Sample records for genotype error detection
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Douglas, Julie A.; Skol, Andrew D.; Boehnke, Michael
2002-01-01
Gene-mapping studies routinely rely on checking for Mendelian transmission of marker alleles in a pedigree, as a means of screening for genotyping errors and mutations, with the implicit assumption that, if a pedigree is consistent with Mendel’s laws of inheritance, then there are no genotyping errors. However, the occurrence of inheritance inconsistencies alone is an inadequate measure of the number of genotyping errors, since the rate of occurrence depends on the number and relationships of genotyped pedigree members, the type of errors, and the distribution of marker-allele frequencies. In this article, we calculate the expected probability of detection of a genotyping error or mutation as an inheritance inconsistency in nuclear-family data, as a function of both the number of genotyped parents and offspring and the marker-allele frequency distribution. Through computer simulation, we explore the sensitivity of our analytic calculations to the underlying error model. Under a random-allele–error model, we find that detection rates are 51%–77% for multiallelic markers and 13%–75% for biallelic markers; detection rates are generally lower when the error occurs in a parent than in an offspring, unless a large number of offspring are genotyped. Errors are especially difficult to detect for biallelic markers with equally frequent alleles, even when both parents are genotyped; in this case, the maximum detection rate is 34% for four-person nuclear families. Error detection in families in which parents are not genotyped is limited, even with multiallelic markers. Given these results, we recommend that additional error checking (e.g., on the basis of multipoint analysis) be performed, beyond routine checking for Mendelian consistency. Furthermore, our results permit assessment of the plausibility of an observed number of inheritance inconsistencies for a family, allowing the detection of likely pedigree—rather than genotyping—errors in the early stages of a genome scan. Such early assessments are valuable in either the targeting of families for resampling or discontinued genotyping. PMID:11791214
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Detecting genotyping errors and describing black bear movement in northern Idaho
Michael K. Schwartz; Samuel A. Cushman; Kevin S. McKelvey; Jim Hayden; Cory Engkjer
2006-01-01
Non-invasive genetic sampling has become a favored tool to enumerate wildlife. Genetic errors, caused by poor quality samples, can lead to substantial biases in numerical estimates of individuals. We demonstrate how the computer program DROPOUT can detect amplification errors (false alleles and allelic dropout) in a black bear (Ursus americanus) dataset collected in...
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Dye shift: a neglected source of genotyping error in molecular ecology.
Sutton, Jolene T; Robertson, Bruce C; Jamieson, Ian G
2011-05-01
Molecular ecologists must be vigilant in detecting and accounting for genotyping error, yet potential errors stemming from dye-induced mobility shift (dye shift) may be frequently neglected and largely unknown to researchers who employ 3-primer systems with automated genotyping. When left uncorrected, dye shift can lead to mis-scoring alleles and even to falsely calling new alleles if different dyes are used to genotype the same locus in subsequent reactions. When we used four different fluorophore labels from a standard dye set to genotype the same set of loci, differences in the resulting size estimates for a single allele ranged from 2.07 bp to 3.68 bp. The strongest effects were associated with the fluorophore PET, and relative degree of dye shift was inversely related to locus size. We found little evidence in the literature that dye shift is regularly accounted for in 3-primer studies, despite knowledge of this phenomenon existing for over a decade. However, we did find some references to erroneous standard correction factors for the same set of dyes that we tested. We thus reiterate the need for strict quality control when attempting to reduce possible sources of genotyping error, and in cases where different dyes are applied to a single locus, perhaps mistakenly, we strongly discourage researchers from assuming generic correction patterns. © 2011 Blackwell Publishing Ltd.
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Evaluating mixed samples as a source of error in non-invasive genetic studies using microsatellites
Roon, David A.; Thomas, M.E.; Kendall, K.C.; Waits, L.P.
2005-01-01
The use of noninvasive genetic sampling (NGS) for surveying wild populations is increasing rapidly. Currently, only a limited number of studies have evaluated potential biases associated with NGS. This paper evaluates the potential errors associated with analysing mixed samples drawn from multiple animals. Most NGS studies assume that mixed samples will be identified and removed during the genotyping process. We evaluated this assumption by creating 128 mixed samples of extracted DNA from brown bear (Ursus arctos) hair samples. These mixed samples were genotyped and screened for errors at six microsatellite loci according to protocols consistent with those used in other NGS studies. Five mixed samples produced acceptable genotypes after the first screening. However, all mixed samples produced multiple alleles at one or more loci, amplified as only one of the source samples, or yielded inconsistent electropherograms by the final stage of the error-checking process. These processes could potentially reduce the number of individuals observed in NGS studies, but errors should be conservative within demographic estimates. Researchers should be aware of the potential for mixed samples and carefully design gel analysis criteria and error checking protocols to detect mixed samples.
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Correcting for Sample Contamination in Genotype Calling of DNA Sequence Data
Flickinger, Matthew; Jun, Goo; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min
2015-01-01
DNA sample contamination is a frequent problem in DNA sequencing studies and can result in genotyping errors and reduced power for association testing. We recently described methods to identify within-species DNA sample contamination based on sequencing read data, showed that our methods can reliably detect and estimate contamination levels as low as 1%, and suggested strategies to identify and remove contaminated samples from sequencing studies. Here we propose methods to model contamination during genotype calling as an alternative to removal of contaminated samples from further analyses. We compare our contamination-adjusted calls to calls that ignore contamination and to calls based on uncontaminated data. We demonstrate that, for moderate contamination levels (5%–20%), contamination-adjusted calls eliminate 48%–77% of the genotyping errors. For lower levels of contamination, our contamination correction methods produce genotypes nearly as accurate as those based on uncontaminated data. Our contamination correction methods are useful generally, but are particularly helpful for sample contamination levels from 2% to 20%. PMID:26235984
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Creel, Scott; Spong, Goran; Sands, Jennifer L; Rotella, Jay; Zeigle, Janet; Joe, Lawrence; Murphy, Kerry M; Smith, Douglas
2003-07-01
Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.
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Wu, Da-lin; Ling, Han-xin; Tang, Hao
2004-11-01
To evaluate the accuracy of PCR with sequence-specific primers (PCR-SSP) for HLA-I genotyping and analyze the causes of the errors occurring in the genotyping. DNA samples and were obtained from 34 clinical patients, and serological typing with monoclonal antibody (mAb) and HLA-A and, B antigen genotyping with PCR-SSP were performed. HLA-A and, B alleles were successfully typed in 34 clinical samples by mAb and PCR-SSP. No false positive or false negative results were found, and the erroneous and missed diagnosis rates were obviously higher in serological detection, being 23.5% for HLA-A and 26.5% for HLA-B. Error or confusion was more likely to occur in the antigens of A2 and A68, A32 and A33, B5, B60 and B61. DNA typing for HLA-I class (A, B antigens) by PCR-SSP has high resolution, high specificity, and good reproducibility, which is more suitable for clinical application than serological typing. PCR-SSP may accurately detect the alleles that are easily missed or mistaken in serological typing.
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Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe
2010-01-01
Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome. PMID:20543950
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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...
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Yan, Song; Li, Yun
2014-02-15
Despite its great capability to detect rare variant associations, next-generation sequencing is still prohibitively expensive when applied to large samples. In case-control studies, it is thus appealing to sequence only a subset of cases to discover variants and genotype the identified variants in controls and the remaining cases under the reasonable assumption that causal variants are usually enriched among cases. However, this approach leads to inflated type-I error if analyzed naively for rare variant association. Several methods have been proposed in recent literature to control type-I error at the cost of either excluding some sequenced cases or correcting the genotypes of discovered rare variants. All of these approaches thus suffer from certain extent of information loss and thus are underpowered. We propose a novel method (BETASEQ), which corrects inflation of type-I error by supplementing pseudo-variants while keeps the original sequence and genotype data intact. Extensive simulations and real data analysis demonstrate that, in most practical situations, BETASEQ leads to higher testing powers than existing approaches with guaranteed (controlled or conservative) type-I error. BETASEQ and associated R files, including documentation, examples, are available at http://www.unc.edu/~yunmli/betaseq
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Darvasi, A.; Soller, M.
1994-01-01
Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: ``selective DNA pooling'') takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F(2) and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions. PMID:7896115
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Hühn, M; Lotito, S; Piepho, H P
1993-09-01
Multilocation trials in plant breeding lead to cross-classified data sets with rows=genotypes and columns=environments, where the breeder is particularly interested in the rank orders of the genotypes in the different environments. Non-identical rank orders are the result of genotype x environment interactions. Not every interaction, however, causes rank changes among the genotypes (rank-interaction). From a breeder's point of view, interaction is tolerable only as long as it does not affect the rank orders. Therefore, the question arises of under which circumstances does interaction become rank-interaction. This paper contributes to our understanding of this topic. In our study we emphasized the detection of relationships between the similarity of the rank orders (measured by Kendall's coefficient of concordance W) and the functions of the diverse variance components (genotypes, environments, interaction, error). On the basis of extensive data sets on different agricultural crops (faba bean, fodder beet, sugar beet, oats, winter rape) obtained from registration trials (1985-1989) carried out in the Federal Republic of Germany, we obtained the following as main result: W ≅ σ 2 (g) /(σ 2 (g) + σ 2 (v) ) where σ 2 (g) =genotypic variance and σ 2 (v) = σ 2 (ge) + σ 2 (o) /L with σ 2 (ge) =interaction variance, σ 2 (o) =error variance and L=number of replications.
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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.
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Escott-Price, Valentina; Ghodsi, Mansoureh; Schmidt, Karl Michael
2014-04-01
We evaluate the effect of genotyping errors on the type-I error of a general association test based on genotypes, showing that, in the presence of errors in the case and control samples, the test statistic asymptotically follows a scaled non-central $\\chi ^2$ distribution. We give explicit formulae for the scaling factor and non-centrality parameter for the symmetric allele-based genotyping error model and for additive and recessive disease models. They show how genotyping errors can lead to a significantly higher false-positive rate, growing with sample size, compared with the nominal significance levels. The strength of this effect depends very strongly on the population distribution of the genotype, with a pronounced effect in the case of rare alleles, and a great robustness against error in the case of large minor allele frequency. We also show how these results can be used to correct $p$-values.
