii) ascertain the relative contributions of the variables evaluated; and iv) identify the most promising hybrids to produce superior segregating populations. The trial was conducted in 2015 at the State University of Mato Grosso do Sul, Brazil. We used a randomized block design with three replications, and recorded the days to emergence, days to flowering, days to maturity, plant height, number of branches, number of pods, number of seeds per pod, weight of 100 grains, and productivity. The genetic diversity of the genotypes was determined by cluster analysis using two dissimilarity measures: the Euclidean distance and the standardized mean Mahalanobis distance using the Ward hierarchical method. The genotypes 'CNFC 10762', 'IAC Dawn', and 'BRS Style' had the highest grain yields, and clusters that were based on the Euclidean distance differed from those based on the Mahalanobis distance, the second being more precise. The yield grain character has greater relevance to the dispute. Hybrids with a high heterotic effect can be obtained by crossing 'IAC Alvorada' with 'CNFC 10762', 'IAC Alvorada' with 'CNFC 10764', and 'BRS Style' with 'IAC Alvorada'.

Mathematical Ability and Socio-Economic Background: IRT Modeling to Estimate Genotype by Environment Interaction.

PubMed

Schwabe, Inga; Boomsma, Dorret I; van den Berg, Stéphanie M

2017-12-01

Genotype by environment interaction in behavioral traits may be assessed by estimating the proportion of variance that is explained by genetic and environmental influences conditional on a measured moderating variable, such as a known environmental exposure. Behavioral traits of interest are often measured by questionnaires and analyzed as sum scores on the items. However, statistical results on genotype by environment interaction based on sum scores can be biased due to the properties of a scale. This article presents a method that makes it possible to analyze the actually observed (phenotypic) item data rather than a sum score by simultaneously estimating the genetic model and an item response theory (IRT) model. In the proposed model, the estimation of genotype by environment interaction is based on an alternative parametrization that is uniquely identified and therefore to be preferred over standard parametrizations. A simulation study shows good performance of our method compared to analyzing sum scores in terms of bias. Next, we analyzed data of 2,110 12-year-old Dutch twin pairs on mathematical ability. Genetic models were evaluated and genetic and environmental variance components estimated as a function of a family's socio-economic status (SES). Results suggested that common environmental influences are less important in creating individual differences in mathematical ability in families with a high SES than in creating individual differences in mathematical ability in twin pairs with a low or average SES.

Transfer of a Mycobacterium tuberculosis Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System

PubMed Central

Cowan, Lauren S.; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T.

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay. PMID:14715809

Changing practice: red blood cell typing by molecular methods for patients with sickle cell disease.

PubMed

Casas, Jessica; Friedman, David F; Jackson, Tannoa; Vege, Sunitha; Westhoff, Connie M; Chou, Stella T

2015-06-01

Extended red blood cell (RBC) antigen matching is recommended to limit alloimmunization in patients with sickle cell disease (SCD). DNA-based testing to predict blood group phenotypes has enhanced availability of antigen-negative donor units and improved typing of transfused patients, but replacement of routine serologic typing for non-ABO antigens with molecular typing for patients has not been reported. This study compared the historical RBC antigen phenotypes obtained by hemagglutination methods with genotype predictions in 494 patients with SCD. For discrepant results, repeat serologic testing was performed and/or investigated by gene sequencing for silent or variant alleles. Seventy-one typing discrepancies were identified among 6360 antigen comparisons (1.1%). New specimens for repeat serologic testing were obtained for 66 discrepancies and retyping agreed with the genotype in 64 cases. One repeat Jk(b-) serologic phenotype, predicted Jk(b+) by genotype, was found by direct sequencing of JK to be a silenced allele, and one N typing discrepancy remains under investigation. Fifteen false-negative serologic results were associated with alleles encoding weak antigens or single-dose Fy(b) expression. DNA-based RBC typing provided improved accuracy and expanded information on RBC antigens compared to hemagglutination methods, leading to its implementation as the primary method for extended RBC typing for patients with SCD at our institution. © 2015 AABB.

Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

PubMed Central

Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto

2017-01-01

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. PMID:28235086

Emergence of Cryptosporidium hominis Monkey Genotype II and Novel Subtype Family Ik in the Squirrel Monkey (Saimiri sciureus) in China.

PubMed

Liu, Xuehan; Xie, Na; Li, Wei; Zhou, Ziyao; Zhong, Zhijun; Shen, Liuhong; Cao, Suizhong; Yu, Xingming; Hu, Yanchuan; Chen, Weigang; Peng, Gangneng

2015-01-01

A single Cryptosporidium isolate from a squirrel monkey with no clinical symptoms was obtained from a zoo in Ya'an city, China, and was genotyped by PCR amplification and DNA sequencing of the small-subunit ribosomal RNA (SSU rRNA), 70-kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein, and actin genes. This multilocus genetic characterization determined that the isolate was Cryptosporidium hominis, but carried 2, 10, and 6 nucleotide differences in the SSU rRNA, HSP70, and actin loci, respectively, which is comparable to the variations at these loci between C. hominis and the previously reported monkey genotype (2, 3, and 3 nucleotide differences). Phylogenetic studies, based on neighbor-joining and maximum likelihood methods, showed that the isolate identified in the current study had a distinctly discordant taxonomic status, distinct from known C. hominis and also from the monkey genotype, with respect to the three loci. Restriction fragment length polymorphisms of the SSU rRNA gene obtained from this study were similar to those of known C. hominis but clearly differentiated from the monkey genotype. Further subtyping was performed by sequence analysis of the gene encoding the 60-kDa glycoprotein (gp60). Maximum homology of only 88.3% to C. hominis subtype IdA10G4 was observed for the current isolate, and phylogenetic analysis demonstrated that this particular isolate belonged to a novel C. hominis subtype family, IkA7G4. This study is the first to report C. hominis infection in the squirrel monkey and, based on the observed genetic characteristics, confirms a new C. hominis genotype, monkey genotype II. Thus, these results provide novel insights into genotypic variation in C. hominis.

An XML-based interchange format for genotype-phenotype data.

PubMed

Whirl-Carrillo, M; Woon, M; Thorn, C F; Klein, T E; Altman, R B

2008-02-01

Recent advances in high-throughput genotyping and phenotyping have accelerated the creation of pharmacogenomic data. Consequently, the community requires standard formats to exchange large amounts of diverse information. To facilitate the transfer of pharmacogenomics data between databases and analysis packages, we have created a standard XML (eXtensible Markup Language) schema that describes both genotype and phenotype data as well as associated metadata. The schema accommodates information regarding genes, drugs, diseases, experimental methods, genomic/RNA/protein sequences, subjects, subject groups, and literature. The Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB; www.pharmgkb.org) has used this XML schema for more than 5 years to accept and process submissions containing more than 1,814,139 SNPs on 20,797 subjects using 8,975 assays. Although developed in the context of pharmacogenomics, the schema is of general utility for exchange of genotype and phenotype data. We have written syntactic and semantic validators to check documents using this format. The schema and code for validation is available to the community at http://www.pharmgkb.org/schema/index.html (last accessed: 8 October 2007). (c) 2007 Wiley-Liss, Inc.

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies.

PubMed

Peippo, Jaana; Viitala, Sirja; Virta, Jouni; Räty, Mervi; Tammiranta, Niina; Lamminen, Terttu; Aro, Johanna; Myllymäki, Hannu; Vilkki, Johanna

2007-11-01

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy. (c) 2007 Wiley-Liss, Inc.

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

PubMed

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

PubMed

Prince, Linda M

2015-01-01

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

Simple SNP-based minimal marker genotyping for Humulus lupulus L. identification and variety validation.

PubMed

Henning, John A; Coggins, Jamie; Peterson, Matthew

2015-10-06

Hop is an economically important crop for the Pacific Northwest USA as well as other regions of the world. It is a perennial crop with rhizomatous or clonal propagation system for varietal distribution. A big concern for growers as well as brewers is variety purity and questions are regularly posed to public agencies concerning the availability of genotype testing. Current means for genotyping are based upon 25 microsatellites that provides relatively accurate genotyping but cannot always differentiate sister-lines. In addition, numerous PCR runs (25) are required to complete this process and only a few laboratories exist that perform this service. A genotyping protocol based upon SNPs would enable rapid accurate genotyping that can be assayed at any laboratory facility set up for SNP-based genotyping. The results of this study arose from a larger project designed for whole genome association studies upon the USDA-ARS hop germplasm collection consisting of approximately 116 distinct hop varieties and germplasm (female lines) from around the world. The original dataset that arose from partial sequencing of 121 genotypes resulted in the identification of 374,829 SNPs using TASSEL-UNEAK pipeline. After filtering out genotypes with more than 50% missing data (5 genotypes) and SNP markers with more than 20% missing data, 32,206 highly filtered SNP markers across 116 genotypes were identified and considered for this study. Minor allele frequency (MAF) was calculated for each SNP and ranked according to the most informative to least informative. Only those markers without missing data across genotypes as well as 60% or less heterozygous gamete calls were considered for further analysis. Genetic distances among individuals in the study were calculated using the marker with the highest MAF value, then by using a combination of the two markers with highest MAF values and so on. This process was reiterated until a set of markers was identified that allowed for all genotypes in the study to be genetically differentiated from each other. Next, we compared genetic matrices calculated from the minimal marker sets [(Table 2; 6-, 7-, 8-, 10- and 12-marker set matrices] and that of a matrix calculated from a set of markers with no missing data across all 116 samples (1006 SNP markers). The minimum number of markers required to meet both specifications was a set of 7-markers (Table 3). These seven SNPs were then aligned with a genome assembly, and DNA sequence both upstream and downstream were used to identify primer sequences that can be used to develop seven amplicons for high resolution melting curve PCR detection or other SNP-based PCR detection methods. This study identifies a set of 7 SNP markers that may prove useful for the identification and validation of hop varieties and accessions. Variety validation of unknown samples assumes that the variety under question has been included a priori in a discovery panel. These results are based upon in silica studies and markers need to be validated using different SNP marker technology upon a differential set of hop genotypes. The marker sequence data and suggested primer sets provide potential means to fingerprint hop varieties in most genetic laboratories utilizing SNP-marker technology.

A tool for selecting SNPs for association studies based on observed linkage disequilibrium patterns.

PubMed

De La Vega, Francisco M; Isaac, Hadar I; Scafe, Charles R

2006-01-01

The design of genetic association studies using single-nucleotide polymorphisms (SNPs) requires the selection of subsets of the variants providing high statistical power at a reasonable cost. SNPs must be selected to maximize the probability that a causative mutation is in linkage disequilibrium (LD) with at least one marker genotyped in the study. The HapMap project performed a genome-wide survey of genetic variation with about a million SNPs typed in four populations, providing a rich resource to inform the design of association studies. A number of strategies have been proposed for the selection of SNPs based on observed LD, including construction of metric LD maps and the selection of haplotype tagging SNPs. Power calculations are important at the study design stage to ensure successful results. Integrating these methods and annotations can be challenging: the algorithms required to implement these methods are complex to deploy, and all the necessary data and annotations are deposited in disparate databases. Here, we present the SNPbrowser Software, a freely available tool to assist in the LD-based selection of markers for association studies. This stand-alone application provides fast query capabilities and swift visualization of SNPs, gene annotations, power, haplotype blocks, and LD map coordinates. Wizards implement several common SNP selection workflows including the selection of optimal subsets of SNPs (e.g. tagging SNPs). Selected SNPs are screened for their conversion potential to either TaqMan SNP Genotyping Assays or the SNPlex Genotyping System, two commercially available genotyping platforms, expediting the set-up of genetic studies with an increased probability of success.

DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method.

PubMed

Johanson, Helene C; Hyland, Valentine; Wicking, Carol; Sturm, Richard A

2009-04-01

We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buccal swab sample collected, when used for nucleic acid PCR and genotyping.

A comparison of hydroponic and soil-based screening methods to identify salt tolerance in the field in barley

PubMed Central

Tavakkoli, Ehsan; Fatehi, Foad; Rengasamy, Pichu; McDonald, Glenn K.

2012-01-01

Success in breeding crops for yield and other quantitative traits depends on the use of methods to evaluate genotypes accurately under field conditions. Although many screening criteria have been suggested to distinguish between genotypes for their salt tolerance under controlled environmental conditions, there is a need to test these criteria in the field. In this study, the salt tolerance, ion concentrations, and accumulation of compatible solutes of genotypes of barley with a range of putative salt tolerance were investigated using three growing conditions (hydroponics, soil in pots, and natural saline field). Initially, 60 genotypes of barley were screened for their salt tolerance and uptake of Na+, Cl–, and K+ at 150 mM NaCl and, based on this, a subset of 15 genotypes was selected for testing in pots and in the field. Expression of salt tolerance in saline solution culture was not a reliable indicator of the differences in salt tolerance between barley plants that were evident in saline soil-based comparisons. Significant correlations were observed in the rankings of genotypes on the basis of their grain yield production at a moderately saline field site and their relative shoot growth in pots at ECe 7.2 [Spearman’s rank correlation (rs)=0.79] and ECe 15.3 (rs=0.82) and the crucial parameter of leaf Na+ (rs=0.72) and Cl– (rs=0.82) concentrations at ECe 7.2 dS m−1. This work has established screening procedures that correlated well with grain yield at sites with moderate levels of soil salinity. This study also showed that both salt exclusion and osmotic tolerance are involved in salt tolerance and that the relative importance of these traits may differ with the severity of the salt stress. In soil, ion exclusion tended to be more important at low to moderate levels of stress but osmotic stress became more important at higher stress levels. Salt exclusion coupled with a synthesis of organic solutes were shown to be important components of salt tolerance in the tolerant genotypes and further field tests of these plants under stress conditions will help to verify their potential utility in crop-improvement programmes. PMID:22442423

Evaluation of mailed pediatric buccal cytobrushes for use in a case-control study of birth defects.

PubMed

Gallagher, Margaret L; Sturchio, Cynthia; Smith, Ashley; Koontz, Deborah; Jenkins, Mary M; Honein, Margaret A; Rasmussen, Sonja A

2011-07-01

Buccal cell collection is a convenient DNA collection method; however, little attention has been given to the quality of DNA obtained from pediatric populations. The purpose of this study was to determine the effect of a modified cytobrush collection method on the yield and quality of infant buccal DNA collected as part of a population-based case-control study of birth defects. METHODS Cytobrushes were collected from infants, mothers, and fathers using a standard collection method in 1997 to 2003 and a modified protocol that allows air-drying of the cytobrushes after collection from 2003 to the present. Yield and quality of DNA from 1057 cytobrushes was assessed by quantitative PCR and short tandem repeat (STR) genotyping, respectively. RESULTS Air-dried cytobrushes from infants had higher median DNA yields (1300 ng) and STR completion rates (99.5%) than standard collection method cytobrushes (60 ng and 59.5%, respectively). A subset of DNA aliquots was genotyped for six single nucleotide polymorphisms (SNPs). Aliquots from both collection methods that passed the quality protocol (DNA concentration >1 ng/μl, and successful amplification of ≥1 STR) had high genotype completion rates (99-100%). The median DNA yield following whole genome amplification was more than twofold higher for air-dried than standard collection specimens (p < 0.001). CONCLUSION Yield and quality of buccal DNA collected from infants are improved by using a method that incorporates air-drying; however, DNA collected by both methods is suitable for genotyping if stringent quality control procedures are instituted. These findings may be helpful for future epidemiologic studies of birth defects and other adverse pediatric outcomes. Copyright © 2011 Wiley-Liss, Inc.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

PubMed

Schön, T; Miotto, P; Köser, C U; Viveiros, M; Böttger, E; Cambau, E

2017-03-01

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Genomic Selection in Plant Breeding: Methods, Models, and Perspectives.

PubMed

Crossa, José; Pérez-Rodríguez, Paulino; Cuevas, Jaime; Montesinos-López, Osval; Jarquín, Diego; de Los Campos, Gustavo; Burgueño, Juan; González-Camacho, Juan M; Pérez-Elizalde, Sergio; Beyene, Yoseph; Dreisigacker, Susanne; Singh, Ravi; Zhang, Xuecai; Gowda, Manje; Roorkiwal, Manish; Rutkoski, Jessica; Varshney, Rajeev K

2017-11-01

Genomic selection (GS) facilitates the rapid selection of superior genotypes and accelerates the breeding cycle. In this review, we discuss the history, principles, and basis of GS and genomic-enabled prediction (GP) as well as the genetics and statistical complexities of GP models, including genomic genotype×environment (G×E) interactions. We also examine the accuracy of GP models and methods for two cereal crops and two legume crops based on random cross-validation. GS applied to maize breeding has shown tangible genetic gains. Based on GP results, we speculate how GS in germplasm enhancement (i.e., prebreeding) programs could accelerate the flow of genes from gene bank accessions to elite lines. Recent advances in hyperspectral image technology could be combined with GS and pedigree-assisted breeding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Superior Root Hair Formation Confers Root Efficiency in Some, But Not All, Rice Genotypes upon P Deficiency.

PubMed

Nestler, Josefine; Wissuwa, Matthias

2016-01-01

Root hairs are a low-cost way to extend root surface area (RSA), water and nutrient acquisition. This study investigated to what extend variation exists for root hair formation in rice in dependence of genotype, phosphorus (P) supply, growth medium, and root type. In general, genotypic variation was found for three root hair properties: root hair length, density, and longevity. In low P nutrient solution more than twofold genotypic difference was detected for root hair length while only onefold variation was found in low P soil. These differences were mostly due to the ability of some genotypes to increase root hair length in response to P deficiency. In addition, we were able to show that a higher proportion of root hairs remain viable even in mature, field-grown plants under low P conditions. All investigated root hair parameters exhibited high correlations across root types which were always higher in the low P conditions compared to the high P controls. Therefore we hypothesize that a low P response leads to a systemic signal in the entire root system. The genotype DJ123 consistently had the longest root hairs under low P conditions and we estimated that, across the field-grown root system, root hairs increased the total RSA by 31% in this genotype. This would explain why DJ123 is considered to be very root efficient in P uptake and suggests that DJ123 should be utilized as a donor in breeding for enhanced P uptake. Surprisingly, another root and P efficient genotype seemed not to rely on root hair growth upon P deficiency and therefore must contain different methods of low P adaptation. Genotypic ranking of root hair properties did change substantially with growth condition highlighting the need to phenotype plants in soil-based conditions or at least to validate results obtained in solution-based growth conditions.

Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study.

PubMed

Jajou, Rana; de Neeling, Albert; van Hunen, Rianne; de Vries, Gerard; Schimmel, Henrieke; Mulder, Arnout; Anthony, Richard; van der Hoek, Wim; van Soolingen, Dick

2018-01-01

Patients with Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping. VNTR genotyping and WGS were performed in parallel on all Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated. In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%). When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.

Using Extended Genealogy to Estimate Components of Heritability for 23 Quantitative and Dichotomous Traits

PubMed Central

Zaitlen, Noah; Kraft, Peter; Patterson, Nick; Pasaniuc, Bogdan; Bhatia, Gaurav; Pollack, Samuela; Price, Alkes L.

2013-01-01

Important knowledge about the determinants of complex human phenotypes can be obtained from the estimation of heritability, the fraction of phenotypic variation in a population that is determined by genetic factors. Here, we make use of extensive phenotype data in Iceland, long-range phased genotypes, and a population-wide genealogical database to examine the heritability of 11 quantitative and 12 dichotomous phenotypes in a sample of 38,167 individuals. Most previous estimates of heritability are derived from family-based approaches such as twin studies, which may be biased upwards by epistatic interactions or shared environment. Our estimates of heritability, based on both closely and distantly related pairs of individuals, are significantly lower than those from previous studies. We examine phenotypic correlations across a range of relationships, from siblings to first cousins, and find that the excess phenotypic correlation in these related individuals is predominantly due to shared environment as opposed to dominance or epistasis. We also develop a new method to jointly estimate narrow-sense heritability and the heritability explained by genotyped SNPs. Unlike existing methods, this approach permits the use of information from both closely and distantly related pairs of individuals, thereby reducing the variance of estimates of heritability explained by genotyped SNPs while preventing upward bias. Our results show that common SNPs explain a larger proportion of the heritability than previously thought, with SNPs present on Illumina 300K genotyping arrays explaining more than half of the heritability for the 23 phenotypes examined in this study. Much of the remaining heritability is likely to be due to rare alleles that are not captured by standard genotyping arrays. PMID:23737753

Using extended genealogy to estimate components of heritability for 23 quantitative and dichotomous traits.

PubMed

Zaitlen, Noah; Kraft, Peter; Patterson, Nick; Pasaniuc, Bogdan; Bhatia, Gaurav; Pollack, Samuela; Price, Alkes L

2013-05-01

Important knowledge about the determinants of complex human phenotypes can be obtained from the estimation of heritability, the fraction of phenotypic variation in a population that is determined by genetic factors. Here, we make use of extensive phenotype data in Iceland, long-range phased genotypes, and a population-wide genealogical database to examine the heritability of 11 quantitative and 12 dichotomous phenotypes in a sample of 38,167 individuals. Most previous estimates of heritability are derived from family-based approaches such as twin studies, which may be biased upwards by epistatic interactions or shared environment. Our estimates of heritability, based on both closely and distantly related pairs of individuals, are significantly lower than those from previous studies. We examine phenotypic correlations across a range of relationships, from siblings to first cousins, and find that the excess phenotypic correlation in these related individuals is predominantly due to shared environment as opposed to dominance or epistasis. We also develop a new method to jointly estimate narrow-sense heritability and the heritability explained by genotyped SNPs. Unlike existing methods, this approach permits the use of information from both closely and distantly related pairs of individuals, thereby reducing the variance of estimates of heritability explained by genotyped SNPs while preventing upward bias. Our results show that common SNPs explain a larger proportion of the heritability than previously thought, with SNPs present on Illumina 300K genotyping arrays explaining more than half of the heritability for the 23 phenotypes examined in this study. Much of the remaining heritability is likely to be due to rare alleles that are not captured by standard genotyping arrays.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

PubMed Central

Owen, Joseph R.; Noyes, Noelle; Young, Amy E.; Prince, Daniel J.; Blanchard, Patricia C.; Lehenbauer, Terry W.; Aly, Sharif S.; Davis, Jessica H.; O’Rourke, Sean M.; Abdo, Zaid; Belk, Keith; Miller, Michael R.; Morley, Paul; Van Eenennaam, Alison L.

2017-01-01

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. PMID:28739600

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

PubMed

Owen, Joseph R; Noyes, Noelle; Young, Amy E; Prince, Daniel J; Blanchard, Patricia C; Lehenbauer, Terry W; Aly, Sharif S; Davis, Jessica H; O'Rourke, Sean M; Abdo, Zaid; Belk, Keith; Miller, Michael R; Morley, Paul; Van Eenennaam, Alison L

2017-09-07

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates ( Histophilus somni , Mycoplasma bovis , Mannheimia haemolytica , and Pasteurella multocida ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni , M. haemolytica , and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. Copyright © 2017 Owen et al.

High proportion of subgroup A' (genotype A) among Brazilian isolates of Hepatitis B virus.

PubMed

Araujo, N M; Mello, F C A; Yoshida, C F T; Niel, C; Gomes, S A

2004-07-01

Hepatitis B virus (HBV) genotype A has been divided recently into two subgroups, designated A-A' (genotype A excluding A') and A'. Isolates belonging to subgroup A' have been identified in Africa. A new genotyping method, based on PCR amplification of the pre-S/S genome region and subsequent restriction fragment length polymorphism (RFLP) analysis, was developed, that established a correlation between RFLP subtypes and subgroups within genotype A. To investigate the occurrence of subgroup A' in South America, 119 Brazilian HBV isolates were analyzed. Ninety-three (78%) of them belonged to genotype A, with three predominating RFLP subtypes: 44 (37%) isolates were classified as AI, 30 (25%) were AII, and 18 (15%) were AIII. Pre-S/S nucleotide sequences of 15 genotype A isolates were determined. Phylogenetic analysis performed with these 15 and an additional 41 sequences revealed that isolates AI and AII clustered in subgroup A', whereas isolates AIII were classified into subgroup A-A'. The correlation RFLP subtypes-subgroups was confirmed by the presence of amino acid residues specific for subgroup A' in the surface antigens and polymerase of isolates AI and AII. The high proportion (63%) of isolates from subgroup A' suggested an African origin for a large number of Brazilian HBVs.

Biallelic and Triallelic 5-Hydroxytyramine Transporter Gene-Linked Polymorphic Region (5-HTTLPR) Polymorphisms and Their Relationship with Lifelong Premature Ejaculation: A Case-Control Study in a Chinese Population

PubMed Central

Huang, Yuanyuan; Zhang, Xiansheng; Gao, Jingjing; Tang, Dongdong; Gao, Pan; Li, Chao; Liu, Weiqun; Liang, Chaozhao

2016-01-01

Background This study aimed to explore the relationship between premature ejaculation (PE) and the serotonin transporter gene-linked polymorphic region (5-HTTLPR) with respect to the biallelic and triallelic classifications. Material/Methods A total of 115 outpatients who complained of ejaculating prematurely and who were diagnosed as having lifelong premature ejaculation (LPE) and 101 controls without PE complaint were recruited. All subjects completed a detailed questionnaire and were genotyped for 5-HTTLPR polymorphism using PCR-based technology. We evaluated the associations between 5-HTTLPR allelic and genotypic frequencies and their association with LPE, as well as the intravaginal ejaculation latency time (IELT) of different 5-HTTLPR genotypes among LPE patients. Results The patients and controls did not differ significantly in terms of any characteristic except age. The results showed no significant difference regarding biallelic 5-HTTLPR. According to the triallelic classification, no significant difference was found when comparing the genotypic distribution (P=0.091). However, the distribution of the S, LG, and LA alleles in the cases was significantly different from the controls (P=0.018). We found a significantly lower frequency of LA allele and higher frequency of LG allele in patients. Based on another classification by expression, we found a significantly lower frequency of the L’L’ genotype (OR=0.37; 95%CI=0.15–0.91, P=0.025) in patients with LPE. No significant association was detected between IELT of LPE and different genotypes. Conclusions Contrary to the general classification based on S/L alleles, triallelic 5-HTTLPR was associated with LPE. Triallelic 5-HTTLPR may be a promising field for genetic research in PE to avoid false-negative results in future studies. PMID:27311544

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping.

PubMed

Sariguzel, Fatma Mutlu; Berk, Elife; Gokahmetoglu, Selma; Ercal, Baris Derya; Celik, Ilhami

2015-01-01

The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.

Genotypic Detection of Antibiotic Resistance in "Escherichia Coli.": A Classroom Exercise

ERIC Educational Resources Information Center

Longtin, Sarah; Guilfoile, Patrick; Asper, Andrea

2004-01-01

Bacterial antibiotic resistance remains a problem of clinical importance. Current microbiological methods for determining antibiotic resistance are based on culturing bacteria, and may require up to 48 hours to complete. Molecular methods are increasingly being developed to speed the identification of antibiotic resistance and to determine its…

Comparison on genomic predictions using three GBLUP methods and two single-step blending methods in the Nordic Holstein population

PubMed Central

2012-01-01

Background A single-step blending approach allows genomic prediction using information of genotyped and non-genotyped animals simultaneously. However, the combined relationship matrix in a single-step method may need to be adjusted because marker-based and pedigree-based relationship matrices may not be on the same scale. The same may apply when a GBLUP model includes both genomic breeding values and residual polygenic effects. The objective of this study was to compare single-step blending methods and GBLUP methods with and without adjustment of the genomic relationship matrix for genomic prediction of 16 traits in the Nordic Holstein population. Methods The data consisted of de-regressed proofs (DRP) for 5 214 genotyped and 9 374 non-genotyped bulls. The bulls were divided into a training and a validation population by birth date, October 1, 2001. Five approaches for genomic prediction were used: 1) a simple GBLUP method, 2) a GBLUP method with a polygenic effect, 3) an adjusted GBLUP method with a polygenic effect, 4) a single-step blending method, and 5) an adjusted single-step blending method. In the adjusted GBLUP and single-step methods, the genomic relationship matrix was adjusted for the difference of scale between the genomic and the pedigree relationship matrices. A set of weights on the pedigree relationship matrix (ranging from 0.05 to 0.40) was used to build the combined relationship matrix in the single-step blending method and the GBLUP method with a polygenetic effect. Results Averaged over the 16 traits, reliabilities of genomic breeding values predicted using the GBLUP method with a polygenic effect (relative weight of 0.20) were 0.3% higher than reliabilities from the simple GBLUP method (without a polygenic effect). The adjusted single-step blending and original single-step blending methods (relative weight of 0.20) had average reliabilities that were 2.1% and 1.8% higher than the simple GBLUP method, respectively. In addition, the GBLUP method with a polygenic effect led to less bias of genomic predictions than the simple GBLUP method, and both single-step blending methods yielded less bias of predictions than all GBLUP methods. Conclusions The single-step blending method is an appealing approach for practical genomic prediction in dairy cattle. Genomic prediction from the single-step blending method can be improved by adjusting the scale of the genomic relationship matrix. PMID:22455934

High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.

PubMed

Tamburro, M; Ripabelli, G

2017-01-01

Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools. To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health. A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available. HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations. Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.

Genotyping for Human Papillomavirus (HPV) 16/18/52/58 Has a Higher Performance than HPV16/18 Genotyping in Triaging Women with Positive High-risk HPV Test in Northern Thailand

PubMed Central

Khunamornpong, Surapan; Settakorn, Jongkolnee; Sukpan, Kornkanok; Suprasert, Prapaporn; Srisomboon, Jatupol; Intaraphet, Suthida; Siriaunkgul, Sumalee

2016-01-01

Background Testing for high-risk human papillomavirus DNA (HPV test) has gained increasing acceptance as an alternative method to cytology in cervical cancer screening. Compared to cytology, HPV test has a higher sensitivity for the detection of histologic high-grade squamous intraepithelial lesion or worse (HSIL+), but this could lead to a large colposcopy burden. Genotyping for HPV16/18 has been recommended in triaging HPV-positive women. This study was aimed to evaluate the screening performance of HPV testing and the role of genotyping triage in Northern Thailand. Methods A population-based cervical screening program was performed in Chiang Mai (Northern Thailand) using cytology (conventional Pap test) and HPV test (Hybrid Capture 2). Women who had abnormal cytology or were HPV-positive were referred for colposcopy. Cervical samples from these women were genotyped using the Linear Array assay. Results Of 5,456 women, 2.0% had abnormal Pap test results and 6.5% tested positive with Hybrid Capture 2. Of 5,433 women eligible for analysis, 355 with any positive test had histologic confirmation and 57 of these had histologic HSIL+. The sensitivity for histologic HSIL+ detection was 64.9% for Pap test and 100% for Hybrid Capture 2, but the ratio of colposcopy per detection of each HSIL+ was more than two-fold higher with Hybrid Capture 2 than Pap test (5.9 versus 2.8). Genotyping results were available in 316 samples. HPV52, HPV16, and HPV58 were the three most common genotypes among women with histologic HSIL+. Performance of genotyping triage using HPV16/18/52/58 was superior to that of HPV16/18, with a higher sensitivity (85.7% versus 28.6%) and negative predictive value (94.2% versus 83.9%). Conclusions In Northern Thailand, HPV testing with genotyping triage shows better screening performance than cervical cytology alone. In this region, the addition of genotyping for HPV52/58 to HPV16/18 is deemed necessary in triaging women with positive HPV test. PMID:27336913

Interrelationship of hepatitis C virus genotypes with patient characteristics in Bahrain

PubMed Central

Abdulla, Maheeba A; Murad, Eman A; Aljenaidi, Hend A; Aljowder, Duha R; Aljeeran, Omar IK; Farid, Eman; Al Qamish, Jehad R

2017-01-01

Aim Hepatitis C virus (HCV) shows genotype-based variation in prevalence across geographical regions. This study was conducted to understand the clinical interrelationship of HCV genotypes with patient characteristics. Methods Medical records of 122 patients positive for HCV RNA test collected during 2013 and 2014 were included for analysis. Only adults were included in the study. HCV RNA extraction and genotyping was done as part of the routine diagnostic requirements. The association of continuous and categorical variables with genotypes was analyzed through analysis of variance and chi-square tests, respectively. Results Of the 122 patients selected, 103 were Bahrainis, 18 non-Bahrainis, and 1 was unregistered. Genotype 1 was the predominant (53%) one, followed by types 3 (23%) and 4 (20%). Classical symptoms, clinical signs, liver function test, and ultrasonographic results were recorded. Cirrhosis and ascites showed significant variation across genotypes. Although alanine transaminase, total bilirubin, and albumin levels were increased, gamma-glutamyltransferase and alkaline phosphatase levels were normal. About 12% of the subjects were alcohol users, 4% were positive for HIV infection and 2.4% were positive for hepatitis B virus infection. The circulating HCV RNA load was at medium-level in the study cohort and showed significant association with the HCV genotypes and subtypes. Patients with genotype 1a had 6 times more load than patients with type 4 (P<0.05). Conclusion This study reconfirmed the incidence and distribution of different genotypes in Bahrain population, and delineated the relationship of HCV RNA viral load with the severity of liver disease in our cohort. PMID:28280398

Genotype analysis, using PCR with type-specific primers, of hepatitis B virus isolates from patients coinfected with hepatitis delta virus genotype II from Miyako Island, Japan.

PubMed

Moriyama, Moriyama; Taira, Masaaki; Matsumura, Hiroshi; Aoki, Hiroshi; Mikuni, Morio; Kaneko, Miki; Shioda, Atsuo; Iwaguchi, Kayo; Arai, Shinobu; Ichijima, Sagiri; Iwasaki, Hiroko; Tanaka, Naohide; Abe, Kenji; Arakawa, Yasuyuki

2003-01-01

The aims of this study were to determine the hepatitis B virus (HBV) genotypes in hepatitis delta virus (HDV) RNA-positive patients and to characterize the HBV nucleotide sequences that may be found on a distant island of Japan. This study included three patients with chronic hepatitis who were positive for hepatitis B surface antigen (enzyme-linked immunosorbent assay; ELISA), HDV antibody (ELISA) and HDV RNA by polymerase chain reaction (PCR). The HBV genotype was determined by nested PCR using type-specific primers. The first-round PCR products from two patients were sequenced, followed by an investigation of nucleotide homology. Viruses from all three patients in this study were classified as HBV genotype B. Comparison with HBV isolates from geographically neighboring regions revealed that the two HBV isolates had 97.9-98.6% identity at the nucleotide level to a Chinese isolate, 98.3-98.6% identity to the Okinawa isolate and 98.6-98.8% identity to a Japanese isolate of genotype B. On phylogenetic analysis, the HBV isolates from the two patients were classified as HBV genotype B. The HBV isolates of cases 1 and 3 clustered in the same group as isolates from the Chinese mainland and Japanese mainland, which are geographically near Miyako Island. The HBV isolates coinfected with HDV found on Miyako Island were of genotype B. The PCR method based on genotype-specific primers was useful in determining HBV genotypes. Copyright 2003 S. Karger AG, Basel

Short-Read Whole-Genome Sequencing for Laboratory-Based Surveillance of Bordetella pertussis.

PubMed

Marchand-Austin, Alex; Tsang, Raymond S W; Guthrie, Jennifer L; Ma, Jennifer H; Lim, Gillian H; Crowcroft, Natasha S; Deeks, Shelley L; Farrell, David J; Jamieson, Frances B

2017-05-01

Bordetella pertussis is a Gram-negative bacterium that causes respiratory infections in humans. Ongoing molecular surveillance of B. pertussis acellular vaccine (aP) antigens is critical for understanding the interaction between evolutionary pressures, disease pathogenesis, and vaccine effectiveness. Methods currently used to characterize aP components are relatively labor-intensive and low throughput. To address this challenge, we sought to derive aP antigen genotypes from minimally processed short-read whole-genome sequencing data generated from 40 clinical B. pertussis isolates and analyzed using the SRST2 bioinformatic package. SRST2 was able to identify aP antigen genotypes for all antigens with the exception of pertactin, possibly due to low read coverage in GC-rich low-complexity regions of variation. Two main genotypes were observed in addition to a singular third genotype that contained an 84-bp deletion that was identified by SRST2 despite the issues in allele calling. This method has the potential to generate large pools of B. pertussis molecular data that can be linked to clinical and epidemiological information to facilitate research of vaccine effectiveness and disease severity in the context of emerging vaccine antigen-deficient strains. Copyright © 2017 American Society for Microbiology.

The advantages of haplotype analysis of the promoter region of the human apolipoprotein E gene.