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Impact and quantification of the sources of error in DNA pooling designs.
Jawaid, A; Sham, P
2009-01-01
The analysis of genome wide variation offers the possibility of unravelling the genes involved in the pathogenesis of disease. Genome wide association studies are also particularly useful for identifying and validating targets for therapeutic intervention as well as for detecting markers for drug efficacy and side effects. The cost of such large-scale genetic association studies may be reduced substantially by the analysis of pooled DNA from multiple individuals. However, experimental errors inherent in pooling studies lead to a potential increase in the false positive rate and a loss in power compared to individual genotyping. Here we quantify various sources of experimental error using empirical data from typical pooling experiments and corresponding individual genotyping counts using two statistical methods. We provide analytical formulas for calculating these different errors in the absence of complete information, such as replicate pool formation, and for adjusting for the errors in the statistical analysis. We demonstrate that DNA pooling has the potential of estimating allele frequencies accurately, and adjusting the pooled allele frequency estimates for differential allelic amplification considerably improves accuracy. Estimates of the components of error show that differential allelic amplification is the most important contributor to the error variance in absolute allele frequency estimation, followed by allele frequency measurement and pool formation errors. Our results emphasise the importance of minimising experimental errors and obtaining correct error estimates in genetic association studies.
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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
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A video method to study Drosophila sleep.
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.
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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.
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Powerful Inference with the D-Statistic on Low-Coverage Whole-Genome Data
Soraggi, Samuele; Wiuf, Carsten; Albrechtsen, Anders
2017-01-01
The detection of ancient gene flow between human populations is an important issue in population genetics. A common tool for detecting ancient admixture events is the D-statistic. The D-statistic is based on the hypothesis of a genetic relationship that involves four populations, whose correctness is assessed by evaluating specific coincidences of alleles between the groups. When working with high-throughput sequencing data, calling genotypes accurately is not always possible; therefore, the D-statistic currently samples a single base from the reads of one individual per population. This implies ignoring much of the information in the data, an issue especially striking in the case of ancient genomes. We provide a significant improvement to overcome the problems of the D-statistic by considering all reads from multiple individuals in each population. We also apply type-specific error correction to combat the problems of sequencing errors, and show a way to correct for introgression from an external population that is not part of the supposed genetic relationship, and how this leads to an estimate of the admixture rate. We prove that the D-statistic is approximated by a standard normal distribution. Furthermore, we show that our method outperforms the traditional D-statistic in detecting admixtures. The power gain is most pronounced for low and medium sequencing depth (1–10×), and performances are as good as with perfectly called genotypes at a sequencing depth of 2×. We show the reliability of error correction in scenarios with simulated errors and ancient data, and correct for introgression in known scenarios to estimate the admixture rates. PMID:29196497
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Accurate typing of short tandem repeats from genome-wide sequencing data and its applications.
Fungtammasan, Arkarachai; Ananda, Guruprasad; Hile, Suzanne E; Su, Marcia Shu-Wei; Sun, Chen; Harris, Robert; Medvedev, Paul; Eckert, Kristin; Makova, Kateryna D
2015-05-01
Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution. © 2015 Fungtammasan et al.; Published by Cold Spring Harbor Laboratory Press.
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Powerful Inference with the D-Statistic on Low-Coverage Whole-Genome Data.
Soraggi, Samuele; Wiuf, Carsten; Albrechtsen, Anders
2018-02-02
The detection of ancient gene flow between human populations is an important issue in population genetics. A common tool for detecting ancient admixture events is the D-statistic. The D-statistic is based on the hypothesis of a genetic relationship that involves four populations, whose correctness is assessed by evaluating specific coincidences of alleles between the groups. When working with high-throughput sequencing data, calling genotypes accurately is not always possible; therefore, the D-statistic currently samples a single base from the reads of one individual per population. This implies ignoring much of the information in the data, an issue especially striking in the case of ancient genomes. We provide a significant improvement to overcome the problems of the D-statistic by considering all reads from multiple individuals in each population. We also apply type-specific error correction to combat the problems of sequencing errors, and show a way to correct for introgression from an external population that is not part of the supposed genetic relationship, and how this leads to an estimate of the admixture rate. We prove that the D-statistic is approximated by a standard normal distribution. Furthermore, we show that our method outperforms the traditional D-statistic in detecting admixtures. The power gain is most pronounced for low and medium sequencing depth (1-10×), and performances are as good as with perfectly called genotypes at a sequencing depth of 2×. We show the reliability of error correction in scenarios with simulated errors and ancient data, and correct for introgression in known scenarios to estimate the admixture rates. Copyright © 2018 Soraggi et al.
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Shifflett, Benjamin; Huang, Rong; Edland, Steven D
2017-01-01
Genotypic association studies are prone to inflated type I error rates if multiple hypothesis testing is performed, e.g., sequentially testing for recessive, multiplicative, and dominant risk. Alternatives to multiple hypothesis testing include the model independent genotypic χ 2 test, the efficiency robust MAX statistic, which corrects for multiple comparisons but with some loss of power, or a single Armitage test for multiplicative trend, which has optimal power when the multiplicative model holds but with some loss of power when dominant or recessive models underlie the genetic association. We used Monte Carlo simulations to describe the relative performance of these three approaches under a range of scenarios. All three approaches maintained their nominal type I error rates. The genotypic χ 2 and MAX statistics were more powerful when testing a strictly recessive genetic effect or when testing a dominant effect when the allele frequency was high. The Armitage test for multiplicative trend was most powerful for the broad range of scenarios where heterozygote risk is intermediate between recessive and dominant risk. Moreover, all tests had limited power to detect recessive genetic risk unless the sample size was large, and conversely all tests were relatively well powered to detect dominant risk. Taken together, these results suggest the general utility of the multiplicative trend test when the underlying genetic model is unknown.
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Genetic mapping in the presence of genotyping errors.
Cartwright, Dustin A; Troggio, Michela; Velasco, Riccardo; Gutin, Alexander
2007-08-01
Genetic maps are built using the genotypes of many related individuals. Genotyping errors in these data sets can distort genetic maps, especially by inflating the distances. We have extended the traditional likelihood model used for genetic mapping to include the possibility of genotyping errors. Each individual marker is assigned an error rate, which is inferred from the data, just as the genetic distances are. We have developed a software package, called TMAP, which uses this model to find maximum-likelihood maps for phase-known pedigrees. We have tested our methods using a data set in Vitis and on simulated data and confirmed that our method dramatically reduces the inflationary effect caused by increasing the number of markers and leads to more accurate orders.
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Genetic Mapping in the Presence of Genotyping Errors
Cartwright, Dustin A.; Troggio, Michela; Velasco, Riccardo; Gutin, Alexander
2007-01-01
Genetic maps are built using the genotypes of many related individuals. Genotyping errors in these data sets can distort genetic maps, especially by inflating the distances. We have extended the traditional likelihood model used for genetic mapping to include the possibility of genotyping errors. Each individual marker is assigned an error rate, which is inferred from the data, just as the genetic distances are. We have developed a software package, called TMAP, which uses this model to find maximum-likelihood maps for phase-known pedigrees. We have tested our methods using a data set in Vitis and on simulated data and confirmed that our method dramatically reduces the inflationary effect caused by increasing the number of markers and leads to more accurate orders. PMID:17277374
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Estimating genotype error rates from high-coverage next-generation sequence data.
Wall, Jeffrey D; Tang, Ling Fung; Zerbe, Brandon; Kvale, Mark N; Kwok, Pui-Yan; Schaefer, Catherine; Risch, Neil
2014-11-01
Exome and whole-genome sequencing studies are becoming increasingly common, but little is known about the accuracy of the genotype calls made by the commonly used platforms. Here we use replicate high-coverage sequencing of blood and saliva DNA samples from four European-American individuals to estimate lower bounds on the error rates of Complete Genomics and Illumina HiSeq whole-genome and whole-exome sequencing. Error rates for nonreference genotype calls range from 0.1% to 0.6%, depending on the platform and the depth of coverage. Additionally, we found (1) no difference in the error profiles or rates between blood and saliva samples; (2) Complete Genomics sequences had substantially higher error rates than Illumina sequences had; (3) error rates were higher (up to 6%) for rare or unique variants; (4) error rates generally declined with genotype quality (GQ) score, but in a nonlinear fashion for the Illumina data, likely due to loss of specificity of GQ scores greater than 60; and (5) error rates increased with increasing depth of coverage for the Illumina data. These findings, especially (3)-(5), suggest that caution should be taken in interpreting the results of next-generation sequencing-based association studies, and even more so in clinical application of this technology in the absence of validation by other more robust sequencing or genotyping methods. © 2014 Wall et al.; Published by Cold Spring Harbor Laboratory Press.
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Promises and pitfalls of Illumina sequencing for HIV resistance genotyping.
Brumme, Chanson J; Poon, Art F Y
2017-07-15
Genetic sequencing ("genotyping") plays a critical role in the modern clinical management of HIV infection. This virus evolves rapidly within patients because of its error-prone reverse transcriptase and short generation time. Consequently, HIV variants with mutations that confer resistance to one or more antiretroviral drugs can emerge during sub-optimal treatment. There are now multiple HIV drug resistance interpretation algorithms that take the region of the HIV genome encoding the major drug targets as inputs; expert use of these algorithms can significantly improve to clinical outcomes in HIV treatment. Next-generation sequencing has the potential to revolutionize HIV resistance genotyping by lowering the threshold that rare but clinically significant HIV variants can be detected reproducibly, and by conferring improved cost-effectiveness in high-throughput scenarios. In this review, we discuss the relative merits and challenges of deploying the Illumina MiSeq instrument for clinical HIV genotyping. Copyright © 2016 Elsevier B.V. All rights reserved.
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Vašek, Jakub; Viehmannová, Iva; Ocelák, Martin; Cachique Huansi, Danter; Vejl, Pavel
2017-01-01
An analysis of the population structure and genetic diversity for any organism often depends on one or more molecular marker techniques. Nonetheless, these techniques are not absolutely reliable because of various sources of errors arising during the genotyping process. Thus, a complex analysis of genotyping error was carried out with the AFLP method in 169 samples of the oil seed plant Plukenetia volubilis L. from small isolated subpopulations in the Peruvian Amazon. Samples were collected in nine localities from the region of San Martin. Analysis was done in eight datasets with a genotyping error from 0 to 5%. Using eleven primer combinations, 102 to 275 markers were obtained according to the dataset. It was found that it is only possible to obtain the most reliable and robust results through a multiple-level filtering process. Genotyping error and software set up influence both the estimation of population structure and genetic diversity, where in our case population number (K) varied between 2–9 depending on the dataset and statistical method used. Surprisingly, discrepancies in K number were caused more by statistical approaches than by genotyping errors themselves. However, for estimation of genetic diversity, the degree of genotyping error was critical because descriptive parameters (He, FST, PLP 5%) varied substantially (by at least 25%). Due to low gene flow, P. volubilis mostly consists of small isolated subpopulations (ΦPT = 0.252–0.323) with some degree of admixture given by socio-economic connectivity among the sites; a direct link between the genetic and geographic distances was not confirmed. The study illustrates the successful application of AFLP to infer genetic structure in non-model plants. PMID:28910307
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Sun, Lei; Dimitromanolakis, Apostolos
2014-01-01
Pedigree errors and cryptic relatedness often appear in families or population samples collected for genetic studies. If not identified, these issues can lead to either increased false negatives or false positives in both linkage and association analyses. To identify pedigree errors and cryptic relatedness among individuals from the 20 San Antonio Family Studies (SAFS) families and cryptic relatedness among the 157 putatively unrelated individuals, we apply PREST-plus to the genome-wide single-nucleotide polymorphism (SNP) data and analyze estimated identity-by-descent (IBD) distributions for all pairs of genotyped individuals. Based on the given pedigrees alone, PREST-plus identifies the following putative pairs: 1091 full-sib, 162 half-sib, 360 grandparent-grandchild, 2269 avuncular, 2717 first cousin, 402 half-avuncular, 559 half-first cousin, 2 half-sib+first cousin, 957 parent-offspring and 440,546 unrelated. Using the genotype data, PREST-plus detects 7 mis-specified relative pairs, with their IBD estimates clearly deviating from the null expectations, and it identifies 4 cryptic related pairs involving 7 individuals from 6 families.
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Quality control and quality assurance in genotypic data for genome-wide association studies
Laurie, Cathy C.; Doheny, Kimberly F.; Mirel, Daniel B.; Pugh, Elizabeth W.; Bierut, Laura J.; Bhangale, Tushar; Boehm, Frederick; Caporaso, Neil E.; Cornelis, Marilyn C.; Edenberg, Howard J.; Gabriel, Stacy B.; Harris, Emily L.; Hu, Frank B.; Jacobs, Kevin; Kraft, Peter; Landi, Maria Teresa; Lumley, Thomas; Manolio, Teri A.; McHugh, Caitlin; Painter, Ian; Paschall, Justin; Rice, John P.; Rice, Kenneth M.; Zheng, Xiuwen; Weir, Bruce S.
2011-01-01
Genome-wide scans of nucleotide variation in human subjects are providing an increasing number of replicated associations with complex disease traits. Most of the variants detected have small effects and, collectively, they account for a small fraction of the total genetic variance. Very large sample sizes are required to identify and validate findings. In this situation, even small sources of systematic or random error can cause spurious results or obscure real effects. The need for careful attention to data quality has been appreciated for some time in this field, and a number of strategies for quality control and quality assurance (QC/QA) have been developed. Here we extend these methods and describe a system of QC/QA for genotypic data in genome-wide association studies. This system includes some new approaches that (1) combine analysis of allelic probe intensities and called genotypes to distinguish gender misidentification from sex chromosome aberrations, (2) detect autosomal chromosome aberrations that may affect genotype calling accuracy, (3) infer DNA sample quality from relatedness and allelic intensities, (4) use duplicate concordance to infer SNP quality, (5) detect genotyping artifacts from dependence of Hardy-Weinberg equilibrium (HWE) test p-values on allelic frequency, and (6) demonstrate sensitivity of principal components analysis (PCA) to SNP selection. The methods are illustrated with examples from the ‘Gene Environment Association Studies’ (GENEVA) program. The results suggest several recommendations for QC/QA in the design and execution of genome-wide association studies. PMID:20718045
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QuASAR: quantitative allele-specific analysis of reads
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
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Analysis of case-only studies accounting for genotyping error.
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.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Geraldes, Armando; Hannemann, Jan; Grassa, Chris
2013-01-01
Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. Despite the declining costs of genotyping by sequencing, for most studies, the use of large SNP genotyping arrays still offers the most cost-effective solution for large-scale targeted genotyping. Here we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species range. Due to the rapid decay of linkage disequilibrium in P. trichocarpa we adopted a candidate gene approach to the arraymore » design that resulted in the selection of 34,131 SNPs, the majority of which are located in, or within 2 kb, of 3,543 candidate genes. A subset of the SNPs (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%, indicating that high-quality data are generated with this array. We demonstrate that even among small numbers of samples (n=10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that due to ascertainment bias the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca (P. balsamifera and P. angustifolia). Finally, we provide evidence for the utility of the array for intraspecific studies of genetic differentiation and for species assignment and the detection of natural hybrids.« less
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Software for Quantifying and Simulating Microsatellite Genotyping Error
Johnson, Paul C.D.; Haydon, Daniel T.
2007-01-01
Microsatellite genetic marker data are exploited in a variety of fields, including forensics, gene mapping, kinship inference and population genetics. In all of these fields, inference can be thwarted by failure to quantify and account for data errors, and kinship inference in particular can benefit from separating errors into two distinct classes: allelic dropout and false alleles. Pedant is MS Windows software for estimating locus-specific maximum likelihood rates of these two classes of error. Estimation is based on comparison of duplicate error-prone genotypes: neither reference genotypes nor pedigree data are required. Other functions include: plotting of error rate estimates and confidence intervals; simulations for performing power analysis and for testing the robustness of error rate estimates to violation of the underlying assumptions; and estimation of expected heterozygosity, which is a required input. The program, documentation and source code are available from http://www.stats.gla.ac.uk/~paulj/pedant.html. PMID:20066126
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A Video Method to Study Drosophila Sleep
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
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Geraldes, A; Difazio, S P; Slavov, G T; Ranjan, P; Muchero, W; Hannemann, J; Gunter, L E; Wymore, A M; Grassa, C J; Farzaneh, N; Porth, I; McKown, A D; Skyba, O; Li, E; Fujita, M; Klápště, J; Martin, J; Schackwitz, W; Pennacchio, C; Rokhsar, D; Friedmann, M C; Wasteneys, G O; Guy, R D; El-Kassaby, Y A; Mansfield, S D; Cronk, Q C B; Ehlting, J; Douglas, C J; Tuskan, G A
2013-03-01
Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids. © 2013 Blackwell Publishing Ltd.
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QuASAR: quantitative allele-specific analysis of reads.
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.
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Blue, Elizabeth Marchani; Sun, Lei; Tintle, Nathan L.; Wijsman, Ellen M.
2014-01-01
When analyzing family data, we dream of perfectly informative data, even whole genome sequences (WGS) for all family members. Reality intervenes, and we find next-generation sequence (NGS) data have error, and are often too expensive or impossible to collect on everyone. Genetic Analysis Workshop 18 groups “Quality Control” and “Dropping WGS through families using GWAS framework” focused on finding, correcting, and using errors within the available sequence and family data, developing methods to infer and analyze missing sequence data among relatives, and testing for linkage and association with simulated blood pressure. We found that single nucleotide polymorphisms, NGS, and imputed data are generally concordant, but that errors are particularly likely at rare variants, homozygous genotypes, within regions with repeated sequences or structural variants, and within sequence data imputed from unrelateds. Admixture complicated identification of cryptic relatedness, but information from Mendelian transmission improved error detection and provided an estimate of the de novo mutation rate. Both genotype and pedigree errors had an adverse effect on subsequent analyses. Computationally fast rules-based imputation was accurate, but could not cover as many loci or subjects as more computationally demanding probability-based methods. Incorporating population-level data into pedigree-based imputation methods improved results. Observed data outperformed imputed data in association testing, but imputed data were also useful. We discuss the strengths and weaknesses of existing methods, and suggest possible future directions. Topics include improving communication between those performing data collection and analysis, establishing thresholds for and improving imputation quality, and incorporating error into imputation and analytical models. PMID:25112184
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Sethi, Suresh; Linden, Daniel; Wenburg, John; Lewis, Cara; Lemons, Patrick R.; Fuller, Angela K.; Hare, Matthew P.
2016-01-01
Error-tolerant likelihood-based match calling presents a promising technique to accurately identify recapture events in genetic mark–recapture studies by combining probabilities of latent genotypes and probabilities of observed genotypes, which may contain genotyping errors. Combined with clustering algorithms to group samples into sets of recaptures based upon pairwise match calls, these tools can be used to reconstruct accurate capture histories for mark–recapture modelling. Here, we assess the performance of a recently introduced error-tolerant likelihood-based match-calling model and sample clustering algorithm for genetic mark–recapture studies. We assessed both biallelic (i.e. single nucleotide polymorphisms; SNP) and multiallelic (i.e. microsatellite; MSAT) markers using a combination of simulation analyses and case study data on Pacific walrus (Odobenus rosmarus divergens) and fishers (Pekania pennanti). A novel two-stage clustering approach is demonstrated for genetic mark–recapture applications. First, repeat captures within a sampling occasion are identified. Subsequently, recaptures across sampling occasions are identified. The likelihood-based matching protocol performed well in simulation trials, demonstrating utility for use in a wide range of genetic mark–recapture studies. Moderately sized SNP (64+) and MSAT (10–15) panels produced accurate match calls for recaptures and accurate non-match calls for samples from closely related individuals in the face of low to moderate genotyping error. Furthermore, matching performance remained stable or increased as the number of genetic markers increased, genotyping error notwithstanding.