PubMed

McLaughlin, D P; Sharma, A; McGinley, A; Samra, G S

2001-12-15

Polymorphisms in the regulatory region of the human apolipoprotein E gene (gene, APOE; protein, apoE) have been implicated in Alzheimer's disease. Here we describe in detail the advantages of a simple method for haplotype analysis of this region (at -491 and -427 bases relative to the transcription start site of the gene). The promoter region of the APOE gene was amplified by polymerase chain reaction (PCR) and this fragment was then used as a template for PCR with "nested" primers to generate a 228-bp product incorporating both the -491 and the -427 loci. PCR products were then digested with DraI and AluI together and subjected to polyacrylamide gel electrophoresis. The distinct pattern of bands appearing on the gel was then used to ascribe [-491,-427] haplotypes to each subject, from which -491 and -427 genotypes were inferred. -491 and -427 genotypes were also confirmed by digestion with DraI alone or AluI alone. Haplotype analysis was successful in all 20 samples analyzed and was 100% consistent with genotyping. We suggest that this is a reliable, time-saving method that the will be useful in large-scale APOE promoter genotyping studies. (c)2001 Elsevier Science.

Molecular typing of Legionella pneumophila from air-conditioning cooling waters using mip gene, SBT, and FAFLP methods.

PubMed

Gong, Xiangli; Li, Juntao; Zhang, Ying; Hou, Shuiping; Qu, Pinghua; Yang, Zhicong; Chen, Shouyi

2017-08-01

Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila (L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires. Copyright © 2017 Elsevier B.V. All rights reserved.

Lactase persistence genotyping on whole blood by loop-mediated isothermal amplification and melting curve analysis.

PubMed

Abildgaard, Anders; Tovbjerg, Sara K; Giltay, Axel; Detemmerman, Liselot; Nissen, Peter H

2018-03-26

The lactase persistence phenotype is controlled by a regulatory enhancer region upstream of the Lactase (LCT) gene. In northern Europe, specifically the -13910C > T variant has been associated with lactase persistence whereas other persistence variants, e.g. -13907C > G and -13915 T > G, have been identified in Africa and the Middle East. The aim of the present study was to compare a previously developed high resolution melting assay (HRM) with a novel method based on loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) with both whole blood and DNA as input material. To evaluate the LAMP-MC method, we used 100 whole blood samples and 93 DNA samples in a two tiered study. First, we studied the ability of the LAMP-MC method to produce specific melting curves for several variants of the LCT enhancer region. Next, we performed a blinded comparison between the LAMP-MC method and our existing HRM method with clinical samples of unknown genotype. The LAMP-MC method produced specific melting curves for the variants at position -13909, -13910, -13913 whereas the -13907C > G and -13915 T > G variants produced indistinguishable melting profiles. The LAMP-MC assay is a simple method for lactase persistence genotyping and compares well with our existing HRM method. Copyright © 2018. Published by Elsevier B.V.

A multiplex method for detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations.

PubMed

Zhang, L; Yang, Y; Liu, R; Li, Q; Yang, F; Ma, L; Liu, H; Chen, X; Yang, Z; Cui, L; He, Y

2015-12-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations. © 2015 John Wiley & Sons Ltd.

UGT1A1 gene polymorphism: Impact on toxicity and efficacy of irinotecan-based regimens in metastatic colorectal cancer

PubMed Central

Schulz, Christoph; Heinemann, Volker; Schalhorn, Andreas; Moosmann, Nikolas; Zwingers, Thomas; Boeck, Stefan; Giessen, Clemens; Stemmler, Hans-Joachim

2009-01-01

AIM: To investigate the correlation between uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) gene polymorphisms and irinotecan-associated side effects and parameters of drug efficacy in patients with metastatic colorectal cancer (mCRC) receiving a low-dose weekly irinotecan chemotherapeutic regimen. METHODS: Genotypes were retrospectively evaluated by gene scan analysis on the ABI 310 sequencer of the TATAA box in the promoter region of the UGT1A1 gene in blood samples from 105 patients who had received 1st line irinotecan-based chemotherapy for mCRC. RESULTS: The distribution of the genotypes was as follows: wild type genotype (WT) (6/6) 39.0%, heterozygous genotype (6/7) 49.5%, and homozygous genotype (7/7) 9.5%. The overall response rate (OR) was similar between patients carrying the (6/7, 7/7) or the WT genotype (6/6) (44.3% vs 43.2%, P = 0.75). Neither time to progression [(TTP) 8.1 vs 8.2 mo, P = 0.97] nor overall survival [(OS) 21.2 vs 18.9 mo, P = 0.73] differed significantly in patients who carried the (6/6) when compared to the (6/7, 7/7) genotype. No significant differences in toxicity were observed: Grade 3 and 4 delayed diarrhoea [(6/7, 7/7) vs (6/6); 13.0% vs 6.2%, P = 0.08], treatment delays [(6/7, 7/7) vs (6/6); 25.1% vs 19.3%, P =0.24] or dose reductions [(6/7, 7/7) vs (6/6); 21.5% vs 27.2%, P = 0.07]. CONCLUSION: This analysis demonstrates the non-significant influence of the UGT1A1 gene polymorphism on efficacy and rate of irinotecan-associated toxicity in mCRC patients receiving low-dose irinotecan based chemotherapy. PMID:19859999

[Current knowledge on the strain typing of the pathogenic fungus Histoplasma capsulatum var. capsulatum: a review of the findings].

PubMed

Reyes-Montes, M del R; Taylor, M L; Curiel-Quesada, E; Mesa-Arango, A C

2000-12-01

The classification of microbial strains is currently based on different typing methods, which must meet certain criteria in order to be widely used. Phenotypic and genotypic methods are being employed in the epidemiology of several fungal diseases. However, some problems associated to the phenotypic methods have fostered genotyping procedures, from DNA polymorphic diversity to gene sequencing studies, all aiming to differentiate and to relate fungal isolates or strains. Through these studies, it is possible to identify outbreaks, to detect nosocomial infection transmission, and to determine the source of infection, as well as to recognize virulent isolates. This paper is aimed at analyzing the methods recently used to type Histoplasma capsulatum, causative agent of the systemic mycosis known as histoplasmosis, in order to recommend those that yield reproducible and accurate results.

RECQ1 A159C Polymorphism Is Associated With Overall Survival of Patients With Resected Pancreatic Cancer: A Replication Study in NRG Oncology Radiation Therapy Oncology Group 9704

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Donghui, E-mail: dli@mdanderson.org; Moughan, Jennifer; Crane, Christopher

Purpose: To confirm whether a previously observed association between RECQ1 A159C variant and clinical outcome of resectable pancreatic cancer patients treated with preoperative chemoradiation is reproducible in another patient population prospectively treated with postoperative chemoradiation. Methods and Materials: Patients were selected, according to tissue availability, from eligible patients with resected pancreatic cancer who were enrolled on the NRG Oncology Radiation Therapy Oncology Group 9704 trial of 5-fluorouacil (5-FU)-based chemoradiation preceded and followed by 5-FU or gemcitabine. Deoxyribonucleic acid was extracted from paraffin-embedded tissue sections, and genotype was determined using the Taqman method. The correlation between genotype and overall survival wasmore » analyzed using a Kaplan-Meier plot, log-rank test, and multivariate Cox proportional hazards models. Results: In the 154 of the study's 451 eligible patients with evaluable tissue, genotype distribution followed Hardy-Weinberg equilibrium (ie, 37% had genotype AA, 43% AC, and 20% CC). The RECQ1 variant AC/CC genotype carriers were associated with being node positive compared with the AA carrier (P=.03). The median survival times (95% confidence interval [CI]) for AA, AC, and CC carriers were 20.6 (16.3-26.1), 18.8 (14.2-21.6), and 14.2 (10.3-21.0) months, respectively. On multivariate analysis, patients with the AC/CC genotypes were associated with worse survival than patients with the AA genotype (hazard ratio [HR] 1.54, 95% CI 1.07-2.23, P=.022). This result seemed slightly stronger for patients on the 5-FU arm (n=82) (HR 1.64, 95% CI 0.99-2.70, P=.055) than for patients on the gemcitabine arm (n=72, HR 1.46, 95% CI 0.81-2.63, P=.21). Conclusions: Results of this study suggest that the RECQ1 A159C genotype may be a prognostic or predictive factor for resectable pancreatic cancer patients who are treated with adjuvant 5-FU before and after 5-FU-based chemoradiation. Further study is needed in patients treated with gemcitabine to determine whether an association exists.« less

Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method

PubMed Central

Geens, Tom; Desplanques, Ann; Van Loock, Marnix; Bönner, Brigitte M.; Kaleta, Erhard F.; Magnino, Simone; Andersen, Arthur A.; Everett, Karin D. E.; Vanrompay, Daisy

2005-01-01

Twenty-one avian Chlamydophila psittaci isolates from different European countries were characterized using ompA restriction fragment length polymorphism, ompA sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method. PMID:15872282

Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

PubMed Central

2013-01-01

Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome. PMID:24010766

Molecular Methods for the Detection of Mycoplasma and Ureaplasma Infections in Humans

PubMed Central

Waites, Ken B.; Xiao, Li; Paralanov, Vanya; Viscardi, Rose M.; Glass, John I.

2012-01-01

Mycoplasma and Ureaplasma species are well-known human pathogens responsible for a broad array of inflammatory conditions involving the respiratory and urogenital tracts of neonates, children, and adults. Greater attention is being given to these organisms in diagnostic microbiology, largely as a result of improved methods for their laboratory detection, made possible by powerful molecular-based techniques that can be used for primary detection in clinical specimens. For slow-growing species, such as Mycoplasma pneumoniae and Mycoplasma genitalium, molecular-based detection is the only practical means for rapid microbiological diagnosis. Most molecular-based methods used for detection and characterization of conventional bacteria have been applied to these organisms. A complete genome sequence is available for one or more strains of all of the important human pathogens in the Mycoplasma and Ureaplasma genera. Information gained from genome analyses and improvements in efficiency of DNA sequencing are expected to significantly advance the field of molecular detection and genotyping during the next few years. This review provides a summary and critical review of methods suitable for detection and characterization of mycoplasmas and ureaplasmas of humans, with emphasis on molecular genotypic techniques. PMID:22819362

A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA+252 C>T SNP genotyping.

PubMed

Soler, Stephan; Rittore, Cécile; Touitou, Isabelle; Philibert, Laurent

2011-02-20

From the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening. We set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers. Allele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis. This work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping. Copyright © 2010 Elsevier B.V. All rights reserved.

Multiplexed microsatellite recovery using massively parallel sequencing

Treesearch

T.N. Jennings; B.J. Knaus; T.D. Mullins; S.M. Haig; R.C. Cronn

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of...

Simultaneous detection of Hb constant spring (α142, TAA>CAA, α2) and the α2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran.

PubMed

Akhavan-Niaki, Haleh; Banihashemi, Ali; Mostafazadeh, Amrollah; Kholghi Oskooei, Vahid; Azizi, Mandana; Youssefi Kamangar, Reza; Elmi, Maryam Mitra

2012-01-01

Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) (HBA2:c.427T>C) and α2 IVS-I donor site (GAGGTGAGG>GAGG - - - - -) (HBA2:c.95+2_95+6delTGAGG) are nondeletional α-thalassemia (α-thal) mutations found all over the world. Identification of α-thal genotypes in at-risk couples for severe anemia or in highly heterogeneous populations requires rapid, accurate and cost-effective genotyping methods. In this study, a pair of primers were used to specifically amplify an 883 bp fragment from the α2-globin gene in order to simultaneously identify these two mutations by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We determined the genotypic frequencies of Hb CS and the α2 IVS-I donor site mutations after amplification and enzymatic digestion with Tru9I in 238 northern Iranian samples referred for α-thal testing. Hb CS and the α2 IVS-I donor site mutations accounted for 21 (8.8%) and 29 (12.2%) of the nondeletional cases. This genotyping assay has proven to be a rapid, reliable and useful diagnostic tool for simultaneous detection of these two anomalies for genetic counseling or further prenatal diagnosis.

High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples.

PubMed

Abal-Fabeiro, J L; Maside, X; Llovo, J; Bello, X; Torres, M; Treviño, M; Moldes, L; Muñoz, A; Carracedo, A; Bartolomé, C

2014-04-01

The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.

PubMed

Gentilini, Fabio; Turba, Maria E

2014-01-01

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis for genotyping Duffy blood group in inhabitants of Sudan, the Fourth Cataract of the Nile

PubMed Central

2012-01-01

Background Genetic polymophisms of the Duffy antigen receptor for the chemokines (DARC) gene successfully protected against blood stage infection by Plasmodium vivax infection. The Fy (a-, b-) phenotype is predominant among African populations, particularly those originating from West Africa, and it is rare among non-African populations. The aim of this study was to analyse the frequency of four Duffy blood groups based on SNPs (T-33C, G125A, G298A and C5411T) in two local tribes of Sudanese Arabs, the Shagia and Manasir, which are both from the region of the Fourth Nile cataract in Sudan. Methods An analysis of polymorphisms was performed on 217 individuals (126 representatives of the Shagia tribe and 91 of the Manasir). Real-time PCR and TaqMan Genotyping Assays were used to study the prevalence of alleles and genotypes. Results The analysis of allelic and genotype frequency in the T-33C polymorphisms demonstrated a significant dominance of the C allele and CC genotype (OR = 0.53 [0.32-0.88]; p = 0.02) in both tribes. The G125A polymorphism is associated with phenotype Fy(a-, b-) and was identified in 83% of Shagia and 77% of Manasir. With regard to G298A polymorphisms, the genotype frequencies were different between the tribes (p = 0,002) and no single AA homozygote was found. Based on four SNPs examined, 20 combinations of genotypes for the Shagia and Manasir tribes were determined. The genotype CC/AA/GG/CT occurred most often in Shagia tribe (45.9%) but was rare in the Manasir tribe (6.6%) (p < 0.001 Shagia versus Manasir). The FY*AES allele was identified in both analysed tribes. The presence of individuals with the FY*A/FY*A genotype was demonstrated only in the Shagia tribe. Conclusion This is probably the first report showing genotypically Duffy-negative people who carry both FY*BES and FY*AES. The identification of the FY*AES allele in both tribes may be due to admixture of the non-African genetic background. Taken as a whole, allele and genotype frequencies between the Shagia and the Manasir were statistically different. However, the presence of individuals with the FY*A/FY*A genotype was demonstrated only in the Shagia tribe. PMID:22510366

Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal.

PubMed

Rodrigues, Sueli G; Nunes, Márcio R T; Casseb, Samir M M; Prazeres, Assis S C; Rodrigues, Daniela S G; Silva, Mayra O; Cruz, Ana C R; Tavares-Neto, José C; Vasconcelos, Pedro F C

2010-10-01

Saint Louis encephalitis virus (SLEV), a member of the genus Flavivirus (family Flaviviridae), is an encephalitogenic arbovirus broadly distributed in the Americas. Phylogenetic analysis based on the full-length E gene sequences obtained for 30 Brazilian SLEV strains was performed using different methods including Bayesian and relaxed molecular clock approaches. A new genetic lineage was suggested, hereafter named genotype VIII, which co-circulates with the previously described genotype V in the Brazilian Amazon region. Genotypes II and III were restricted to São Paulo state (South-east Atlantic rainforest ecosystem). The analysis also suggested the emergence of an SLEV common ancestor between 1875 and 1973 (mean of 107 years ago), giving rise to two major genetic groups: genotype II, more prevalent in the North America, and a second group comprising the other genotypes (I and III-VIII), broadly dispersed throughout the Americas, suggesting that SLEV initially emerged in South America and spread to North America. In conclusion, the current study demonstrates the high genetic variability of SLEV and its geographical dispersion in Brazil and other New World countries.

Bias Characterization in Probabilistic Genotype Data and Improved Signal Detection with Multiple Imputation

PubMed Central

Palmer, Cameron; Pe’er, Itsik

2016-01-01

Missing data are an unavoidable component of modern statistical genetics. Different array or sequencing technologies cover different single nucleotide polymorphisms (SNPs), leading to a complicated mosaic pattern of missingness where both individual genotypes and entire SNPs are sporadically absent. Such missing data patterns cannot be ignored without introducing bias, yet cannot be inferred exclusively from nonmissing data. In genome-wide association studies, the accepted solution to missingness is to impute missing data using external reference haplotypes. The resulting probabilistic genotypes may be analyzed in the place of genotype calls. A general-purpose paradigm, called Multiple Imputation (MI), is known to model uncertainty in many contexts, yet it is not widely used in association studies. Here, we undertake a systematic evaluation of existing imputed data analysis methods and MI. We characterize biases related to uncertainty in association studies, and find that bias is introduced both at the imputation level, when imputation algorithms generate inconsistent genotype probabilities, and at the association level, when analysis methods inadequately model genotype uncertainty. We find that MI performs at least as well as existing methods or in some cases much better, and provides a straightforward paradigm for adapting existing genotype association methods to uncertain data. PMID:27310603

One year nationwide evaluation of 24-locus MIRU-VNTR genotyping on Slovenian Mycobacterium tuberculosis isolates.

PubMed

Bidovec-Stojkovic, Urska; Zolnir-Dovc, Manca; Supply, Philip

2011-10-01

Slovenia is one of the few countries where IS6110 RFLP is applied for genotyping M. tuberculosis at a nationwide level, which has been in effect since 2000. Based on S6110 RFLP clustering, typical risk factors and routes of M. tuberculosis transmission were identified, such as alcohol abuse, homelessness, and bars. However, IS6110 RFLP typing suffers from important limitations including a long wait for results, which reduces the potential benefit of molecular-guided tuberculosis (TB) control. PCR-based 24-locus MIRU-VNTR typing combined with spoligotyping has recently emerged as a potential alternative for faster, large-scale genotyping of M. tuberculosis. We compared these genotyping methods for analyzing 196 Slovenian Mycobacterium tuberculosis isolates representing 97.5% of all culture-positive cases included in the Slovenian TB Registry in 2008. IS6110 RFLP and 24-locus MIRU-VNTR typing combined with spoligotyping identified 157 and 155 distinct profiles, 135 and 125 unique isolates, and 61 and 71 clustered isolates grouped into 22 and 29 clusters, respectively. The discriminatory indexes were very close, at 0.9963 and 0.9965, respectively. The majority of the molecular clusters defined by either of the two methods were identical, including in the few cases for which epidemiological links were available. The differences frequently consisted of single-band changes in IS6170-RFLP profiles subdividing a MIRU-VNTR/spoligotype-based cluster. Our one-year nationwide study showed that the results of 24-locus MIRU-VNTR typing combined with spoligotyping reached a high level of concordance with those obtained from IS6110 RFLP typing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Diversity of Lactobacilli in the Oral Cavities of Young Women with Dental Caries

PubMed Central

Caufield, P.W.; Li, Y.; Dasanayake, A.; Saxena, D.

2009-01-01

For nearly a century, lactobacilli (LB) in the oral cavity have been generally associated with dental caries. Here, we characterized the LB isolated from the saliva of 6 women with active caries using genetic-based taxonomical identification methods. From each subject, 30 isolates growing on Rogosa medium and presumed to be LB were analyzed. Of the 180 isolates, 176 were further characterized by biotyping, DNA melting points, DNA chromosomal fingerprinting, genotyping, and phylogenetic cluster assessment. We found a total of 30 unique genotypes of LB in the saliva of caries-active women, with each woman harboring between 2 and 8 distinct genotypes. Although Lactobacillus vaginalis, Lactobacillus fermentum, and Lactobacillus salivarius were found in 4 of 6 of the subjects, results from other studies using comparable methods show an entirely different array of LB associated with caries. These collective observations lead us to surmise that LB associated with dental caries are likely exogenous and opportunistic colonizers, arising from food or other reservoirs outside the oral cavity. PMID:17167253

Scoring Methods for Building Genotypic Scores: An Application to Didanosine Resistance in a Large Derivation Set

PubMed Central

Houssaini, Allal; Assoumou, Lambert; Miller, Veronica; Calvez, Vincent; Marcelin, Anne-Geneviève; Flandre, Philippe

2013-01-01

Background Several attempts have been made to determine HIV-1 resistance from genotype resistance testing. We compare scoring methods for building weighted genotyping scores and commonly used systems to determine whether the virus of a HIV-infected patient is resistant. Methods and Principal Findings Three statistical methods (linear discriminant analysis, support vector machine and logistic regression) are used to determine the weight of mutations involved in HIV resistance. We compared these weighted scores with known interpretation systems (ANRS, REGA and Stanford HIV-db) to classify patients as resistant or not. Our methodology is illustrated on the Forum for Collaborative HIV Research didanosine database (N = 1453). The database was divided into four samples according to the country of enrolment (France, USA/Canada, Italy and Spain/UK/Switzerland). The total sample and the four country-based samples allow external validation (one sample is used to estimate a score and the other samples are used to validate it). We used the observed precision to compare the performance of newly derived scores with other interpretation systems. Our results show that newly derived scores performed better than or similar to existing interpretation systems, even with external validation sets. No difference was found between the three methods investigated. Our analysis identified four new mutations associated with didanosine resistance: D123S, Q207K, H208Y and K223Q. Conclusions We explored the potential of three statistical methods to construct weighted scores for didanosine resistance. Our proposed scores performed at least as well as already existing interpretation systems and previously unrecognized didanosine-resistance associated mutations were identified. This approach could be used for building scores of genotypic resistance to other antiretroviral drugs. PMID:23555613

Pharmacogenomics of platinum-based chemotherapy response in NSCLC: a genotyping study and a pooled analysis

PubMed Central

Chen, Juan; Wang, Zhan; Zou, Ting; Cui, Jiajia; Yin, Jiye; Zheng, Wei; Jiang, Wuzhong; Zhou, Honghao; Liu, Zhaoqian

2016-01-01

Published data showed inconsistent results about associations of extensively studied polymorphisms with platinum-based chemotherapy response. Our study aimed to provide reliable conclusions of these associations by detecting genotypes of the SNPs in a larger sample size and summarizing a comprehensive pooled analysis. 13 SNPs in 8 genes were genotyped in 1024 NSCLC patients by SequenomMassARRAY. 39 published studies and our study were included in meta-analysis. Patients with GA or GG genotypes of XRCC1 G1196 had better response than AA genotype carriers (Genotyping study: OR = 0.72, 95%CI: 0.53-0.96, P = 0.028; Meta-analysis: OR = 0.74, 95%CI: 0.62-0.89, P = 0.001). Patients carrying CT or TT genotypes of XRCC1 C580T could be more sensitive to platinum-based chemotherapy compared to patients with CC genotype (OR = 0.54, 95%CI: 0.37-0.80, P = 0.002). CC genotype of XRCC3 C18067T carriers showed more resistance to platinum-based chemotherapy when compared to those with CT or TT genotypes (OR = 0.69, 95%CI: 0.52-0.91, P = 0.009). Our study indicated that XRCC1 G1196A/C580T and XRCC3 C18067T should be paid attention for personalized platinum-based chemotherapy in NSCLC patients. PMID:27248474

A pharmacogenetics study to predict outcome in patients receiving anti-VEGF therapy in age related macular degeneration

PubMed Central

Kitchens, John W; Kassem, Nawal; Wood, William; Stone, Thomas W; Isernhagen, Rick; Wood, Edward; Hancock, Brad A; Radovich, Milan; Waymire, Josh; Li, Lang; Schneider, Bryan P

2013-01-01

Purpose To ascertain whether single nucleotide polymorphisms (SNPs) in the Vascular Endothelial Growth factor (VEGFA), Complement Factor H (CFH), and LOC387715 genes could predict outcome to anti-VEGF therapy for patients with age related macular degeneration (AMD). Methods Patients with “wet” AMD were identified by chart review. Baseline optical coherence tomography (OCT) and visual acuity (VA) data, and at least 6 months of clinical follow up after 3 initial monthly injections of bevacizumab or ranibizumab were required for inclusion. Based on OCT and VA, patients were categorized into two possible clinical outcomes: (a) responders and (b) non-responders. DNA was extracted from saliva and genotyped for candidate SNPs in the VEGFA, LOC387715, and CFH genes. Clinical outcomes were statistically compared to patient genotypes. Results 101 patients were recruited, and one eye from each patient was included in the analysis. 97% of samples were successfully genotyped for all SNPs. We found a statistically significant association between the LOC387715 A69S TT genotype and outcome based on OCT. Conclusion Genetic variation may be associated with outcome in patients receiving anti-VEGF therapy. PMID:24143065

A molecular switch sensor for detection of PRSS1 genotype based on site-specific DNA cleavage of restriction endonuclease.

PubMed

Liu, Qicai; Gao, Feng; Weng, Shaohuang; Peng, Huaping; Lin, Liqing; Zhao, Chengfei; Lin, Xinhua

2015-01-01

PRSS1 mutations or polymorphism in the peripheral blood of patients can be used as susceptible molecular markers to pancreatic cancer. A sensor for selective electrochemical detection of PRSS1 genotypes was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. A mercapto-modified hairpin probe was immobilized on a gold electrode. The probe's neck can be cleaved by EcoRI in the absence of rs10273639 C/C of PRSS1 genotype, but it cannot be cleaved in the presence of T/T. The difference in quantity of electric charge was monitored by biosensors before and after enzymatic cleavage. Electrochemical signals are generated by differential pulse voltammetry interrogation of methylene blue (MB) that quantitatively binds to surface-confined hairpin probe via electrostatic interactions. The results suggested this method had a good specificity in distinguishing PRSS1 genotypes. There was a good linear relationship between the charge and the logarithmic function of PRSS1 rs10273639 T/T type DNA concentration (current=120.6303+8.8512log C, R=0.9942). The detection limit was estimated at 0.5 fM. The molecular switch sensor has several advantages, and it is possible to qualitatively, quantitatively, and noninvasively detect PRSS1 genotypes in the blood of patients with pancreatic cancer. © 2015 by the Association of Clinical Scientists, Inc.

Detection of neonatal unit clusters of Candida parapsilosis fungaemia by microsatellite genotyping: Results from laboratory-based sentinel surveillance, South Africa, 2009-2010.

PubMed

Magobo, Rindidzani E; Naicker, Serisha D; Wadula, Jeannette; Nchabeleng, Maphoshane; Coovadia, Yacoob; Hoosen, Anwar; Lockhart, Shawn R; Govender, Nelesh P

2017-05-01

Neonatal candidaemia is a common, deadly and costly hospital-associated disease. To determine the genetic diversity of Candida parapsilosis causing fungaemia in South African neonatal intensive care units (NICUs). From February 2009 through to August 2010, cases of candidaemia were reported through laboratory-based surveillance. C. parapsilosis isolates from neonatal cases were submitted for identification by internal transcribed spacer (ITS) region sequencing, antifungal susceptibility testing and microsatellite genotyping. Cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Of 1671 cases with a viable Candida isolate, 393 (24%) occurred among neonates. Isolates from 143 neonatal cases were confirmed as C. parapsilosis sensu stricto. Many isolates were resistant to fluconazole (77/143; 54%) and voriconazole (20/143; 14%). Of 79 closely-related genotypes, 18 were represented by ≥2 isolates; 61 genotypes had a single isolate each. Seven clusters, comprised of 82 isolates, were identified at five hospitals in three provinces. Isolates belonging to certain clusters were significantly more likely to be fluconazole resistant: all cluster 7 isolates and the majority of cluster 4 (78%), 5 (89%) and 6 (67%) isolates (P<.001). Candida parapsilosis-associated candidaemia in public-sector NICUs was caused by closely related genotypes and there was molecular evidence of undetected outbreaks as well as intra-hospital transmission. © 2017 Blackwell Verlag GmbH.

Minimum Information for Reporting Next Generation Sequence Genotyping (MIRING): Guidelines for Reporting HLA and KIR Genotyping via Next Generation Sequencing

PubMed Central

Mack, Steven J.; Milius, Robert P.; Gifford, Benjamin D.; Sauter, Jürgen; Hofmann, Jan; Osoegawa, Kazutoyo; Robinson, James; Groeneweg, Mathijs; Turenchalk, Gregory S.; Adai, Alex; Holcomb, Cherie; Rozemuller, Erik H.; Penning, Maarten T.; Heuer, Michael L.; Wang, Chunlin; Salit, Marc L.; Schmidt, Alexander H.; Parham, Peter R.; Müller, Carlheinz; Hague, Tim; Fischer, Gottfried; Fernandez-Viňa, Marcelo; Hollenbach, Jill A; Norman, Paul J.; Maiers, Martin

2015-01-01

The development of next-generation sequencing (NGS) technologies for HLA and KIR genotyping is rapidly advancing knowledge of genetic variation of these highly polymorphic loci. NGS genotyping is poised to replace older methods for clinical use, but standard methods for reporting and exchanging these new, high quality genotype data are needed. The Immunogenomic NGS Consortium, a broad collaboration of histocompatibility and immunogenetics clinicians, researchers, instrument manufacturers and software developers, has developed the Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines. MIRING is a checklist that specifies the content of NGS genotyping results as well as a set of messaging guidelines for reporting the results. A MIRING message includes five categories of structured information – message annotation, reference context, full genotype, consensus sequence and novel polymorphism – and references to three categories of accessory information – NGS platform documentation, read processing documentation and primary data. These eight categories of information ensure the long-term portability and broad application of this NGS data for all current histocompatibility and immunogenetics use cases. In addition, MIRING can be extended to allow the reporting of genotype data generated using pre-NGS technologies. Because genotyping results reported using MIRING are easily updated in accordance with reference and nomenclature databases, MIRING represents a bold departure from previous methods of reporting HLA and KIR genotyping results, which have provided static and less-portable data. More information about MIRING can be found online at miring.immunogenomics.org. PMID:26407912

Using DNA fingerprints to infer familial relationships within NHANES III households

PubMed Central

Katki, Hormuzd A.; Sanders, Christopher L.; Graubard, Barry I.; Bergen, Andrew W.

2009-01-01

Developing, targeting, and evaluating genomic strategies for population-based disease prevention require population-based data. In response to this urgent need, genotyping has been conducted within the Third National Health and Nutrition Examination (NHANES III), the nationally-representative household-interview health survey in the U.S. However, before these genetic analyses can occur, family relationships within households must be accurately ascertained. Unfortunately, reported family relationships within NHANES III households based on questionnaire data are incomplete and inconclusive with regards to actual biological relatedness of family members. We inferred family relationships within households using DNA fingerprints (Identifiler®) that contain the DNA loci used by law enforcement agencies for forensic identification of individuals. However, performance of these loci for relationship inference is not well understood. We evaluated two competing statistical methods for relationship inference on pairs of household members: an exact likelihood ratio relying on allele frequencies to an Identical By State (IBS) likelihood ratio that only requires matching alleles. We modified these methods to account for genotyping errors and population substructure. The two methods usually agree on the rankings of the most likely relationships. However, the IBS method underestimates the likelihood ratio by not accounting for the informativeness of matching rare alleles. The likelihood ratio is sensitive to estimates of population substructure, and parent-child relationships are sensitive to the specified genotyping error rate. These loci were unable to distinguish second-degree relationships and cousins from being unrelated. The genetic data is also useful for verifying reported relationships and identifying data quality issues. An important by-product is the first explicitly nationally-representative estimates of allele frequencies at these ubiquitous forensic loci. PMID:20664713

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

PubMed

Tong, Steven Y C; Xie, Shirley; Richardson, Leisha J; Ballard, Susan A; Dakh, Farshid; Grabsch, Elizabeth A; Grayson, M Lindsay; Howden, Benjamin P; Johnson, Paul D R; Giffard, Philip M

2011-01-01

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Genotype imputation in the domestic dog

PubMed Central

Meurs, K. M.

2016-01-01

Application of imputation methods to accurately predict a dense array of SNP genotypes in the dog could provide an important supplement to current analyses of array-based genotyping data. Here, we developed a reference panel of 4,885,283 SNPs in 83 dogs across 15 breeds using whole genome sequencing. We used this panel to predict the genotypes of 268 dogs across three breeds with 84,193 SNP array-derived genotypes as inputs. We then (1) performed breed clustering of the actual and imputed data; (2) evaluated several reference panel breed combinations to determine an optimal reference panel composition; and (3) compared the accuracy of two commonly used software algorithms (Beagle and IMPUTE2). Breed clustering was well preserved in the imputation process across eigenvalues representing 75 % of the variation in the imputed data. Using Beagle with a target panel from a single breed, genotype concordance was highest using a multi-breed reference panel (92.4 %) compared to a breed-specific reference panel (87.0 %) or a reference panel containing no breeds overlapping with the target panel (74.9 %). This finding was confirmed using target panels derived from two other breeds. Additionally, using the multi-breed reference panel, genotype concordance was slightly higher with IMPUTE2 (94.1 %) compared to Beagle; Pearson correlation coefficients were slightly higher for both software packages (0.946 for Beagle, 0.961 for IMPUTE2). Our findings demonstrate that genotype imputation from SNP array-derived data to whole genome-level genotypes is both feasible and accurate in the dog with appropriate breed overlap between the target and reference panels. PMID:27129452

The Role of G22 A Adenosine Deaminase 1 Gene Polymorphism and the Activities of ADA Isoenzymes in Fertile and Infertile Men.

PubMed

Fattahi, Amir; Khodadadi, Iraj; Amiri, Iraj; Latifi, Zeinab; Ghorbani, Marzieh; Tavilani, Heidar

2015-10-01

To evaluate frequency distribution of adenosine deaminase 1 (ADA1) G22 A alleles and genotypes in fertile and infertile men. In this study we evaluate frequency distribution of ADA1 G22 A alleles and genotypes in 200 fertile and 200 infertile men. The polymerase chain reaction-restriction fragment length polymorphism technique was used for determining ADA1 G22 A variants. In addition, ADA isoenzymes activities (ADA1 and ADA2) were measured using colorimetric method. The frequency of GG genotype was significantly higher and GA genotype was lower in infertile males compared with fertile men (P = .048 and P = .045, respectively). However, there was not any noticeable difference in allele distribution between groups (P >.05). Based on logistic regression analysis, the GA genotype has a protective role and can decrease the risk of male infertility 1.7 times (P = .046). There were significantly higher activities of ADAT and its isoenzymes in infertile males compared with fertile men (P <.05). Also, the ADA1 activity with GG genotype was higher than GA carriers in all population (P = .001). Our results revealed that the activity of ADA isoenzymes and distribution of ADA1 G22 A genotypes were different among fertile and infertile men and more likely the GA genotype, which had lower ADA1 activity and was higher in fertile men is a protective factor against infertility. Copyright © 2015 Elsevier Inc. All rights reserved.

A microsphere-based assay for mutation analysis of the biotinidase gene using dried blood spots

PubMed Central

Lindau-Shepard, Barbara; Janik, David K.; Pass, Kenneth A.

2012-01-01

Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay. PMID:27625817

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

PubMed

Martinez-Serra, Jordi; Robles, Juan; Nicolàs, Antoni; Gutierrez, Antonio; Ros, Teresa; Amat, Juan Carlos; Alemany, Regina; Vögler, Oliver; Abelló, Aina; Noguera, Aina; Besalduch, Joan

2014-01-01

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism.

PubMed

Tsartsianidou, V; Triantafillidou, D; Karaiskou, N; Tarantili, P; Triantafillidis, G; Georgakis, E; Triantafyllidis, A

2017-05-01

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland.

PubMed

Dobec, Marinko; Bannwart, Fridolin; Kaeppeli, Franz; Cassinotti, Pascal

2009-05-01

There is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses. To optimize the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz) in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities in Switzerland. 680 cervical cell specimens with normal cytology, obtained from women undergoing routine cervical screening by liquid-based Pap smear, were analyzed by the LA HPV genotyping test for HPV-DNA. The automation of the LA HPV genotyping test resulted in a total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Any of 37 HPV genotypes were detected in 117 (17.2%) and high-risk (HR) HPV in 55 (8.1%) of 680 women with normal cytology. The highest prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21-30 and showed a continuous decrease in older age-groups. The most common HR-HPV genotypes were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). The optimization and automation of the LA HPV genotyping test makes it suited for high throughput HPV detection and typing. The epidemiological data provides information about distribution of HPV genotypes in women without cytological abnormalities in Switzerland and may be important for determining the future impact of vaccines and potential changes in the country's epidemiological HPV profile.

Genetic predisposition for adult lactose intolerance and relation to diet, bone density, and bone fractures.

PubMed

Obermayer-Pietsch, Barbara M; Bonelli, Christine M; Walter, Daniela E; Kuhn, Regina J; Fahrleitner-Pammer, Astrid; Berghold, Andrea; Goessler, Walter; Stepan, Vinzenz; Dobnig, Harald; Leb, Georg; Renner, Wilfried

2004-01-01

Evidence that genetic disposition for adult lactose intolerance significantly affects calcium intake, bone density, and fractures in postmenopausal women is presented. PCR-based genotyping of lactase gene polymorphisms may complement diagnostic procedures to identify persons at risk for both lactose malabsorption and osteoporosis. Lactase deficiency is a common autosomal recessive condition resulting in decreased intestinal lactose degradation. A -13910 T/C dimorphism (LCT) near the lactase phlorizin hydrolase gene, reported to be strongly associated with adult lactase nonpersistence, may have an impact on calcium supply, bone density, and osteoporotic fractures in the elderly. We determined LCT genotypes TT, TC, and CC in 258 postmenopausal women using a polymerase chain reaction-based assay. Genotypes were related to milk intolerance, nutritional calcium intake, intestinal calcium absorption, bone mineral density (BMD), and nonvertebral fractures. Twenty-four percent of all women were found to have CC genotypes and genetic lactase deficiency. Age-adjusted BMD at the hip in CC genotypes and at the spine in CC and TC genotypes was reduced by -7% to -11% depending on the site measured (p = 0.04). LCT(T/C-13910) polymorphisms alone accounted for 2-4% of BMD in a multiple regression model. Bone fracture incidence was significantly associated with CC genotypes (p = 0.001). Milk calcium intake was significantly lower (-55%, p = 0.004) and aversion to milk consumption was significantly higher (+166%, p = 0.01) in women with the CC genotype, but there were no differences in overall dietary calcium intake or in intestinal calcium absorption test values. The LCT(T/C-13910) polymorphism is associated with subjective milk intolerance, reduced milk calcium intake, and reduced BMD at the hip and the lumbar spine and may predispose to bone fractures. Genetic testing for lactase deficiency may complement indirect methods in the detection of individuals at risk for both lactose malabsorption and osteoporosis.

Differential interactions between Curtobacterium flaccumfaciens pv. flaccumfaciens and common bean.

PubMed

Valdo, S C D; Wendland, A; Araújo, L G; Melo, L C; Pereira, H S; Melo, P G; Faria, L C

2016-11-21

Bacterial wilt of common bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is an important disease in terms of economic importance. It reduces grain yield by colonizing xylem vessels, subsequently impeding the translocation of water and nutrients to the superior plant parts. The existence of physiological races in C. flaccumfaciens pv. flaccumfaciens has not so far been reported. The objective of the present investigation was to identify physiological races, evaluate differential interaction, and select resistant genotypes of common bean. Initially, 30 genotypes of common bean were inoculated with eight isolates exhibiting different levels of aggressiveness, under controlled greenhouse conditions. Disease was assessed 15 days after inoculation. The existence of differential interactions between C. flaccumfaciens pv. flaccumfaciens isolates and common bean genotypes were identified by utilizing partial diallel analysis. The most aggressive isolates were BRM 14939 and BRM 14942 and the least aggressive isolates were BRM 14941 and BRM 14946. The genotypes IPA 9, Ouro Branco, and Michelite were selected as more resistant among the test isolates. The genotypes IAC Carioca Akytã, BRS Notável, Pérola, IAC Carioca Aruã, and Coquinho contributed more to the isolate x genotype interaction according to the ecovalence method of estimation, and were, therefore, indicated as differentials. Based on these results, it was possible to conclude that physiological races of the pathogen exist, to select resistant genotypes, and to propose a set of differentials.

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

PubMed

Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

2010-10-01

To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

Diversity of Mycobacterium tuberculosis lineages in French Polynesia.

PubMed

Osman, Djaltou Aboubaker; Phelippeau, Michael; Drancourt, Michel; Musso, Didier

2017-04-01

French Polynesia is an overseas territory located in the South Pacific. The incidence of tuberculosis in French Polynesia has been stable since 2000 with an average of 20 cases/y/100,000 inhabitants. Molecular epidemiology of Mycobacterium tuberculosis in French Polynesia is unknown because M. tuberculosis isolates have not been routinely genotyped. From 2009 to 2012, 34 isolates collected from 32 French Polynesian patients were identified as M. tuberculosis by probe hybridization. These isolates were genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units (MIRUs)-variable number of tandem repeat (VNTR). Spoligotype patterns obtained using commercial kits were compared with the online international database SITVIT. MIRU-VNTR genotyping was performed using an in-house protocol based on capillary electrophoresis sizing for 24-loci MIRU-VNTR genotyping. The results of the spoligotyping method revealed that 25 isolates grouped into six previously described spoligotypes [H1, H3, U likely (S), T1, Manu, and Beijing] and nine isolates grouped into six new spoligotypes. Comparison with the international database MIRU-VNTRplus distributed 30 isolates into five lineages (Haarlem, Latin American Mediterranean, S, X, and Beijing) and four as unassigned isolates. Genotyping identified four phylogenetic lineages belonging to the modern Euro-American subgroup, one Beijing genotype responsible for worldwide pandemics, including remote islands in the South Pacific, and one Manu genotype of the ancestral lineage of M. tuberculosis. Copyright © 2015. Published by Elsevier B.V.

Population Structure of Candida parapsilosis: No Genetic Difference Between French and Uruguayan Isolates Using Microsatellite Length Polymorphism.

PubMed

Desnos-Ollivier, Marie; Bórmida, Victoria; Poirier, Philippe; Nourrisson, Céline; Pan, Dinorah; Bretagne, Stéphane; Puime, Andrès; Dromer, Françoise

2018-04-01

Candida parapsilosis is a human commensal yeast, frequently involved in infection worldwide and especially in neonates. It is the second species responsible for bloodstream infections in Uruguay and the third species in France. We were interested in knowing whether the population structure of isolates responsible for candidemia in France and in Uruguay was different. Genotyping methods based on microsatellite length polymorphism (MLP) have been described and are especially used for investigation of local outbreaks. We therefore determined the genotypes of 159 C. parapsilosis isolates recovered from 122 patients (84 French patients from 43 hospitals and 38 Uruguayan patients from 10 hospitals) using three microsatellites markers previously described. Our results confirmed that C. parapsilosis population has a high genetic diversity, clonal inheritance and that majority of patients were infected by a single isolate. But we described recurrent infections due to related or unrelated genotypes resulting from isolates harboring loss or gain of heterozygosity. We also described three cases of coinfections due to unrelated genotypes. We did not uncover geographic specificity but observed two linked genotypes that seem to be associated with voriconazole resistance. Finally, among eight isolates involved in grouped cases, the genotypes were similar in six cases supporting the hypothesis of inter-patient transmission. These results confirmed the usefulness of performing MLP genotyping analysis for grouped cases of C. parapsilosis isolates in order to reinforce preventive hygiene measures.

A detailed view on Model-Based Multifactor Dimensionality Reduction for detecting gene-gene interactions in case-control data in the absence and presence of noise

PubMed Central

CATTAERT, TOM; CALLE, M. LUZ; DUDEK, SCOTT M.; MAHACHIE JOHN, JESTINAH M.; VAN LISHOUT, FRANÇOIS; URREA, VICTOR; RITCHIE, MARYLYN D.; VAN STEEN, KRISTEL

2010-01-01

SUMMARY Analyzing the combined effects of genes and/or environmental factors on the development of complex diseases is a great challenge from both the statistical and computational perspective, even using a relatively small number of genetic and non-genetic exposures. Several data mining methods have been proposed for interaction analysis, among them, the Multifactor Dimensionality Reduction Method (MDR), which has proven its utility in a variety of theoretical and practical settings. Model-Based Multifactor Dimensionality Reduction (MB-MDR), a relatively new MDR-based technique that is able to unify the best of both non-parametric and parametric worlds, was developed to address some of the remaining concerns that go along with an MDR-analysis. These include the restriction to univariate, dichotomous traits, the absence of flexible ways to adjust for lower-order effects and important confounders, and the difficulty to highlight epistasis effects when too many multi-locus genotype cells are pooled into two new genotype groups. Whereas the true value of MB-MDR can only reveal itself by extensive applications of the method in a variety of real-life scenarios, here we investigate the empirical power of MB-MDR to detect gene-gene interactions in the absence of any noise and in the presence of genotyping error, missing data, phenocopy, and genetic heterogeneity. For the considered simulation settings, we show that the power is generally higher for MB-MDR than for MDR, in particular in the presence of genetic heterogeneity, phenocopy, or low minor allele frequencies. PMID:21158747

Rapid multiplexed genotyping for hereditary thrombophilia by SELDI-TOF mass spectrometry.

PubMed

Yang, Shangbin; Xu, Lihui; Wu, Haifeng M

2010-03-01

Approximately 50% of patients with venous thromboembolism also present with hereditary predisposition. The most common genetic factors are single nucleotide polymorphisms (SNPs) of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T. Genotyping these SNPs helps clinicians to correctly diagnose the disease and properly manage patients. In this study, we report a novel method using surface-enhanced laser desorption and ionization time of flight mass spectrometry to rapidly genotype, in a multiplex fashion, 3 SNPs that predispose patients to thrombosis. First, patient DNA samples were subjected to polymerase chain reaction to amplify and extend the DNA products with masses corresponding to specific genotypes. Polymerase chain reaction products were then applied to Q10 anionic protein chips, undergoing on-chip sample enrichment and clean-up. Finally, the genotypes of the SNPs were determined by surface-enhanced laser desorption and ionization time of flight mass spectrometry. This method offers a rapid turnaround time of less than 5 hours from sample collection to result reporting. The analytical accuracy of each SNP genotyping result has been confirmed by DNA sequencing. In addition, the genotype results produced by this method were validated by comparing them with results obtained by the approved method in the clinical reference laboratory. This novel method is fast, accurate, and reproducible, and thus provides an excellent platform to promote personalized medicine in the management of clotting disorders.

Genetic Markers Analyses and Bioinformatic Approaches to Distinguish Between Olive Tree (Olea europaea L.) Cultivars.

PubMed

Ben Ayed, Rayda; Ben Hassen, Hanen; Ennouri, Karim; Rebai, Ahmed

2016-12-01

The genetic diversity of 22 olive tree cultivars (Olea europaea L.) sampled from different Mediterranean countries was assessed using 5 SNP markers (FAD2.1; FAD2.3; CALC; SOD and ANTHO3) located in four different genes. The genotyping analysis of the 22 cultivars with 5 SNP loci revealed 11 alleles (average 2.2 per allele). The dendrogram based on cultivar genotypes revealed three clusters consistent with the cultivars classification. Besides, the results obtained with the five SNPs were compared to those obtained with the SSR markers using bioinformatic analyses and by computing a cophenetic correlation coefficient, indicating the usefulness of the UPGMA method for clustering plant genotypes. Based on principal coordinate analysis using a similarity matrix, the first two coordinates, revealed 54.94 % of the total variance. This work provides a more comprehensive explanation of the diversity available in Tunisia olive cultivars, and an important contribution for olive breeding and olive oil authenticity.

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

NASA Astrophysics Data System (ADS)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Impact of the method of G6PD deficiency assessment on genetic association studies of malaria susceptibility.

PubMed

Johnson, Marla K; Clark, Tamara D; Njama-Meya, Denise; Rosenthal, Philip J; Parikh, Sunil

2009-09-30

Clinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency. We compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31-0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70-1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37-0.88 for females). This study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

PubMed

Hardy, M Y; Ontiveros, N; Varney, M D; Tye-Din, J A

2018-04-01

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Phenotypic and genotypic data integration and exploration through a web-service architecture.

PubMed

Nuzzo, Angelo; Riva, Alberto; Bellazzi, Riccardo

2009-10-15

Linking genotypic and phenotypic information is one of the greatest challenges of current genetics research. The definition of an Information Technology infrastructure to support this kind of studies, and in particular studies aimed at the analysis of complex traits, which require the definition of multifaceted phenotypes and the integration genotypic information to discover the most prevalent diseases, is a paradigmatic goal of Biomedical Informatics. This paper describes the use of Information Technology methods and tools to develop a system for the management, inspection and integration of phenotypic and genotypic data. We present the design and architecture of the Phenotype Miner, a software system able to flexibly manage phenotypic information, and its extended functionalities to retrieve genotype information from external repositories and to relate it to phenotypic data. For this purpose we developed a module to allow customized data upload by the user and a SOAP-based communications layer to retrieve data from existing biomedical knowledge management tools. In this paper we also demonstrate the system functionality by an example application of the system in which we analyze two related genomic datasets. In this paper we show how a comprehensive, integrated and automated workbench for genotype and phenotype integration can facilitate and improve the hypothesis generation process underlying modern genetic studies.

Rapid genotyping of common MeCP2 mutations with an electronic DNA microchip using serial differential hybridization.

PubMed

Thistlethwaite, William A; Moses, Linda M; Hoffbuhr, Kristen C; Devaney, Joseph M; Hoffman, Eric P

2003-05-01

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.

Enterovirus-71 genotype C isolated in Peru between 2006 and 2009.

PubMed

Huaman, Jose L; Carrion, Gladys; Ampuero, Julia S; Ocaña, Victor; Laguna-Torres, V Alberto; Hontz, Robert D

2016-12-01

Enterovirus-71 (EV71) was first isolated in California, United States in 1969, belongs to the genus Enterovirus, family Picornaviridae. Although infection normally causes mild, often undiagnosed illness, it can cause central nervous system infections that could turn fatal. Based on VP1 gene analysis, EV71 has been classified into six separate genotypes. Although the molecular epidemiology of EV71 has been well described via studies originating from Asia and Europe, it is mostly unknown in South America. From our study, four EV71 isolates from Peru were characterized using phylogenetic methods to determine their relationship with known reference strains. These four Peruvian EV71 isolates from between 2006 and 2009 were analyzed by RT-PCR using primers capable of amplifying the entire VP1 gene. Reference strains representing all six known genotypes were used to determine any recognizable phylogenetic relationships. In fact, all of our isolates clustered together within the genotype C1 lineage- separate from Asian, European, North American, and Australian strains. We present evidence that EV71 genotype C1 exists in Peru, and this is the first such report documenting EV71 genotype C1 circulating in South America. Gathering additional isolates will help elucidate a more complete global epidemiological picture of EV71 infections. Published by Elsevier B.V.

Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.

PubMed

Biedrzycka, Aleksandra; Sebastian, Alvaro; Migalska, Magdalena; Westerdahl, Helena; Radwan, Jacek

2017-07-01

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage. © 2016 John Wiley & Sons Ltd.

Using artificial neural networks to select upright cowpea (Vigna unguiculata) genotypes with high productivity and phenotypic stability.

PubMed

Barroso, L M A; Teodoro, P E; Nascimento, M; Torres, F E; Nascimento, A C C; Azevedo, C F; Teixeira, F R F

2016-11-03

Cowpea (Vigna unguiculata) is grown in three Brazilian regions: the Midwest, North, and Northeast, and is consumed by people on low incomes. It is important to investigate the genotype x environment (GE) interaction to provide accurate recommendations for farmers. The aim of this study was to identify cowpea genotypes with high adaptability and phenotypic stability for growing in the Brazilian Cerrado, and to compare the use of artificial neural networks with the Eberhart and Russell (1966) method. Six trials with upright cowpea genotypes were conducted in 2005 and 2006 in the States of Mato Grosso do Sul and Mato Grosso. The data were subjected to adaptability and stability analysis by the Eberhart and Russell (1966) method and artificial neural networks. The genotypes MNC99-537F-4 and EVX91-2E-2 provided grain yields above the overall environment means, and exhibited high stability according to both methods. Genotype IT93K-93-10 was the most suitable for unfavorable environments. There was a high correlation between the results of both methods in terms of classifying the genotypes by their adaptability and stability. Therefore, this new approach would be effective in quantifying the GE interaction in upright cowpea breeding programs.

European external quality control study on the competence of laboratories to recognize rare sequence variants resulting in unusual genotyping results.

PubMed

Márki-Zay, János; Klein, Christoph L; Gancberg, David; Schimmel, Heinz G; Dux, László

2009-04-01

Depending on the method used, rare sequence variants adjacent to the single nucleotide polymorphism (SNP) of interest may cause unusual or erroneous genotyping results. Because such rare variants are known for many genes commonly tested in diagnostic laboratories, we organized a proficiency study to assess their influence on the accuracy of reported laboratory results. Four external quality control materials were processed and sent to 283 laboratories through 3 EQA organizers for analysis of the prothrombin 20210G>A mutation. Two of these quality control materials contained sequence variants introduced by site-directed mutagenesis. One hundred eighty-nine laboratories participated in the study. When samples gave a usual result with the method applied, the error rate was 5.1%. Detailed analysis showed that more than 70% of the failures were reported from only 9 laboratories. Allele-specific amplification-based PCR had a much higher error rate than other methods (18.3% vs 2.9%). The variants 20209C>T and [20175T>G; 20179_20180delAC] resulted in unusual genotyping results in 67 and 85 laboratories, respectively. Eighty-three (54.6%) of these unusual results were not recognized, 32 (21.1%) were attributed to technical issues, and only 37 (24.3%) were recognized as another sequence variant. Our findings revealed that some of the participating laboratories were not able to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our study indicates that the majority of the failures could be avoided by improved training and careful selection and validation of the methods applied.

The Vitamin D Receptor (VDR) Gene Polymorphisms in Turkish Brain Cancer Patients

PubMed Central

Toptaş, Bahar; Kafadar, Ali Metin; Cacina, Canan; Turan, Saime; Yurdum, Leman Melis; Yiğitbaşı, Nihal; Gökçe, Muhammed Oğuz; Zeybek, Ümit; Yaylım, Ilhan

2013-01-01

Objective. It has been stated that brain cancers are an increasingly serious issue in many parts of the world. The aim of our study was to determine a possible relationship between Vitamin D receptor (VDR) gene polymorphisms and the risk of glioma and meningioma. Methods. We investigated the VDR Taq-I and VDR Fok-I gene polymorphisms in 100 brain cancer patients (including 44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjects. This study was performed by polymerase chain reaction-based restriction fragment length polymorphism (RF LP). Results. VDR Fok-I ff genotype was significantly increased in meningioma patients (15.9%) compared with controls (2.5%), and carriers of Fok-I ff genotype had a 6.47-fold increased risk for meningioma cases. There was no significant difference between patients and controls for VDR Taq-I genotypes and alleles. Conclusions. We suggest that VDR Fok-I genotypes might affect the development of meningioma. PMID:23691496

“Forward Genetics” as a Method to Maximize Power and Cost-Efficiency in Studies of Human Complex Traits

PubMed Central

Derks, E. M.; Dolan, C. V.; Kahn, R. S.; Ophoff, R. A.

2010-01-01

There is increasing interest in methods to disentangle the relationship between genotype and (endo)phenotypes in human complex traits. We present a population-based method of increasing the power and cost-efficiency of studies by selecting random individuals with a particular genotype and then assessing the accompanying quantitative phenotypes. Using statistical derivations, power- and cost graphs we show that such a “forward genetics” approach can lead to a marked reduction in sample size and costs. This approach is particularly apt for implementing in epidemiological studies for which DNA is already available but the phenotyping costs are high. Electronic supplementary material The online version of this article (doi:10.1007/s10519-010-9348-y) contains supplementary material, which is available to authorized users. PMID:20232132

An Efficient, Simple, and Noninvasive Procedure for Genotyping Aquatic and Nonaquatic Laboratory Animals.

PubMed

Okada, Morihiro; Miller, Thomas C; Roediger, Julia; Shi, Yun-Bo; Schech, Joseph Mat

2017-09-01

Various animal models are indispensible in biomedical research. Increasing awareness and regulations have prompted the adaptation of more humane approaches in the use of laboratory animals. With the development of easier and faster methodologies to generate genetically altered animals, convenient and humane methods to genotype these animals are important for research involving such animals. Here, we report skin swabbing as a simple and noninvasive method for extracting genomic DNA from mice and frogs for genotyping. We show that this method is highly reliable and suitable for both immature and adult animals. Our approach allows a simpler and more humane approach for genotyping vertebrate animals.

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

PubMed Central

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz

2017-01-01

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup. PMID:29099791

AML outcome: role of nucleotide excision repair polymorphisms in intermediate risk patients

PubMed Central

Strom, Sara S; Estey, Elihu H; Outschoorn, Ubaldo Martinez; Guillermo, Garcia-Manero

2010-01-01

Purpose Acute Myeloid Leukemia (AML) is frequently associated with genetic abnormalities. Based on pre-treatment cytogenetics, patients are classified into favorable, intermediate and poor subgroups. Cytogenetics predicts treatment outcome for the favorable and poor subgroups but not for the intermediate subgroup. Polymorphisms within the nucleotide excision repair (NER) pathway may lead to inter-individual differences in DNA repair capacity (DRC) which could influence outcome. Methods We studied the role of 6 polymorphisms (ERCC1 Gln504Lys, XPD Lys751Gln, XPC Ala499Val, XPC Lys939Gln, XPG Asp1104His, and CCNH Val270Ala) within NER pathway on overall and disease-free survival among 170 adult de-novo AML patients with intermediate cytogenetics [diploid (n=117); non-diploid (n=53)], treated with induction chemotherapy. Kaplan-Meier methods and Cox proportional hazards models were performed. Results Diploid patients with the XPD AC/CC genotype survived shorter than those with the wild-type (AA) genotype (median survival 22 vs. 40 months, log-rank p = 0.03). Similarly diploid patients with XPC CT/TT genotype survived shorter than those with the wild-type (CC) genotype (median survival 15 vs. 30 months, log-rank p = 0.02). Among diploid patients, after adjusting for clinical and socio-demographic variables, patients carrying both XPD AC/CC and XPC CT/TT had a greater than two-fold increased risk of dying compared to those with the wild-type genotypes (HR=2.49; 95%CI: 1.06–5.85). No significant associations were observed for disease-free survival in AML patients. Conclusion By reduced DRC, this combined genotype may result in greater susceptibility to treatment effects decreasing overall survival. These findings could in the future help in selecting treatment strategies for patients with normal cytogenetics. PMID:20141440

Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, J.M.

The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR{beta} chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation {chi}{sup 2} method was used to evaluate evidence for linkagemore » disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal {chi}{sup 2}. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401, *0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women. 67 refs., 7 tabs.« less

A Simple PCR Method for Rapid Genotype Analysis of Mycobacterium ulcerans

PubMed Central

Stinear, Timothy; Davies, John K.; Jenkin, Grant A.; Portaels, Françoise; Ross, Bruce C.; OppEdIsano, Frances; Purcell, Maria; Hayman, John A.; Johnson, Paul D. R.

2000-01-01

Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype. PMID:10747130

Novel approach to differentiate subclades of varicella-zoster virus genotypes E1 and E2 in Germany.

PubMed

Schmidt-Chanasit, Jonas; Olschläger, Stephan; Bialonski, Alexandra; Heinemann, Patrick; Bleymehl, Karoline; Gross, Gerd; Günther, Stephan; Ulrich, Rainer G; Doerr, Hans Wilhelm

2009-11-01

Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) in children and reactivation of VZV in elderly or immunocompromised persons can cause shingles (zoster). A subclade differentiation of the most prevalent VZV genotypes E1 and E2 in Germany was not possible with the current genotyping methods in use, but is highly important to understand the VZV molecular evolution in more detail and especially to follow up the routes of infection. Therefore the objective of this study was to develop a simple PCR-based method for differentiation of E1 and E2 subclades. Viral DNA was isolated from vesicle fluid samples of six selected German zoster patients and used to amplify nine complete open reading frames (ORFs) of the VZV genome by different PCR assays. Phylogenetic analysis was performed by a Bayesian approach. Based on the analysis of a total of nine ORFs, a 7482 bp stretch consisting of ORFs 5, 37 and 62 contained informative sites for identification of novel subclades E1a, E2a and E2b for VZV genotypes E1 and E2. Specific single nucleotide polymorphisms (SNPs) were demonstrated for subclades E2a and E2b within the ORFs 5, 37 and 62, whereas a subclade E1a-specific SNP was found in ORF 56. The classification of E1 and E2 subclades may facilitate a more exact and in-depth monitoring of the molecular evolution of VZV in Germany in the future.

Development of a genotyping microarray for Usher syndrome

PubMed Central

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-01-01

Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

PubMed

Mallory, Melanie A; Lucic, Danijela X; Sears, Mitchell T; Cloherty, Gavin A; Hillyard, David R

2014-05-01

HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method. Copyright © 2014 Elsevier B.V. All rights reserved.

Knowing your genes: does this impact behaviour change?

PubMed

O'Donovan, Clare B; Walsh, Marianne C; Gibney, Michael J; Brennan, Lorraine; Gibney, Eileen R

2017-08-01

It is postulated that knowledge of genotype may be more powerful than other types of personalised information in terms of motivating behaviour change. However, there is also a danger that disclosure of genetic risk may promote a fatalistic attitude and demotivate individuals. The original concept of personalised nutrition (PN) focused on genotype-based tailored dietary advice; however, PN can also be delivered based on assessment of dietary intake and phenotypic measures. Whilst dietitians currently provide PN advice based on diet and phenotype, genotype-based PN advice is not so readily available. The aim of this review is to examine the evidence for genotype-based personalised information on motivating behaviour change, and factors which may affect the impact of genotype-based personalised advice. Recent findings in PN will also be discussed, with respect to a large European study, Food4Me, which investigated the impact of varying levels of PN advice on motivating behaviour change. The researchers reported that PN advice resulted in greater dietary changes compared with general healthy eating advice, but no additional benefit was observed for PN advice based on phenotype and genotype information. Within Food4Me, work from our group revealed that knowledge of MTHFR genotype did not significantly improve intakes of dietary folate. In general, evidence is weak with regard to genotype-based PN advice. For future work, studies should test the impact of PN advice developed on a strong nutrigenetic evidence base, ensure an appropriate study design for the research question asked, and incorporate behaviour change techniques into the intervention.

Genotype-based gene signature of glioma risk.

PubMed

Huang, Yen-Tsung; Zhang, Yi; Wu, Zhijin; Michaud, Dominique S

2017-07-01

Glioma accounts for 80% of malignant brain tumors, but its etiologic determinants remain elusive. Despite genetic susceptibility loci identified by genome-wide association study (GWAS), the agnostic approach leaves open the possibility that other susceptibility genes remain to be discovered. Here we conduct a gene-centric integrative GWAS (iGWAS) of glioma risk that combines transcriptomics and genetics. We synthesized a brain transcriptomics dataset (n = 354), a GWAS dataset (n = 4203), and an advanced glioma tumor transcriptomic dataset (n = 483) to conduct an iGWAS. Using the expression quantitative trait loci (eQTL) dataset, we built models to predict gene expression for the GWAS data, based on eQTL genotypes. With the predicted gene expression, iGWAS analyses were performed using a novel statistical method. Gene signature risk score was constructed using a penalized logistic regression model. A total of 30527 transcripts were analyzed using the iGWAS approach. Four novel glioma susceptibility genes were identified with internal and external validation, including DRD5 (P = 3.0 × 10-79), WDR1 (P = 8.4 × 10-77), NOMO1 (P = 1.3 × 10-25), and PDXDC1 (P = 8.3 × 10-24). The genotype-predicted transcription pattern between cases and controls is consistent with that between tumor and its matched normal tissue. The genotype-based 4-gene signature improved the classification between glioma cases and controls based on age, gender, and population stratification, with area under the receiver operating characteristic curve increasing from 0.77 to 0.85 (P = 8.1 × 10-23). A new genotype-based gene signature of glioma was identified using a novel iGWAS approach, which integrates multiplatform genomic data as well as different genetic association studies. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes

PubMed Central

Pasquali, Matias; Beyer, Marco; Logrieco, Antonio; Audenaert, Kris; Balmas, Virgilio; Basler, Ryan; Boutigny, Anne-Laure; Chrpová, Jana; Czembor, Elżbieta; Gagkaeva, Tatiana; González-Jaén, María T.; Hofgaard, Ingerd S.; Köycü, Nagehan D.; Hoffmann, Lucien; Lević, Jelena; Marin, Patricia; Miedaner, Thomas; Migheli, Quirico; Moretti, Antonio; Müller, Marina E. H.; Munaut, Françoise; Parikka, Päivi; Pallez-Barthel, Marine; Piec, Jonathan; Scauflaire, Jonathan; Scherm, Barbara; Stanković, Slavica; Thrane, Ulf; Uhlig, Silvio; Vanheule, Adriaan; Yli-Mattila, Tapani; Vogelgsang, Susanne

2016-01-01

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000–2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified. PMID:27092107

A novel Tetra-primer ARMS-PCR based assay for genotyping SNP rs12303764(G/T) of human Unc-51 like kinase 1 gene.

PubMed

Randhawa, Rohit; Duseja, Ajay; Changotra, Harish

2017-02-01

Various case-control studies have shown association of single nucleotide polymorphism rs12303764(G/T) in ULK1 with crohn's disease. The techniques used in these studies were time consuming, complicated and require sophisticated/expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective Tetra-primer ARMS-PCR assay to genotype single nucleotide polymorphism rs12303764(G/T) of ULK1 gene. We manually designed allele specific primers. DNA fragment amplified using outer primers was sequenced to obtain samples with known genotypes (GG, GT and TT) for further use in the development of T-ARMS-PCR assay. Amplification conditions were optimized for parameters; annealing temperature, Taq DNA polymerase and primers. The developed T-ARMS-PCR assay was applied to genotype one hundred samples from healthy individuals. Genotyping results of 10 DNA samples from healthy individuals for rs12303764(G/T) by T-ARMS-PCR assay and sequencing were concordant. The newly developed assay was further applied to genotype samples from 100 healthy individuals of North Indian origin. Genotype frequencies were 9, 34 and 57 % for GG, GT and TT, respectively. Allele frequencies were 0.26 and 0.74 for G and T, respectively. The allele frequencies were in Hardy-Weinberg's equilibrium (p = 0.2443). T-ARMS-PCR assay developed in our laboratory for genotyping rs12303764 (G/T) of ULK1 gene is time saving and cost-effective as compared to the available methods. Furthermore, this is the first study reporting allelic and genotype frequencies of ULK1 rs12303764 (G/T) variants in North Indian population.

Warfarin genotyping in a single PCR reaction for microchip electrophoresis.

PubMed

Poe, Brian L; Haverstick, Doris M; Landers, James P

2012-04-01

Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

Human Papillomavirus Genotype Prevalence in Invasive Penile Cancers from a Registry-Based United States Population

PubMed Central

Hernandez, Brenda Y.; Goodman, Marc T.; Unger, Elizabeth R.; Steinau, Martin; Powers, Amy; Lynch, Charles F.; Cozen, Wendy; Saber, Maria Sibug; Peters, Edward S.; Wilkinson, Edward J.; Copeland, Glenn; Hopenhayn, Claudia; Huang, Youjie; Watson, Meg; Altekruse, Sean F.; Lyu, Christopher; Saraiya, Mona

2013-01-01

Background: Human papillomavirus (HPV) is estimated to play an etiologic role in 40–50% of penile cancers worldwide. Estimates of HPV prevalence in U.S. penile cancer cases are limited. Methods: HPV DNA was evaluated in tumor tissue from 79 invasive penile cancer patients diagnosed in 1998–2005 within the catchment areas of seven U.S. cancer registries. HPV was genotyped using PCR-based Linear Array and INNO-LiPA assays and compared by demographic, clinical, and pathologic characteristics and survival. Histological classification was also obtained by independent pathology review. Results: HPV DNA was present in 50 of 79 (63%) of invasive penile cancer cases. Sixteen viral genotypes were detected. HPV 16, found in 46% (36/79) of all cases (72% of HPV-positive cases) was the most prevalent genotype followed equally by HPV 18, 33, and 45, each of which comprised 5% of all cases. Multiple genotypes were detected in 18% of viral positive cases. HPV prevalence did not significantly vary by age, race/ethnicity, population size of geographic region, cancer stage, histology, grade, penile subsite, or prior cancer history. Penile cases diagnosed in more recent years were more likely to be HPV-positive. Overall survival did not significantly vary by HPV status. Conclusion: The relatively high prevalence of HPV in our study population provides limited evidence of a more prominent and, possibly, increasing role of infection in penile carcinogenesis in the U.S. compared to other parts of the world. PMID:24551592

Manganese superoxide dismutase Ala-9Val polymorphism and risk of breast cancer in a population-based case–control study of African Americans and whites

PubMed Central

Millikan, Robert C; Player, Jon; de Cotret, Allan René; Moorman, Patricia; Pittman, Gary; Vannappagari, Vani; Tse, Chiu-Kit J; Keku, Temitope

2004-01-01

Introduction A polymorphism in the manganese superoxide dismutase (MnSOD) gene, Ala-9Val, has been examined in association with breast cancer risk in several epidemiologic studies. Results suggest that the Ala allele increases the risk of breast cancer and modifies the effects of environmental exposures that produce oxidative damage to DNA. Methods We examined the role of the MnSOD Ala-9Val polymorphism in a population-based case–control study of invasive and in situ breast cancer in North Carolina. Genotypes were evaluated for 2025 cases (760 African Americans and 1265 whites) and for 1812 controls (677 African Americans and 1135 whites). Results The odds ratio for MnSOD Ala/Ala versus any MnSOD Val genotypes was not elevated in African Americans (odds ratio = 0.9, 95% confidence interval = 0.7–1.2) or in whites (odds ratio = 1.0, 95% confidence interval = 0.8–1.2). Greater than additive joint effects were observed for the Ala/Ala genotype and smoking, radiation to the chest, and occupational exposure to ionizing radiation. Antagonism was observed between the Ala/Ala genotype and the use of nonsteroidal anti-inflammatory drugs. Conclusions The MnSOD genotype may contribute to an increased risk of breast cancer in the presence of specific environmental exposures. These results provide further evidence for the importance of reactive oxygen species and of oxidative DNA damage in the etiology of breast cancer. PMID:15217492

[Prevalence of occult hepatitis B virus infection and its phylogenetic features among mother-teenager pairs].

PubMed

Dong, Xiao-lian; Yao, Qing-qing; Wang, Xue-cai; Xu, Hai-tao; Wang, Xiao-li; Chen, Sheng-yu; Tang, Zhi-feng; Zheng, Ying-Jie

2013-03-01

Prevalence of occult hepatitis B virus (HBV) infection (OBI) was investigated in a paired mother-teenager population and HBV S gene variation including overt and occult HBV, was determined. A follow-up study based on an initial survey of 135 mother-teenager pairs was carried out through collection of questionnaires and blood samples HBsAg were detected by ELISA method, viral load by PCR amplification and HBV S gene by phylogenetic analysis. 102 pairs of subjects were followed-up. Blood samples from 94 mothers and 101 children were collected. OBI prevalence in mothers was 10.0% (6/60), significantly higher than 2.0% (2/101) in teenagers. Medians of viral load were 399.9 IU/ml and 247.6 IU/ml in overt and occult HBV strains, but without significant difference. 1 occult HBV strain belonged to genotype B with serotype adw while the other 7 were genotype C with serotype adr. 15 of the overt HBV strains belonged to genotype B with serotype adw and the other 8 were genotype C with serotype adr. Proportions of genotype-C strains were significantly higher in occult HBV strains than in overt HBV strains. OBI was seen in teenage-mother population.

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current genotyping procedures: a population-based study in northeast Mexico.

PubMed

Peñuelas-Urquides, Katia; Martínez-Rodríguez, Herminia Guadalupe; Enciso-Moreno, José Antonio; Molina-Salinas, Gloria María; Silva-Ramírez, Beatriz; Padilla-Rivas, Gerardo Raymundo; Vera-Cabrera, Lucio; Torres-de-la-Cruz, Víctor Manuel; Martínez-Martínez, Yazmin Berenice; Ortega-García, Jorge Luis; Garza-Treviño, Elsa Nancy; Enciso-Moreno, Leonor; Saucedo-Cárdenas, Odila; Becerril-Montes, Pola; Said-Fernández, Salvador

2014-09-01

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman's rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Paraoxonase-1 gene Q192R polymorphism and reactive oxygen metabolites.

PubMed

Kotani, K; Tsuzaki, K; Sakane, N

2012-01-01

Paraoxonase-1 (PON1) is a high-density lipoprotein-associated antioxidant enzyme. The Q192R polymorphism of the PON1 gene can protect against oxidative conditions, but the relationship between Q192R polymorphism and oxidative stress-related markers remains controversial. In this study, the diacron reactive oxygen metabolites (d-ROMs) test was used to investigate the relationship between Q192R polymorphism and oxidative stress-related markers in Japanese subjects. Patients without a history of overt cardiovascular disease who were not receiving antioxidant medication were enrolled in a cross-sectional clinic-based study. An allele-specific polymerase chain reaction method was used to assess the PON1 Q192R polymorphism and compare the level of d-ROMs between genotypes. A total of 103 subjects were analysed. The RR genotype was associated with a significantly lower level of d-ROMs than the RQ and QQ genotypes. After multivariate analysis the relationship between the genotypes and level of d-ROMs remained independently significant. The RR genotype may be protective against oxidative stress in cardiovascular diseasefree Japanese subjects. In addition, the d-ROMs test can be useful for examining the role of the PON1 Q192R polymorphism under oxidative conditions.