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VIPER: a visualisation tool for exploring inheritance inconsistencies in genotyped pedigrees
2012-01-01
Background Pedigree genotype datasets are used for analysing genetic inheritance and to map genetic markers and traits. Such datasets consist of hundreds of related animals genotyped for thousands of genetic markers and invariably contain multiple errors in both the pedigree structure and in the associated individual genotype data. These errors manifest as apparent inheritance inconsistencies in the pedigree, and invalidate analyses of marker inheritance patterns across the dataset. Cleaning raw datasets of bad data points (incorrect pedigree relationships, unreliable marker assays, suspect samples, bad genotype results etc.) requires expert exploration of the patterns of exposed inconsistencies in the context of the inheritance pedigree. In order to assist this process we are developing VIPER (Visual Pedigree Explorer), a software tool that integrates an inheritance-checking algorithm with a novel space-efficient pedigree visualisation, so that reported inheritance inconsistencies are overlaid on an interactive, navigable representation of the pedigree structure. Methods and results This paper describes an evaluation of how VIPER displays the different scales and types of dataset that occur experimentally, with a description of how VIPER's display interface and functionality meet the challenges presented by such data. We examine a range of possible error types found in real and simulated pedigree genotype datasets, demonstrating how these errors are exposed and explored using the VIPER interface and we evaluate the utility and usability of the interface to the domain expert. Evaluation was performed as a two stage process with the assistance of domain experts (geneticists). The initial evaluation drove the iterative implementation of further features in the software prototype, as required by the users, prior to a final functional evaluation of the pedigree display for exploring the various error types, data scales and structures. Conclusions The VIPER display was shown to effectively expose the range of errors found in experimental genotyped pedigrees, allowing users to explore the underlying causes of reported inheritance inconsistencies. This interface will provide the basis for a full data cleaning tool that will allow the user to remove isolated bad data points, and reversibly test the effect of removing suspect genotypes and pedigree relationships. PMID:22607476
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Haplotypic Analysis of Wellcome Trust Case Control Consortium Data
Browning, Brian L.; Browning, Sharon R.
2008-01-01
We applied a recently developed multilocus association testing method (localized haplotype clustering) to Wellcome Trust Case Control Consortium data (14,000 cases of seven common diseases and 3,000 shared controls genotyped on the Affymetrix 500K array). After rigorous data quality filtering, we identified three disease-associated loci with strong statistical support from localized haplotype cluster tests but with only marginal significance in single marker tests. These loci are chromosomes 10p15.1 with type 1 diabetes (p = 5.1 × 10-9), 12q15 with type 2 diabetes (p = 1.9 × 10-7) and 15q26.2 with hypertension (p = 2.8 × 10-8). We also detected the association of chromosome 9p21.3 with type 2 diabetes (p = 2.8 × 10-8), although this locus did not pass our stringent genotype quality filters. The association of 10p15.1 with type 1 diabetes and 9p21.3 with type 2 diabetes have both been replicated in other studies using independent data sets. Overall, localized haplotype cluster analysis had better success detecting disease associated variants than a previous single-marker analysis of imputed HapMap SNPs. We found that stringent application of quality score thresholds to genotype data substantially reduced false-positive results arising from genotype error. In addition, we demonstrate that it is possible to simultaneously phase 16,000 individuals genotyped on genome-wide data (450K markers) using the Beagle software package. PMID:18224336
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Li, J L; Deng, H; Lai, D B; Xu, F; Chen, J; Gao, G; Recker, R R; Deng, H W
2001-07-01
To efficiently manipulate large amounts of genotype data generated with fluorescently labeled dinucleotide markers, we developed a Microsoft database management system, named. offers several advantages. First, it accommodates the dynamic nature of the accumulations of genotype data during the genotyping process; some data need to be confirmed or replaced by repeat lab procedures. By using, the raw genotype data can be imported easily and continuously and incorporated into the database during the genotyping process that may continue over an extended period of time in large projects. Second, almost all of the procedures are automatic, including autocomparison of the raw data read by different technicians from the same gel, autoadjustment among the allele fragment-size data from cross-runs or cross-platforms, autobinning of alleles, and autocompilation of genotype data for suitable programs to perform inheritance check in pedigrees. Third, provides functions to track electrophoresis gel files to locate gel or sample sources for any resultant genotype data, which is extremely helpful for double-checking consistency of raw and final data and for directing repeat experiments. In addition, the user-friendly graphic interface of renders processing of large amounts of data much less labor-intensive. Furthermore, has built-in mechanisms to detect some genotyping errors and to assess the quality of genotype data that then are summarized in the statistic reports automatically generated by. The can easily handle >500,000 genotype data entries, a number more than sufficient for typical whole-genome linkage studies. The modules and programs we developed for the can be extended to other database platforms, such as Microsoft SQL server, if the capability to handle still greater quantities of genotype data simultaneously is desired.
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snpAD: An ancient DNA genotype caller.
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.
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The Bloodgen Project of the European Union, 2003–2009
Avent, Neil D.; Martinez, Antonio; Flegel, Willy A.; Olsson, Martin L.; Scott, Marion L.; Nogués, Núria; Písăcka, Martin; Daniels, Geoff L.; Muñiz-Diaz, Eduardo; Madgett, Tracey E.; Storry, Jill R.; Beiboer, Sigrid; Maaskant-van Wijk, Petra M.; von Zabern, Inge; Jiménez, Elisa; Tejedor, Diego; López, Monica; Camacho, Emma; Cheroutre, Goedele; Hacker, Anita; Jinoch, Pavel; Svobodova, Irena; van der Schoot, Ellen; de Haas, Masja
2009-01-01
Summary The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism. PMID:21113258
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High-density marker imputation accuracy in sixteen French cattle breeds.
Hozé, Chris; Fouilloux, Marie-Noëlle; Venot, Eric; Guillaume, François; Dassonneville, Romain; Fritz, Sébastien; Ducrocq, Vincent; Phocas, Florence; Boichard, Didier; Croiseau, Pascal
2013-09-03
Genotyping with the medium-density Bovine SNP50 BeadChip® (50K) is now standard in cattle. The high-density BovineHD BeadChip®, which contains 777,609 single nucleotide polymorphisms (SNPs), was developed in 2010. Increasing marker density increases the level of linkage disequilibrium between quantitative trait loci (QTL) and SNPs and the accuracy of QTL localization and genomic selection. However, re-genotyping all animals with the high-density chip is not economically feasible. An alternative strategy is to genotype part of the animals with the high-density chip and to impute high-density genotypes for animals already genotyped with the 50K chip. Thus, it is necessary to investigate the error rate when imputing from the 50K to the high-density chip. Five thousand one hundred and fifty three animals from 16 breeds (89 to 788 per breed) were genotyped with the high-density chip. Imputation error rates from the 50K to the high-density chip were computed for each breed with a validation set that included the 20% youngest animals. Marker genotypes were masked for animals in the validation population in order to mimic 50K genotypes. Imputation was carried out using the Beagle 3.3.0 software. Mean allele imputation error rates ranged from 0.31% to 2.41% depending on the breed. In total, 1980 SNPs had high imputation error rates in several breeds, which is probably due to genome assembly errors, and we recommend to discard these in future studies. Differences in imputation accuracy between breeds were related to the high-density-genotyped sample size and to the genetic relationship between reference and validation populations, whereas differences in effective population size and level of linkage disequilibrium showed limited effects. Accordingly, imputation accuracy was higher in breeds with large populations and in dairy breeds than in beef breeds. More than 99% of the alleles were correctly imputed if more than 300 animals were genotyped at high-density. No improvement was observed when multi-breed imputation was performed. In all breeds, imputation accuracy was higher than 97%, which indicates that imputation to the high-density chip was accurate. Imputation accuracy depends mainly on the size of the reference population and the relationship between reference and target populations.
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High-density marker imputation accuracy in sixteen French cattle breeds
2013-01-01
Background Genotyping with the medium-density Bovine SNP50 BeadChip® (50K) is now standard in cattle. The high-density BovineHD BeadChip®, which contains 777 609 single nucleotide polymorphisms (SNPs), was developed in 2010. Increasing marker density increases the level of linkage disequilibrium between quantitative trait loci (QTL) and SNPs and the accuracy of QTL localization and genomic selection. However, re-genotyping all animals with the high-density chip is not economically feasible. An alternative strategy is to genotype part of the animals with the high-density chip and to impute high-density genotypes for animals already genotyped with the 50K chip. Thus, it is necessary to investigate the error rate when imputing from the 50K to the high-density chip. Methods Five thousand one hundred and fifty three animals from 16 breeds (89 to 788 per breed) were genotyped with the high-density chip. Imputation error rates from the 50K to the high-density chip were computed for each breed with a validation set that included the 20% youngest animals. Marker genotypes were masked for animals in the validation population in order to mimic 50K genotypes. Imputation was carried out using the Beagle 3.3.0 software. Results Mean allele imputation error rates ranged from 0.31% to 2.41% depending on the breed. In total, 1980 SNPs had high imputation error rates in several breeds, which is probably due to genome assembly errors, and we recommend to discard these in future studies. Differences in imputation accuracy between breeds were related to the high-density-genotyped sample size and to the genetic relationship between reference and validation populations, whereas differences in effective population size and level of linkage disequilibrium showed limited effects. Accordingly, imputation accuracy was higher in breeds with large populations and in dairy breeds than in beef breeds. More than 99% of the alleles were correctly imputed if more than 300 animals were genotyped at high-density. No improvement was observed when multi-breed imputation was performed. Conclusion In all breeds, imputation accuracy was higher than 97%, which indicates that imputation to the high-density chip was accurate. Imputation accuracy depends mainly on the size of the reference population and the relationship between reference and target populations. PMID:24004563
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Integrated digital error suppression for improved detection of circulating tumor DNA
Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.