Application of genotyping-by-sequencing for mapping disease resistance in grapevine breeding families

USDA-ARS?s Scientific Manuscript database

Genotyping-by-Sequencing (GBS) is a low-cost, high-throughput, method for genome-wide polymorphism discovery and genotyping adjacent to restriction sites. Since 2010, GBS has been applied for the genotyping of over 12,000 grape breeding lines, with a primary focus on identifying markers predictive ...

Development of a cost-efficient novel method for rapid, concurrent genotyping of five common single nucleotide polymorphisms of the brain derived neurotrophic factor (BDNF) gene by tetra-primer amplification refractory mutation system.

PubMed

Wang, Cathy K; Xu, Michael S; Ross, Colin J; Lo, Ryan; Procyshyn, Ric M; Vila-Rodriguez, Fidel; White, Randall F; Honer, William G; Barr, Alasdair M

2015-09-01

Brain derived neurotrophic factor (BDNF) is a molecular trophic factor that plays a key role in neuronal survival and plasticity. Single nucleotide polymorphisms (SNPs) of the BDNF gene have been associated with specific phenotypic traits in a large number of neuropsychiatric disorders and the response to psychotherapeutic medications in patient populations. Nevertheless, due to study differences and occasionally contrasting findings, substantial further research is required to understand in better detail the association between specific BDNF SNPs and these psychiatric disorders. While considerable progress has been made recently in developing advanced genotyping platforms of SNPs, many high-throughput probe- or array-based detection methods currently available are limited by high costs, slow processing times or access to advanced instrumentation. The polymerase chain reaction (PCR)-based, tetra-primer amplification refractory mutation system (T-ARMS) method is a potential alternative technique for detecting SNP genotypes efficiently, quickly, easily, and cheaply. As a tool in psychopathology research, T-ARMS was shown to be capable of detecting five common SNPs in the BDNF gene (rs6265, rs988748, rs11030104, 11757G/C and rs7103411), which are all SNPs with previously demonstrated clinical relevance to schizophrenia and depression. The present technique therefore represents a suitable protocol for many research laboratories to study the genetic correlates of BDNF in psychiatric disorders. Copyright Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Genetic variation in Southern USA rice genotypes for seedling salinity tolerance

PubMed Central

De Leon, Teresa B.; Linscombe, Steven; Gregorio, Glenn; Subudhi, Prasanta K.

2015-01-01

The success of a rice breeding program in developing salt tolerant varieties depends on genetic variation and the salt stress response of adapted and donor rice germplasm. In this study, we used a combination of morphological and physiological traits in multivariate analyses to elucidate the phenotypic and genetic variation in salinity tolerance of 30 Southern USA rice genotypes, along with 19 donor genotypes with varying degree of tolerance. Significant genotypic variation and correlations were found among the salt injury score (SIS), ion leakage, chlorophyll reduction, shoot length reduction, shoot K+ concentration, and shoot Na+/K+ ratio. Using these parameters, the combined methods of cluster analysis and discriminant analysis validated the salinity response of known genotypes and classified most of the USA varieties into sensitive groups, except for three and seven varieties placed in the tolerant and moderately tolerant groups, respectively. Discriminant function and MANOVA delineated the differences in tolerance and suggested no differences between sensitive and highly sensitive (HS) groups. DNA profiling using simple sequence repeat markers showed narrow genetic diversity among USA genotypes. However, the overall genetic clustering was mostly due to subspecies and grain type differentiation and not by varietal grouping based on salinity tolerance. Among the donor genotypes, Nona Bokra, Pokkali, and its derived breeding lines remained the donors of choice for improving salinity tolerance during the seedling stage. However, due to undesirable agronomic attributes and photosensitivity of these donors, alternative genotypes such as TCCP266, Geumgangbyeo, and R609 are recommended as useful and novel sources of salinity tolerance for USA rice breeding programs. PMID:26074937

SensiScreen® KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations

PubMed Central

Guldmann-Christensen, Mariann; Hauge Kyneb, Majbritt; Voogd, Kirsten; Andersen, Christina; Epistolio, Samantha; Merlo, Elisabetta; Yding Wolff, Tine; Hamilton-Dutoit, Stephen; Lorenzen, Jan; Christensen, Ulf Bech

2017-01-01

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase’s proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25–1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods. PMID:28636636

A phasing and imputation method for pedigreed populations that results in a single-stage genomic evaluation

PubMed Central

2012-01-01

Background Efficient, robust, and accurate genotype imputation algorithms make large-scale application of genomic selection cost effective. An algorithm that imputes alleles or allele probabilities for all animals in the pedigree and for all genotyped single nucleotide polymorphisms (SNP) provides a framework to combine all pedigree, genomic, and phenotypic information into a single-stage genomic evaluation. Methods An algorithm was developed for imputation of genotypes in pedigreed populations that allows imputation for completely ungenotyped animals and for low-density genotyped animals, accommodates a wide variety of pedigree structures for genotyped animals, imputes unmapped SNP, and works for large datasets. The method involves simple phasing rules, long-range phasing and haplotype library imputation and segregation analysis. Results Imputation accuracy was high and computational cost was feasible for datasets with pedigrees of up to 25 000 animals. The resulting single-stage genomic evaluation increased the accuracy of estimated genomic breeding values compared to a scenario in which phenotypes on relatives that were not genotyped were ignored. Conclusions The developed imputation algorithm and software and the resulting single-stage genomic evaluation method provide powerful new ways to exploit imputation and to obtain more accurate genetic evaluations. PMID:22462519

The Tubulin-Based-Polymorphism Method Provides a Simple and Effective Alternative to the Genomic Profiling of Grape

PubMed Central

Mastromauro, Francesco; Gianì, Silvia; Morello, Laura

2016-01-01

The TBP (Tubulin-Based-Polymorphism) method, based on a nuclear ILP (Intron-Length-Polymorphism) molecular marker, has been used for genotyping 37 accessions of the genus Vitis inclusive of different species, rootstocks, wild and cultivated subspecies. A distinct DNA barcode made up by a different number of amplicons, was attributed to each of the different accessions. TBP data were compared with those obtained, with the use of an internationally validated set of six SSR markers. Genetic relationships among the different accessions, dendrogram distributions, correlation values and polymorphic index values (PICs) were definitively comparable when not in favor of TBP. Such an experimental consistency is based upon a genomic organization of the multiple members of the β-tubulin gene family, the targets of TBP-mediated amplification, that is conserved in Vitis as in any other plant species. The TBP amplicons can actually be used as a useful source of sequence polymorphisms for generating primer pairs capable of identifying specific cultivars in a simple assay. An example for the identification of the ‘Sangiovese’ cv. is reported. More generally, these data are discussed in terms of the actual advantages that the introduction of the TBP method in the field of grape characterization and genotyping can provide. PMID:27643687

Vitis Phylogenomics: Hybridization Intensities from a SNP Array Outperform Genotype Calls

PubMed Central

Miller, Allison J.; Matasci, Naim; Schwaninger, Heidi; Aradhya, Mallikarjuna K.; Prins, Bernard; Zhong, Gan-Yuan; Simon, Charles; Buckler, Edward S.; Myles, Sean

2013-01-01

Understanding relationships among species is a fundamental goal of evolutionary biology. Single nucleotide polymorphisms (SNPs) identified through next generation sequencing and related technologies enable phylogeny reconstruction by providing unprecedented numbers of characters for analysis. One approach to SNP-based phylogeny reconstruction is to identify SNPs in a subset of individuals, and then to compile SNPs on an array that can be used to genotype additional samples at hundreds or thousands of sites simultaneously. Although powerful and efficient, this method is subject to ascertainment bias because applying variation discovered in a representative subset to a larger sample favors identification of SNPs with high minor allele frequencies and introduces bias against rare alleles. Here, we demonstrate that the use of hybridization intensity data, rather than genotype calls, reduces the effects of ascertainment bias. Whereas traditional SNP calls assess known variants based on diversity housed in the discovery panel, hybridization intensity data survey variation in the broader sample pool, regardless of whether those variants are present in the initial SNP discovery process. We apply SNP genotype and hybridization intensity data derived from the Vitis9kSNP array developed for grape to show the effects of ascertainment bias and to reconstruct evolutionary relationships among Vitis species. We demonstrate that phylogenies constructed using hybridization intensities suffer less from the distorting effects of ascertainment bias, and are thus more accurate than phylogenies based on genotype calls. Moreover, we reconstruct the phylogeny of the genus Vitis using hybridization data, show that North American subgenus Vitis species are monophyletic, and resolve several previously poorly known relationships among North American species. This study builds on earlier work that applied the Vitis9kSNP array to evolutionary questions within Vitis vinifera and has general implications for addressing ascertainment bias in array-enabled phylogeny reconstruction. PMID:24236035

The Polymorphism of YWHAE, a Gene Encoding 14-3-3Epsilon, and Brain Morphology in Schizophrenia: A Voxel-Based Morphometric Study

PubMed Central

Nemoto, Kiyotaka; Takahashi, Tsutomu; Aleksic, Branko; Furuichi, Atsushi; Nakamura, Yumiko; Ikeda, Masashi; Noguchi, Kyo; Kaibuchi, Kozo; Iwata, Nakao; Ozaki, Norio; Suzuki, Michio

2014-01-01

Background YWHAE is a possible susceptibility gene for schizophrenia that encodes 14-3-3epsilon, a Disrupted-in-Schizophrenia 1 (DISC1)-interacting molecule, but the effect of variation in its genotype on brain morphology remains largely unknown. Methods In this voxel-based morphometric magnetic resonance imaging study, we conducted whole-brain analyses regarding the effects of YWHAE single-nucleotide polymorphisms (SNPs) (rs28365859, rs11655548, and rs9393) and DISC1 SNP (rs821616) on gray matter volume in a Japanese sample of 72 schizophrenia patients and 86 healthy controls. On the basis of a previous animal study, we also examined the effect of rs28365859 genotype specifically on hippocampal volume. Results Whole-brain analyses showed no significant genotype effect of these SNPs on gray matter volume in all subjects, but we found significant genotype-by-diagnosis interaction for rs28365859 in the left insula and right putamen. The protective C allele carriers of rs28365859 had a significantly larger left insula than the G homozygotes only for schizophrenia patients, while the controls with G allele homozygosity had a significantly larger right putamen than the C allele carriers. The C allele carriers had a larger right hippocampus than the G allele homozygotes in schizophrenia patients, but not in healthy controls. No significant interaction was found between rs28365859 and DISC1 SNP on gray matter volume. Conclusions These different effects of the YWHAE (rs28365859) genotype on brain morphology in schizophrenia and healthy controls suggest that variation in its genotype might be, at least partly, related to the abnormal neurodevelopment, including in the limbic regions, reported in schizophrenia. Our results also suggest its specific role among YWHAE SNPs in the pathophysiology of schizophrenia. PMID:25105667

Common Polymorphisms in GSTA1, GSTM1 and GSTT1 Are Associated with Susceptibility to Urinary Bladder Cancer in Individuals from Balkan Endemic Nephropathy Areas of Serbia.

PubMed

Matic, Marija; Dragicevic, Biljana; Pekmezovic, Tatjana; Suvakov, Sonja; Savic-Radojevic, Ana; Pljesa-Ercegovac, Marija; Dragicevic, Dejan; Smiljic, Jelena; Simic, Tatjana

2016-09-01

Balkan endemic nephropathy (BEN) is a chronic familial form of interstitial nephritis that might eventually lead to end stage renal disease. This nephropathy affects individuals living along of the Danube River and its tributaries in Serbia, Bosnia, Croatia, Bulgaria and Romania. The increased incidence of urinary tract tumors in the BEN areas is well described, but its specific genetic predisposition is still unclear. Certain nephrocarcinogenic compounds, including those associated with BEN, are metabolized by glutathione S-transferase (GST) superfamily of phase II detoxication enzymes. Importantly, the GST-mediated detoxification may result in formation of more toxic compounds. We examined the association of common GST polymorphisms and bladder cancer (BC) risk in individuals from BEN areas in Serbia. A hospital-based case-control study included 201 BC cases (67 from BEN region) and 122 controls. Each polymorphism was identified by a PCR-based method. Individuals from BEN region with low-expression GSTA1 genotype (AB+BB) exhibited a 2.6-fold higher BC risk compared to those with GSTA1 (AA) genotype who were from non-BEN region (OR = 2.60, p = 0.015). In contrast, carriers of GSTM1-active genotype from BEN region had a 2.9-fold increased BC risk compared to those with GSTM1-active genotype from non-BEN region (OR = 2.90, p = 0.010). Likewise, carriers with GSTT1-active genotype from BEN region exhibited 2.1-fold higher BC risk compared to those from non-BEN region with GSTT1-active genotype (OR = 2.10, p = 0.027). Thus, common polymorphisms in GSTA1, GSTM1 and GSTT1 are associated with susceptibility to BC in individuals from BEN areas of Serbia.

[Individual Identification of Cartilage by Direct Amplification in Mass Disasters].

PubMed

Wang, C H; Xu, C; Li, X Q; Wu, Y; Du, Z

2017-06-01

To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification. Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method. In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent. Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters. Copyright© by the Editorial Department of Journal of Forensic Medicine

Genotypic diversity of european Phytophthora ramorum isolates based on SSR analysis

Treesearch

Kris Van Poucke; Annelies Vercauteren; Martine Maes; Sabine Werres; Kurt Heungens

2013-01-01

in Scotland were genotyped using seven microsatellite markers as described by Vercauteren et al. (2010). Thirty multilocus genotypes were identified within the Scottish population, with 51 percent of the isolates belonging to the main European genotype EU1MG1 and 13 unique detected genotypes. Ten of those genotypes were site specific, often represented by...

HIV coreceptor phenotyping in the clinical setting.

PubMed

Low, Andrew J; Swenson, Luke C; Harrigan, P Richard

2008-01-01

The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.

SNP Data Quality Control in a National Beef and Dairy Cattle System and Highly Accurate SNP Based Parentage Verification and Identification

PubMed Central

McClure, Matthew C.; McCarthy, John; Flynn, Paul; McClure, Jennifer C.; Dair, Emma; O'Connell, D. K.; Kearney, John F.

2018-01-01

A major use of genetic data is parentage verification and identification as inaccurate pedigrees negatively affect genetic gain. Since 2012 the international standard for single nucleotide polymorphism (SNP) verification in Bos taurus cattle has been the ISAG SNP panels. While these ISAG panels provide an increased level of parentage accuracy over microsatellite markers (MS), they can validate the wrong parent at ≤1% misconcordance rate levels, indicating that more SNP are needed if a more accurate pedigree is required. With rapidly increasing numbers of cattle being genotyped in Ireland that represent 61 B. taurus breeds from a wide range of farm types: beef/dairy, AI/pedigree/commercial, purebred/crossbred, and large to small herd size the Irish Cattle Breeding Federation (ICBF) analyzed different SNP densities to determine that at a minimum ≥500 SNP are needed to consistently predict only one set of parents at a ≤1% misconcordance rate. For parentage validation and prediction ICBF uses 800 SNP (ICBF800) selected based on SNP clustering quality, ISAG200 inclusion, call rate (CR), and minor allele frequency (MAF) in the Irish cattle population. Large datasets require sample and SNP quality control (QC). Most publications only deal with SNP QC via CR, MAF, parent-progeny conflicts, and Hardy-Weinberg deviation, but not sample QC. We report here parentage, SNP QC, and a genomic sample QC pipelines to deal with the unique challenges of >1 million genotypes from a national herd such as SNP genotype errors from mis-tagging of animals, lab errors, farm errors, and multiple other issues that can arise. We divide the pipeline into two parts: a Genotype QC and an Animal QC pipeline. The Genotype QC identifies samples with low call rate, missing or mixed genotype classes (no BB genotype or ABTG alleles present), and low genotype frequencies. The Animal QC handles situations where the genotype might not belong to the listed individual by identifying: >1 non-matching genotypes per animal, SNP duplicates, sex and breed prediction mismatches, parentage and progeny validation results, and other situations. The Animal QC pipeline make use of ICBF800 SNP set where appropriate to identify errors in a computationally efficient yet still highly accurate method. PMID:29599798

Cholesterol, APOE genotype, and Alzheimer disease

PubMed Central

Hall, K.; Murrell, J.; Ogunniyi, A.; Deeg, M.; Baiyewu, O.; Gao, S.; Gureje, O.; Dickens, J.; Evans, R.; Smith-Gamble, V.; Unverzagt, F.W.; Shen, J.; Hendrie, H.

2010-01-01

Objective To examine the relationship between cholesterol and other lipids, APOE genotype, and risk of Alzheimer disease (AD) in a population-based study of elderly Yoruba living in Ibadan, Nigeria. Methods Blood samples and clinical data were collected from Yoruba study participants aged 70 years and older (N = 1,075) as part of the Indianapolis-Ibadan Dementia Project, a longitudinal epidemiologic study of AD. Cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglyceride levels were measured in fasting blood samples. DNA was extracted and APOE was genotyped. Diagnoses of AD were made by consensus using National Institute of Neurologic Disorders/Stroke-Alzheimer's Disease and Related Disorders Association criteria. Results Logistic regression models showed interaction after adjusting for age and gender between APOE-ε4 genotype and biomarkers in the risk of AD cholesterol*genotype (p = 0.022), LDL*genotype (p = 0.018), and triglyceride*genotype (p = 0.036). Increasing levels of cholesterol and LDL were associated with increased risk of AD in individuals without the APOE-ε4 allele, but not in those with APOE-ε4. There was no significant association between levels of triglycerides and AD risk in those without APOE-ε4. Conclusions There was a significant interaction between cholesterol, APOE-ε4, and the risk of Alzheimer disease (AD) in the Yoruba, a population that has lower cholesterol levels and lower incidence rates of AD compared to African Americans. APOE status needs to be considered when assessing the relationship between lipid levels and AD risk in population studies. PMID:16434658

A parsimonious tree-grow method for haplotype inference.

PubMed

Li, Zhenping; Zhou, Wenfeng; Zhang, Xiang-Sun; Chen, Luonan

2005-09-01

Haplotype information has become increasingly important in analyzing fine-scale molecular genetics data, such as disease genes mapping and drug design. Parsimony haplotyping is one of haplotyping problems belonging to NP-hard class. In this paper, we aim to develop a novel algorithm for the haplotype inference problem with the parsimony criterion, based on a parsimonious tree-grow method (PTG). PTG is a heuristic algorithm that can find the minimum number of distinct haplotypes based on the criterion of keeping all genotypes resolved during tree-grow process. In addition, a block-partitioning method is also proposed to improve the computational efficiency. We show that the proposed approach is not only effective with a high accuracy, but also very efficient with the computational complexity in the order of O(m2n) time for n single nucleotide polymorphism sites in m individual genotypes. The software is available upon request from the authors, or from http://zhangroup.aporc.org/bioinfo/ptg/ chen@elec.osaka-sandai.ac.jp Supporting materials is available from http://zhangroup.aporc.org/bioinfo/ptg/bti572supplementary.pdf

Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients.

PubMed

Malapelle, Umberto; Pisapia, Pasquale; Rocco, Danilo; Smeraglio, Riccardo; di Spirito, Maria; Bellevicine, Claudio; Troncone, Giancarlo

2016-10-01

The advent of genomic based personalized medicine has led to multiple advances in the molecular characterization of many tumor types, such as non-small cell lung cancer (NSCLC). NSCLC is diagnosed in most cases on small tissue samples that may be not always sufficient for EGFR mutational assessment to select patients for first and second generations' tyrosine kinase inhibitors (TKIs) therapy. In patients without tissue availability at presentation, the analysis of cell free DNA (cfDNA) derived from liquid biopsy samples, in particular from plasma, represent an established alternative to provide EGFR mutational testing for treatment decision making. In addition, a new paradigm for TKIs resistance management was recently approved by Food and Drug Administration, supporting the liquid biopsy based genotyping prior to tissue based genotyping for the detection of T790M mutation to select patients for third generation TKIs. In these settings, real time PCR (RT-PCR) and digital PCR 'targeted' methods, which detect known mutations by specific probes, have extensively been adopted. Taking into account the restricted reference range and the limited multiplexing power of these targeted methods, the performance of liquid biopsy analyses may be further improved by next generation sequencing (NGS). While most tissue based NGS genotyping is well established, liquid biopsy NGS application is challenging, requiring a careful validation of the whole process, from blood collection to variant calling. Here we review this evolving field, highlighting those methodological points that are crucial to accurately select NSCLC patients for TKIs treatment administration by NGS on cfDNA.

HLA-A, -B, -DRB1 allele and haplotype frequencies of 920 cord blood units from Central Chile.

PubMed

Schäfer, Christian; Sauter, Jürgen; Riethmüller, Tobias; Kashi, Zahra Mehdizadeh; Schmidt, Alexander H; Barriga, Francisco J

2016-08-01

We present human leukocyte antigen (HLA) haplotype and allele/antigenic group frequencies derived from a data set of 920 umbilical cord blood units collected in Central Chile. HLA-A and -B genotypes were typed using sequence specific oligonucleotide probe methods while HLA-DRB1 genotypes were obtained from sequencing-based typing. The most frequent haplotype is A*29~B*44~DRB1*07:01 with an estimated frequency of 2.1%. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

'Neisseria skkuensis' sp. nov., isolated from the blood of a diabetic patient with a foot ulcer.

PubMed

Lee, Mi Young; Park, Eu Gene; Choi, Ji Young; Cheong, Hae Suk; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon; Ko, Kwan Soo

2010-07-01

A Gram-negative bacterium was isolated from the blood of a patient with diabetes mellitus. However, it could not be identified by conventional microbiological methods, and so was characterized by phenotypic and genotypic analyses. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Neisseria. Based on the phenotypic and genotypic characteristics, we propose that strain SMC-A9199(T) (=KCTC 22696(T)=JCM 16127(T)) should be classified as a novel species, 'Neisseria skkuensis' sp. nov. The patient was further treated with amoxicillin-clavulanate and ciprofloxacin for 3 weeks.

Pharmacogenetic-Based Efavirenz Dose Modification: Suggestions for an African Population and the Different CYP2B6 Genotypes

PubMed Central

Mukonzo, Jackson K.; Owen, Joel S.; Ogwal-Okeng, Jasper; Kuteesa, Ronald B.; Nanzigu, Sarah; Sewankambo, Nelson; Thabane, Lehana; Gustafsson, Lars L.; Ross, Colin; Aklillu, Eleni

2014-01-01

Background Pharmacogenetics contributes to inter-individual variability in pharmacokinetics (PK) of efavirenz (EFV), leading to variations in both efficacy and toxicity. The purpose of this study was to assess the effect of genetic factors on EFV pharmacokinetics, treatment outcomes and genotype based EFV dose recommendations for adult HIV-1 infected Ugandans. Methods In total, 556 steady-state plasma EFV concentrations from 99 HIV infected patients (64 female) treated with EFV/lamivudine/zidovidine were analyzed. Patient genotypes for CYP2B6 (*6 & *11), CYP3A5 (*3,*6 & *7) and ABCB1 c.4046A>G, baseline biochemistries and CD4 and viral load change from baseline were determined. A one-compartment population PK model with first-order absorption (NONMEM) was used to estimate genotype effects on EFV pharmacokinetics. PK simulations were performed based upon population genotype frequencies. Predicted AUCs were compared between the product label and simulations for doses of 300 mg, 450 mg, and 600 mg. Results EFV apparent clearance (CL/F) was 2.2 and 1.74 fold higher in CYP2B6*6 (*1/*1) and CYP2B6*6 (*1/*6) compared CYP2B6*6 (*6/*6) carriers, while a 22% increase in F1 was observed for carriers of ABCB1 c.4046A>G variant allele. Higher mean AUC was attained in CYP2B6 *6/*6 genotypes compared to CYP2B6 *1/*1 (p<0.0001). Simulation based AUCs for 600 mg doses were 1.25 and 2.10 times the product label mean AUC for the Ugandan population in general and CYP2B6*6/*6 genotypes respectively. Simulated exposures for EFV daily doses of 300 mg and 450 mg are comparable to the product label. Viral load fell precipitously on treatment, with only six patients having HIV RNA >40 copies/mL after 84 days of treatment. No trend with exposure was noted for these six patients. Conclusion Results of this study suggest that daily doses of 450 mg and 300 mg might meet the EFV treatment needs of HIV-1 infected Ugandans in general and individuals homozygous for CYP2B6*6 mutation, respectively. PMID:24497997

Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

PubMed Central

Unemo, Magnus; Dillon, Jo-Anne R.

2011-01-01

Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

Usefulness of hepatitis C virus RNA counts by second generation HCV bDNA-probe in chronic hepatitis C based on the HCV genotype.

PubMed

Kobayashi, M; Kumada, H; Arase, Y; Chayama, K; Kobayashi, M; Tsubota, A; Koida, I; Saitoh, S; Suzuki, Y; Murashima, N; Ikeda, K; Miyano, Y; Mizoshita, K; Matsuda, M; Koike, H; Hashimoto, M

1998-04-01

Detection of hepatitis C virus (HCV) RNA by a second generation (ver 2) HCV bDNA-probe method (bDNA-probe) was compared with detection by the first generation (ver 1) assay. The two assays were performed simultaneously with the same serum samples of HCV genotypes 1b, 2a, 2b, 3a, and 3b. The positive rates with ver 1 were 82% for HCV genotype 1b (type 1b), 57.6% for HCV genotype 2a (type 2a), 75.0% for HCV genotype 2b (type 2b), 55.6% for HCV genotype 3a (type 3a), and 93.8% for HCV genotype 3b (type 3b). The positive rates with ver 2 were 95.0% for type 1b, 93.9% for type 2a, 83.3% for type 2b, 100% for type 3a, and 93.8% for type 3b. With Fisher's exact test, the detection rate for type 2a was significantly higher (P = 0.001) with ver 2 than with ver 1. We obtained regression lines using the HCV counts measured by bDNA-probe on the y axis and the HCV counts obtained by an HCV reverse transcriptase (RT)-competitive polymerase chain reaction method (competitive PCR) on the x axis. The gradients for types 1b, 2a, and 3b were greater with ver 2 compared to ver 1. The gradients for types 2a and 3b were the highest: for type 2a, y = 0.135x + 0.6 with ver 1 and y = 0.248x + 0.1 with ver 2; for type 3b, y = 0.366x + 0.1 with ver 1 and y = 0.727x + 0.3 for ver 2. In addition, HCV-RNA counts for all the genotypes tested in this study were significantly higher with ver 2 than with ver 1. Hence, we conclude that ver 2 of the bDNA-probe measures HCV-RNA counts closer to those obtained with competitive PCR than the ver 1 assay.

mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

PubMed

Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

2017-03-01

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cytological Evaluation and REBA HPV-ID HPV Testing of Newly Developed Liquid-Based Cytology, EASYPREP: Comparison with SurePath.

PubMed

Lee, Youn Soo; Gong, Gyungyub; Sohn, Jin Hee; Ryu, Ki Sung; Lee, Jung Hun; Khang, Shin Kwang; Cho, Kyung-Ja; Kim, Yong-Man; Kang, Chang Suk

2013-06-01

The objective of this study was to evaluate a newly-developed EASYPREP liquid-based cytology method in cervicovaginal specimens and compare it with SurePath. Cervicovaginal specimens were prospectively collected from 1,000 patients with EASYPREP and SurePath. The specimens were first collected by brushing for SurePath and second for EASYPREP. The specimens of both methods were diagnosed according to the Bethesda System. Additionally, we performed to REBA HPV-ID genotyping and sequencing analysis for human papillomavirus (HPV) on 249 specimens. EASYPREP and SurePath showed even distribution of cells and were equal in cellularity and staining quality. The diagnostic agreement between the two methods was 96.5%. Based on the standard of SurePath, the sensitivity, specificity, positive predictive value, and negative predictive value of EASYPREP were 90.7%, 99.2%, 94.8%, and 98.5%, respectively. The positivity of REBA HPV-ID was 49.4% and 95.1% in normal and abnormal cytological samples, respectively. The result of REBA HPV-ID had high concordance with sequencing analysis. EASYPREP provided comparable results to SurePath in the diagnosis and staining quality of cytology examinations and in HPV testing with REBA HPV-ID. EASYPREP could be another LBC method choice for the cervicovaginal specimens. Additionally, REBA HPV-ID may be a useful method for HPV genotyping.

Large-scale parentage inference with SNPs: an efficient algorithm for statistical confidence of parent pair allocations.

PubMed

Anderson, Eric C

2012-11-08

Advances in genotyping that allow tens of thousands of individuals to be genotyped at a moderate number of single nucleotide polymorphisms (SNPs) permit parentage inference to be pursued on a very large scale. The intergenerational tagging this capacity allows is revolutionizing the management of cultured organisms (cows, salmon, etc.) and is poised to do the same for scientific studies of natural populations. Currently, however, there are no likelihood-based methods of parentage inference which are implemented in a manner that allows them to quickly handle a very large number of potential parents or parent pairs. Here we introduce an efficient likelihood-based method applicable to the specialized case of cultured organisms in which both parents can be reliably sampled. We develop a Markov chain representation for the cumulative number of Mendelian incompatibilities between an offspring and its putative parents and we exploit it to develop a fast algorithm for simulation-based estimates of statistical confidence in SNP-based assignments of offspring to pairs of parents. The method is implemented in the freely available software SNPPIT. We describe the method in detail, then assess its performance in a large simulation study using known allele frequencies at 96 SNPs from ten hatchery salmon populations. The simulations verify that the method is fast and accurate and that 96 well-chosen SNPs can provide sufficient power to identify the correct pair of parents from amongst millions of candidate pairs.

Assessment of spatial discordance of primary and effective seed dispersal of European beech (Fagus sylvatica L.) by ecological and genetic methods.

PubMed

Millerón, M; López de Heredia, U; Lorenzo, Z; Alonso, J; Dounavi, A; Gil, L; Nanos, N

2013-03-01

Spatial discordance between primary and effective dispersal in plant populations indicates that postdispersal processes erase the seed rain signal in recruitment patterns. Five different models were used to test the spatial concordance of the primary and effective dispersal patterns in a European beech (Fagus sylvatica) population from central Spain. An ecological method was based on classical inverse modelling (SSS), using the number of seed/seedlings as input data. Genetic models were based on direct kernel fitting of mother-to-offspring distances estimated by a parentage analysis or were spatially explicit models based on the genotype frequencies of offspring (competing sources model and Moran-Clark's Model). A fully integrated mixed model was based on inverse modelling, but used the number of genotypes as input data (gene shadow model). The potential sources of error and limitations of each seed dispersal estimation method are discussed. The mean dispersal distances for seeds and saplings estimated with these five methods were higher than those obtained by previous estimations for European beech forests. All the methods show strong discordance between primary and effective dispersal kernel parameters, and for dispersal directionality. While seed rain was released mostly under the canopy, saplings were established far from mother trees. This discordant pattern may be the result of the action of secondary dispersal by animals or density-dependent effects; that is, the Janzen-Connell effect. © 2013 Blackwell Publishing Ltd.

Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

PubMed

Hu, Jian; Zhou, Yi-ren; Ding, Jia-lin; Wang, Zhi-yuan; Liu, Ling; Wang, Ye-kai; Lou, Hui-ling; Qiao, Shou-yi; Wu, Yan-hua

2017-05-20

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, I A , I B and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

Identification of Characteristic Macromolecules of Escherichia coli Genotypes by Atomic Force Microscope Nanoscale Mechanical Mapping

NASA Astrophysics Data System (ADS)

Chang, Alice Chinghsuan; Liu, Bernard Haochih

2018-02-01

The categorization of microbial strains is conventionally based on the molecular method, and seldom are the morphological characteristics in the bacterial strains studied. In this research, we revealed the macromolecular structures of the bacterial surface via AFM mechanical mapping, whose resolution was not only determined by the nanoscale tip size but also the mechanical properties of the specimen. This technique enabled the nanoscale study of membranous structures of microbial strains with simple specimen preparation and flexible working environments, which overcame the multiple restrictions in electron microscopy and label-enable biochemical analytical methods. The characteristic macromolecules located among cellular surface were considered as surface layer proteins and were found to be specific to the Escherichia coli genotypes, from which the averaged molecular sizes were characterized with diameters ranging from 38 to 66 nm, and the molecular shapes were kidney-like or round. In conclusion, the surface macromolecular structures have unique characteristics that link to the E. coli genotype, which suggests that the genomic effects on cellular morphologies can be rapidly identified using AFM mechanical mapping. [Figure not available: see fulltext.

Association of CHEK2 polymorphisms with the efficacy of platinum-based chemotherapy for advanced non-small-cell lung cancer in Chinese never-smoking women

PubMed Central

Xu, Wen; Liu, Di; Yang, Yang; Ding, Xi; Sun, Yifeng; Zhang, Baohong

2016-01-01

Background Cell cycle checkpoint kinase 2 (CHEK2) plays an essential role in the repair of DNA damage. Single nucleotide polymorphisms (SNPs) in DNA repair genes are thought to influence treatment effects and survival of cancer patients. This study aimed to investigate the relationship between polymorphisms in the CHEK2 gene and efficacy of platinum-based doublet chemotherapy in never-smoking Chinese female patients with advanced non-small-cell lung cancer (NSCLC). Methods Using DNA from blood samples of 272 Chinese advanced NSCLC non-smoking female patients treated with first-line platinum-based chemotherapy, we have analyzed the relationships between four SNPs in the CHEK2 gene and clinical outcomes. Results We found that overall survival (OS) was significantly associated with CHEK2 rs4035540 (Log-Rank P=0.020), as well as the CHEK2 rs4035540 dominant model (Log-Rank P=0.026), especially in the lung adenocarcinoma group. After multivariate analysis, patients with rs4035540 A/G genotype had a significantly better OS than those with the G/G genotype (HR =0.67, 95% CI, 0.48–0.93; P=0.016). In the toxicity analysis, it was observed that patients with the CHEK2 rs4035540 A/A genotype had a higher risk of gastrointestinal toxicity than the G/G genotype group (P=0.009). However, there are no significant associations between chemotherapy treatments and genetic variations. Conclusions Our findings indicate that SNPs in CHEK2 are related to Chinese advanced NSCLC never-smoking female patients receiving platinum-based doublet chemotherapy in China. Patients with rs4035540 A/G genotype have a better OS. And patients with rs4035540 A/A genotype have a higher risk of gastrointestinal toxicity. These results point to a direction for predicting the prognosis for Chinese never-smoking NSCLC female patients. However, there are no significant associations between chemotherapy treatments and SNPs in CHEK2, which need more samples to the further study. PMID:27747004

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

PubMed Central

Sampath, Rangarajan; Russell, Kevin L.; Massire, Christian; Eshoo, Mark W.; Harpin, Vanessa; Blyn, Lawrence B.; Melton, Rachael; Ivy, Cristina; Pennella, Thuy; Li, Feng; Levene, Harold; Hall, Thomas A.; Libby, Brian; Fan, Nancy; Walcott, Demetrius J.; Ranken, Raymond; Pear, Michael; Schink, Amy; Gutierrez, Jose; Drader, Jared; Moore, David; Metzgar, David; Addington, Lynda; Rothman, Richard; Gaydos, Charlotte A.; Yang, Samuel; St. George, Kirsten; Fuschino, Meghan E.; Dean, Amy B.; Stallknecht, David E.; Goekjian, Ginger; Yingst, Samuel; Monteville, Marshall; Saad, Magdi D.; Whitehouse, Chris A.; Baldwin, Carson; Rudnick, Karl H.; Hofstadler, Steven A.; Lemon, Stanley M.; Ecker, David J.

2007-01-01

Background Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance. PMID:17534439

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

PubMed Central

2012-01-01

Background Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population. PMID:22656068

Phased genotyping-by-sequencing enhances analysis of genetic diversity and reveals divergent copy number variants in maize

USDA-ARS?s Scientific Manuscript database

High-throughput sequencing of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken fr...

Ontology-guided data preparation for discovering genotype-phenotype relationships.

PubMed

Coulet, Adrien; Smaïl-Tabbone, Malika; Benlian, Pascale; Napoli, Amedeo; Devignes, Marie-Dominique

2008-04-25

Complexity and amount of post-genomic data constitute two major factors limiting the application of Knowledge Discovery in Databases (KDD) methods in life sciences. Bio-ontologies may nowadays play key roles in knowledge discovery in life science providing semantics to data and to extracted units, by taking advantage of the progress of Semantic Web technologies concerning the understanding and availability of tools for knowledge representation, extraction, and reasoning. This paper presents a method that exploits bio-ontologies for guiding data selection within the preparation step of the KDD process. We propose three scenarios in which domain knowledge and ontology elements such as subsumption, properties, class descriptions, are taken into account for data selection, before the data mining step. Each of these scenarios is illustrated within a case-study relative to the search of genotype-phenotype relationships in a familial hypercholesterolemia dataset. The guiding of data selection based on domain knowledge is analysed and shows a direct influence on the volume and significance of the data mining results. The method proposed in this paper is an efficient alternative to numerical methods for data selection based on domain knowledge. In turn, the results of this study may be reused in ontology modelling and data integration.

Laser capture microdissection as a tool to evaluate human papillomavirus genotyping and methylation as biomarkers of persistence and progression of anal lesions

PubMed Central

Cornall, Alyssa M; Roberts, Jennifer M; Molano, Monica; Machalek, Dorothy A; Phillips, Samuel; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Garland, Suzanne M; Tabrizi, Sepehr N

2015-01-01

Introduction Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, high-grade squamous intraepithelial lesion (HSIL). Detection of specific HPV genotypes and HPV-related biomarkers may be an option for primary anal screening. However, more data on the natural history of HPV-related anal lesions are required. The outcomes from this study will enhance our understanding of the clinical and biological behaviour of HPV-related anal lesions and inform the development of future HPV genotype and/or biomarker screening tests. Methods and analysis HIV-negative and HIV-positive men who have sex with men, aged 35 years and over, recruited from community-based settings in Sydney, Australia, attend 6 clinic visits over 3 years. At the first 5 visits, participants undergo a digital anorectal examination, an anal swab for HPV genotyping and anal cytology, and high-resolution anoscopy with directed biopsy of any visible abnormalities that are suggestive of any abnormality suspicious of SIL. Tissue sections from participants diagnosed with histologically confirmed HSIL at the baseline clinic visit will undergo laser capture microdissection, HPV detection and genotyping, and quantitation of CpG methylation in baseline and follow-up biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as non-persistent and persistent based on their status at 12 months. The performance of HPV genotype and methylation status in predicting disease persistence at 12 months will be assessed, along with associations with HIV status and other covariates such as age. Ethics and dissemination The St Vincent's Hospital Ethics Committee granted ethics approval for the study. Written informed consent is obtained from all individuals before any study-specific procedures are performed. Findings from this study will be disseminated to participants and the community through study newsletters, and through peer-reviewed publications and international conferences. PMID:26310402

Imputation across genotyping arrays for genome-wide association studies: assessment of bias and a correction strategy.

PubMed

Johnson, Eric O; Hancock, Dana B; Levy, Joshua L; Gaddis, Nathan C; Saccone, Nancy L; Bierut, Laura J; Page, Grier P

2013-05-01

A great promise of publicly sharing genome-wide association data is the potential to create composite sets of controls. However, studies often use different genotyping arrays, and imputation to a common set of SNPs has shown substantial bias: a problem which has no broadly applicable solution. Based on the idea that using differing genotyped SNP sets as inputs creates differential imputation errors and thus bias in the composite set of controls, we examined the degree to which each of the following occurs: (1) imputation based on the union of genotyped SNPs (i.e., SNPs available on one or more arrays) results in bias, as evidenced by spurious associations (type 1 error) between imputed genotypes and arbitrarily assigned case/control status; (2) imputation based on the intersection of genotyped SNPs (i.e., SNPs available on all arrays) does not evidence such bias; and (3) imputation quality varies by the size of the intersection of genotyped SNP sets. Imputations were conducted in European Americans and African Americans with reference to HapMap phase II and III data. Imputation based on the union of genotyped SNPs across the Illumina 1M and 550v3 arrays showed spurious associations for 0.2 % of SNPs: ~2,000 false positives per million SNPs imputed. Biases remained problematic for very similar arrays (550v1 vs. 550v3) and were substantial for dissimilar arrays (Illumina 1M vs. Affymetrix 6.0). In all instances, imputing based on the intersection of genotyped SNPs (as few as 30 % of the total SNPs genotyped) eliminated such bias while still achieving good imputation quality.

Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria

PubMed Central

Efstratiou, Androulla; Engler, Kathryn H.; Dawes, Charlotte S.; Sesardic, Dorothea

1998-01-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended. PMID:9774560

Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria.

PubMed

Efstratiou, A; Engler, K H; Dawes, C S; Sesardic, D

1998-11-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.

Genetic characterization and improved genotyping of the dysferlin-deficient mouse strain Dysf (tm1Kcam).

PubMed

Wiktorowicz, Tatiana; Kinter, Jochen; Kobuke, Kazuhiro; Campbell, Kevin P; Sinnreich, Michael

2015-01-01

Mouse models of dysferlinopathies are valuable tools with which to investigate the pathomechanisms underlying these diseases and to test novel therapeutic strategies. One such mouse model is the Dysf (tm1Kcam) strain, which was generated using a targeting vector to replace a 12-kb region of the dysferlin gene and which features a progressive muscular dystrophy. A prerequisite for successful animal studies using genetic mouse models is an accurate genotyping protocol. Unfortunately, the lack of robustness of currently available genotyping protocols for the Dysf (tm1Kcam) mouse has prevented efficient colony management. Initial attempts to improve the genotyping protocol based on the published genomic structure failed. These difficulties led us to analyze the targeted locus of the dysferlin gene of the Dysf (tm1Kcam) mouse in greater detail. In this study we resequenced and analyzed the targeted locus of the Dysf (tm1Kcam) mouse and developed a novel PCR protocol for genotyping. We found that instead of a deletion, the dysferlin locus in the Dysf (tm1Kcam) mouse carries a targeted insertion. This genetic characterization enabled us to establish a reliable method for genotyping of the Dysf (tm1Kcam) mouse, and thus has made efficient colony management possible. Our work will make the Dysf (tm1Kcam) mouse model more attractive for animal studies of dysferlinopathies.

Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

PubMed

De Santis, Riccardo; Ancora, Massimo; De Massis, Fabrizio; Ciammaruconi, Andrea; Zilli, Katiuscia; Di Giannatale, Elisabetta; Pittiglio, Valentina; Fillo, Silvia; Lista, Florigio

2013-10-01

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Genetic diversity of Echinococcus granulosus in center of Iran.

PubMed

Pestechian, Nader; Hosseini Safa, Ahmad; Tajedini, Mohammadhasan; Rostami-Nejad, Mohammad; Mousavi, Mohammad; Yousofi, Hosseinali; Haghjooy Javanmard, Shaghayegh

2014-08-01

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

Multiplex genotyping system for efficient inference of matrilineal genetic ancestry with continental resolution

PubMed Central

2011-01-01

Background In recent years, phylogeographic studies have produced detailed knowledge on the worldwide distribution of mitochondrial DNA (mtDNA) variants, linking specific clades of the mtDNA phylogeny with certain geographic areas. However, a multiplex genotyping system for the detection of the mtDNA haplogroups of major continental distribution that would be desirable for efficient DNA-based bio-geographic ancestry testing in various applications is still missing. Results Three multiplex genotyping assays, based on single-base primer extension technology, were developed targeting a total of 36 coding-region mtDNA variants that together differentiate 43 matrilineal haplo-/paragroups. These include the major diagnostic haplogroups for Africa, Western Eurasia, Eastern Eurasia and Native America. The assays show high sensitivity with respect to the amount of template DNA: successful amplification could still be obtained when using as little as 4 pg of genomic DNA and the technology is suitable for medium-throughput analyses. Conclusions We introduce an efficient and sensitive multiplex genotyping system for bio-geographic ancestry inference from mtDNA that provides resolution on the continental level. The method can be applied in forensics, to aid tracing unknown suspects, as well as in population studies, genealogy and personal ancestry testing. For more complete inferences of overall bio-geographic ancestry from DNA, the mtDNA system provided here can be combined with multiplex systems for suitable autosomal and, in the case of males, Y-chromosomal ancestry-sensitive DNA markers. PMID:21429198

Evaluation of Machine Learning and Rules-Based Approaches for Predicting Antimicrobial Resistance Profiles in Gram-negative Bacilli from Whole Genome Sequence Data.

PubMed

Pesesky, Mitchell W; Hussain, Tahir; Wallace, Meghan; Patel, Sanket; Andleeb, Saadia; Burnham, Carey-Ann D; Dantas, Gautam

2016-01-01

The time-to-result for culture-based microorganism recovery and phenotypic antimicrobial susceptibility testing necessitates initial use of empiric (frequently broad-spectrum) antimicrobial therapy. If the empiric therapy is not optimal, this can lead to adverse patient outcomes and contribute to increasing antibiotic resistance in pathogens. New, more rapid technologies are emerging to meet this need. Many of these are based on identifying resistance genes, rather than directly assaying resistance phenotypes, and thus require interpretation to translate the genotype into treatment recommendations. These interpretations, like other parts of clinical diagnostic workflows, are likely to be increasingly automated in the future. We set out to evaluate the two major approaches that could be amenable to automation pipelines: rules-based methods and machine learning methods. The rules-based algorithm makes predictions based upon current, curated knowledge of Enterobacteriaceae resistance genes. The machine-learning algorithm predicts resistance and susceptibility based on a model built from a training set of variably resistant isolates. As our test set, we used whole genome sequence data from 78 clinical Enterobacteriaceae isolates, previously identified to represent a variety of phenotypes, from fully-susceptible to pan-resistant strains for the antibiotics tested. We tested three antibiotic resistance determinant databases for their utility in identifying the complete resistome for each isolate. The predictions of the rules-based and machine learning algorithms for these isolates were compared to results of phenotype-based diagnostics. The rules based and machine-learning predictions achieved agreement with standard-of-care phenotypic diagnostics of 89.0 and 90.3%, respectively, across twelve antibiotic agents from six major antibiotic classes. Several sources of disagreement between the algorithms were identified. Novel variants of known resistance factors and incomplete genome assembly confounded the rules-based algorithm, resulting in predictions based on gene family, rather than on knowledge of the specific variant found. Low-frequency resistance caused errors in the machine-learning algorithm because those genes were not seen or seen infrequently in the test set. We also identified an example of variability in the phenotype-based results that led to disagreement with both genotype-based methods. Genotype-based antimicrobial susceptibility testing shows great promise as a diagnostic tool, and we outline specific research goals to further refine this methodology.

[Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique].

PubMed

Zhang, Yinfeng; Luo, Haining; Zhang, Yunshan

2015-12-01

To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

PubMed

Daniels, Rachel; Hamilton, Elizabeth J; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F; Hartl, Daniel L; Volkman, Sarah K

2015-11-10

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs. This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

Identifying and mitigating batch effects in whole genome sequencing data.

PubMed

Tom, Jennifer A; Reeder, Jens; Forrest, William F; Graham, Robert R; Hunkapiller, Julie; Behrens, Timothy W; Bhangale, Tushar R

2017-07-24

Large sample sets of whole genome sequencing with deep coverage are being generated, however assembling datasets from different sources inevitably introduces batch effects. These batch effects are not well understood and can be due to changes in the sequencing protocol or bioinformatics tools used to process the data. No systematic algorithms or heuristics exist to detect and filter batch effects or remove associations impacted by batch effects in whole genome sequencing data. We describe key quality metrics, provide a freely available software package to compute them, and demonstrate that identification of batch effects is aided by principal components analysis of these metrics. To mitigate batch effects, we developed new site-specific filters that identified and removed variants that falsely associated with the phenotype due to batch effect. These include filtering based on: a haplotype based genotype correction, a differential genotype quality test, and removing sites with missing genotype rate greater than 30% after setting genotypes with quality scores less than 20 to missing. This method removed 96.1% of unconfirmed genome-wide significant SNP associations and 97.6% of unconfirmed genome-wide significant indel associations. We performed analyses to demonstrate that: 1) These filters impacted variants known to be disease associated as 2 out of 16 confirmed associations in an AMD candidate SNP analysis were filtered, representing a reduction in power of 12.5%, 2) In the absence of batch effects, these filters removed only a small proportion of variants across the genome (type I error rate of 3%), and 3) in an independent dataset, the method removed 90.2% of unconfirmed genome-wide SNP associations and 89.8% of unconfirmed genome-wide indel associations. Researchers currently do not have effective tools to identify and mitigate batch effects in whole genome sequencing data. We developed and validated methods and filters to address this deficiency.

Field-based phenomics for plant genetics research

USDA-ARS?s Scientific Manuscript database

Perhaps the greatest challenge for crop research in the 21st century is how to predict crop performance as a function of genetic architecture and climate change. Advances in “next generation” DNA sequencing have greatly reduced genotyping costs. Methods for characterization of plant traits (phenotyp...

High Resolution Melting (HRM) applied to wine authenticity.

PubMed

Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

2017-02-01

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

PubMed

Royo, L J; Fernández, I; Azor, P J; Alvarez, I; Pérez-Pardal, L; Goyache, F

2008-06-01

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

PubMed Central

2012-01-01

Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as limiting factors in genetic studies. We have conducted the first formal analysis of the effect of novel variants on genotyping arrays, and we have shown that these variants account for a large portion of miscalled and uncalled genotypes. Genetic studies will benefit from substantial improvements in the accuracy of their results by incorporating VINOs in their analyses. PMID:22260749

Novel rope-based sampling of classical swine fever shedding in a group of wild boar showing low contagiosity upon experimental infection with a classical swine fever field strain of genotype 2.3.

PubMed

Mouchantat, Susan; Globig, Anja; Böhle, Wolfgang; Petrov, Anja; Strebelow, Heinz-Günther; Mettenleiter, Thomas C; Depner, Klaus

2014-06-04

Several classical swine fever (CSF) epidemics in wild boar and domestic pigs in Europe during the last decades have been caused by CSF virus (CSFV) strains of genotype 2.3. This genotype is known to be virulent leading to high morbidity and mortality. We experimentally infected two eight months old wild boar with 10(5,5) TCID50 of CSFV genotype 2.3 and kept the animals together with five noninoculated wild boar of the same age. Our original purpose was to evaluate a non-invasive sampling method based on saliva collection using "rope-in-a-bait" sampling baits. While expecting high morbidity, high level of virus shedding and some mortality, we actually observed a subclinical course of infection with an unexpected low contagiosity. The two inoculated animals infected only three contact animals while two contact animals remained uninfected. These findings substantially add to our epidemiological understanding of CSFV circulation in wild boar populations. CSFV infected animals older than six months and in good condition may not shed sufficient virus to transmit infection to all seronegative in-contact animals. The contagiosity in relation to the animal's age is discussed. This supports the hypothesis of silent perpetuation of CSFV in wild boar populations for several months if the wild boar density is sufficiently high. The feasibility of the "rope-in-a-bait" sampling method could be proven during the short viraemic phase of infected animals during the second week of infection. Copyright © 2014 Elsevier B.V. All rights reserved.

[Association between 5-hydroxytryptamine 2A receptor gene polymorphisms and susceptibility to occupational stress in oilfield workers].

PubMed

Jiang, Y; Palizhati, Abudoureyimu; Gao, X Y; Guan, S Z; Liu, J W

2016-10-20

Objective: To investigate the association between 5-hydroxytryptamine 2A (5-HT2A) receptor gene polymorphisms and occupational stress in oilfield workers. Methods: Cluster sampling was used to select 826 oilfield workers from January to August, 2013. The SNaPshot single nucleotide polymorphism (SNP) genotyping method was used to determine the genotypes of rs6313, rs1923884, and rs2070040 in 5-HT2A receptor gene, and the Occupational Stress Inventory-Revised Edition was used to analyze occupational stress in these workers. Results: There were no significant differences in occupational stress between groups with different individual characteristics ( P >0.05 ) . As for the comparison of occupational stress scores between workers with different genotypes of each SNP of 5-HT2A receptor gene, the workers with CC and CT genotypes of rs6313 had significantly higher role boundary scores than those with TT genotype ( P <0.05) , and the workers with CC genotype had a significantly higher vocational stress score than those with CT genotype ( P <0.05) ; the workers with CT genotype of rs1923884 had a significantly higher occupational role score than those with CC genotype ( P <0.05) and a significantly higher coping resources score than those with CC and TT genotypes ( P <0.05) ; the workers with AG genotype of rs2070040 had a significantly higher vocational stress score than those with AA genotype ( P <0.05) . The ordinal multinomial logistic regression analysis showed that workers with CT genotype of rs1923884 were susceptible to occupational stress ( OR =1.56, 95% CI 1.10~2.20) . Conclusion: CT genotype of rs1923884 in 5-HT2A receptor gene may be associated with the susceptibility to occupational stress in oilfield workers.

KRAS polymorphisms are associated with survival of CRC in Chinese population.

PubMed

Dai, Qiong; Wei, Hui Lian; Huang, Juan; Zhou, Tie Jun; Chai, Li; Yang, Zhi-Hui

2016-04-01

rs12245, rs12587, rs9266, rs1137282, rs61764370, and rs712 of KRAS oncogene are characterized in the 3'UTR. The study highlights the important role of these polymorphisms playing in the susceptibility, oxaliplatin-based chemotherapy sensitivity, progression, and prognosis of CRC. Improved multiplex ligation detection reaction (iMLDR) technique is used for genotyping. An unconditional logistic regression model was used to estimate the association of certain polymorphism and CRC risk. The Kaplan-Meier method, log-rank test, and Cox regression model were used to evaluate the effects of polymorphisms on survival analysis. Results demonstrated that TT genotype and T allele of rs712 were associated with the increased risk of CRC; the patients with GG genotype and G allele of rs61764370 had a shorter survival and a higher risk of relapse or metastasis of CRC. Our studies supported the conclusions that rs61764370 and rs712 polymorphisms of the KRAS are functional and it may play an important role in the development of CRC and oxaliplatin-based chemotherapy efficiency and prognosis of CRC.

Scaffolding as an effort for thinking process optimization on heredity

NASA Astrophysics Data System (ADS)

Azizah, N. R.; Masykuri, M.; Prayitno, B. A.

2018-04-01

Thinking is an activity and process of manipulating and transforming data or information into memory. Thinking process is different between one and other person. Thinking process can be developed by interaction between student and their environment, such as scaffolding. Given scaffolding is based on each student necessity. There are 2 level on scaffolding such as explaining, reviewing, and restructuring; and developing conceptual thinking. This research is aimed to describe student’s thinking process on heredity especially on inheritance that is before and after scaffolding. This research used descriptive qualitative method. There were three kinds of subject degree such as the students with high, middle, and low achieving students. The result showed that subjects had some difficulty in dihybrid inheritance question in different place. Most difficulty was on determining the number of different characteristic, parental genotype, gamete, and ratio of genotype and phenotype F2. Based on discussed during scaffolding showed that the subjects have some misunderstanding terms and difficulty to determine parental, gamete, genotype, and phenotype. Final result in this research showed that the subjects develop thinking process higher after scaffolding. Therefore the subjects can solve question properly.

Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

PubMed

Catalano, Valentina; Moreno-Sanz, Paula; Lorenzi, Silvia; Grando, Maria Stella

2016-09-21

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.

[Phenotypic and genotypic characteristics of Mycobacterium kansasii strains isolated in Spain (2000-2003)].

PubMed

Jiménez-Pajares, María Soledad; Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan Antonio

2005-05-01

Mycobacterium kansasii is an opportunistic pathogen that mainly causes pulmonary infections. This species accounted for 9.7% of Mycobacteria other than tuberculosis complex identified in the reference laboratory of the Spanish Centro Nacional de Microbiologia during the period of 2000-2003. In this study we analyzed the phenotypic and genotypic characteristics of 298 M. kansasii strains isolated over this 4-year period. The phenotypic characteristics were determined by conventional methods: biochemical testing, culture and morphological study. Genotypic characteristics were studied using PCR restriction fragment analysis of a fragment of the hsp65 gene and digestion with BstEII and HaeIII, according to the method of Telenti. Among the total of tested strains, 57.4% had the typical phenotypic characteristics described for M. kansasii. The rest had atypical patterns that we grouped into 17 biotypes. Strains belonging to six of the seven described genotypes were identified, with 86.6% of the strains falling into genotype I. Analysis of the phenotypic characteristics of M. kansasii showed a higher discrimination index for intraspecific differentiation than genotypic methods. Nevertheless, the high variability of phenotypic characteristics, some of which were very specific for the species (e.g., photochromogenicity), could complicate their identification. Hence both conventional and molecular methods should be used to accurately identify the atypical isolates.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

PubMed

Nooshadokht, Maryam; Kalantari-Khandani, Behjat; Sharifi, Iraj; Kamyabi, Hossein; Liyanage, Namal P M; Lagenaur, Laurel A; Kagnoff, Martin F; Singer, Steven M; Babaei, Zahra; Solaymani-Mohammadi, Shahram

2017-10-01

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer. Copyright © 2017. Published by Elsevier B.V.

Evaluation of Genome-Enabled Selection for Bacterial Cold Water Disease Resistance Using Progeny Performance Data in Rainbow Trout: Insights on Genotyping Methods and Genomic Prediction Models.

PubMed

Vallejo, Roger L; Leeds, Timothy D; Fragomeni, Breno O; Gao, Guangtu; Hernandez, Alvaro G; Misztal, Ignacy; Welch, Timothy J; Wiens, Gregory D; Palti, Yniv

2016-01-01

Bacterial cold water disease (BCWD) causes significant economic losses in salmonid aquaculture, and traditional family-based breeding programs aimed at improving BCWD resistance have been limited to exploiting only between-family variation. We used genomic selection (GS) models to predict genomic breeding values (GEBVs) for BCWD resistance in 10 families from the first generation of the NCCCWA BCWD resistance breeding line, compared the predictive ability (PA) of GEBVs to pedigree-based estimated breeding values (EBVs), and compared the impact of two SNP genotyping methods on the accuracy of GEBV predictions. The BCWD phenotypes survival days (DAYS) and survival status (STATUS) had been recorded in training fish (n = 583) subjected to experimental BCWD challenge. Training fish, and their full sibs without phenotypic data that were used as parents of the subsequent generation, were genotyped using two methods: restriction-site associated DNA (RAD) sequencing and the Rainbow Trout Axiom® 57 K SNP array (Chip). Animal-specific GEBVs were estimated using four GS models: BayesB, BayesC, single-step GBLUP (ssGBLUP), and weighted ssGBLUP (wssGBLUP). Family-specific EBVs were estimated using pedigree and phenotype data in the training fish only. The PA of EBVs and GEBVs was assessed by correlating mean progeny phenotype (MPP) with mid-parent EBV (family-specific) or GEBV (animal-specific). The best GEBV predictions were similar to EBV with PA values of 0.49 and 0.46 vs. 0.50 and 0.41 for DAYS and STATUS, respectively. Among the GEBV prediction methods, ssGBLUP consistently had the highest PA. The RAD genotyping platform had GEBVs with similar PA to those of GEBVs from the Chip platform. The PA of ssGBLUP and wssGBLUP methods was higher with the Chip, but for BayesB and BayesC methods it was higher with the RAD platform. The overall GEBV accuracy in this study was low to moderate, likely due to the small training sample used. This study explored the potential of GS for improving resistance to BCWD in rainbow trout using, for the first time, progeny testing data to assess the accuracy of GEBVs, and it provides the basis for further investigation on the implementation of GS in commercial rainbow trout populations.

Evaluation of Genome-Enabled Selection for Bacterial Cold Water Disease Resistance Using Progeny Performance Data in Rainbow Trout: Insights on Genotyping Methods and Genomic Prediction Models

PubMed Central

Vallejo, Roger L.; Leeds, Timothy D.; Fragomeni, Breno O.; Gao, Guangtu; Hernandez, Alvaro G.; Misztal, Ignacy; Welch, Timothy J.; Wiens, Gregory D.; Palti, Yniv

2016-01-01

Bacterial cold water disease (BCWD) causes significant economic losses in salmonid aquaculture, and traditional family-based breeding programs aimed at improving BCWD resistance have been limited to exploiting only between-family variation. We used genomic selection (GS) models to predict genomic breeding values (GEBVs) for BCWD resistance in 10 families from the first generation of the NCCCWA BCWD resistance breeding line, compared the predictive ability (PA) of GEBVs to pedigree-based estimated breeding values (EBVs), and compared the impact of two SNP genotyping methods on the accuracy of GEBV predictions. The BCWD phenotypes survival days (DAYS) and survival status (STATUS) had been recorded in training fish (n = 583) subjected to experimental BCWD challenge. Training fish, and their full sibs without phenotypic data that were used as parents of the subsequent generation, were genotyped using two methods: restriction-site associated DNA (RAD) sequencing and the Rainbow Trout Axiom® 57 K SNP array (Chip). Animal-specific GEBVs were estimated using four GS models: BayesB, BayesC, single-step GBLUP (ssGBLUP), and weighted ssGBLUP (wssGBLUP). Family-specific EBVs were estimated using pedigree and phenotype data in the training fish only. The PA of EBVs and GEBVs was assessed by correlating mean progeny phenotype (MPP) with mid-parent EBV (family-specific) or GEBV (animal-specific). The best GEBV predictions were similar to EBV with PA values of 0.49 and 0.46 vs. 0.50 and 0.41 for DAYS and STATUS, respectively. Among the GEBV prediction methods, ssGBLUP consistently had the highest PA. The RAD genotyping platform had GEBVs with similar PA to those of GEBVs from the Chip platform. The PA of ssGBLUP and wssGBLUP methods was higher with the Chip, but for BayesB and BayesC methods it was higher with the RAD platform. The overall GEBV accuracy in this study was low to moderate, likely due to the small training sample used. This study explored the potential of GS for improving resistance to BCWD in rainbow trout using, for the first time, progeny testing data to assess the accuracy of GEBVs, and it provides the basis for further investigation on the implementation of GS in commercial rainbow trout populations. PMID:27303436

Antioxidant capacity of cornelian cherry (Cornus mas L.) - comparison between permanganate reducing antioxidant capacity and other antioxidant methods.

PubMed

Popović, Boris M; Stajner, Dubravka; Slavko, Kevrešan; Sandra, Bijelić

2012-09-15

Ethanol extracts (80% in water) of 10 cornelian cherry (Cornus mas L.) genotypes were studied for antioxidant properties, using methods including DPPH(), ()NO, O(2)(-) and ()OH antiradical powers, FRAP, total phenolic and anthocyanin content (TPC and ACC) and also one relatively new, permanganate method (permanganate reducing antioxidant capacity-PRAC). Lipid peroxidation (LP) was also determined as an indicator of oxidative stress. The data from different procedures were compared and analysed by multivariate techniques (correlation matrix calculation and principal component analysis (PCA)). Significant positive correlations were obtained between TPC, ACC and DPPH(), ()NO, O(2)(-), and ()OH antiradical powers, and also between PRAC and TPC, ACC and FRAP. PCA found two major clusters of cornelian cherry, based on antiradical power, FRAP and PRAC and also on chemical composition. Chemometric evaluation showed close interdependence between PRAC method and FRAP and ACC. There was a huge variation between C. mas genotypes in terms of antioxidant activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Negative social acts and pain: evidence of a workplace bullying and 5-HTT genotype interaction.

PubMed

Jacobsen, Daniel Pitz; Nielsen, Morten Birkeland; Einarsen, Ståle; Gjerstad, Johannes

2018-05-01

Objectives Long-term exposure to systematic negative acts at work, usually labeled workplace bullying, is a prevalent problem at many workplaces. The adverse effects of such exposure may range from psychological symptoms, such as depression and anxiety to somatic ailments like cardiovascular disease and musculoskeletal complaints. In this study, we examined the relationships among exposure to negative acts, genetic variability in the 5-HTT gene SLC6A4 and pain. Methods The study was based on a nationally representative survey of 987 Norwegian employees drawn from the Norwegian Central Employee Register by Statistics Norway. Exposure to bullying in the workplace was measured with the 9-item version of the Negative Acts Questionnaire - Revised (NAQ-R) inventory. Pain was rated using an 11-point (0-10) numeric rating scale (NRS). Genotyping with regard to SLC6A4 was carried out using a combination of gel-electrophoresis and TaqMan assay. Results The data revealed a significant interaction between exposure to negative acts and the SLC6A4 genotype with regard to pain (linear regression with 5000 resamples; age, sex, tobacco use and education were included as covariates). The relationship between negative acts and pain intensity was significantly stronger for subjects with the LALA genotype than for subjects with the SLA/LALG/SLG genotype. No significant difference between subjects with the LALA genotype and SS genotype was observed. Conclusions Our data demonstrated that the relationship between bullying and pain was modified by the 5-HTT genotype, ie, genetic variation in SLC6A4. The association between negative acts and health among vulnerable individuals appeared more potent than previously reported.

Comparison of AC electronic monitoring and field data for estimating tolerance to Empoasca kraemeri (Homoptera: Cicadellidae) in common bean genotypes.

PubMed

Serrano, M S; Backus, E A; Cardona, C

2000-12-01

Two methods for estimating the tolerance of common bean genotypes to Empoasca kraemeri Ross & Moore were compared, using a yield trial carried out at Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, versus stylet penetration tactics measured by AC electronic feeding monitors. A stylet penetration index was devised based on principal component scores of three penetration tactics identified (pulsing laceration, cell rupturing, and lancing sap ingestion), combined with knowledge of the hopperburn symptoms caused by each tactic. Tolerant genotypes, as classified by the CIAT yield index, showed significantly more unprotected yield and lower hopperburn scores than the susceptible control. They also induced performance of less pulsing laceration (the tactic considered most damaging to the plant), and more of the other two, mitigating tactics, especially cell rupturing. When index values were calculated for each genotype, stylet penetration index values matched those of the yield index for three out of five genotypes: two EMP-coded tolerant lines ('EMP 385' and 'EMP 392') and the susceptible control 'BAT 41'. Thus, for these three genotypes, all subsequent hoppereburn symptoms are predictable by the type of feeding behavior performed on them. 'Porrillo Sintético' and 'EMP 84', considered borderline genotypes by the yield index, were overestimated and underestimated respectively, by the stylet penetration index. We postulate that, for these two genotypes, plant physiological responses to feeding (either compensatory or heightened sensitivity, respectively) synergize with type of feeding performed to generate the overall hopperburn condition. This multivariate analysis of electronic monitoring data was successfully used to devise an index of resistance. The implications of using the stylet penetration index and the advantages of using electronic monitoring in a bean-breeding program are discussed.

A robust multifactor dimensionality reduction method for detecting gene-gene interactions with application to the genetic analysis of bladder cancer susceptibility

PubMed Central

Gui, Jiang; Andrew, Angeline S.; Andrews, Peter; Nelson, Heather M.; Kelsey, Karl T.; Karagas, Margaret R.; Moore, Jason H.

2010-01-01

A central goal of human genetics is to identify and characterize susceptibility genes for common complex human diseases. An important challenge in this endeavor is the modeling of gene-gene interaction or epistasis that can result in non-additivity of genetic effects. The multifactor dimensionality reduction (MDR) method was developed as machine learning alternative to parametric logistic regression for detecting interactions in absence of significant marginal effects. The goal of MDR is to reduce the dimensionality inherent in modeling combinations of polymorphisms using a computational approach called constructive induction. Here, we propose a Robust Multifactor Dimensionality Reduction (RMDR) method that performs constructive induction using a Fisher’s Exact Test rather than a predetermined threshold. The advantage of this approach is that only those genotype combinations that are determined to be statistically significant are considered in the MDR analysis. We use two simulation studies to demonstrate that this approach will increase the success rate of MDR when there are only a few genotype combinations that are significantly associated with case-control status. We show that there is no loss of success rate when this is not the case. We then apply the RMDR method to the detection of gene-gene interactions in genotype data from a population-based study of bladder cancer in New Hampshire. PMID:21091664

Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase

PubMed Central

Sysa-Shah, Polina; Sørensen, Lars L; Abraham, M Roselle; Gabrielson, Kathleen L

2015-01-01

Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2tg) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2tg mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2tg data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2tg from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species. PMID:26310459

STR melting curve analysis as a genetic screening tool for crime scene samples.

PubMed

Nguyen, Quang; McKinney, Jason; Johnson, Donald J; Roberts, Katherine A; Hardy, Winters R

2012-07-01

In this proof-of-concept study, high-resolution melt curve (HRMC) analysis was investigated as a postquantification screening tool to discriminate human CSF1PO and THO1 genotypes amplified with mini-STR primers in the presence of SYBR Green or LCGreen Plus dyes. A total of 12 CSF1PO and 11 HUMTHO1 genotypes were analyzed on the LightScanner HR96 and LS-32 systems and were correctly differentiated based upon their respective melt profiles. Short STR amplicon melt curves were affected by repeat number, and single-source and mixed DNA samples were additionally differentiated by the formation of heteroduplexes. Melting curves were shown to be unique and reproducible from DNA quantities ranging from 20 to 0.4 ng and distinguished identical from nonidentical genotypes from DNA derived from different biological fluids and compromised samples. Thus, a method is described which can assess both the quantity and the possible probative value of samples without full genotyping. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

Internal validation of STRmix™ for the interpretation of single source and mixed DNA profiles.

PubMed

Moretti, Tamyra R; Just, Rebecca S; Kehl, Susannah C; Willis, Leah E; Buckleton, John S; Bright, Jo-Anne; Taylor, Duncan A; Onorato, Anthony J

2017-07-01

The interpretation of DNA evidence can entail analysis of challenging STR typing results. Genotypes inferred from low quality or quantity specimens, or mixed DNA samples originating from multiple contributors, can result in weak or inconclusive match probabilities when a binary interpretation method and necessary thresholds (such as a stochastic threshold) are employed. Probabilistic genotyping approaches, such as fully continuous methods that incorporate empirically determined biological parameter models, enable usage of more of the profile information and reduce subjectivity in interpretation. As a result, software-based probabilistic analyses tend to produce more consistent and more informative results regarding potential contributors to DNA evidence. Studies to assess and internally validate the probabilistic genotyping software STRmix™ for casework usage at the Federal Bureau of Investigation Laboratory were conducted using lab-specific parameters and more than 300 single-source and mixed contributor profiles. Simulated forensic specimens, including constructed mixtures that included DNA from two to five donors across a broad range of template amounts and contributor proportions, were used to examine the sensitivity and specificity of the system via more than 60,000 tests comparing hundreds of known contributors and non-contributors to the specimens. Conditioned analyses, concurrent interpretation of amplification replicates, and application of an incorrect contributor number were also performed to further investigate software performance and probe the limitations of the system. In addition, the results from manual and probabilistic interpretation of both prepared and evidentiary mixtures were compared. The findings support that STRmix™ is sufficiently robust for implementation in forensic laboratories, offering numerous advantages over historical methods of DNA profile analysis and greater statistical power for the estimation of evidentiary weight, and can be used reliably in human identification testing. With few exceptions, likelihood ratio results reflected intuitively correct estimates of the weight of the genotype possibilities and known contributor genotypes. This comprehensive evaluation provides a model in accordance with SWGDAM recommendations for internal validation of a probabilistic genotyping system for DNA evidence interpretation. Copyright © 2017. Published by Elsevier B.V.

Multiplexed microsatellite recovery using massively parallel sequencing

USGS Publications Warehouse

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

PubMed Central

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

Current methodologies on genotyping for nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA).

PubMed

Miao, Jian; Chen, Lequn; Wang, Jingwen; Wang, Wenxin; Chen, Dingqiang; Li, Lin; Li, Bing; Deng, Yang; Xu, Zhenbo

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen in hospitals and the community. As the rapid spread and wide distribution of antimicrobial resistance (such as MRSA), treatment for infectious diseases caused by microorganisms has become a vital threat. Thus, early identification and genotyping are essential for further therapeutic treatment and the control of rapid expansion of MRSA. In combination with applications and data feedbacks, this review focused on the currently available molecular-based assays on their utility and performance for rapid typing of MRSA, especially on effective molecular-based methods. Besides, a common mobile element SCCmec and prevalence of HA-MRSA, LA-MRSA and CA-MRSA were introduced in this review in order to provide a more complete profile of MRSA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Use of partial least squares regression to impute SNP genotypes in Italian cattle breeds.

PubMed

Dimauro, Corrado; Cellesi, Massimo; Gaspa, Giustino; Ajmone-Marsan, Paolo; Steri, Roberto; Marras, Gabriele; Macciotta, Nicolò P P

2013-06-05

The objective of the present study was to test the ability of the partial least squares regression technique to impute genotypes from low density single nucleotide polymorphisms (SNP) panels i.e. 3K or 7K to a high density panel with 50K SNP. No pedigree information was used. Data consisted of 2093 Holstein, 749 Brown Swiss and 479 Simmental bulls genotyped with the Illumina 50K Beadchip. First, a single-breed approach was applied by using only data from Holstein animals. Then, to enlarge the training population, data from the three breeds were combined and a multi-breed analysis was performed. Accuracies of genotypes imputed using the partial least squares regression method were compared with those obtained by using the Beagle software. The impact of genotype imputation on breeding value prediction was evaluated for milk yield, fat content and protein content. In the single-breed approach, the accuracy of imputation using partial least squares regression was around 90 and 94% for the 3K and 7K platforms, respectively; corresponding accuracies obtained with Beagle were around 85% and 90%. Moreover, computing time required by the partial least squares regression method was on average around 10 times lower than computing time required by Beagle. Using the partial least squares regression method in the multi-breed resulted in lower imputation accuracies than using single-breed data. The impact of the SNP-genotype imputation on the accuracy of direct genomic breeding values was small. The correlation between estimates of genetic merit obtained by using imputed versus actual genotypes was around 0.96 for the 7K chip. Results of the present work suggested that the partial least squares regression imputation method could be useful to impute SNP genotypes when pedigree information is not available.