2016-01-01
High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799
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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.
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Lobach, Irvna; Fan, Ruzone; Carroll, Raymond T.
2011-01-01
With the advent of dense single nucleotide polymorphism genotyping, population-based association studies have become the major tools for identifying human disease genes and for fine gene mapping of complex traits. We develop a genotype-based approach for association analysis of case-control studies of gene-environment interactions in the case when environmental factors are measured with error and genotype data are available on multiple genetic markers. To directly use the observed genotype data, we propose two genotype-based models: genotype effect and additive effect models. Our approach offers several advantages. First, the proposed risk functions can directly incorporate the observed genotype data while modeling the linkage disequihbrium information in the regression coefficients, thus eliminating the need to infer haplotype phase. Compared with the haplotype-based approach, an estimating procedure based on the proposed methods can be much simpler and significantly faster. In addition, there is no potential risk due to haplotype phase estimation. Further, by fitting the proposed models, it is possible to analyze the risk alleles/variants of complex diseases, including their dominant or additive effects. To model measurement error, we adopt the pseudo-likelihood method by Lobach et al. [2008]. Performance of the proposed method is examined using simulation experiments. An application of our method is illustrated using a population-based case-control study of association between calcium intake with the risk of colorectal adenoma development. PMID:21031455
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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.
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Hou, Lin; Sun, Ning; Mane, Shrikant; Sayward, Fred; Rajeevan, Nallakkandi; Cheung, Kei-Hoi; Cho, Kelly; Pyarajan, Saiju; Aslan, Mihaela; Miller, Perry; Harvey, Philip D.; Gaziano, J. Michael; Concato, John; Zhao, Hongyu
2017-01-01
A key step in genomic studies is to assess high throughput measurements across millions of markers for each participant’s DNA, either using microarrays or sequencing techniques. Accurate genotype calling is essential for downstream statistical analysis of genotype-phenotype associations, and next generation sequencing (NGS) has recently become a more common approach in genomic studies. How the accuracy of variant calling in NGS-based studies affects downstream association analysis has not, however, been studied using empirical data in which both microarrays and NGS were available. In this article, we investigate the impact of variant calling errors on the statistical power to identify associations between single nucleotides and disease, and on associations between multiple rare variants and disease. Both differential and nondifferential genotyping errors are considered. Our results show that the power of burden tests for rare variants is strongly influenced by the specificity in variant calling, but is rather robust with regard to sensitivity. By using the variant calling accuracies estimated from a substudy of a Cooperative Studies Program project conducted by the Department of Veterans Affairs, we show that the power of association tests is mostly retained with commonly adopted variant calling pipelines. An R package, GWAS.PC, is provided to accommodate power analysis that takes account of genotyping errors (http://zhaocenter.org/software/). PMID:28019059
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Kammers, Kai; Taub, Margaret A.; Ruczinski, Ingo; Martin, Joshua; Yanek, Lisa R.; Frazee, Alyssa; Gao, Yongxing; Hoyle, Dixie; Faraday, Nauder; Becker, Diane M.; Cheng, Linzhao; Wang, Zack Z.; Leek, Jeff T.; Becker, Lewis C.; Mathias, Rasika A.
2017-01-01
Previously, we have described our feeder-free, xeno-free approach to generate megakaryocytes (MKs) in culture from human induced pluripotent stem cells (iPSCs). Here, we focus specifically on the integrity of these MKs using: (1) genotype discordance between parent cell DNA to iPSC cell DNA and onward to the differentiated MK DNA; (2) genomic structural integrity using copy number variation (CNV); and (3) transcriptomic signatures of the derived MK lines compared to the iPSC lines. We detected a very low rate of genotype discordance; estimates were 0.0001%-0.01%, well below the genotyping error rate for our assay (0.37%). No CNVs were generated in the iPSCs that were subsequently passed on to the MKs. Finally, we observed highly biologically relevant gene sets as being upregulated in MKs relative to the iPSCs: platelet activation, blood coagulation, megakaryocyte development, platelet formation, platelet degranulation, and platelet aggregation. These data strongly support the integrity of the derived MK lines. PMID:28107356
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Kernel machine methods for integrative analysis of genome-wide methylation and genotyping studies.
Zhao, Ni; Zhan, Xiang; Huang, Yen-Tsung; Almli, Lynn M; Smith, Alicia; Epstein, Michael P; Conneely, Karen; Wu, Michael C
2018-03-01
Many large GWAS consortia are expanding to simultaneously examine the joint role of DNA methylation in addition to genotype in the same subjects. However, integrating information from both data types is challenging. In this paper, we propose a composite kernel machine regression model to test the joint epigenetic and genetic effect. Our approach works at the gene level, which allows for a common unit of analysis across different data types. The model compares the pairwise similarities in the phenotype to the pairwise similarities in the genotype and methylation values; and high correspondence is suggestive of association. A composite kernel is constructed to measure the similarities in the genotype and methylation values between pairs of samples. We demonstrate through simulations and real data applications that the proposed approach can correctly control type I error, and is more robust and powerful than using only the genotype or methylation data in detecting trait-associated genes. We applied our method to investigate the genetic and epigenetic regulation of gene expression in response to stressful life events using data that are collected from the Grady Trauma Project. Within the kernel machine testing framework, our methods allow for heterogeneity in effect sizes, nonlinear, and interactive effects, as well as rapid P-value computation. © 2017 WILEY PERIODICALS, INC.
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Leung, Ross Ka-Kit; Dong, Zhi Qiang; Sa, Fei; Chong, Cheong Meng; Lei, Si Wan; Tsui, Stephen Kwok-Wing; Lee, Simon Ming-Yuen
2014-02-01
Minor variants have significant implications in quasispecies evolution, early cancer detection and non-invasive fetal genotyping but their accurate detection by next-generation sequencing (NGS) is hampered by sequencing errors. We generated sequencing data from mixtures at predetermined ratios in order to provide insight into sequencing errors and variations that can arise for which simulation cannot be performed. The information also enables better parameterization in depth of coverage, read quality and heterogeneity, library preparation techniques, technical repeatability for mathematical modeling, theory development and simulation experimental design. We devised minor variant authentication rules that achieved 100% accuracy in both testing and validation experiments. The rules are free from tedious inspection of alignment accuracy, sequencing read quality or errors introduced by homopolymers. The authentication processes only require minor variants to: (1) have minimum depth of coverage larger than 30; (2) be reported by (a) four or more variant callers, or (b) DiBayes or LoFreq, plus SNVer (or BWA when no results are returned by SNVer), and with the interassay coefficient of variation (CV) no larger than 0.1. Quantification accuracy undermined by sequencing errors could neither be overcome by ultra-deep sequencing, nor recruiting more variant callers to reach a consensus, such that consistent underestimation and overestimation (i.e. low CV) were observed. To accommodate stochastic error and adjust the observed ratio within a specified accuracy, we presented a proof of concept for the use of a double calibration curve for quantification, which provides an important reference towards potential industrial-scale fabrication of calibrants for NGS.
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An efficient study design to test parent-of-origin effects in family trios.
Yu, Xiaobo; Chen, Gao; Feng, Rui
2017-11-01
Increasing evidence has shown that genes may cause prenatal, neonatal, and pediatric diseases depending on their parental origins. Statistical models that incorporate parent-of-origin effects (POEs) can improve the power of detecting disease-associated genes and help explain the missing heritability of diseases. In many studies, children have been sequenced for genome-wide association testing. But it may become unaffordable to sequence their parents and evaluate POEs. Motivated by the reality, we proposed a budget-friendly study design of sequencing children and only genotyping their parents through single nucleotide polymorphism array. We developed a powerful likelihood-based method, which takes into account both sequence reads and linkage disequilibrium to infer the parental origins of children's alleles and estimate their POEs on the outcome. We evaluated the performance of our proposed method and compared it with an existing method using only genotypes, through extensive simulations. Our method showed higher power than the genotype-based method. When either the mean read depth or the pair-end length was reasonably large, our method achieved ideal power. When single parents' genotypes were unavailable or parental genotypes at the testing locus were not typed, both methods lost power compared with when complete data were available; but the power loss from our method was smaller than the genotype-based method. We also extended our method to accommodate mixed genotype, low-, and high-coverage sequence data from children and their parents. At presence of sequence errors, low-coverage parental sequence data may lead to lower power than parental genotype data. © 2017 WILEY PERIODICALS, INC.
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Marks, Clive A; Obendorf, David; Pereira, Filipe; Edwards, Ivo; Hall, Graham P
2014-08-01
Models used for resource allocation in eradication programmes must be based on replicated data of known quality and have proven predictive accuracy, or they may provide a false indication of species presence and/or distribution. In the absence of data corroborating the presence of extant foxes Vulpes vulpes in Tasmania, a habitat-specific model based upon mtDNA data (Sarre et al . 2012. Journal Applied Ecology , 50, 459-468) implied that foxes were widespread. Overall, 61 of 9940 (0·6%) surveyed scats were assigned as mtDNA fox positive by the fox eradication programme (FEP). We investigated the spatiotemporal distribution of the 61 mtDNA-assigned fox scats and modelled the probability of replicating scat detection in independent surveys using detection dogs based upon empirically derived probabilities of scat detection success obtained by the FEP using imported fox scats. In a prior mainland study, fox genotypes were recurrently detected in a consecutive four-day pool of scats. In Tasmania, only three contemporaneously collected scat pairs of unknown genotype were detected by the FEP within an area corresponding to a conservatively large mainland fox home range (639 ha) in a decade. Nearest neighbour pairs were widely spaced (mean = 7·0 km; circular area = 153 km 2 ) and generated after a mean of 281 days. The majority of assigned mtDNA positive scats were found in urban and peri-urban environments corresponding to small mainland fox home ranges (30-45 ha) that imply higher scat density and more certain replication. Using the lowest empirically determined scat detection success for dogs, the failure to replicate fox scat detection on 34 of 36 occasions in a large (639 ha) home range is highly improbable ( P = 0·00001) and suggestive of Type I error. Synthesis and applications . Type I error, which may have various sources, should be considered when scat mtDNA data are few, accumulated over many years, uncorroborated by observations of extant specimens, inadequately replicated in independent surveys within an expected spatiotemporal scale and reported in geographically isolated environments unlikely to have been colonized.