Molecular and agronomic analysis of intraspecific variability in Capsicum baccatum var. pendulum accessions.

PubMed

Leite, P S S; Rodrigues, R; Silva, R N O; Pimenta, S; Medeiros, A M; Bento, C S; Gonçalves, L S A

2016-10-05

Capsicum baccatum is one of the most important chili peppers in South America, since this region is considered to be the center of origin and diversity of this species. In Brazil, C. baccatum has been widely explored by family farmers and there are different local names for each fruit phenotype, such as cambuci and dedo-de-moça (lady's finger). Although very popular among farmers and consumers, C. baccatum has been less extensively studied than other Capsicum species. This study describes the phenotypic and genotypic variability in C. baccatum var. pendulum accessions. Twenty-nine accessions from the Universidade Estadual do Norte Fluminense Darcy Ribeiro gene bank, and one commercial genotype ('BRS-Mari') were evaluated for 53 morphoagronomic descriptors (31 qualitative and 22 quantitative traits). In addition, accessions were genotyped using 30 microsatellite primers. Three accessions from the C. annuum complex were included in the molecular characterization. Nine of 31 qualitative descriptors were monomorphic, while all quantitative descriptors were highly significant different between accessions (P < 0.01). Using the unweighted pair group method using arithmetic averages, four groups were obtained based on multicategoric variables and five groups were obtained based on quantitative variables. In the genotyping analysis, 12 polymorphic simple sequence repeat primers amplified in C. baccatum with dissimilarity between accessions ranging from 0.13 to 0.91, permitting the formation of two distinct groups for Bayesian analysis. These results indicate wide variability among the accessions comparing phenotypic and genotypic data and revealed distinct patterns of dissimilarity between matrices, indicating that both steps are valuable for the characterization of C. baccatum var. pendulum accessions.

Simple SNP-based minimal marker genotyping for (Humulus lupulus L.) identification and variety validation

USDA-ARS?s Scientific Manuscript database

Hop is a perennial crop with clonal propagation system for varietal distribution. Brewers and growers are highly concerned about variety purity and regularly seek genotype testing. Current means for genotyping are based upon SSRs OR AFLPs that are relatively accurate but cannot differentiate close...

Improvement of Predictive Ability by Uniform Coverage of the Target Genetic Space

PubMed Central

Bustos-Korts, Daniela; Malosetti, Marcos; Chapman, Scott; Biddulph, Ben; van Eeuwijk, Fred

2016-01-01

Genome-enabled prediction provides breeders with the means to increase the number of genotypes that can be evaluated for selection. One of the major challenges in genome-enabled prediction is how to construct a training set of genotypes from a calibration set that represents the target population of genotypes, where the calibration set is composed of a training and validation set. A random sampling protocol of genotypes from the calibration set will lead to low quality coverage of the total genetic space by the training set when the calibration set contains population structure. As a consequence, predictive ability will be affected negatively, because some parts of the genotypic diversity in the target population will be under-represented in the training set, whereas other parts will be over-represented. Therefore, we propose a training set construction method that uniformly samples the genetic space spanned by the target population of genotypes, thereby increasing predictive ability. To evaluate our method, we constructed training sets alongside with the identification of corresponding genomic prediction models for four genotype panels that differed in the amount of population structure they contained (maize Flint, maize Dent, wheat, and rice). Training sets were constructed using uniform sampling, stratified-uniform sampling, stratified sampling and random sampling. We compared these methods with a method that maximizes the generalized coefficient of determination (CD). Several training set sizes were considered. We investigated four genomic prediction models: multi-locus QTL models, GBLUP models, combinations of QTL and GBLUPs, and Reproducing Kernel Hilbert Space (RKHS) models. For the maize and wheat panels, construction of the training set under uniform sampling led to a larger predictive ability than under stratified and random sampling. The results of our methods were similar to those of the CD method. For the rice panel, all training set construction methods led to similar predictive ability, a reflection of the very strong population structure in this panel. PMID:27672112

N-acetyltransferase gene polymorphisms & plasma isoniazid concentrations in patients with tuberculosis

PubMed Central

Hemanth Kumar, A. K.; Ramesh, K.; Kannan, T.; Sudha, V.; Haribabu, Hemalatha; Lavanya, J.; Swaminathan, Soumya; Ramachandran, Geetha

2017-01-01

Background & objectives: Variations in the N-acetyltransferase (NAT2) gene among different populations could affect the metabolism and disposition of isoniazid (INH). This study was performed to genotype NAT2 gene polymorphisms in tuberculosis (TB) patients from Chennai, India, and compare plasma INH concentrations among the different genotypes. Methods: Adult patients with TB treated in the Revised National TB Control Programme (RNTCP) in Chennai, Tamil Nadu, were genotyped for NAT2 gene polymorphism, and two-hour post-dosing INH concentrations were compared between the different genotypes. Plasma INH was determined by high-performance liquid chromatography. Genotyping of the NAT2 gene polymorphism was performed by real-time polymerase chain reaction method. Results: Among the 326 patients genotyped, there were 189 (58%), 114 (35%) and 23 (7%) slow, intermediate and fast acetylators, respectively. The median two-hour INH concentrations in slow, intermediate and fast acetylators were 10.2, 8.1 and 4.1 μg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P<0.001). Interpretation & conclusions: Genotyping of TB patients from south India for NAT2 gene polymorphism revealed that 58 per cent of the study population comprised slow acetylators. Two-hour INH concentrations differed significantly among the three genotypes. PMID:28574024

Genetic diversity studies in pea (Pisum sativum L.) using simple sequence repeat markers.

PubMed

Kumari, P; Basal, N; Singh, A K; Rai, V P; Srivastava, C P; Singh, P K

2013-03-13

The genetic diversity among 28 pea (Pisum sativum L.) genotypes was analyzed using 32 simple sequence repeat markers. A total of 44 polymorphic bands, with an average of 2.1 bands per primer, were obtained. The polymorphism information content ranged from 0.657 to 0.309 with an average of 0.493. The variation in genetic diversity among these cultivars ranged from 0.11 to 0.73. Cluster analysis based on Jaccard's similarity coefficient using the unweighted pair-group method with arithmetic mean (UPGMA) revealed 2 distinct clusters, I and II, comprising 6 and 22 genotypes, respectively. Cluster II was further differentiated into 2 subclusters, IIA and IIB, with 12 and 10 genotypes, respectively. Principal component (PC) analysis revealed results similar to those of UPGMA. The first, second, and third PCs contributed 21.6, 16.1, and 14.0% of the variation, respectively; cumulative variation of the first 3 PCs was 51.7%.

Spatial and temporal variability of microgeographic genetic structure in white-tailed deer

USGS Publications Warehouse

Scribner, Kim T.; Smith, Michael H.; Chesser, Ronald K.

1997-01-01

Techniques are described that define contiguous genetic subpopulations of white-tailed deer (Odocoileus virginianus) based on the spatial dispersion of 4,749 individuals that possessed discrete character values (alleles or genotypes) during each of 6 years (1974-1979). White-tailed deer were not uniformly distributed in space, but exhibited considerable spatial genetic structuring. Significant non-random clusters of individuals were documented during each year based on specific alleles and genotypes at the Sdh locus. Considerable temporal variation was observed in the position and genetic composition of specific clusters, which reflected changes in allele frequency in small geographic areas. The position of clusters did not consistently correspond with traditional management boundaries based on major discontinuities in habitat (swamp versus upland) and hunt compartments that were defined by roads and streams. Spatio-temporal stability of observed genetic contiguous clusters was interpreted relative to method and intensity of harvest, movements, and breeding ecology.

Improving Genomic Prediction in Cassava Field Experiments by Accounting for Interplot Competition

PubMed Central

Elias, Ani A.; Rabbi, Ismail; Kulakow, Peter; Jannink, Jean-Luc

2018-01-01

Plants competing for available resources is an unavoidable phenomenon in a field. We conducted studies in cassava (Manihot esculenta Crantz) in order to understand the pattern of this competition. Taking into account the competitive ability of genotypes while selecting parents for breeding advancement or commercialization can be very useful. We assumed that competition could occur at two levels: (i) the genotypic level, which we call interclonal, and (ii) the plot level irrespective of the type of genotype, which we call interplot competition or competition error. Modification in incidence matrices was applied in order to relate neighboring genotype/plot to the performance of a target genotype/plot with respect to its competitive ability. This was added into a genomic selection (GS) model to simultaneously predict the direct and competitive ability of a genotype. Predictability of the models was tested through a 10-fold cross-validation method repeated five times. The best model was chosen as the one with the lowest prediction root mean squared error (pRMSE) compared to that of the base model having no competitive component. Results from our real data studies indicated that <10% increase in accuracy was achieved with GS-interclonal competition model, but this value reached up to 25% with a GS-competition error model. We also found that the competitive influence of a cassava clone is not just limited to the adjacent neighbors but spreads beyond them. Through simulations, we found that a 26% increase of accuracy in estimating trait genotypic effect can be achieved even in the presence of high competitive variance. PMID:29358232

Improving Genomic Prediction in Cassava Field Experiments by Accounting for Interplot Competition.

PubMed

Elias, Ani A; Rabbi, Ismail; Kulakow, Peter; Jannink, Jean-Luc

2018-03-02

Plants competing for available resources is an unavoidable phenomenon in a field. We conducted studies in cassava ( Manihot esculenta Crantz) in order to understand the pattern of this competition. Taking into account the competitive ability of genotypes while selecting parents for breeding advancement or commercialization can be very useful. We assumed that competition could occur at two levels: (i) the genotypic level, which we call interclonal, and (ii) the plot level irrespective of the type of genotype, which we call interplot competition or competition error. Modification in incidence matrices was applied in order to relate neighboring genotype/plot to the performance of a target genotype/plot with respect to its competitive ability. This was added into a genomic selection (GS) model to simultaneously predict the direct and competitive ability of a genotype. Predictability of the models was tested through a 10-fold cross-validation method repeated five times. The best model was chosen as the one with the lowest prediction root mean squared error (pRMSE) compared to that of the base model having no competitive component. Results from our real data studies indicated that <10% increase in accuracy was achieved with GS-interclonal competition model, but this value reached up to 25% with a GS-competition error model. We also found that the competitive influence of a cassava clone is not just limited to the adjacent neighbors but spreads beyond them. Through simulations, we found that a 26% increase of accuracy in estimating trait genotypic effect can be achieved even in the presence of high competitive variance. Copyright © 2018 Elias et al.

A genotype probability index for multiple alleles and haplotypes.

PubMed

Percy, A; Kinghorn, B P

2005-12-01

We use linear algebra to calculate an index of information content in genotype probabilities which has previously been calculated using trigonometry. The new method can be generalized allowing the index to be calculated for loci with more than two alleles. Applications of this index include its use in genotyping strategies, strategies to manage genetic disorders and in estimation of genotype effects.

Detection and genotyping of human papilloma virus in cervical cancer specimens from Saudi patients.

PubMed

Al-Badawi, Ismail A; Al-Suwaine, Abdulrahman; Al-Aker, Murad; Asaad, Lina; Alaidan, Alwaleed; Tulbah, Asma; Fe Bohol, Marie; Munkarah, Adnan R

2011-07-01

To determine the rates and types of human papilloma virus (HPV) infection in cervical cancer specimens from Saudi patients. One hundred specimens were randomly selected and retrieved from the achieved samples stored in the pathology department accessioned under the diagnosis of cervical cancer and carcinoma in situ between the years 1997 and 2007. Human papilloma virus in the clinical samples was detected using polymerase chain reaction amplification methods. Two primer systems are commonly used: the MY09-MY11 primers and the GP5+-GP6+ that amplify a wide range of HPV genotypes. Human papilloma virus isolates were genotyped using DNA sequencing and reverse line blot hybridization assay to identify the high-risk HPV genotypes. Ninety cases fulfilled the diagnostic criteria and were analyzed. The rate of HPV genotype detection among cervical cancer samples was 95.5%. The most common HPV genotype detected by both methods was HPV-16 (63.4%), followed by HPV-18 (11.1%), HPV-45 (4.5%), HPV-33 (3.3%), and HPV-31, HPV-52, HPV-53, HPV-58, HPV-59, and HPV-66 with 2.2% prevalence rate each. Prevalence of HPV genotypes among patients with cervical cancer in Saudi Arabia is comparable to the international rates. The use of the reverse line blot hybridization assay genotyping method could be useful for classifying oncogenic HPV-positive women. It is relatively inexpensive and reliable and can be performed in routine practice or epidemiological study compared with the available standard commercial kits.

Classification of cassava genotypes based on qualitative and quantitative data.

PubMed

Oliveira, E J; Oliveira Filho, O S; Santos, V S

2015-02-02

We evaluated the genetic variation of cassava accessions based on qualitative (binomial and multicategorical) and quantitative traits (continuous). We characterized 95 accessions obtained from the Cassava Germplasm Bank of Embrapa Mandioca e Fruticultura; we evaluated these accessions for 13 continuous, 10 binary, and 25 multicategorical traits. First, we analyzed the accessions based only on quantitative traits; next, we conducted joint analysis (qualitative and quantitative traits) based on the Ward-MLM method, which performs clustering in two stages. According to the pseudo-F, pseudo-t2, and maximum likelihood criteria, we identified five and four groups based on quantitative trait and joint analysis, respectively. The smaller number of groups identified based on joint analysis may be related to the nature of the data. On the other hand, quantitative data are more subject to environmental effects in the phenotype expression; this results in the absence of genetic differences, thereby contributing to greater differentiation among accessions. For most of the accessions, the maximum probability of classification was >0.90, independent of the trait analyzed, indicating a good fit of the clustering method. Differences in clustering according to the type of data implied that analysis of quantitative and qualitative traits in cassava germplasm might explore different genomic regions. On the other hand, when joint analysis was used, the means and ranges of genetic distances were high, indicating that the Ward-MLM method is very useful for clustering genotypes when there are several phenotypic traits, such as in the case of genetic resources and breeding programs.

Mechanisms of Oryza sativa (Poaceae) resistance to Tagosodes orizicolus (Homoptera: Delphacidae) under greenhouse condition in Venezuela.

PubMed

González, Alex; Labrín, Natalia; Alvarez, Rosa M; Jayaro, Yorman; Gamboa, Carlos; Reyes, Edicta; Barrientos, Venancio

2012-03-01

Tagosodes orizicolus is one of the main plagues of rice in tropical America causing two types of damages, the direct one, feeding and oviposition effect, and an indirect one, by the transmission of the "Rice hoja blanca virus". During 2006-2007 we carried out research under greenhouse conditions at Fundaci6n Danac, Venezuela, in order to determine the mechanisms of antixenosis, antibiosis and tolerance to T. orizicolus, which could be acting in commercial varieties and advanced lines of the rice genetic breeding programs of INIA and Fundaci6n Danac. The method of free feeding was used for the antixenosis evaluation, whereas the method of forced feeding was used for antibiosis evaluation (effect on survival and oviposition). Additionally, we used the indirect method based on biomass depression to estimate the tolerance. Some of the evaluated traits included: grade of damage, number of insects settling on rice plants, percentage of sogata mortality at the mature state, number of eggs in the leaf midrib and an index of tolerance. The results showed that rice genotypes possess different combinations of resistance mechanisms, as well as different grades of reactions. The susceptible control 'Bluebonnet 50' was consistently susceptible across experiments and the resistant control 'Makalioka' had high antixenosis and high antibiosis based on survival and oviposition. The rest of the genotypes presented lower or higher degrees of antixenosis and antibiosis for survival and oviposition. The genotype 'FD0241-M-17-6-1-1-1-1' was identified with possible tolerance to the direct damage of sogata.

Use of a rep-PCR system to predict species in the Aspergillus section Nigri.

PubMed

Palencia, Edwin R; Klich, Maren A; Glenn, Anthony E; Bacon, Charles W

2009-10-01

The Aspergillus niger aggregate within the A. section Nigri is a group of black-spored aspergilli of great agro-economic importance whose well defined taxonomy has been elusive. Rep-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the data obtained from ITS and partial calmodulin regions. For this purpose, 20 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library in this barcoding system that served to identify 34 field isolates. A pair-wise similarity matrix was calculated using the cone-based Pearson correlation method and the dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA), illustrating four different clustered groups: the uniseriate cluster (I), the Aspergillus carbonarius cluster (II), and. the two A. niger aggregate clusters (named III.A and III.B). Rep-PCR showed higher resolution than the ITS and the partial calmodulin gene analytical procedures. The data of the 34 unknown field isolates, collected from different locations in the United States, indicated that only 12% of the field isolates were >95% similar to one of the genotypes included in the A. section Nigri library. However, 64% of the field isolates matched genotypes with the reference library (similarity values >90%). Based on these results, this barcoding procedure has the potential for use as a reproducible tool for identifying the black-spored aspergilli.

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

rs11613352 polymorphism (TT genotype) associates with a decrease of triglycerides and an increase of HDL in familial hypercholesterolemia patients.

PubMed

Aledo, Rosa; Padró, Teresa; Mata, Pedro; Alonso, Rodrigo; Badimon, Lina

2015-04-01

Recent genome-wide association studies have identified a locus on chromosome 12q13.3 associated with plasma levels of triglyceride and high-density lipoprotein cholesterol, with rs11613352 being the lead single nucleotide polymorphism in this genome-wide association study locus. The aim of the study is to investigate the involvement of rs11613352 in a population with high cardiovascular risk due to familial hypercholesterolemia. The single nucleotide polymorphism was genotyped by Taqman(®) assay in a cohort of 601 unrelated familial hypercholesterolemia patients and its association with plasma triglyceride and high-density lipoprotein cholesterol levels was analyzed by multivariate methods based on linear regression. Minimal allele frequency was 0.17 and genotype frequencies were 0.69, 0.27, and 0.04 for CC, CT, and TT genotypes, respectively. The polymorphism is associated in a recessive manner (TT genotype) with a decrease in triglyceride levels (P=.002) and with an increase in high-density lipoprotein cholesterol levels (P=.021) after adjusting by age and sex. The polymorphism rs11613352 may contribute to modulate the cardiovascular risk by modifying plasma lipid levels in familial hypercholesterolemia patients. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

High clustering rates of multidrug-resistant Mycobacterium tuberculosis genotypes in Panama

PubMed Central

2013-01-01

Background Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype. Conclusion Our findings suggest that MDR Mycobacterium tuberculosis is highly clustered in Panama’s metropolitan area corresponding to Panama City and Colon City, and our study reveals the genotype distribution across the country. PMID:24053690

Clonality and Micro-Diversity of a Nationwide Spreading Genotype of Mycobacterium tuberculosis in Japan

PubMed Central

Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Seto, Junji; Ahiko, Tadayuki; Yamamoto, Kaori; Hase, Atushi; Maeda, Shinji; Yamamoto, Taro

2015-01-01

Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as ‘M-strain’ has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB. PMID:25734518

Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing.

PubMed

Mallory, Melanie A; Lucic, Danijela; Ebbert, Mark T W; Cloherty, Gavin A; Toolsie, Dan; Hillyard, David R

2017-05-01

HCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6. To evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing. This study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database. Agreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven "not detected" by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure. The PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results. Copyright © 2017 Elsevier B.V. All rights reserved.

A Unified Approach to Genotype Imputation and Haplotype-Phase Inference for Large Data Sets of Trios and Unrelated Individuals

PubMed Central

Browning, Brian L.; Browning, Sharon R.

2009-01-01

We present methods for imputing data for ungenotyped markers and for inferring haplotype phase in large data sets of unrelated individuals and parent-offspring trios. Our methods make use of known haplotype phase when it is available, and our methods are computationally efficient so that the full information in large reference panels with thousands of individuals is utilized. We demonstrate that substantial gains in imputation accuracy accrue with increasingly large reference panel sizes, particularly when imputing low-frequency variants, and that unphased reference panels can provide highly accurate genotype imputation. We place our methodology in a unified framework that enables the simultaneous use of unphased and phased data from trios and unrelated individuals in a single analysis. For unrelated individuals, our imputation methods produce well-calibrated posterior genotype probabilities and highly accurate allele-frequency estimates. For trios, our haplotype-inference method is four orders of magnitude faster than the gold-standard PHASE program and has excellent accuracy. Our methods enable genotype imputation to be performed with unphased trio or unrelated reference panels, thus accounting for haplotype-phase uncertainty in the reference panel. We present a useful measure of imputation accuracy, allelic R2, and show that this measure can be estimated accurately from posterior genotype probabilities. Our methods are implemented in version 3.0 of the BEAGLE software package. PMID:19200528

Agreement for HPV genotyping detection between self-collected specimens on a FTA cartridge and clinician-collected specimens

PubMed Central

Guan, YaoYao; Gravitt, Patti E.; Howard, Roslyn; Eby, Yolanda J.; Wang, Shaoming; Li, Belinda; Feng, Changyan; Qiao, You-Lin; Castle, Philip E.

2016-01-01

The current method of transporting self-collected cervicovaginal specimen for HPV DNA testing relies on liquid based medium, which is challenging and expensive to transport. A novel, dry storage and transportation device, Whatman indicating FTA™ Elute Cartridge, avoids some of the pitfalls of liquid-based medium. This method has been shown to be comparable to liquid-based collection medium, but relative performance of self-collected (SC) and clinician-collected (CC) samples onto FTA cards has not been reported. The objective of this study is to compare the analytic performance of self- and clinician-collected samples onto FTA cartridges for the detection of carcinogenic HPV using Linear Array. There was a 91% agreement, 69% positive agreement, and kappa of 0.75 between the clinician-collected and self-collected specimens for detection of any carcinogenic HPV genotype. When the HPV results were categorized hierarchically according to cervical cancer risk, there was no difference in the distribution of the HPV results for the clinician- and self-collected specimens (p = 0.7). This study concludes that FTA elute cartridge is a promising method of specimen transport for cervical cancer screening programs considering using self-collected specimen and HPV testing. Larger studies with clinical endpoints are now needed to assess the clinical performance. PMID:23370404

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

PubMed

Kinkar, Liina; Laurimäe, Teivi; Acosta-Jamett, Gerardo; Andresiuk, Vanessa; Balkaya, Ibrahim; Casulli, Adriano; Gasser, Robin B; González, Luis Miguel; Haag, Karen L; Zait, Houria; Irshadullah, Malik; Jabbar, Abdul; Jenkins, David J; Manfredi, Maria Teresa; Mirhendi, Hossein; M'rad, Selim; Rostami-Nejad, Mohammad; Oudni-M'rad, Myriam; Pierangeli, Nora Beatriz; Ponce-Gordo, Francisco; Rehbein, Steffen; Sharbatkhori, Mitra; Kia, Eshrat Beigom; Simsek, Sami; Soriano, Silvia Viviana; Sprong, Hein; Šnábel, Viliam; Umhang, Gérald; Varcasia, Antonio; Saarma, Urmas

2018-06-21

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1. Copyright © 2018. Published by Elsevier B.V.

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio.

PubMed

Zivcak, Marek; Brückova, Klaudia; Sytar, Oksana; Brestic, Marian; Olsovska, Katarina; Allakhverdiev, Suleyman I

2017-06-01

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique. The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

PubMed

Girault, G; Wattiau, P; Saqib, M; Martin, B; Vorimore, F; Singha, H; Engelsma, M; Roest, H J; Spicic, S; Grunow, R; Vicari, N; De Keersmaecker, S C J; Roosens, N H C; Fabbi, M; Tripathi, B N; Zientara, S; Madani, N; Laroucau, K

2018-05-08

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei. Copyright © 2018 Elsevier B.V. All rights reserved.

Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience.

PubMed

Fasano, Ross M; Sullivan, Harold Cliff; Bray, Robert A; Gebel, Howard M; Meyer, Erin K; Winkler, Annie M; Josephson, Cassandra D; Stowell, Sean R; Sandy Duncan, Alexander; Roback, John D

2017-03-01

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.

Phenylalanine hydroxylase deficiency in Mexico: genotype-phenotype correlations, BH4 responsiveness and evidence of a founder effect.

PubMed

Vela-Amieva, M; Abreu-González, M; González-del Angel, A; Ibarra-González, I; Fernández-Lainez, C; Barrientos-Ríos, R; Monroy-Santoyo, S; Guillén-López, S; Alcántara-Ortigoza, M A

2015-07-01

The mutational spectrum of the phenylalanine hydroxylase gene (PAH) in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype-phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype-phenotype correlations and genotype-based prediction of BH4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5G>T being the most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1A>T and c.969+6T>C. The genotype-phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH4 testing and therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of genotype x environment interactions in cotton using the method proposed by Eberhart and Russell and reaction norm models.

PubMed

Alves, R S; Teodoro, P E; Farias, F C; Farias, F J C; Carvalho, L P; Rodrigues, J I S; Bhering, L L; Resende, M D V

2017-08-17

Cotton produces one of the most important textile fibers of the world and has great relevance in the world economy. It is an economically important crop in Brazil, which is the world's fifth largest producer. However, studies evaluating the genotype x environment (G x E) interactions in cotton are scarce in this country. Therefore, the goal of this study was to evaluate the G x E interactions in two important traits in cotton (fiber yield and fiber length) using the method proposed by Eberhart and Russell (simple linear regression) and reaction norm models (random regression). Eight trials with sixteen upland cotton genotypes, conducted in a randomized block design, were used. It was possible to identify a genotype with wide adaptability and stability for both traits. Reaction norm models have excellent theoretical and practical properties and led to more informative and accurate results than the method proposed by Eberhart and Russell and should, therefore, be preferred. Curves of genotypic values as a function of the environmental gradient, which predict the behavior of the genotypes along the environmental gradient, were generated. These curves make possible the recommendation to untested environmental levels.

Spectral reflectance indices as a selection criterion for yield improvement in wheat

NASA Astrophysics Data System (ADS)

Babar, Md. Ali

2005-11-01

Scope and methods of study. Yield in wheat ( Triticum aestivum L.) is a complex trait and influenced by many environmental factors, and yield improvement is a daunting task for wheat breeders. Spectral reflectance indices (SRIs) have been used to study different physiological traits in wheat. SRIs have the potential to differentiate genotypes for grain yield. SRIs strongly associated with grain yield can be used to achieve effective genetic gain in wheat under different environments. Three experiments (15 adapted genotypes, 25 and 36 random sister lines derived from two different crosses) under irrigated conditions, and three experiments (each with 30 advanced genotypes) under water-limited conditions were conducted in three successive years in Northwest Mexico at the CIMMYT (International Maize and wheat Improvement Center) experimental station. SRIs and different agronomic data were collected for three years, and biomass was harvested for two years. Phenotypic and genetic correlations between SRIs and grain yield, between SRIs and biomass, realized and broad sense heritability, direct and correlated selection responses for grain yield, and SRIs were calculated. Findings and conclusion. Seven SRIs were calculated, and three near infrared based indices (WI, NWI-1 and NWI-2) showed higher level of genetic and phenotypic correlations with grain yield, yield components and biomass than other SRIs (PRI, RNDVI, GNDVI, and SR) under both irrigated and water limiting environments. Moderate to high realized and broad sense heritability, and selection response were demonstrated by the three NIR based indices. High efficiency of correlated response for yield estimation was demonstrated by the three NIR based indices. The ratio between the correlated response to grain yield based on the three NIR based indices and direct selection response for grain yield was very close to one. The NIR based indices showed very high accuracy in selecting superior genotypes for grain yield under both well-watered and water-limited conditions. These results demonstrated that effective genetic gain in grain yield improvement can be achieved by making selections with the three NIR based indices.

Comparison of three PCR-based assays for SNP genotyping in sugar beet

USDA-ARS?s Scientific Manuscript database

Background: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved t...

Determining the relative susceptibility of four prion protein genotypes to atypical scrapie

USDA-ARS?s Scientific Manuscript database

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). We describe a mass spectrometry-based method of detecting and quantifying the polymorphisms of sheep P...

The potential for using canopy spectral reflectance as an indirect selection tool for yield improvement in winter wheat

NASA Astrophysics Data System (ADS)

Prasad, Bishwajit

Scope and methods of study. Complementing breeding effort by deploying alternative methods of identifying higher yielding genotypes in a wheat breeding program is important for obtaining greater genetic gains. Spectral reflectance indices (SRI) are one of the many indirect selection tools that have been reported to be associated with different physiological process of wheat. A total of five experiments (a set of 25 released cultivars from winter wheat breeding programs of the U.S. Great Plains and four populations of randomly derived recombinant inbred lines having 25 entries in each population) were conducted in two years under Great Plains winter wheat rainfed environments at Oklahoma State University research farms. Grain yield was measured in each experiment and biomass was measured in three experiments at three growth stages (booting, heading, and grainfilling). Canopy spectral reflectance was measured at three growth stages and eleven SRI were calculated. Correlation (phenotypic and genetic) between grain yield and SRI, biomass and SRI, heritability (broad sense) of the SRI and yield, response to selection and correlated response, relative selection efficiency of the SRI, and efficiency in selecting the higher yielding genotypes by the SRI were assessed. Findings and conclusions. The genetic correlation coefficients revealed that the water based near infrared indices (WI and NWI) were strongly associated with grain yield and biomass production. The regression analysis detected a linear relationship between the water based indices with grain yield and biomass. The two newly developed indices (NWI-3 and NWI-4) gave higher broad sense heritability than grain yield, higher direct response to selection compared to grain yield, correlated response equal to or higher than direct response for grain yield, relative selection efficiency greater than one, and higher efficiency in selecting higher yielding genotypes. Based on the overall genetic analysis required to establish any trait as an efficient indirect selection tool, the water based SRI (especially NWI-3 and NWI-4) have the potential to complement the classical breeding effort for selecting genotypes with higher yield potential in a winter wheat breeding program.

Pedigree data analysis with crossover interference.

PubMed Central

Browning, Sharon

2003-01-01

We propose a new method for calculating probabilities for pedigree genetic data that incorporates crossover interference using the chi-square models. Applications include relationship inference, genetic map construction, and linkage analysis. The method is based on importance sampling of unobserved inheritance patterns conditional on the observed genotype data and takes advantage of fast algorithms for no-interference models while using reweighting to allow for interference. We show that the method is effective for arbitrarily many markers with small pedigrees. PMID:12930760

Detection and Genotyping of Human Papillomavirus in Self-Obtained Cervicovaginal Samples by Using the FTA Cartridge: New Possibilities for Cervical Cancer Screening ▿

PubMed Central

Lenselink, Charlotte H.; de Bie, Roosmarie P.; van Hamont, Dennis; Bakkers, Judith M. J. E.; Quint, Wim G. V.; Massuger, Leon F. A. G.; Bekkers, Ruud L. M.; Melchers, Willem J. G.

2009-01-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF10-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening. PMID:19553570

Detection and genotyping of human papillomavirus in self-obtained cervicovaginal samples by using the FTA cartridge: new possibilities for cervical cancer screening.

PubMed

Lenselink, Charlotte H; de Bie, Roosmarie P; van Hamont, Dennis; Bakkers, Judith M J E; Quint, Wim G V; Massuger, Leon F A G; Bekkers, Ruud L M; Melchers, Willem J G

2009-08-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF(10)-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

PubMed

Odegaard, Justin I; Vincent, John J; Mortimer, Stefanie; Vowles, James V; Ulrich, Bryan C; Banks, Kimberly C; Fairclough, Stephen R; Zill, Oliver A; Sikora, Marcin; Mokhtari, Reza; Abdueva, Diana; Nagy, Rebecca J; Lee, Christine E; Kiedrowski, Lesli A; Paweletz, Cloud P; Eltoukhy, Helmy; Lanman, Richard B; Chudova, Darya I; Talasaz, AmirAli

2018-04-24

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 1-11. ©2018 AACR. ©2018 American Association for Cancer Research.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

PubMed

Zheng, Xuelian; Yang, Shixin; Zhang, Dengwei; Zhong, Zhaohui; Tang, Xu; Deng, Kejun; Zhou, Jianping; Qi, Yiping; Zhang, Yong

2016-07-01

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

Evaluation of red blood cell and platelet antigen genotyping platforms (ID CORE XT/ID HPA XT) in routine clinical practice.

PubMed

Finning, Kirstin; Bhandari, Radhika; Sellers, Fiona; Revelli, Nicoletta; Villa, Maria Antonietta; Muñiz-Díaz, Eduardo; Nogués, Núria

2016-03-01

High-throughput genotyping platforms enable simultaneous analysis of multiple polymorphisms for blood group typing. BLOODchip® ID is a genotyping platform based on Luminex® xMAP technology for simultaneous determination of 37 red blood cell (RBC) antigens (ID CORE XT) and 18 human platelet antigens (HPA) (ID HPA XT) using the BIDS XT software. In this international multicentre study, the performance of ID CORE XT and ID HPA XT, using the centres' current genotyping methods as the reference for comparison, and the usability and practicality of these systems, were evaluated under working laboratory conditions. DNA was extracted from whole blood in EDTA with Qiagen methodologies. Ninety-six previously phenotyped/genotyped samples were processed per assay: 87 testing samples plus five positive controls and four negative controls. Results were available for 519 samples: 258 with ID CORE XT and 261 with ID HPA XT. There were three "no calls" that were either caused by human error or resolved after repeating the test. Agreement between the tests and reference methods was 99.94% for ID CORE XT (9,540/9,546 antigens determined) and 100% for ID HPA XT (all 4,698 alleles determined). There were six discrepancies in antigen results in five RBC samples, four of which (in VS, N, S and Do(a)) could not be investigated due to lack of sufficient sample to perform additional tests and two of which (in S and C) were resolved in favour of ID CORE XT (100% accuracy). The total hands-on time was 28-41 minutes for a batch of 16 samples. Compared with the reference platforms, ID CORE XT and ID HPA XT were considered simpler to use and had shorter processing times. ID CORE XT and ID HPA XT genotyping platforms for RBC and platelet systems were accurate and user-friendly in working laboratory settings.

Cow genotyping strategies for genomic selection in a small dairy cattle population.

PubMed

Jenko, J; Wiggans, G R; Cooper, T A; Eaglen, S A E; Luff, W G de L; Bichard, M; Pong-Wong, R; Woolliams, J A

2017-01-01

This study compares how different cow genotyping strategies increase the accuracy of genomic estimated breeding values (EBV) in dairy cattle breeds with low numbers. In these breeds, few sires have progeny records, and genotyping cows can improve the accuracy of genomic EBV. The Guernsey breed is a small dairy cattle breed with approximately 14,000 recorded individuals worldwide. Predictions of phenotypes of milk yield, fat yield, protein yield, and calving interval were made for Guernsey cows from England and Guernsey Island using genomic EBV, with training sets including 197 de-regressed proofs of genotyped bulls, with cows selected from among 1,440 genotyped cows using different genotyping strategies. Accuracies of predictions were tested using 10-fold cross-validation among the cows. Genomic EBV were predicted using 4 different methods: (1) pedigree BLUP, (2) genomic BLUP using only bulls, (3) univariate genomic BLUP using bulls and cows, and (4) bivariate genomic BLUP. Genotyping cows with phenotypes and using their data for the prediction of single nucleotide polymorphism effects increased the correlation between genomic EBV and phenotypes compared with using only bulls by 0.163±0.022 for milk yield, 0.111±0.021 for fat yield, and 0.113±0.018 for protein yield; a decrease of 0.014±0.010 for calving interval from a low base was the only exception. Genetic correlation between phenotypes from bulls and cows were approximately 0.6 for all yield traits and significantly different from 1. Only a very small change occurred in correlation between genomic EBV and phenotypes when using the bivariate model. It was always better to genotype all the cows, but when only half of the cows were genotyped, a divergent selection strategy was better compared with the random or directional selection approach. Divergent selection of 30% of the cows remained superior for the yield traits in 8 of 10 folds. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes

PubMed Central

Ahlawat, Sonika; Sharma, Rekha; Maitra, A.; Roy, Manoranjan; Tantia, M.S.

2014-01-01

New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes. PMID:25606428

Final Report of Unmet Needs of Interferon-Based Therapy for Chronic Hepatitis C in Korea: Basis for Moving into the Direct-Acting Antiviral Era

PubMed Central

Jang, Eun Sun; Kim, Young Seok; Kim, Kyung-Ah; Lee, Youn Jae; Chung, Woo Jin; Kim, In Hee; Lee, Byung Seok; Jeong, Sook-Hyang

2017-01-01

Background/Aims To evaluate the era of direct acting antivirals (DAAs), we must understand the treatment patterns and outcomes of interferon-based therapy for hepatitis C virus (HCV) infection. We aimed to elucidate the treatment rate, factors affecting treatment decisions, and efficacy of interferon-based therapy in a real-world setting. Methods This nationwide cohort study included 1,191 newly diagnosed patients with chronic HCV infection at seven tertiary hospitals in South Korea. Subjects were followed retrospectively until March 2015, which was just before the approval of DAA therapy. Results In total, 48.2% and 49.3% of the patients had HCV genotypes 1 and 2, respectively. Interferon-based therapy was initiated in 541 patients (45.4%). The major reasons for no treatment included ineligibility (18.9%), concern about adverse events (22.3%), cost (21.5%), and an age >75 years (19.5%). Interferon-based therapy was discontinued (18.5%) mainly due to adverse events (n=66). The intent-to-treat analysis found that the sustained virologic response (SVR) rate was 58.3% in genotype 1 patients and 74.7% in non-genotype 1 patients. Conclusions Approximately one-third of newly diagnosed HCV patients in South Korea received interferon-based therapy and showed a suboptimal SVR rate. Diagnosis of patients at younger ages and with a less advanced liver status and reducing the DAA therapy cost may fulfill unmet needs. PMID:28506027

Gene-gene interactions of CYP2A6 and MAOA polymorphisms on smoking behavior in Chinese male population.

PubMed

Tang, Xun; Guo, Song; Sun, Hongqiang; Song, Xuemei; Jiang, Zuonin; Sheng, Lixiang; Zhou, Dongfeng; Hu, Yonghua; Chen, Dafang

2009-05-01

Nicotine is the major psychoactive ingredient in tobacco, and is responsible for dependence through the nicotine-stimulated reward pathway mediated by the central dopaminergic system. Consequently, genetic polymorphisms in both nicotine metabolism and dopamine catabolism genes may influence smoking behavior, and interact with each other resulting in risk modulation. In this study, we investigated the association and multilocus gene-gene interactions of cytochrome P450 2A6 (CYP2A6), dopamine beta-hydroxylase (DBH), catechol O-methyl transferase (COMT), and monoamine oxidase A (MAOA) polymorphisms with smoking behavior in a community-based Chinese male population. The polymorphisms were genotyped in 203 current smokers, 66 former smokers, and 102 never smokers. Multivariate logistic regression models and the multifactor dimensionality reduction method were used to analyze the association and multilocus gene-gene interactions. Statistically significant trends were shown for increased risk of smoking initiation in participants with CYP2A6*1B/CYP2A6*1B genotypes compared with those with CYP2A6*1A/CYP2A6*1A genotypes [odds ratio (OR)=3.5, 95% confidence interval (CI)= 1.5-8.1], and participants with CYP2A6*1/CYP2A6*1 genotypes were at higher risk of smoking initiation (OR=2.4, 95% CI=1.2-4.5) and smoking persistence (OR=4.0, 95% CI=1.5-10.3) than those who have CYP2A6*4C genotypes. Moreover, the best model involved a gene-gene interaction between MAOA and CYP2A6 was characterized by the multifactor dimensionality reduction method (64.11% accuracy, P<0.001), and indicated that carriers of the combined 1460 T/O genotype for MAOA EcoRV and CYP2A6*1/CYP2A6*1 genotypes were at higher risk of smoking (OR=15.4, 95% CI=4.5-52.5). These findings suggested a substantial influence of CYP2A6 polymorphism as well as the interaction with MAOA resulting in risk modulation on smoking behavior in Chinese male population.

High frequency of DD polymorphism of the angiotensin-converting enzyme gene in Turkish asthmatic patients.

PubMed

Urhan, Meral; Degirmenci, Irfan; Harmanci, Emel; Gunes, Hasan V; Metintas, Muzaffer; Basaran, Ayse

2004-01-01

The aim of this study is to detect the incidence of the angiotensin-converting enzyme (ACE) gene polymorphism in Turkish asthmatic patients and to examine whether there is an association between the disease and ACE gene polymorphism. In our study, the genomic DNA of 100 asthmatic patients and 88 healthy subjects was analyzed Genomic DNA was isolated from peripheral blood by using standard methods. The intron 16 of the ACE gene was amplified by the polymerase chain reaction (PCR) method using primers ACE and ACEX to examine the presence and absence of a 287-base pair (bp) DNA fragment that showed I/D polymorphism genotypes. PCR products were separated by agarose gel electrophoresis and were visualized by a charge-coupled device camera. Serum ACE activities were measured using an ACE kit. The results were evaluated statistically using the chi-square test and one-way analysis of variance. Although the population of patients with asthma was characterized by a higher frequency (30%) of the DD genotype of ACE, they were characterized by lower frequency (48%) of the ID genotype of ACE (DD, 16%, and ID, 64%, in healthy control subjects). The frequency of the I and D alleles of the ACE gene was not significantly different between asthmatic patients (0.46/0.54) and healthy controls (0.52/ 0.48). In addition, in both asthmatic patients and controls, there was a significant decrease of the levels of ACE activity in individuals that have II genotypes when compared with individuals that have DD genotypes. ACE activities were increased significantly in all asthmatic patients (67.20 +/- 1.95 IU/L) compared with all healthy controls (60.90 +/- 2.12 IU/L).

N-acetyltransferase 2 gene polymorphism as a biomarker for susceptibility to bladder cancer in Bangladeshi population.

PubMed

Hosen, Md Bayejid; Islam, Jahidul; Salam, Md Abdus; Islam, Md Fakhrul; Hawlader, M Zakir Hossain; Kabir, Yearul

2015-03-01

To investigate the association between the three most common single nucleotide polymorphisms of the N-acetyltransferase 2 gene together with cigarette smoking and the risk of developing bladder cancer and its aggressiveness. A case-control study on 102 bladder cancer patients and 140 control subjects was conducted. The genomic DNA was extracted from peripheral white blood cells and N-acetyltransferase 2 alleles were differentiated by polymerase chain reaction-based restriction fragment length polymorphism methods. Bladder cancer risk was estimated as odds ratio and 95% confidence interval using binary logistic regression models adjusting for age and gender. Overall, N-acetyltransferase 2 slow genotypes were associated with bladder cancer risk (odds ratio=4.45; 95% confidence interval=2.26-8.77). The cigarette smokers with slow genotypes were found to have a sixfold increased risk to develop bladder cancer (odds ratio=6.05; 95% confidence interval=2.23-15.82). Patients with slow acetylating genotypes were more prone to develop high-grade (odds ratio=6.63; 95% confidence interval=1.15-38.13; P<0.05) and invasive (odds ratio=10.6; 95% confidence interval=1.00-111.5; P=0.05) tumor. N-acetyltransferase 2 slow genotype together with tobacco smoking increases bladder cancer risk. Patients with N-acetyltransferase 2 slow genotypes were more likely to develop a high-grade and invasive tumor. N-acetyltransferase 2 slow genotype is an important genetic determinant for bladder cancer in Bangladesh population. © 2014 Wiley Publishing Asia Pty Ltd.

Human Papilloma Virus Genotype Distribution in Cervical lesions in Zanjan, Iran

PubMed

Ahmadi, Shahrzad; Goudarzi, Hossein; Jalilvand, Ahmad; Esmaeilzadeh, Abdolreza

2017-12-29

Objective: Cervical cancer is one of the most common cancers among women all over the world, and main cause is persistent infection with high risk human papillomavirus (HPV) strains. It has been reported that the distribution and prevalence of HPV types varies by geographical region, so that this is important for prevention by type-specific vaccines. The aim of current study was to determine the genotype distribution of HPV using the INNO-LiPA genotyping assay in Zanjan province, North West Iran. Methods: A total of 112 formalin-fixed paraffin embedded (FFPE) tissue samples from cases of low-grade intraepithelial lesion (LSIL), high-grade intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC) were collected. The polymerase chain reaction (PCR) was used to amplify DNA for genotyping. Results: Among the 112 samples from females (ranging from 20 to 69 years, mean age 43.8 ± 10.1) tested for HPV DNA, 50 samples were positive. Based on results of genotyping, most common HPV genotypes were HPV18 (48%) followed by HPV-6 (24%), HPV73 (16%), HPV-51(8%), HPV-31(8%), HPV-16 (8%), HPV-56 (4%), HPV-44 (4%). Conclusion: While HPV infection is the major etiological factor for cervical cancer, presence was relatively low in our survey. In the positive cases, however, HPV18 was the most common in line with many other populations. The fact that types vary among different populations must clearly be taken into account in design of vaccines for our country. Creative Commons Attribution License

Retreatment of hepatitis C patients with pegylated interferon combined with ribavirin in non-responders to interferon plus ribavirin. Is it different in real life?

PubMed Central

2010-01-01

Background More than 50% of hepatitis C viruses (HCV)-infected patients do not respond to the classical Interferon (IFN)/Ribavirin (RBV) combination therapy. The aim of this study was to evaluate the efficacy of retreatment with Peg-Interferon alpha-2b (PEG-IFN alpha-2b) plus RBV, in patients with HCV, genotypes 1 or 3, who were non-responders to the previous standard treatment with IFN/RBV. Methods In the period 2005-2007, a total of 238 HCV chronic patients were non-responders to previous treatment with IFN plus RBV. Of these 130 agreed to be retreated with PEG-IFN alpha-2b and participated in this evaluation (90 with genotype 1 HCV and 40 with genotype 3 HCV). Patients were retreated at assisted IFN application hubs in compliance with the country's public health system rules. They received subcutaneous PEG-IFN alpha-2b, 1.5 μg, once weekly, associated with RBV, through the oral route, with doses determined according to weight (1,000 mg if weight ≤ 75 kg and 1,250 mg if > 75 kg). Patients with genotype 1 HCV were retreated for over 48 weeks and patients with genotype 3 HCV for over 24 weeks. HCV-RNA was tested by polymerase chain reaction (PCR) at baseline, at week 12, at the end of the treatment, and 6 months thereafter. The predictiveness of week 12 in the development of a sustained virologic response (SVR) was also evaluated. Patients with negative HCV-RNA at week 12 were considered as early virologic responders (EVR). Results EVR was observed in 25% of the patients with genotype 1 HCV and in 64% of the patients genotype 3 HCV (risk = 2.075 and p-value = 0.0414). SVR was observed in 22.2% of the patients with genotype 1 HCV and in 40% with genotype 3 HCV (intention-to-treat analysis). The positive predictive value (PPV) of the HCV-RNA testing at week 12, in order to obtain the SVR, was 65% for genotype 1 and 56% for genotype 3, and the negative predictive value (NPV) was 88% for genotype 1 and 89% for genotype 3. Conclusions PEG-IFN alpha-2b plus weight-based ribavirin is effective in re-treating previous interferon-α plus RBV failure; 22.2% of the patients with genotype 1 HCV and 40% of patients with genotype 3 HCV achieved SVR. PMID:20646277

Assessing and comparison of different machine learning methods in parent-offspring trios for genotype imputation.

PubMed

Mikhchi, Abbas; Honarvar, Mahmood; Kashan, Nasser Emam Jomeh; Aminafshar, Mehdi

2016-06-21

Genotype imputation is an important tool for prediction of unknown genotypes for both unrelated individuals and parent-offspring trios. Several imputation methods are available and can either employ universal machine learning methods, or deploy algorithms dedicated to infer missing genotypes. In this research the performance of eight machine learning methods: Support Vector Machine, K-Nearest Neighbors, Extreme Learning Machine, Radial Basis Function, Random Forest, AdaBoost, LogitBoost, and TotalBoost compared in terms of the imputation accuracy, computation time and the factors affecting imputation accuracy. The methods employed using real and simulated datasets to impute the un-typed SNPs in parent-offspring trios. The tested methods show that imputation of parent-offspring trios can be accurate. The Random Forest and Support Vector Machine were more accurate than the other machine learning methods. The TotalBoost performed slightly worse than the other methods.The running times were different between methods. The ELM was always most fast algorithm. In case of increasing the sample size, the RBF requires long imputation time.The tested methods in this research can be an alternative for imputation of un-typed SNPs in low missing rate of data. However, it is recommended that other machine learning methods to be used for imputation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters.

PubMed

Tamura, Miki; Kawasaki, Hiroko; Sugiyama, Junta

1999-02-01

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.

Stepwise Distributed Open Innovation Contests for Software Development: Acceleration of Genome-Wide Association Analysis

PubMed Central

Hill, Andrew; Loh, Po-Ru; Bharadwaj, Ragu B.; Pons, Pascal; Shang, Jingbo; Guinan, Eva; Lakhani, Karim; Kilty, Iain

2017-01-01

Abstract Background: The association of differing genotypes with disease-related phenotypic traits offers great potential to both help identify new therapeutic targets and support stratification of patients who would gain the greatest benefit from specific drug classes. Development of low-cost genotyping and sequencing has made collecting large-scale genotyping data routine in population and therapeutic intervention studies. In addition, a range of new technologies is being used to capture numerous new and complex phenotypic descriptors. As a result, genotype and phenotype datasets have grown exponentially. Genome-wide association studies associate genotypes and phenotypes using methods such as logistic regression. As existing tools for association analysis limit the efficiency by which value can be extracted from increasing volumes of data, there is a pressing need for new software tools that can accelerate association analyses on large genotype-phenotype datasets. Results: Using open innovation (OI) and contest-based crowdsourcing, the logistic regression analysis in a leading, community-standard genetics software package (PLINK 1.07) was substantially accelerated. OI allowed us to do this in <6 months by providing rapid access to highly skilled programmers with specialized, difficult-to-find skill sets. Through a crowd-based contest a combination of computational, numeric, and algorithmic approaches was identified that accelerated the logistic regression in PLINK 1.07 by 18- to 45-fold. Combining contest-derived logistic regression code with coarse-grained parallelization, multithreading, and associated changes to data initialization code further developed through distributed innovation, we achieved an end-to-end speedup of 591-fold for a data set size of 6678 subjects by 645 863 variants, compared to PLINK 1.07's logistic regression. This represents a reduction in run time from 4.8 hours to 29 seconds. Accelerated logistic regression code developed in this project has been incorporated into the PLINK2 project. Conclusions: Using iterative competition-based OI, we have developed a new, faster implementation of logistic regression for genome-wide association studies analysis. We present lessons learned and recommendations on running a successful OI process for bioinformatics. PMID:28327993

Stepwise Distributed Open Innovation Contests for Software Development: Acceleration of Genome-Wide Association Analysis.

PubMed

Hill, Andrew; Loh, Po-Ru; Bharadwaj, Ragu B; Pons, Pascal; Shang, Jingbo; Guinan, Eva; Lakhani, Karim; Kilty, Iain; Jelinsky, Scott A

2017-05-01

The association of differing genotypes with disease-related phenotypic traits offers great potential to both help identify new therapeutic targets and support stratification of patients who would gain the greatest benefit from specific drug classes. Development of low-cost genotyping and sequencing has made collecting large-scale genotyping data routine in population and therapeutic intervention studies. In addition, a range of new technologies is being used to capture numerous new and complex phenotypic descriptors. As a result, genotype and phenotype datasets have grown exponentially. Genome-wide association studies associate genotypes and phenotypes using methods such as logistic regression. As existing tools for association analysis limit the efficiency by which value can be extracted from increasing volumes of data, there is a pressing need for new software tools that can accelerate association analyses on large genotype-phenotype datasets. Using open innovation (OI) and contest-based crowdsourcing, the logistic regression analysis in a leading, community-standard genetics software package (PLINK 1.07) was substantially accelerated. OI allowed us to do this in <6 months by providing rapid access to highly skilled programmers with specialized, difficult-to-find skill sets. Through a crowd-based contest a combination of computational, numeric, and algorithmic approaches was identified that accelerated the logistic regression in PLINK 1.07 by 18- to 45-fold. Combining contest-derived logistic regression code with coarse-grained parallelization, multithreading, and associated changes to data initialization code further developed through distributed innovation, we achieved an end-to-end speedup of 591-fold for a data set size of 6678 subjects by 645 863 variants, compared to PLINK 1.07's logistic regression. This represents a reduction in run time from 4.8 hours to 29 seconds. Accelerated logistic regression code developed in this project has been incorporated into the PLINK2 project. Using iterative competition-based OI, we have developed a new, faster implementation of logistic regression for genome-wide association studies analysis. We present lessons learned and recommendations on running a successful OI process for bioinformatics. © The Author 2017. Published by Oxford University Press.

Possible Synergistic Interactions Among Multiple HPV Genotypes in Women Suffering from Genital Neoplasia

PubMed

Hajia, Massoud; Sohrabi, Amir

2018-03-27

Objective: Persistence of HPV infection is the true cause of cervical disorders. It is reported that competition may exist among HPV genotypes for colonization. This survey was designed to establish the multiple HPV genotype status in our community and the probability of multiple HPV infections involvement. Methods: All multiple HPV infections were selected for investigation in women suffering from genital infections referred to private laboratories in Tehran, Iran. A total of 160 multi HPV positive specimens from cervical scraping were identified by the HPV genotyping methods, "INNO-LiPA and Geno Array". Result: In present study, HPV 6 (LR), 16 (HR), 53 (pHR), 31 (HR) and 11 (LR) were included in 48.8% of detected infections as the most five dominant genotypes. HPV 16 was detected at the highest rate with genotypes 53, 31 and 52, while HPV 53 appeared linked with HPV 16, 51 and 56 in concurrent infections. It appears that HPV 16 and 53 may have significant tendencies to associate with each other rather than with other genotypes. Analysis of the data revealed there may be some synergistic interactions with a few particular genotypes such as "HPV 53". Conclusion: Multiple HPV genotypes appear more likely to be linked with development of cervical abnormalities especially in patients with genital infections. Since, there are various patterns of dominant HPV genotypes in different regions of world, more investigations of this type should be performed for careHPV programs in individual countries. Creative Commons Attribution License

Cryptosporidium source tracking in the Potomac River watershed.

PubMed

Yang, Wenli; Chen, Plato; Villegas, Eric N; Landy, Ronald B; Kanetsky, Charles; Cama, Vitaliano; Dearen, Theresa; Schultz, Cherie L; Orndorff, Kenneth G; Prelewicz, Gregory J; Brown, Miranda H; Young, Kim Roy; Xiao, Lihua

2008-11-01

To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.

Genotyping the factor VIII intron 22 inversion locus using fluorescent in situ hybridization.

PubMed

Sheen, Campbell R; McDonald, Margaret A; George, Peter M; Smith, Mark P; Morris, Christine M

2011-02-15

The factor VIII intron 22 inversion is the most common cause of hemophilia A, accounting for approximately 40% of all severe cases of the disease. Southern hybridization and multiplex long distance PCR are the most commonly used techniques to detect the inversion in a diagnostic setting, although both have significant limitations. Here we describe our experience establishing a multicolor fluorescent in situ hybridization (FISH) based assay as an alternative to existing methods for genetic diagnosis of the inversion. Our assay was designed to apply three differentially labelled BAC DNA probes that when hybridized to interphase nuclei would exhibit signal patterns that are consistent with the normal or the inversion locus. When the FISH assay was applied to five normal and five inversion male samples, the correct genotype was assignable with p<0.001 for all samples. When applied to carrier female samples the assay could not assign a genotype to all female samples, probably due to a lower proportion of informative nuclei in female samples caused by the added complexity of a second X chromosome. Despite this complication, these pilot findings show that the assay performs favourably compared to the commonly used methods. Copyright © 2010 Elsevier Inc. All rights reserved.

A Unified Framework for Association Analysis with Multiple Related Phenotypes

PubMed Central

Stephens, Matthew

2013-01-01

We consider the problem of assessing associations between multiple related outcome variables, and a single explanatory variable of interest. This problem arises in many settings, including genetic association studies, where the explanatory variable is genotype at a genetic variant. We outline a framework for conducting this type of analysis, based on Bayesian model comparison and model averaging for multivariate regressions. This framework unifies several common approaches to this problem, and includes both standard univariate and standard multivariate association tests as special cases. The framework also unifies the problems of testing for associations and explaining associations – that is, identifying which outcome variables are associated with genotype. This provides an alternative to the usual, but conceptually unsatisfying, approach of resorting to univariate tests when explaining and interpreting significant multivariate findings. The method is computationally tractable genome-wide for modest numbers of phenotypes (e.g. 5–10), and can be applied to summary data, without access to raw genotype and phenotype data. We illustrate the methods on both simulated examples, and to a genome-wide association study of blood lipid traits where we identify 18 potential novel genetic associations that were not identified by univariate analyses of the same data. PMID:23861737

A new method for studying population genetics of cyst nematodes based on Pool-Seq and genomewide allele frequency analysis.

PubMed

Mimee, Benjamin; Duceppe, Marc-Olivier; Véronneau, Pierre-Yves; Lafond-Lapalme, Joël; Jean, Martine; Belzile, François; Bélair, Guy

2015-11-01

Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time-consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping-by-sequencing and Pool-Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well-characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time- and cost-efficient and could be applied to other cyst nematode species. © 2015 Her Majesty the Queen in Right of Canada Molecular Ecology Resources © 2015 John Wiley & Sons Ltd Reproduced with the permission of the Minister of Agriculture and Agri-food.

Marker-assisted selection for recognizing wheat mutant genotypes carrying HMW glutenin alleles related to baking quality.

PubMed

Zamani, Mohammad Javad; Bihamta, Mohammad Reza; Naserian Khiabani, Behnam; Tahernezhad, Zahra; Hallajian, Mohammad Taher; Shamsi, Marzieh Varasteh

2014-01-01

Allelic diversity of HMW glutenin loci in several studies revealed that allelic combinations affect dough quality. Dx5 + Dy10 subunits are related to good baking quality and Dx2 + Dy12 are related to undesirable baking quality. One of the most regular methods to evaluate the baking quality is SDS-PAGE which is used to improve baking quality labs. Marker-assisted selection is the method which can recognize the alleles related to baking quality and this method is based on polymerase chain reaction. 10 pairs of specific primers related to Dx2, Dx2.1, Dx5, Dy10, and Dy12 subunits were used for recognizing baking quality of some wheat varieties and some mutant genotypes. Only 5 pairs of them could show the specific bands. All subunits were recognized by the primers except Dx2.1. Some of the primers were extracted from previous studies and the others were designed based on D genome subunits of wheat. SDS-PAGE method accomplished having confidence in these marker's results. To realize the effect of mutation, seed storage proteins were measured. It showed that mutation had effect on the amount of seed storage protein on the mutant seeds (which showed polymorphism).

Integrated massively parallel sequencing of 15 autosomal STRs and Amelogenin using a simplified library preparation approach.

PubMed

Xue, Jian; Wu, Riga; Pan, Yajiao; Wang, Shunxia; Qu, Baowang; Qin, Ying; Shi, Yuequn; Zhang, Chuchu; Li, Ran; Zhang, Liyan; Zhou, Cheng; Sun, Hongyu

2018-04-02

Massively parallel sequencing (MPS) technologies, also termed as next-generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two-round PCR that requires more steps, making it time-consuming (about 2 days), laborious and expensive. In this study, a 16-plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database-type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS-based STR typing and capillary electrophoresis (CE)-based STR typing. The inconsistency might have been caused by off-ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large-scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation-friendly process flow that saves labor, time and costs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of the LUS in sequence allele designations to facilitate probabilistic genotyping of NGS-based STR typing results.

PubMed

Just, Rebecca S; Irwin, Jodi A

2018-05-01

Some of the expected advantages of next generation sequencing (NGS) for short tandem repeat (STR) typing include enhanced mixture detection and genotype resolution via sequence variation among non-homologous alleles of the same length. However, at the same time that NGS methods for forensic DNA typing have advanced in recent years, many caseworking laboratories have implemented or are transitioning to probabilistic genotyping to assist the interpretation of complex autosomal STR typing results. Current probabilistic software programs are designed for length-based data, and were not intended to accommodate sequence strings as the product input. Yet to leverage the benefits of NGS for enhanced genotyping and mixture deconvolution, the sequence variation among same-length products must be utilized in some form. Here, we propose use of the longest uninterrupted stretch (LUS) in allele designations as a simple method to represent sequence variation within the STR repeat regions and facilitate - in the nearterm - probabilistic interpretation of NGS-based typing results. An examination of published population data indicated that a reference LUS region is straightforward to define for most autosomal STR loci, and that using repeat unit plus LUS length as the allele designator can represent greater than 80% of the alleles detected by sequencing. A proof of concept study performed using a freely available probabilistic software demonstrated that the LUS length can be used in allele designations when a program does not require alleles to be integers, and that utilizing sequence information improves interpretation of both single-source and mixed contributor STR typing results as compared to using repeat unit information alone. The LUS concept for allele designation maintains the repeat-based allele nomenclature that will permit backward compatibility to extant STR databases, and the LUS lengths themselves will be concordant regardless of the NGS assay or analysis tools employed. Further, these biologically based, easy-to-derive designations uphold clear relationships between parent alleles and their stutter products, enabling analysis in fully continuous probabilistic programs that model stutter while avoiding the algorithmic complexities that come with string based searches. Though using repeat unit plus LUS length as the allele designator does not capture variation that occurs outside of the core repeat regions, this straightforward approach would permit the large majority of known STR sequence variation to be used for mixture deconvolution and, in turn, result in more informative mixture statistics in the near term. Ultimately, the method could bridge the gap from current length-based probabilistic systems to facilitate broader adoption of NGS by forensic DNA testing laboratories. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Diversity of Helicobacter pylori genotypes in Iranian patients with different gastroduodenal disorders

PubMed Central

Vaziri, Farzam; Najar Peerayeh, Shahin; Alebouyeh, Masoud; Mirzaei, Tabassom; Yamaoka, Yoshio; Molaei, Mahsa; Maghsoudi, Nader; Zali, Mohammad Reza

2013-01-01

AIM: To investigate the diversity of Helicobacter pylori (H. pylori) genotypes and correlations with disease outcomes in an Iranian population with different gastroduodenal disorders. METHODS: Isolates of H. pylori from patients with different gastroduodenal disorders were analyzed after culture and identification by phenotypic and genotypic methods. Genomic DNA was extracted with the QIAamp DNA mini kit (Qiagen, Germany). After DNA extraction, genotyping was done for cagA, vacA (s and m regions), iceA (iceA1, iceA2) and babA with specific primers for each allele using polymerase chain reaction (PCR). All patients’ pathologic and clinical data and their relation with known genotypes were analyzed by using SPSS version 19.0 software. χ2 test and Fisher’s exact test were used to assess relationships between categorical variables. The level of statistical significance was set at P < 0.05. RESULTS: A total of 71 isolates from 177 patients with different gastroduodenal disorders were obtained. Based on analysis of the cagA gene (positive or negative), vacA s-region (s1 or s2), vacA m-region (m1 or m2), iceA allelic type (iceA1 and iceA2) and babA gene (positive or negative), twenty different genotypic combinations were recognized. The prevalence of cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, iceA1+iceA2 and babA were 62%, 78.9%, 19.7%, 21.1%, 78.9%, 15.5%, 22.5%, 40.8% and 95.8%, respectively. Interestingly, evaluation of PCR results for cagA in 6 patients showed simultaneous existence of cagA variants according to their size diversities that proposed mixed infection in these patients. The most prevalent genotype in cagA-positive isolates was cagA+/vacAs1m2/iceA1+A2/babA+ and in cagA-negative isolates was cagA-/vacAs1m2/iceA-/babA+. There were no relationships between the studied genes and histopathological findings (H. pylori density, neutrophil activity, lymphoid aggregation in lamina propria and glandular atrophy). The strains which carry cagA, vacAs1/m1, iceA2 and babA genes showed significant associations with severe active chronic gastritis (P = 0.011, 0.025, 0.020 and 0.031, respectively). The vacAs1 genotype had significant correlation with the presence of the cagA gene (P = 0.013). Also, babA genotype showed associations with cagA (P = 0.024). In the combined genotypes, only cagA+/vacAs1m1/iceA2/babA+ genotype showed correlation with severe active chronic gastritis (P = 0.025). CONCLUSION: This genotyping panel can be a useful tool for detection of virulent H. pylori isolates and can provide valuable guidance for prediction of the clinical outcomes. PMID:24039362

eCOMPAGT integrates mtDNA: import, validation and export of mitochondrial DNA profiles for population genetics, tumour dynamics and genotype-phenotype association studies.

PubMed

Weissensteiner, Hansi; Schönherr, Sebastian; Specht, Günther; Kronenberg, Florian; Brandstätter, Anita

2010-03-09

Mitochondrial DNA (mtDNA) is widely being used for population genetics, forensic DNA fingerprinting and clinical disease association studies. The recent past has uncovered severe problems with mtDNA genotyping, not only due to the genotyping method itself, but mainly to the post-lab transcription, storage and report of mtDNA genotypes. eCOMPAGT, a system to store, administer and connect phenotype data to all kinds of genotype data is now enhanced by the possibility of storing mtDNA profiles and allowing their validation, linking to phenotypes and export as numerous formats. mtDNA profiles can be imported from different sequence evaluation programs, compared between evaluations and their haplogroup affiliations stored. Furthermore, eCOMPAGT has been improved in its sophisticated transparency (support of MySQL and Oracle), security aspects (by using database technology) and the option to import, manage and store genotypes derived from various genotyping methods (SNPlex, TaqMan, and STRs). It is a software solution designed for project management, laboratory work and the evaluation process all-in-one. The extended mtDNA version of eCOMPAGT was designed to enable error-free post-laboratory data handling of human mtDNA profiles. This software is suited for small to medium-sized human genetic, forensic and clinical genetic laboratories. The direct support of MySQL and the improved database security options render eCOMPAGT a powerful tool to build an automated workflow architecture for several genotyping methods. eCOMPAGT is freely available at http://dbis-informatik.uibk.ac.at/ecompagt.

eCOMPAGT integrates mtDNA: import, validation and export of mitochondrial DNA profiles for population genetics, tumour dynamics and genotype-phenotype association studies

PubMed Central

2010-01-01

Background Mitochondrial DNA (mtDNA) is widely being used for population genetics, forensic DNA fingerprinting and clinical disease association studies. The recent past has uncovered severe problems with mtDNA genotyping, not only due to the genotyping method itself, but mainly to the post-lab transcription, storage and report of mtDNA genotypes. Description eCOMPAGT, a system to store, administer and connect phenotype data to all kinds of genotype data is now enhanced by the possibility of storing mtDNA profiles and allowing their validation, linking to phenotypes and export as numerous formats. mtDNA profiles can be imported from different sequence evaluation programs, compared between evaluations and their haplogroup affiliations stored. Furthermore, eCOMPAGT has been improved in its sophisticated transparency (support of MySQL and Oracle), security aspects (by using database technology) and the option to import, manage and store genotypes derived from various genotyping methods (SNPlex, TaqMan, and STRs). It is a software solution designed for project management, laboratory work and the evaluation process all-in-one. Conclusions The extended mtDNA version of eCOMPAGT was designed to enable error-free post-laboratory data handling of human mtDNA profiles. This software is suited for small to medium-sized human genetic, forensic and clinical genetic laboratories. The direct support of MySQL and the improved database security options render eCOMPAGT a powerful tool to build an automated workflow architecture for several genotyping methods. eCOMPAGT is freely available at http://dbis-informatik.uibk.ac.at/ecompagt. PMID:20214782

Interaction of ACE genotype and salt intake on hypertension among Chinese Kazakhs: results from a population-based cross-sectional study.

PubMed

Wang, Yuyan; Zhang, Biao; Hou, Lei; Han, Wei; Xue, Fang; Wang, Yanhong; Tang, Yong; Liang, Shaohua; Wang, Weizhi; Asaiti, Kuliqian; Wang, Zixing; Hu, Yaoda; Wang, Lei; Qiu, Changchun; Zhang, Mingtao; Jiang, Jingmei

2017-05-17

To explore the effect of interaction between ACE genotype and salt intake on hypertension among Chinese Kazakhs, and to compare applications of interactions between logistic model and generalised partially linear tree-based regression (GPLTR) model. Population-based cross-sectional study. Hong Dun, North Xinjiang, China. Non-consanguineous Chinese Kazakh participants (n=916, 342 men and 574 women) aged ≥30 years. Association between ACE genotype and hypertension, association between salt intake and hypertension, and interaction of ACE genotype and salt intake on hypertension in two models. Associations between salt intake and hypertension were different in ACE genotype of II and ID+DD. Under the logistic models, main and interaction effects were not observed for men, but effects were present in opposite directions for women (main effect of ACE: OR=0.20, p=0.003; interaction effect: OR=1.07, p=0.027). Under the GPLTR model, Bayesian information criterion trees included both salt intake and ACE genotype as split variables. Individuals with a salt intake ≥19.5 g/day and ID+DD genotypes had a 3.99-fold (p=0.004) higher risk of hypertension compared with the II genotype for men, whereas salt intake <20.1 g/day and ID+DD genotypes had an OR=0.55 (p=0.014) compared with the II genotype for women. An interaction of ACE genotype and salt intake on hypertension was observed among Chinese Kazakhs but in different ways according to sex. The GPLTR model appears to be more suitable for an exploration of interactions in complex diseases. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Genotype imputation efficiency in Nelore Cattle

USDA-ARS?s Scientific Manuscript database

Genotype imputation efficiency in Nelore cattle was evaluated in different scenarios of lower density (LD) chips, imputation methods and sets of animals to have their genotypes imputed. Twelve commercial and virtual custom LD chips with densities varying from 7K to 75K SNPs were tested. Customized L...

Flavonoid content and antioxidant capacity of spinach genotypes determined by high-performance liquid chromatography/mass spectrometry

USDA-ARS?s Scientific Manuscript database

Flavonoids in different spinach genotypes were separated, identified, and quantified by a high-performance liquid chromatographic method with photodiode array and mass spectrometric detection. The antioxidant capacities of the genotypes were also measured using two antioxidant assays - oxygen radica...

First-line eradication for Helicobacter pylori-positive gastritis by esomeprazole-based triple therapy is influenced by CYP2C19 genotype

PubMed Central

Saito, Yoshimasa; Serizawa, Hiroshi; Kato, Yukako; Nakano, Masaru; Nakamura, Masahiko; Saito, Hidetsugu; Suzuki, Hidekazu; Kanai, Takanori

2015-01-01

AIM: To evaluate the effect of first line esomeprazole (EPZ)-based triple therapy on Helicobacter pylori (H. pylori) eradication. METHODS: A total of 80 Japanese patients with gastritis who were diagnosed as positive for H. pylori infection by endoscopic biopsy-based or 13C-urea breath tests were included in this study. The average age of the patients was 57.2 years (male/female, 42/38). These patients were treated by first-line eradication therapy with EPZ 40 mg/d, amoxicillin 1500 mg/d, and clarithromycin 400 mg/d for 7 d. All drugs were given twice per day. Correlations between H. pylori eradication, CYP2C19 genotype, and serum pepsinogen (PG) level were analyzed. This study was registered with the UMIN Clinical Trials Registry (UMIN000009642). RESULTS: The H. pylori eradication rates by EPZ-based triple therapy evaluated by intention-to-treat and per protocol were 67.5% and 68.4%, respectively, which were similar to triple therapies with other first-generation proton pump inhibitors (PPIs). The eradication rates in three different CYP2C19 genotypes, described as extensive metabolizer (EM), intermediate metabolizer, and poor metabolizer, were 52.2%, 72.1%, and 84.6%, respectively. The H. pylori eradication rate was significantly lower in EM than non-EM (P < 0.05). The serum PG I level and PG I/II ratio were significantly increased after eradication of H. pylori (P < 0.01), suggesting that gastric atrophy was improved by H. pylori eradication. Thus, first-line eradication by EPZ-based triple therapy for patients with H. pylori-positive gastritis was influenced by CYP2C19 genotype, and the eradication rate was on the same level with other first-generation PPIs in the Japanese population. CONCLUSION: The results from this study suggest that there is no advantage to EPZ-based triple therapy on H. pylori eradication compared to other first-generation PPIs. PMID:26730167

RT-PCR for detection of all seven genotypes of Lyssavirus genus.

PubMed

Vázquez-Morón, S; Avellón, A; Echevarría, J E

2006-08-01

The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

Impact of 6-month frozen storage of cervical specimens in alkaline buffer conditions on human papillomavirus genotyping.