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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
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Genotyping and inflated type I error rate in genome-wide association case/control studies
Sampson, Joshua N; Zhao, Hongyu
2009-01-01
Background One common goal of a case/control genome wide association study (GWAS) is to find SNPs associated with a disease. Traditionally, the first step in such studies is to assign a genotype to each SNP in each subject, based on a statistic summarizing fluorescence measurements. When the distributions of the summary statistics are not well separated by genotype, the act of genotype assignment can lead to more potential problems than acknowledged by the literature. Results Specifically, we show that the proportions of each called genotype need not equal the true proportions in the population, even as the number of subjects grows infinitely large. The called genotypes for two subjects need not be independent, even when their true genotypes are independent. Consequently, p-values from tests of association can be anti-conservative, even when the distributions of the summary statistic for the cases and controls are identical. To address these problems, we propose two new tests designed to reduce the inflation in the type I error rate caused by these problems. The first algorithm, logiCALL, measures call quality by fully exploring the likelihood profile of intensity measurements, and the second algorithm avoids genotyping by using a likelihood ratio statistic. Conclusion Genotyping can introduce avoidable false positives in GWAS. PMID:19236714
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Improving Genomic Prediction in Cassava Field Experiments by Accounting for Interplot Competition
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
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Improving Genomic Prediction in Cassava Field Experiments by Accounting for Interplot Competition.
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.
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Ho, Lindsey A; Lange, Ethan M
2010-12-01
Genome-wide association (GWA) studies are a powerful approach for identifying novel genetic risk factors associated with human disease. A GWA study typically requires the inclusion of thousands of samples to have sufficient statistical power to detect single nucleotide polymorphisms that are associated with only modest increases in risk of disease given the heavy burden of a multiple test correction that is necessary to maintain valid statistical tests. Low statistical power and the high financial cost of performing a GWA study remains prohibitive for many scientific investigators anxious to perform such a study using their own samples. A number of remedies have been suggested to increase statistical power and decrease cost, including the utilization of free publicly available genotype data and multi-stage genotyping designs. Herein, we compare the statistical power and relative costs of alternative association study designs that use cases and screened controls to study designs that are based only on, or additionally include, free public control genotype data. We describe a novel replication-based two-stage study design, which uses free public control genotype data in the first stage and follow-up genotype data on case-matched controls in the second stage that preserves many of the advantages inherent when using only an epidemiologically matched set of controls. Specifically, we show that our proposed two-stage design can substantially increase statistical power and decrease cost of performing a GWA study while controlling the type-I error rate that can be inflated when using public controls due to differences in ancestry and batch genotype effects.
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Bilton, Timothy P.; Schofield, Matthew R.; Black, Michael A.; Chagné, David; Wilcox, Phillip L.; Dodds, Ken G.
2018-01-01
Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species’ genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology (e.g., genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander–Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. PMID:29487138
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Bilton, Timothy P; Schofield, Matthew R; Black, Michael A; Chagné, David; Wilcox, Phillip L; Dodds, Ken G
2018-05-01
Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species' genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology ( e.g. , genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander-Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. Copyright © 2018 Bilton et al.
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USDA-ARS?s Scientific Manuscript database
Genotyping-by-sequencing (GBS) provides an opportunity for fast and inexpensive generation of unbiased SNPs. However, due to its low coverage, GBS SNPs have a higher proportion of missing data and genotyping error associated with heterozygote undercalling than traditional genotyping platforms. These...
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Evaluating the Evidence for Transmission Distortion in Human Pedigrees
Meyer, Wynn K.; Arbeithuber, Barbara; Ober, Carole; Ebner, Thomas; Tiemann-Boege, Irene; Hudson, Richard R.; Przeworski, Molly
2012-01-01
Children of a heterozygous parent are expected to carry either allele with equal probability. Exceptions can occur, however, due to meiotic drive, competition among gametes, or viability selection, which we collectively term “transmission distortion” (TD). Although there are several well-characterized examples of these phenomena, their existence in humans remains unknown. We therefore performed a genome-wide scan for TD by applying the transmission disequilibrium test (TDT) genome-wide to three large sets of human pedigrees of European descent: the Framingham Heart Study (FHS), a founder population of European origin (HUTT), and a subset of the Autism Genetic Resource Exchange (AGRE). Genotyping error is an important confounder in this type of analysis. In FHS and HUTT, despite extensive quality control, we did not find sufficient evidence to exclude genotyping error in the strongest signals. In AGRE, however, many signals extended across multiple SNPs, a pattern highly unlikely to arise from genotyping error. We identified several candidate regions in this data set, notably a locus in 10q26.13 displaying a genome-wide significant TDT in combined female and male transmissions and a signature of recent positive selection, as well as a paternal TD signal in 6p21.1, the same region in which a significant TD signal was previously observed in 30 European males. Neither region replicated in FHS, however, and the paternal signal was not visible in sperm competition assays or as allelic imbalance in sperm. In maternal transmissions, we detected no strong signals near centromeres or telomeres, the regions predicted to be most susceptible to female-specific meiotic drive, but we found a significant enrichment of top signals among genes involved in cell junctions. These results illustrate both the potential benefits and the challenges of using the TDT to study transmission distortion and provide candidates for investigation in future studies. PMID:22377632
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Allelic-based gene-gene interaction associated with quantitative traits.
Jung, Jeesun; Sun, Bin; Kwon, Deukwoo; Koller, Daniel L; Foroud, Tatiana M
2009-05-01
Recent studies have shown that quantitative phenotypes may be influenced not only by multiple single nucleotide polymorphisms (SNPs) within a gene but also by the interaction between SNPs at unlinked genes. We propose a new statistical approach that can detect gene-gene interactions at the allelic level which contribute to the phenotypic variation in a quantitative trait. By testing for the association of allelic combinations at multiple unlinked loci with a quantitative trait, we can detect the SNP allelic interaction whether or not it can be detected as a main effect. Our proposed method assigns a score to unrelated subjects according to their allelic combination inferred from observed genotypes at two or more unlinked SNPs, and then tests for the association of the allelic score with a quantitative trait. To investigate the statistical properties of the proposed method, we performed a simulation study to estimate type I error rates and power and demonstrated that this allelic approach achieves greater power than the more commonly used genotypic approach to test for gene-gene interaction. As an example, the proposed method was applied to data obtained as part of a candidate gene study of sodium retention by the kidney. We found that this method detects an interaction between the calcium-sensing receptor gene (CaSR), the chloride channel gene (CLCNKB) and the Na, K, 2Cl cotransporter gene (CLC12A1) that contributes to variation in diastolic blood pressure.
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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.
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Holcomb, C L; Rastrou, M; Williams, T C; Goodridge, D; Lazaro, A M; Tilanus, M; Erlich, H A
2014-01-01
The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Constantinescu, Dario; Memmah, Mohamed-Mahmoud; Vercambre, Gilles; Génard, Michel; Baldazzi, Valentina; Causse, Mathilde; Albert, Elise; Brunel, Béatrice; Valsesia, Pierre; Bertin, Nadia
2016-01-01
Drought stress is a major abiotic stress threatening plant and crop productivity. In case of fleshy fruits, understanding mechanisms governing water and carbon accumulations and identifying genes, QTLs and phenotypes, that will enable trade-offs between fruit growth and quality under Water Deficit (WD) condition is a crucial challenge for breeders and growers. In the present work, 117 recombinant inbred lines of a population of Solanum lycopersicum were phenotyped under control and WD conditions. Plant water status, fruit growth and composition were measured and data were used to calibrate a process-based model describing water and carbon fluxes in a growing fruit as a function of plant and environment. Eight genotype-dependent model parameters were estimated using a multiobjective evolutionary algorithm in order to minimize the prediction errors of fruit dry and fresh mass throughout fruit development. WD increased the fruit dry matter content (up to 85%) and decreased its fresh weight (up to 60%), big fruit size genotypes being the most sensitive. The mean normalized root mean squared errors of the predictions ranged between 16–18% in the population. Variability in model genotypic parameters allowed us to explore diverse genetic strategies in response to WD. An interesting group of genotypes could be discriminated in which (i) the low loss of fresh mass under WD was associated with high active uptake of sugars and low value of the maximum cell wall extensibility, and (ii) the high dry matter content in control treatment (C) was associated with a slow decrease of mass flow. Using 501 SNP markers genotyped across the genome, a QTL analysis of model parameters allowed to detect three main QTLs related to xylem and phloem conductivities, on chromosomes 2, 4, and 8. The model was then applied to design ideotypes with high dry matter content in C condition and low fresh mass loss in WD condition. The ideotypes outperformed the RILs especially for large and medium fruit-size genotypes, by combining high pedicel conductance and high active uptake of sugars. Interestingly, five small fruit-size RILs were close to the selected ideotypes, and likely bear interesting traits and alleles for adaptation to WD. PMID:28018381
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Genotypic distribution of hepatitis C virus in voluntary blood donors of northern Thailand.
Jutavijittum, Prapan; Jiviriyawat, Yupa; Yousukh, Amnat; Pantip, Chansom; Maneekarn, Niwat; Toriyama, Kan
2009-05-01
The purpose of this study was to determine the prevalence and distribution of HCV genotypes among voluntary blood donors in northern Thailand. From 1998 to 2000, 167 serum samples which tested positive for anti-HCV antibodies in the screening of voluntary blood donors from 5 provinces in northern Thailand were selected for genotyping. Viral RNA was extracted from the sera. The core-E1 region of the HCV-RNA genome was amplified using a OneStep RT-PCR kit. The core-E1 amplicon was sequenced and the HCV genotype was assigned by comparing with the reference sequences available in the GenBank database. Of 167 anti-HCV positive serum samples, 126 (75.4%) contained HCV RNA as detected by PCR. HCV genotype 3 was the most predominant genotype (39.6%), of which 33.3% belonged to genotype 3a and 6.3% to 3b. Genotype 6 was detected in 31%, and genotype 1 was detected in 27.8%. Of the genotype 1 isolates, 14.3% were la, 12.7% were 1b, and 0.8% were 1c. Two HCV isolates detected in the present study were untypeable. About 75% of anti-HCV positive blood donors had chronic HCV infection. In northern Thailand, genotype 3a was the most predominant genotype, while genotype 6, 1a and 1b were also commonly found. The genotypic distribution of HCV isolates from various regions of Thailand were more or less similar. Nevertheless, in this study, the prevalence of HCV genotype 6 (31%) was higher than previously reported by others (8-18%). Phylogenetic analysis of the HCV isolates detected in the present study was also performed.
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Roon, David A.; Waits, L.P.; Kendall, K.C.
2005-01-01
Non-invasive genetic sampling (NGS) is becoming a popular tool for population estimation. However, multiple NGS studies have demonstrated that polymerase chain reaction (PCR) genotyping errors can bias demographic estimates. These errors can be detected by comprehensive data filters such as the multiple-tubes approach, but this approach is expensive and time consuming as it requires three to eight PCR replicates per locus. Thus, researchers have attempted to correct PCR errors in NGS datasets using non-comprehensive error checking methods, but these approaches have not been evaluated for reliability. We simulated NGS studies with and without PCR error and 'filtered' datasets using non-comprehensive approaches derived from published studies and calculated mark-recapture estimates using CAPTURE. In the absence of data-filtering, simulated error resulted in serious inflations in CAPTURE estimates; some estimates exceeded N by ??? 200%. When data filters were used, CAPTURE estimate reliability varied with per-locus error (E??). At E?? = 0.01, CAPTURE estimates from filtered data displayed < 5% deviance from error-free estimates. When E?? was 0.05 or 0.09, some CAPTURE estimates from filtered data displayed biases in excess of 10%. Biases were positive at high sampling intensities; negative biases were observed at low sampling intensities. We caution researchers against using non-comprehensive data filters in NGS studies, unless they can achieve baseline per-locus error rates below 0.05 and, ideally, near 0.01. However, we suggest that data filters can be combined with careful technique and thoughtful NGS study design to yield accurate demographic information. ?? 2005 The Zoological Society of London.
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Identification and Correction of Sample Mix-Ups in Expression Genetic Data: A Case Study
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
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Identification and Correction of Sample Mix-Ups in Expression Genetic Data: A Case Study.
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.
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Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.
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.
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Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009-2010.
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.
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Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009–2010
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
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Paz-García, David A; Munguía-Vega, Adrián; Plomozo-Lugo, Tomas; Weaver, Amy Hudson
2017-04-01
We developed a set of hypervariable microsatellite markers for the Pacific red snapper (Lutjanus peru), an economically important marine fish for small-scale fisheries in the west coast of Mexico. We performed shotgun genome sequencing with the 454 XL titanium chemistry and used bioinformatic tools to search for perfect microsatellite loci. We selected 66 primer pairs that were synthesized and genotyped in an ABI PRISM 3730XL DNA sequencer in 32 individuals from the Gulf of California. We estimated levels of genetic diversity, deviations from linkage and Hardy-Weinberg equilibrium, estimated the frequency of null alleles and the probability of individual identity for the new markers. We reanalyzed 16 loci in 16 individuals to estimate genotyping error rates. Eighteen loci failed to amplify, 16 loci were discarded due to unspecific amplifications and 32 loci (14 tetranucleotide and 18 dinucleotide) were successfully scored. The average number of alleles per locus was 21 (±6.87, SD) and ranged from 8 to 34. The average observed and expected heterozygosities were 0.787 (±0.144 SD, range 0.250-0.935) and 0.909 (±0.122 SD, range 0.381-0.965), respectively. No significant linkage was detected. Eight loci showed deviations from Hardy-Weinberg equilibrium, and from these, four loci showed moderate null allele frequencies (0.104-0.220). The probability of individual identity for the new loci was 1.46 -62 . Genotyping error rates averaged 9.58%. The new markers will be useful to investigate patterns of larval dispersal, metapopulation dynamics, fine-scale genetic structure and diversity aimed to inform the implementation of spatially explicit fisheries management strategies in the Gulf of California.
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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.
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Li, Caixia; Wang, Le; Tong, Hua; Ge, Yiping; Mei, Huan; Chen, Liangyu; Lv, Guixia; Liu, Weida
2016-08-01
To characterize the genotype distribution pattern of Candida albicans associated with vulvovaginal candidiasis (VVC) in Nanjing, China by microsatellite genotyping. A questionnaire was completed by each patient diagnosed with VVC. A total of 208 independent C. albicans was isolated from 208 patients. Microsatellite genotyping characterized the genotype distribution by analysis of the CAI locus marker. PCR of CAI fragments showed the three major genotypes contained 30:45, 21:21 and 32:46 alleles among the 51 genotypes detected, accounting for 29.3, 13.0 and 12.0 % of 208 clinical isolates. Genotype distributions had a similar pattern among different clinical presentations (P = 0.219). In both groups of the (21-30) and (31-40) years, 30:45 was the most frequent genotype allele detected. In the (21-30) year females, 16.5 % of the isolated strains had the genotype 21:21, while the same genotype in the group of (31-40) years was 6.9 %. Genotype distributions were significant difference between the pregnant and non-pregnant women (P < 0.001). 30:45 was detected only one in the 23 pregnant women. The results indicated a unique genotype distribution of C. albicans associated with VVC in Nanjing, eastern China and a different distribution pattern was also detected in pregnant women compared to non-pregnant women.
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Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa.
Forbi, Joseph C; Purdy, Michael A; Campo, David S; Vaughan, Gilberto; Dimitrova, Zoya E; Ganova-Raeva, Lilia M; Xia, Guo-Liang; Khudyakov, Yury E
2012-07-01
We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15% of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n=51; 85%) and genotype 2 (n=9; 15%). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n=43) and genotype 2 (n=6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200-300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.
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Performance evaluation of the Abbott RealTime HCV Genotype II for hepatitis C virus genotyping.
Sohn, Yong-Hak; Ko, Sun-Young; Kim, Myeong Hee; Oh, Heung-Bum
2010-04-01
The Abbott RealTime hepatitis C virus (HCV) Genotype II (Abbott Molecular Inc.) for HCV genotyping, which uses real-time PCR technology, has recently been developed. Accuracy and sensitivity of detection were assessed using the HCV RNA PHW202 performance panel (SeraCare Life Sciences). Consistency with restriction fragment mass polymorphism (RFMP) data, cross-reactivity with other viruses, and the ability to detect minor strains in mixtures of genotypes 1 and 2 were evaluated using clinical samples. All performance panel viruses were correctly genotyped at levels of >500 IU/mL. Results were 100% concordant with RFMP genotypic data (66/66). However, 5% (3/66) of the samples examined displayed probable genotypic cross reactivity. No cross reactivity with other viruses was evident. Minor strains in the mixtures were not effectively distinguished, even at quantities higher than the detection limit. The Abbott RealTime HCV Genotype II assay was very accurate and yielded results consistent with RFMP data. Although the assay has the advantages of automation and short turnaround time, we suggest that further improvements are necessary before it is used routinely in clinical practice. Efforts are needed to decrease cross reactivity among genotypes and to improve the ability to detect minor genotypes in mixed infections.
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Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates▿ †
Fang, Jie; Wichroski, Michael J.; Levine, Steven M.; Baldick, Carl J.; Mazzucco, Charles E.; Walsh, Ann W.; Kienzle, Bernadette K.; Rose, Ronald E.; Pokornowski, Kevin A.; Colonno, Richard J.; Tenney, Daniel J.
2009-01-01
Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy. PMID:19433559
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Detection and molecular characterization of Cryptosporidium and Eimeria species in Philippine bats.
Murakoshi, Fumi; Recuenco, Frances C; Omatsu, Tsutomu; Sano, Kaori; Taniguchi, Satoshi; Masangkay, Joseph S; Alviola, Philip; Eres, Eduardo; Cosico, Edison; Alvarez, James; Une, Yumi; Kyuwa, Shigeru; Sugiura, Yuki; Kato, Kentaro
2016-05-01
The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Bats are naturally infected with zoonotic pathogens; thus, they are potential reservoirs of parasites. We investigated the species and genotype distribution as well as prevalence of Cryptosporidium and Eimeria in Philippine bats. We captured and examined 45 bats; four were positive for Cryptosporidium spp. and seven were positive for Eimeria spp. We detected Cryptosporidium bat genotype II from Ptenochirus jagori. Three other Cryptosporidium sequences, detected from Rhinolophus inops, Cynopterus brachyotis, and Eonycteris spelaea, could not be classified as any known species or genotype; we therefore propose the novel genotype Cryptosporidium bat genotypes V, VI, and VII. Bat genotype V is associated with human cryptosporidiosis clade, and therefore, this genotype may be transmissible to humans. Among the Eimeria sequences, BE3 detected from Scotophilus kuhlii was classified with known bat and rodent clades; however, other sequences detected from C. brachyotis, E. spelaea, Rousettus amplexicaudatus, and R. inops could not be classified with known Eimeria species. These isolates might represent a new genotype. Our findings demonstrate that the bats of the Philippines represent a reservoir of multiple Cryptosporidium and Eimeria spp.