PubMed

LaMere, Brandon J; Howell, Renee; Fetterman, Barbara; Shieh, Jen; Castle, Philip E

2008-08-01

The impact of 6-month storage of cervical specimens under alkaline conditions that occurs as the result of Hybrid Capture 2 testing on human papillomavirus (HPV) genotyping is not well documented. To examine this issue, 143 frozen hc2-positive specimens in specimen transport medium were selected at random from each of the following groups: specimens stored for 6 months, 4 months, and 2.5 months under alkaline pH (pH 12-13) and specimens stored 1 month at neutral pH (pH 6-7) as controls. Specimens were tested in a masked fashion for 20 HPV genotypes (HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) using a prototype, research-use-only GP5+/6+ L1 consensus PCR method and multiplex hybridization using Luminex xMAP for detection of specific HPV genotypes One control specimen had missing test results. There were no statistical differences in the number of HPV genotypes detected, number of carcinogenic HPV genotypes detected, or in the signal strength among HPV-positive results across groups. Six-month frozen storage of cervical specimens at alkaline pH had little impact on testing for HPV genotypes among hc2-positive women using this HPV genotyping method.

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA).

PubMed

Zeng, Weiwei; Yao, Wei; Wang, Yingying; Li, Yingying; Bermann, Sven M; Ren, Yan; Shi, Cunbin; Song, Xinjian; Huang, Qiwen; Zheng, Shuchen; Wang, Qing

2017-05-01

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture. Copyright © 2017 Elsevier B.V. All rights reserved.

Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and the risk of primary Hepatocellular Carcinoma (HCC) in a Chinese population

PubMed Central

Cao, Wei; Zhang, Zuo-Feng; Cai, Lin; Jiang, Qing-Wu; You, Nai-Chieh; Goldstein, Binh Yang; Wei, Guo-Rong; Chen, Chuan-Wei; Lu, Qing-Yi; Zhou, Xue-Fu; Ding, Bao-Guo; Chang, Jun; Yu, Shun-Zhang

2014-01-01

Objectives Methylenetetrahydrofolate reductase (MTHFR), which is expressed in the liver, may be involved in both DNA methylation and DNA synthesis. It is also indicated as a potential risk factor of liver cancer in patients with chronic liver disease. To date, no study has been conducted on MTHFR and hepatocellular carcinoma (HCC) using a population-based design. The objective of this study was to evaluate the effects of polymorphisms of the MTHFR gene on the risk of primary liver cancer and their possible effect modifications on various environmental risk factors. Methods A population-based case–control study was conducted in Taixing, China. MTHFR C677T and A1298C were assayed by PCR-RFLP techniques. Results The frequency of MTHFR 677 C/C wild homo-zygotes genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratios (ORs) for the MTHFR 677 C/T and T/T genotype were 1.66(95% CI: 1.06–2.61), 1.21(95% CI: 0.65–2.28) respectively when compared with the MTHFR 677 C/C genotype. Subjects carrying any T genotype have the increased risk of 1.55(95% CI: 1.01–2.40) for development of primary hepatocellular carcinoma. A high degree of linkage disequilibrium was observed between the C677T and A1298C polymorphisms, with the D′ of 0.887 and p < 0.01. The MTHFR 677 any T genotype was suggested to have potentially more than multiplicative interactions with raw water drinking with p-value for adjusted interaction of 0.03. Conclusion We observed that the MTHFR 677 C/T genotype was associated with an increased risk of primary liver cancer in a Chinese population. The polymorphism of MTHFR 677 might modify the effects of raw water drinking on the risk of primary hepatocellular carcinoma. PMID:17503006

Biotinidase deficiency: Genotype-biochemical phenotype association in Brazilian patients

PubMed Central

Borsatto, Taciane; Sperb-Ludwig, Fernanda; Lima, Samyra E.; S. Carvalho, Maria R.; S. Fonseca, Pablo A.; S. Camelo, José; M. Ribeiro, Erlane; F. V. de Medeiros, Paula; M. Lourenço, Charles; F. M. de Souza, Carolina; Boy, Raquel; Félix, Têmis M.; M. Bittar, Camila; L. C. Pinto, Louise; C. Neto, Eurico; J. Blom, Henk; D. Schwartz, Ida V.

2017-01-01

Introduction The association between the BTD genotype and biochemical phenotype [profound biotinidase deficiency (BD), partial BD or heterozygous activity] is not always consistent. This study aimed to investigate the genotype-biochemical phenotype association in patients with low biotinidase activity. Methods All exons, the 5'UTR and the promoter of the BTD gene were sequenced in 72 Brazilian individuals who exhibited low biotinidase activity. For each patient, the expected biochemical phenotype based on the known genotype was compared with the observed biochemical phenotype. Additional non-genetic factors that could affect the biotinidase activity were also analysed. Results Most individuals were identified by neonatal screening (n = 66/72). When consecutive results for the same patient were compared, age, prematurity and neonatal jaundice appeared to affect the level of biotinidase activity. The biochemical phenotype at the time of the second blood collection changed in 11/22 patients compared to results from the first sample. Three novel variants were found: c.1337T>C (p.L446P), c.1466A>G (p.N489S) and c.962G>A (p.W321*). Some patients with the same genotype presented different biochemical phenotypes. The expected and observed biochemical phenotypes agreed in 68.5% of cases (concordant patients). The non-coding variants c.-183G>A, c.-315A>G and c.-514C>T were present in heterozygosis in 5/17 discordant patients. In addition, c.-183G>A and c.-514C>T were also present in 10/37 concordant patients. Conclusions The variants found in the promoter region do not appear to have a strong impact on biotinidase activity. Since there is a disparity between the BTD genotype and biochemical phenotype, and biotinidase activity may be affected by both genetic and non-genetic factors, we suggest that the diagnosis of BD should be based on more than one measurement of plasma biotinidase activity. DNA analysis can be of additional relevance to differentiate between partial BD and heterozygosity. PMID:28498829

Options in human papillomavirus (HPV) detection for cervical cancer screening: comparison between full genotyping and a rapid qualitative HPV-DNA assay in Ghana.

PubMed

Obiri-Yeboah, Dorcas; Adu-Sarkodie, Yaw; Djigma, Florencia; Akakpo, Kafui; Aniakwa-Bonsu, Ebenezer; Amoako-Sakyi, Daniel; Jacques, Simpore; Mayaud, Philippe

2017-01-01

Modern cervical cancer screening increasingly relies on the use of molecular techniques detecting high-risk oncogenic human papillomavirus (hr-HPV). A major challenge for developing countries like Ghana has been the unavailability and costs of HPV DNA-based testing. This study compares the performance of care HPV, a semi-rapid and affordable qualitative detection assay for 14 hr-HPV genotypes, with HPV genotyping, for the detection of cytological cervical squamous intraepithelial lesions (SIL). A study comparing between frequency matched HIV-1 seropositive and HIV-seronegative women was conducted in the Cape Coast Teaching Hospital, Ghana. A systematic sampling method was used to select women attending clinics in the hospital. Cervical samples were tested for HPV by care HPV and Anyplex-II HPV28 genotyping assay, and by conventional cytology. A total of 175 paired results (94 from HIV-1 seropositive and 81 from HIV-seronegative women) were analyzed based on the ability of both tests to detect the 14 hr-HPV types included in the care HPV assay. The inter-assay concordance was 94.3% (95%CI: 89.7-97.2%, kappa = 0.88), similar by HIV serostatus. The care HPV assay was equally sensitive among HIV-1 seropositive and seronegative women (97.3% vs. 95.7%, p  = 0.50) and slightly more specific among HIV-seronegative women (85.0% vs. 93.1%, p  = 0.10). care HPV had good sensitivity (87.5%) but low specificity (52.1%) for the detection of low SIL or greater lesions, but its performance was superior to genotyping (87.5 and 38.8%, respectively). Reproducibility of care HPV, tested on 97 samples by the same individual was 82.5% (95%CI: 73.4-89.4%). The performance characteristics of care HPV compared to genotyping suggest that this simpler and cheaper HPV detection assay could offer a suitable alternative for HPV screening in Ghana.

Drug-Induced Gingival Overgrowth: The Genetic Dimension

PubMed Central

Charles, Noronha Shyam Curtis; Chavan, Rahul; Moon, Ninad Joshirao; Nalla, Srinivas; Mali, Jaydeepchandra; Prajapati, Anchal

2014-01-01

Background: Currently, the etiology of drug-induced gingival overgrowth is not entirely understood but is clearly multifactorial. Phenytoin, one of the common drugs implicated in gingival enlargement, is metabolized mainly by cytochrome P450 (CYP)2C9 and partly by CYP2C19. The CYP2C9 and CYP2C19 genes are polymorphically expressed and most of the variants result in decreased metabolism of the respective substrates. Aims: The present study was undertaken to investigate the influence of the CYP2C9*2 and *3 variant genotypes on phenytoin hydroxylation in subjects diagnosed with epilepsy from South India, thus establishing the genetic polymorphisms leading to its defective hydroxylation process. Materials and Methods: Fifteen epileptic subjects, age 9 to 60 years were included in the study. Among the study subjects, 8 were males and 7 were females. Genomic DNA was extracted from patients’ blood using Phenol-chloroform method and genotyping was done for CYP2C9 using customized TaqMan genotyping assays on a real time thermocycler, by allelic discrimination method. The genetic polymorphisms *1, *2 and *3 on CYP2C9 were selected based on their function and respective allele frequencies in Asian subcontinent among the Asian populations. Results: CYP2C9*1*2 and CYP2C9*3/*3 were identified with equal frequency in the study population. There were seven subjects with CYP2C9*1/*2 genotype (heterozygous mutant), one subject with CYP2C9*1/*1 (wild type) and seven study subjects with CYP2C9*3/*3 (homozygous mutant). Conclusion: The results obtained in the present study will be helpful in the medical prescription purposes of phenytoin, and a more personalized patient approach with its administration can be advocated. PMID:25317394

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

PubMed Central

Akimoto, Chizuru; Volk, Alexander E; van Blitterswijk, Marka; Van den Broeck, Marleen; Leblond, Claire S; Lumbroso, Serge; Camu, William; Neitzel, Birgit; Onodera, Osamu; van Rheenen, Wouter; Pinto, Susana; Weber, Markus; Smith, Bradley; Proven, Melanie; Talbot, Kevin; Keagle, Pamela; Chesi, Alessandra; Ratti, Antonia; van der Zee, Julie; Alstermark, Helena; Birve, Anna; Calini, Daniela; Nordin, Angelica; Tradowsky, Daniela C; Just, Walter; Daoud, Hussein; Angerbauer, Sabrina; DeJesus-Hernandez, Mariely; Konno, Takuya; Lloyd-Jani, Anjali; de Carvalho, Mamede; Mouzat, Kevin; Landers, John E; Veldink, Jan H; Silani, Vincenzo; Gitler, Aaron D; Shaw, Christopher E; Rouleau, Guy A; van den Berg, Leonard H; Van Broeckhoven, Christine; Rademakers, Rosa; Andersen, Peter M; Kubisch, Christian

2014-01-01

Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting. PMID:24706941

Molecular genotyping of Indian blood group system antigens in Indian blood donors.

PubMed

Gogri, Harita; Pitale, Pranali; Madkaikar, Manisha; Kulkarni, Swati

2018-04-11

Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, In a and In b . The In a /In b polymorphism arises from 252C > G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles. Blood samples were collected from 715 blood donors from Mumbai. DNA was extracted using phenol-chloroform method and genotyping for Indian (In a /IN*01, In b /IN*02) blood group alleles was done by Sequence Specific PCR. Seventeen donors among 715 were heterozygous for In a antigen i.e. In (a+b+). The In a antigen positivity was confirmed serologically, using anti-In a prepared in-house and the genotype-phenotype results were concordant. The frequency of In a (2.37%) was higher than Caucasians and comparable to those reported among Indians of Bombay. This is the first study reporting molecular screening of Indian blood group antigens in Indian population. The frequency of In a and In b antigens was found to be 2.37% and 100% respectively. Red cells of In a positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibodies. Thus, DNA based methods will help in large scale screening of donors to identify rare blood groups, when commercial antisera are unavailable. Copyright © 2018 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chun-Chieh; Department of Medical Imaging and Radiological Science, Chang Gung University School of Medicine, Taoyuan, Taiwan; Lai, Chyong-Huey

Purpose: To study the prognostic value of the human papillomavirus (HPV) genotypes in cervical cancer patients undergoing radiotherapy. Patients and Methods: A total of 1,010 patients with cervical cancer after radiotherapy between 1993 and 2000 were eligible for this study. The HPV genotypes were determined by a genechip, which detects 38 types of HPV. The patient characteristics and treatment outcomes were analyzed using the Cox regression hazard model and classification and regression tree decision tree method. Results: A total of 25 genotypes of HPV were detected in 992 specimens (98.2%). The leading 8 types were HPV16, 58, 18, 33, 52,more » 39, 31, and 45. These types belong to two high-risk HPV species: alpha-7 (HPV18, 39, 45) and alpha-9 (HPV16, 31, 33, 52, 58). Three HPV-based risk groups, which were independent of established prognostic factors, such as International Federation of Gynecology and Obstetrics stage, age, pathologic features, squamous cell carcinoma antigen, and lymph node metastasis, were associated with the survival outcomes. The high-risk group consisted of the patients without HPV infection or the ones infected with the alpha-7 species only. Patients co-infected with the alpha-7 and alpha-9 species belonged to the medium-risk group, and the others were included in the low-risk group. Conclusion: The results of the present study have confirmed the prognostic value of HPV genotypes in cervical cancer treated with radiotherapy. The different effect of the alpha-7 and alpha-9 species on the radiation response deserves additional exploration.« less

Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-05-01

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

Evaluation of redundancy analysis to identify signatures of local adaptation.

PubMed

Capblancq, Thibaut; Luu, Keurcien; Blum, Michael G B; Bazin, Eric

2018-05-26

Ordination is a common tool in ecology that aims at representing complex biological information in a reduced space. In landscape genetics, ordination methods such as principal component analysis (PCA) have been used to detect adaptive variation based on genomic data. Taking advantage of environmental data in addition to genotype data, redundancy analysis (RDA) is another ordination approach that is useful to detect adaptive variation. This paper aims at proposing a test statistic based on RDA to search for loci under selection. We compare redundancy analysis to pcadapt, which is a nonconstrained ordination method, and to a latent factor mixed model (LFMM), which is a univariate genotype-environment association method. Individual-based simulations identify evolutionary scenarios where RDA genome scans have a greater statistical power than genome scans based on PCA. By constraining the analysis with environmental variables, RDA performs better than PCA in identifying adaptive variation when selection gradients are weakly correlated with population structure. Additionally, we show that if RDA and LFMM have a similar power to identify genetic markers associated with environmental variables, the RDA-based procedure has the advantage to identify the main selective gradients as a combination of environmental variables. To give a concrete illustration of RDA in population genomics, we apply this method to the detection of outliers and selective gradients on an SNP data set of Populus trichocarpa (Geraldes et al., 2013). The RDA-based approach identifies the main selective gradient contrasting southern and coastal populations to northern and continental populations in the northwestern American coast. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Comparative genome-wide mapping versus extreme pool-genotyping and development of diagnostic SNP markers linked to QTL for adult plant resistance to stripe rust in common wheat.

PubMed

Wu, Jianhui; Huang, Shuo; Zeng, Qingdong; Liu, Shengjie; Wang, Qilin; Mu, Jingmei; Yu, Shizhou; Han, Dejun; Kang, Zhensheng

2018-06-16

A major stripe rust resistance QTL on chromosome 4BL was localized to a 4.5-Mb interval using comparative QTL mapping methods and validated in 276 wheat genotypes by haplotype analysis. CYMMIT-derived wheat line P10103 was previously identified to have adult plant resistance (APR) to stripe rust in the greenhouse and field. The conventional approach for QTL mapping in common wheat is laborious. Here, we performed QTL detection of APR using a combination of genome-wide scanning and extreme pool-genotyping. SNP-based genetic maps were constructed using the Wheat55 K SNP array to genotype a recombinant inbred line (RIL) population derived from the cross Mingxian 169 × P10103. Five stable QTL were detected across multiple environments. A fter comparing SNP profiles from contrasting, extreme DNA pools of RILs six putative QTL were located to approximate chromosome positions. A major QTL on chromosome 4B was identified in F 2:4 contrasting pools from cross Zhengmai 9023 × P10103. A consensus QTL (LOD = 26-40, PVE = 42-55%), named QYr.nwafu-4BL, was defined and localized to a 4.5-Mb interval flanked by SNP markers AX-110963704 and AX-110519862 in chromosome arm 4BL. Based on stripe rust response, marker genotypes, pedigree analysis and mapping data, QYr.nwafu-4BL is likely to be a new APR QTL. The applicability of the SNP-based markers flanking QYr.nwafu-4BL was validated on a diversity panel of 276 wheat lines. The additional minor QTL on chromosomes 4A, 5A, 5B and 6A enhanced the level of resistance conferred by QYr.nwafu-4BL. Marker-assisted pyramiding of QYr.nwafu-4BL and other favorable minor QTL in new wheat cultivars should improve the level of APR to stripe rust.

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

PubMed

Dündar Yenilmez, Ebru; Kökbaş, Umut; Kartlaşmış, Kezban; Kayrın, Levent; Tuli, Abdullah

2018-01-01

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility.

Spatial overlap links seemingly unconnected genotype-matched TB cases in rural Uganda

PubMed Central

Kato-Maeda, Midori; Emperador, Devy M.; Wandera, Bonnie; Mugagga, Olive; Crandall, John; Janes, Michael; Marquez, Carina; Kamya, Moses R.; Charlebois, Edwin D.; Havlir, Diane V.

2018-01-01

Introduction Incomplete understanding of TB transmission dynamics in high HIV prevalence settings remains an obstacle for prevention. Understanding where transmission occurs could provide a platform for case finding and interrupting transmission. Methods From 2012–2015, we sought to recruit all adults starting TB treatment in a Ugandan community. Participants underwent household (HH) contact investigation, and provided names of social contacts, sites of work, healthcare and socializing, and two sputum samples. Mycobacterium tuberculosis culture-positive specimens underwent 24-loci MIRU-VNTR and spoligotyping. We sought to identify epidemiologic links between genotype-matched cases by analyzing social networks and mapping locations where cases reported spending ≥12 hours over the one-month pre-treatment. Sites of spatial overlap (≤100m) between genotype-matched cases were considered potential transmission sites. We analyzed social networks stratified by genotype clustering status, with cases linked by shared locations, and compared network density by location type between clustered vs. non-clustered cases. Results Of 173 adults with TB, 131 (76%) were enrolled, 108 provided sputum, and 84/131 (78%) were MTB culture-positive: 52% (66/131) tested HIV-positive. Of 118 adult HH contacts, 105 (89%) were screened and 3 (2.5%) diagnosed with active TB. Overall, 33 TB cases (39%) belonged to 15 distinct MTB genotype-matched clusters. Within each cluster, no cases shared a HH or reported shared non-HH contacts. In 6/15 (40%) clusters, potential epidemiologic links were identified by spatial overlap at specific locations: 5/6 involved health care settings. Genotype-clustered TB social networks had significantly greater network density based on shared clinics (p<0.001) and decreased density based on shared marketplaces (p<0.001), compared to non-clustered networks. Conclusions In this molecular epidemiologic study, links between MTB genotype-matched cases were only identifiable via shared locations, healthcare locations in particular, rather than named contacts. This suggests most transmission is occurring between casual contacts, and emphasizes the need for improved infection control in healthcare settings in rural Africa. PMID:29438413

Cytochrome p450 2E1 polymorphisms and the risk of gastric cardia cancer

PubMed Central

Cai, Lin; Zheng, Zong-Li; Zhang, Zuo-Feng

2005-01-01

AIM: Genetic polymorphisms of drug-metabolizing enzymes have recently been shown to affect susceptibility to chemical carcinogenesis. Cytochrome P450 2E1 (CYP2E1) enzyme catalyzes the metabolism of many procarcinogens, such as N-nitrosamines and related compounds. The gene coding for this enzyme is polymorphic and thus may play a role in gastric cardia cancer (GCC) etiology. In this hospital-based case-control study, we evaluate the relationship between genetic polymorphisms of CYP2E1 and the risk of GCC. METHODS: The study subjects comprised 159 histologically confirmed GCC cases identified via hospital cancer registry and surgical records at five hospitals in Fuzhou, Fujian Province, China, between April and November 2001. Controls were 192 patients admitted to the same hospitals for nonmalignant conditions. The genotypes of CYP2E1 were detected by a PCR-based RFLP assay. The odds ratios were estimated by logistic regression analyses and were adjusted for potential confounding factors. RESULTS: The distribution of three genotypes of CYP2E1 in GCC cases and controls was significantly different (χ2 = 16.04, P<0.01). The frequency of the CYP2E1 (c1/c1) genotype in GCC cases and controls was 60.4% and 40.1%, respectively. The CYP2E1 (c1/c1) genotype was associated with an increased risk for GCC (the adjusted (OR) was 2.37, 95% confidence interval (CI): 1.52-3.70). Subjects who carried the CYP2E1 (c1/c1) genotype and were habitual smokers were at a significantly higher risk of developing GCC (OR = 4.68, 95%CI: 2.19-10.04) compared with those who had the CYP2E1 (c1/c2 or c2/c2) genotype and did not smoke. CONCLUSION: These results suggest that the CYP2E1 genotype may influence individual susceptibility to development of GCC, and that the risk increases significantly in smokers. PMID:15793883

[Distribution of hepatitis C virus genotypes among patients with chronic hepatitis C infection in Akdeniz University Hospital, Antalya, Turkey: a five-year evaluation].

PubMed

Sağlik, İmran; Mutlu, Derya; Öngut, Gözde; İnan, Dilara; Öğünç, Dilara; Can Sarinoğlu, Rabia; Özhak Baysan, Betil; Gültekin, Meral; Çolak, Dilek

2014-07-01

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis. It is important to know the genotypes of HCV in the decision of the HCV related chronic hepatitis therapy. The aim of this study was to evaluate the HCV genotypes determined at the Microbiology Laboratory of Akdeniz University Hospital, and to evaluate the changes in the distribution of the genotypes within the last five years. A total of 422 blood samples from HCV-RNA positive chronic hepatitis C patients (219 male, 203 female; age range: 8-79 yrs, mean age 46.3 ± 15.5 yrs) which were sent to our laboratory for genotyping between 2009-2013 period, were analyzed retrospectively. HCV-RNA extractions were performed in an automated system (EZ1 Virus Mini Kit v2.0, Qiagen, Germany), and a commercial reverse hybridization line probe-based assay (LIPA; GEN-C RT-PCR, Italy) was carried out for genotyping, For viral load determinations, a real-time PCR method (Cobas TaqMan HCV, Roche Diagnostics, Germany) was used. Demographic data of the patients were obtained from the hospital information systems and electronic patients' files. Out of the 422 patients, genotype 1b was detected in 63.3% (n= 267), genotype 1a in 14.7% (n= 62), genotype 3a in 11.1% (n= 47), genotype 2b in 0.9% (n= 4), genotype 4e in 0.2% (n= 1). The subtypes couldn't be determined for 5.4% (n= 23), 2.6% (n= 11) and 1.4% (n= 6) of the patients infected with genotype 1, 2 and 4, respectively. One (0.2%) patient, was coinfected with genotype 1 and 4. Of the patients, 40 were foreign-born (16 cases from Russia; 4 of each from Ukraine and Georgia; 3 of each from Turkmenistan, Kyrgyzstan, and Germany; one of each from Tajikistan, Azerbaijan, Uzbekistan, Chechnya, Moldova, Switzerland and Romania) and among these patients genotype 3a (19/40; 47.5%) was the most common genotype followed by genotype 1b (17/40; 42.5%). Median values of HCV viral load were 668.500 IU/ml (range: 2.000-9.630.000) in the whole group; while it was 732.000 IU/ml (range: 2.000-9.630.000) in patients infected with genotype 1 and 444.000 IU/ml (range: 2.650- 8.330.000) in patients infected with the other genotypes (p> 0.05). Patients infected with genotype 1 were found to be older than those infected with other genotypes (47 ± 15.7 and 39.5 ± 12.2, respectively; p< 0.001). Among patients infected with different genotypes, there was no statistically significant difference in terms of genders (p> 0.05). In conclusion, the determination of HCV genotypes is of crucial importance for treatment decision-making of chronic HCV infection. Besides, it also allows monitoring the changes in the epidemiology of HCV. In this study, although genotype 1b was determined as the most common HCV genotype, the detection of other genotypes was remarkable. This finding was attributed to the presence of many foreign national people in Antalya region which was a high capacity tourism area in Turkey.

Detecting Genetic Interactions for Quantitative Traits Using m-Spacing Entropy Measure

PubMed Central

Yee, Jaeyong; Kwon, Min-Seok; Park, Taesung; Park, Mira

2015-01-01

A number of statistical methods for detecting gene-gene interactions have been developed in genetic association studies with binary traits. However, many phenotype measures are intrinsically quantitative and categorizing continuous traits may not always be straightforward and meaningful. Association of gene-gene interactions with an observed distribution of such phenotypes needs to be investigated directly without categorization. Information gain based on entropy measure has previously been successful in identifying genetic associations with binary traits. We extend the usefulness of this information gain by proposing a nonparametric evaluation method of conditional entropy of a quantitative phenotype associated with a given genotype. Hence, the information gain can be obtained for any phenotype distribution. Because any functional form, such as Gaussian, is not assumed for the entire distribution of a trait or a given genotype, this method is expected to be robust enough to be applied to any phenotypic association data. Here, we show its use to successfully identify the main effect, as well as the genetic interactions, associated with a quantitative trait. PMID:26339620

PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature

PubMed Central

Reijnders, Margot R F; Janowski, Robert; Alvi, Mohsan; Self, Jay E; van Essen, Ton J; Vreeburg, Maaike; Rouhl, Rob P W; Stevens, Servi J C; Stegmann, Alexander P A; Schieving, Jolanda; Pfundt, Rolph; van Dijk, Katinke; Smeets, Eric; Stumpel, Connie T R M; Bok, Levinus A; Cobben, Jan Maarten; Engelen, Marc; Mansour, Sahar; Whiteford, Margo; Chandler, Kate E; Douzgou, Sofia; Cooper, Nicola S; Tan, Ene-Choo; Foo, Roger; Lai, Angeline H M; Rankin, Julia; Green, Andrew; Lönnqvist, Tuula; Isohanni, Pirjo; Williams, Shelley; Ruhoy, Ilene; Carvalho, Karen S; Dowling, James J; Lev, Dorit L; Sterbova, Katalin; Lassuthova, Petra; Neupauerová, Jana; Waugh, Jeff L; Keros, Sotirios; Clayton-Smith, Jill; Smithson, Sarah F; Brunner, Han G; van Hoeckel, Ceciel; Anderson, Mel; Clowes, Virginia E; Siu, Victoria Mok; DDD study, The; Selber, Paulo; Leventer, Richard J; Nellaker, Christoffer; Niessing, Dierk; Hunt, David; Baralle, Diana

2018-01-01

Background De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia. Objectives To delineate the clinical spectrum of PURA syndrome and study genotype-phenotype correlations. Methods Diagnostic or research-based exome or Sanger sequencing was performed in individuals with ID. We systematically collected clinical and mutation data on newly ascertained PURA syndrome individuals, evaluated data of previously reported individuals and performed a computational analysis of photographs. We classified mutations based on predicted effect using 3D in silico models of crystal structures of Drosophila-derived Pur-alpha homologues. Finally, we explored genotype-phenotype correlations by analysis of both recurrent mutations as well as mutation classes. Results We report mutations in PURA (purine-rich element binding protein A) in 32 individuals, the largest cohort described so far. Evaluation of clinical data, including 22 previously published cases, revealed that all have moderate to severe ID and neonatal-onset symptoms, including hypotonia (96%), respiratory problems (57%), feeding difficulties (77%), exaggerated startle response (44%), hypersomnolence (66%) and hypothermia (35%). Epilepsy (54%) and gastrointestinal (69%), ophthalmological (51%) and endocrine problems (42%) were observed frequently. Computational analysis of facial photographs showed subtle facial dysmorphism. No strong genotype-phenotype correlation was identified by subgrouping mutations into functional classes. Conclusion We delineate the clinical spectrum of PURA syndrome with the identification of 32 additional individuals. The identification of one individual through targeted Sanger sequencing points towards the clinical recognisability of the syndrome. Genotype-phenotype analysis showed no significant correlation between mutation classes and disease severity. PMID:29097605

Population Structure, Diversity and Trait Association Analysis in Rice (Oryza sativa L.) Germplasm for Early Seedling Vigor (ESV) Using Trait Linked SSR Markers

PubMed Central

Anandan, Annamalai; Anumalla, Mahender; Pradhan, Sharat Kumar; Ali, Jauhar

2016-01-01

Early seedling vigor (ESV) is the essential trait for direct seeded rice to dominate and smother the weed growth. In this regard, 629 rice genotypes were studied for their morphological and physiological responses in the field under direct seeded aerobic situation on 14th, 28th and 56th days after sowing (DAS). It was determined that the early observations taken on 14th and 28th DAS were reliable estimators to study ESV as compared to56th DAS. Further, 96 were selected from 629 genotypes by principal component (PCA) and discriminate function analyses. The selected genotypes were subjected to decipher the pattern of genetic diversity in terms of both phenotypic and genotypic by using ESV QTL linked simple sequence repeat (SSR) markers. To assess the genetic structure, model and distance based approaches were used. Genotyping of 96 rice lines using 39 polymorphic SSRs produced a total of 128 alleles with the phenotypic information content (PIC) value of 0.24. The model based population structure approach grouped the accession into two distinct populations, whereas unrooted tree grouped the genotypes into three clusters. Both model based and structure based approach had clearly distinguished the early vigor genotypes from non-early vigor genotypes. Association analysis revealed that 16 and 10 SSRs showed significant association with ESV traits by general linear model (GLM) and mixed linear model (MLM) approaches respectively. Marker alleles on chromosome 2 were associated with shoot dry weight on 28 DAS, vigor index on 14 and 28 DAS. Improvement in the rate of seedling growth will be useful for identifying rice genotypes acquiescent to direct seeded conditions through marker-assisted selection. PMID:27031620

Association of STin2 Variable Number of Tandem Repeat (VNTR) Polymorphism of Serotonin Transporter Gene with Lifelong Premature Ejaculation: A Case-Control Study in Han Chinese Subjects

PubMed Central

Huang, Yuanyuan; Zhang, Xiansheng; Gao, Jingjing; Tang, Dongdong; Gao, Pan; Peng, Dangwei; Liang, Chaozhao

2016-01-01

Background The STin2 VNTR polymorphism has a variable number of tandem repeats in intron 2 of the serotonin transporter gene. We aimed to explore the relationship between STin2 VNTR polymorphism and lifelong premature ejaculation (LPE). Material/Methods We recruited a total of 115 outpatients who complained of ejaculating prematurely and who were diagnosed as LPE, and 101 controls without PE complaint. Allelic variations of STin2 VNTR were genotyped using PCR-based technology. We evaluated the associations between STin2 VNTR allelic and genotypic frequencies and LPE, as well as the intravaginal ejaculation latency time (IELT) of different STin2 VNTR genotypes among LPE patients. Results The patients and controls did not differ significantly in terms of any characteristic except age. A significantly higher frequency of STin2.12/12 genotype was found among LPE patients versus controls (P=0.026). Frequency of patients carrying at least 1 copy of the 10-repeat allele was significantly lower compared to the control group (28.3% vs. 41.8%, OR=0.55; 95%CI=0.31–0.97, P=0.040). In the LPE group, the mean IELT showed significant difference in STin2.12/12 genotype when compared to those with STin2.12/10 and STin2.10/10 genotypes. The mean IELT in10-repeat allele carriers was 50% longer compared to homozygous carriers of the STin2.12 allele. Conclusions Our results indicate the presence of STin2.10 allele is a protective factor for LPE. Men carrying the higher expression genotype STin2. 12/12 have shorter IELT than 10-repeat allele carriers. PMID:27713390

Case-control approach application for finding a relationship between candidate genes and clinical mastitis in Holstein dairy cattle.

PubMed

Bagheri, Masoumeh; Moradi-Sharhrbabak, M; Miraie-Ashtiani, R; Safdari-Shahroudi, M; Abdollahi-Arpanahi, R

2016-02-01

Mastitis is a major source of economic loss in dairy herds. The objective of this research was to evaluate the association between genotypes within SLC11A1 and CXCR1 candidate genes and clinical mastitis in Holstein dairy cattle using the selective genotyping method. The data set contained clinical mastitis records of 3,823 Holstein cows from two Holstein dairy herds located in two different regions in Iran. Data included the number of cases of clinical mastitis per lactation. Selective genotyping was based on extreme values for clinical mastitis residuals (CMR) from mixed model analyses. Two extreme groups consisting of 135 cows were formed (as cases and controls), and genotyped for the two candidate genes, namely, SLC11A1 and CXCR1, using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), respectively. Associations between single nucleotide polymorphism (SNP) genotypes with CMR and breeding values for milk and protein yield were carried out by applying logistic regression analyses, i.e. estimating the probability of the heterogeneous genotype in the dependency of values for CMR and breeding values (BVs). The sequencing results revealed a novel mutation in 1139 bp of exon 11 of the SLC11A1 gene and this SNP had a significant association with CMR (P < 0.05). PCR-RFLP analysis leads to three banding patterns for CXCR1c.735C>G and these genotypes had significant relationships with CMR. Overall, the results showed that SLC11A1 and CXCR1 are valuable candidate genes for the improvement of mastitis resistance as well as production traits in dairy cattle populations.

Sex steroid metabolism polymorphisms and mammographic density in pre- and early perimenopausal women

PubMed Central

Crandall, Carolyn J; Sehl, Mary E; Crawford, Sybil L; Gold, Ellen B; Habel, Laurel A; Butler, Lesley M; Sowers, MaryFran R; Greendale, Gail A; Sinsheimer, Janet S

2009-01-01

Introduction We examined the association between mammographic density and single-nucleotide polymorphisms (SNPs) in genes encoding CYP1A1, CYP1B1, aromatase, 17β-HSD, ESR1, and ESR2 in pre- and early perimenopausal white, African-American, Chinese, and Japanese women. Methods The Study of Women's Health Across the Nation is a longitudinal community-based cohort study. We analyzed data from 451 pre- and early perimenopausal participants of the ancillary SWAN Mammographic Density study for whom we had complete information regarding mammographic density, genotypes, and covariates. With multivariate linear regression, we examined the relation between percentage mammographic breast density (outcome) and each SNP (primary predictor), adjusting for age, race/ethnicity, parity, cigarette smoking, and body mass index (BMI). Results After multivariate adjustment, the CYP1B1 rs162555 CC genotype was associated with a 9.4% higher mammographic density than the TC/TT genotype (P = 0.04). The CYP19A1 rs936306 TT genotype was associated with 6.2% lower mammographic density than the TC/CC genotype (P = 0.02). The positive association between CYP1A1 rs2606345 and mammographic density was significantly stronger among participants with BMI greater than 30 kg/m2 than among those with BMI less than 25 kg/m2 (Pinteraction = 0.05). Among white participants, the ESR1 rs2234693 CC genotype was associated with a 7.0% higher mammographic density than the CT/TT genotype (P = 0.01). Conclusions SNPs in certain genes encoding sex steroid metabolism enzymes and ESRs were associated with mammographic density. Because the encoded enzymes and ESR1 are expressed in breast tissue, these SNPs may influence breast cancer risk by altering mammographic density. PMID:19630952

Genetic parameters and prediction of genotypic values for root quality traits in cassava using REML/BLUP.

PubMed

Oliveira, E J; Santana, F A; Oliveira, L A; Santos, V S

2014-08-28

The aim of this study was to estimate the genetic parameters and predict the genotypic values of root quality traits in cassava (Manihot esculenta Crantz) using restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP). A total of 471 cassava accessions were evaluated over two years of cultivation. The evaluated traits included amylose content (AML), root dry matter (DMC), cyanogenic compounds (CyC), and starch yield (StYi). Estimates of the individual broad-sense heritability of AML were low (hg(2) = 0.07 ± 0.02), medium for StYi and DMC, and high for CyC. The heritability of AML was substantially improved based on mean of accessions (hm(2) = 0.28), indicating that some strategies such as increasing the number of repetitions can be used to increase the selective efficiency. In general, the observed genotypic values were very close to the predicted average of the improved population, most likely due to the high accuracy (>0.90), especially for DMC, CyC, and StYi. Gains via selection of the 30 best genotypes for each trait were 4.8 and 3.2% for an increase and decrease for AML, respectively, an increase of 10.75 and 74.62% for DMC for StYi, respectively, and a decrease of 89.60% for CyC in relation to the overall mean of the genotypic values. Genotypic correlations between the quality traits of the cassava roots collected were generally favorable, although they were low in magnitude. The REML/BLUP method was adequate for estimating genetic parameters and predicting the genotypic values, making it useful for cassava breeding.