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Li, Yulong; Zhang, Rui; Peng, Rongxue; Ding, Jiansheng; Han, Yanxi; Wang, Guojing; Zhang, Kuo; Lin, Guigao; Li, Jinming
2016-06-01
Currently, several approaches are being used to detect echinoderm microtubule associated protein like 4 gene (EML4)-anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement, but the performance of laboratories in China is unknown. To evaluate the proficiency of different laboratories in detecting EML4-ALK rearrangement, we organized a proficiency test (PT). We prepared formalin-fixed, paraffin-embedded samples derived from the xenograft tumor tissue of three non-small cell lung cancer cell lines with different EML4-ALK rearrangements and used PTs to evaluate the detection performance of laboratories in China. We received results from 94 laboratories that used different methods. Of the participants, 75.53% correctly identified all samples in the PT panel. Among the errors made by participants, false-negative errors were likely to occur. According to the methodology applied, 82.86%, 76.67%, 77.78%, and 66.67% of laboratories using reverse transcriptase polymerase chain reaction, fluorescence in situ hybridization, next-generation sequencing, and immunohistochemical analysis, respectively, could analyze all the samples correctly. Moreover, we have found that the laboratories' genotyping capacity is high, especially for variant 3. Our PT survey revealed that the performance and methodological problems of laboratories must be addressed to further increase the reproducibility and accuracy of detection of EML4-ALK rearrangement to ensure reliable results for selection of appropriate patients. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.
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Fast imputation using medium- or low-coverage sequence data
USDA-ARS?s Scientific Manuscript database
Direct imputation from raw sequence reads can be more accurate than calling genotypes first and then imputing, especially if read depth is low or error rates high, but different imputation strategies are required than those used for data from genotyping chips. A fast algorithm to impute from lower t...
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Zhang, Xiaoyan; Peng, Yanmei; Wang, Ying; Zhang, Zongying; Li, Dawei; Yu, Jialin; Han, Chenggui
2016-11-22
Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV. In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China. The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.
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[Analysis of free foetal DNA in maternal plasma using STR loci].
Vodicka, R; Vrtel, R; Procházka, M; Santavá, A; Dusek, L; Vrbická, D; Singh, R; Krejciríková, E; Schneiderová, E; Santavý, J
2006-01-01
Problems of maternal and foetal genotype differentiation of maternal plasma in pregnant women are solved generally by real-time systems. In this case the specific probes are used to distinguish particular genotype. Mostly gonosomal sequences are utilised to recognise the male foetus. This work describes possibilities in free foetal DNA detection and quantification by STR. Artificial genotype mixtures ranging from 0,2 % to 100 % to simulate maternal and paternal genotypes and 27 DNA samples from pregnant women in different stage of pregnancy were used for DNA quantification and detection. Foetal genotype was confirmed by biological father genotyping. The detection was performed in STR from 21st chromosome Down syndrome (DS) responsible region by innovated (I) QF PCR which allows to reveal and quantify even very rare DNA mosaics. The STR quantification was assessed in artificial mixtures of genotypes and discriminability of particular genotypes was on the level of few percent. Foetal DNA was detected in 74 % of tested samples. The IQF PCR application in quantification and differentiation between maternal and foetal genotypes by STR loci could have importance in non-invasive prenatal diagnostics as another possible marker for DS risk assessment.
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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
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Jun, Goo; Flickinger, Matthew; Hetrick, Kurt N.; Romm, Jane M.; Doheny, Kimberly F.; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min
2012-01-01
DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies. PMID:23103226
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RT-PCR for detection of all seven genotypes of Lyssavirus genus.
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.
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Oddou-Muratorio, S; Houot, M-L; Demesure-Musch, B; Austerlitz, F
2003-12-01
The joint development of polymorphic molecular markers and paternity analysis methods provides new approaches to investigate ongoing patterns of pollen flow in natural plant populations. However, paternity studies are hindered by false paternity assignment and the nondetection of true fathers. To gauge the risk of these two types of errors, we performed a simulation study to investigate the impact on paternity analysis of: (i) the assumed values for the size of the breeding male population (NBMP), and (ii) the rate of scoring error in genotype assessment. Our simulations were based on microsatellite data obtained from a natural population of the entomophilous wild service tree, Sorbus torminalis (L.) Crantz. We show that an accurate estimate of NBMP is required to minimize both types of errors, and we assess the reliability of a technique used to estimate NBMP based on parent-offspring genetic data. We then show that scoring errors in genotype assessment only slightly affect the assessment of paternity relationships, and conclude that it is generally better to neglect the scoring error rate in paternity analyses within a nonisolated population.
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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
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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
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Qu, Conghui; Schuetz, Johanna M.; Min, Jeong Eun; Leach, Stephen; Daley, Denise; Spinelli, John J.; Brooks-Wilson, Angela; Graham, Jinko
2011-01-01
We describe a statistical approach to predict gender-labeling errors in candidate-gene association studies, when Y-chromosome markers have not been included in the genotyping set. The approach adds value to methods that consider only the heterozygosity of X-chromosome SNPs, by incorporating available information about the intensity of X-chromosome SNPs in candidate genes relative to autosomal SNPs from the same individual. To our knowledge, no published methods formalize a framework in which heterozygosity and relative intensity are simultaneously taken into account. Our method offers the advantage that, in the genotyping set, no additional space is required beyond that already assigned to X-chromosome SNPs in the candidate genes. We also show how the predictions can be used in a two-phase sampling design to estimate the gender-labeling error rates for an entire study, at a fraction of the cost of a conventional design. PMID:22303327
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Irías-Mata, Andrea; Stuetz, Wolfgang; Sus, Nadine; Hammann, Simon; Gralla, Katrin; Cordero-Solano, Aracelly; Vetter, Walter; Frank, Jan
2017-08-30
Palm oil is one of the richest sources of tocotrienols and may contain other non-tocopherol vitamin E congeners. The vitamin E profiles of fully ripened fruit mesocarp of three Elaeis guineensis, two Elaeis oleifera, and one hybrid O × G palm fruit genotypes from Costa Rica were analyzed by high-performance liquid chromatography with fluorescence detection and gas chromatography-mass spectrometry after mechanical extraction by a screw press and chemical extraction with hexane. γ-Tocotrienol, α-tocotrienol, and α-tocopherol were the most abundant tocochromanols, while other tocopherols (β-tocopherol, γ-tocopherol, and δ-tocopherol) and α-tocomonoenol were detected at minor concentrations. Significant differences in vitamin E profiles between genotypes were observed, and the variety E. oleifera Quepos (CB9204) had by far the highest content of total tocotrienols (890 μg/g of oil) and total vitamin E (892 μg/g of oil). Chemical extraction with hexane afforded up to 2.5-fold higher vitamin E yields than screw press extraction. α-Tocomonoenol co-eluted with γ-tocopherol in reversed-phase high-performance liquid chromatography analyses and is a possible source of error in the quantification of γ-tocopherol in foods.
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Ahmed, Farhan; El-Kadiki, Alia; Gibbons, Stephen
2017-06-01
Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia.
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AMPLISAS: a web server for multilocus genotyping using next-generation amplicon sequencing data.
Sebastian, Alvaro; Herdegen, Magdalena; Migalska, Magdalena; Radwan, Jacek
2016-03-01
Next-generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus-specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post-processing of NGS data. Amplicon Sequence Assignment (AMPLISAS) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. AMPLISAS is designed as a three-step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. AMPLISAS performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies. © 2015 John Wiley & Sons Ltd.
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Papadakis, Georgina; Chibo, Doris; Druce, Julian; Catton, Michael; Birch, Chris
2014-09-01
Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples. © 2014 Wiley Periodicals, Inc.
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A fast least-squares algorithm for population inference
2013-01-01
Background Population inference is an important problem in genetics used to remove population stratification in genome-wide association studies and to detect migration patterns or shared ancestry. An individual’s genotype can be modeled as a probabilistic function of ancestral population memberships, Q, and the allele frequencies in those populations, P. The parameters, P and Q, of this binomial likelihood model can be inferred using slow sampling methods such as Markov Chain Monte Carlo methods or faster gradient based approaches such as sequential quadratic programming. This paper proposes a least-squares simplification of the binomial likelihood model motivated by a Euclidean interpretation of the genotype feature space. This results in a faster algorithm that easily incorporates the degree of admixture within the sample of individuals and improves estimates without requiring trial-and-error tuning. Results We show that the expected value of the least-squares solution across all possible genotype datasets is equal to the true solution when part of the problem has been solved, and that the variance of the solution approaches zero as its size increases. The Least-squares algorithm performs nearly as well as Admixture for these theoretical scenarios. We compare least-squares, Admixture, and FRAPPE for a variety of problem sizes and difficulties. For particularly hard problems with a large number of populations, small number of samples, or greater degree of admixture, least-squares performs better than the other methods. On simulated mixtures of real population allele frequencies from the HapMap project, Admixture estimates sparsely mixed individuals better than Least-squares. The least-squares approach, however, performs within 1.5% of the Admixture error. On individual genotypes from the HapMap project, Admixture and least-squares perform qualitatively similarly and within 1.2% of each other. Significantly, the least-squares approach nearly always converges 1.5- to 6-times faster. Conclusions The computational advantage of the least-squares approach along with its good estimation performance warrants further research, especially for very large datasets. As problem sizes increase, the difference in estimation performance between all algorithms decreases. In addition, when prior information is known, the least-squares approach easily incorporates the expected degree of admixture to improve the estimate. PMID:23343408
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A fast least-squares algorithm for population inference.
Parry, R Mitchell; Wang, May D
2013-01-23
Population inference is an important problem in genetics used to remove population stratification in genome-wide association studies and to detect migration patterns or shared ancestry. An individual's genotype can be modeled as a probabilistic function of ancestral population memberships, Q, and the allele frequencies in those populations, P. The parameters, P and Q, of this binomial likelihood model can be inferred using slow sampling methods such as Markov Chain Monte Carlo methods or faster gradient based approaches such as sequential quadratic programming. This paper proposes a least-squares simplification of the binomial likelihood model motivated by a Euclidean interpretation of the genotype feature space. This results in a faster algorithm that easily incorporates the degree of admixture within the sample of individuals and improves estimates without requiring trial-and-error tuning. We show that the expected value of the least-squares solution across all possible genotype datasets is equal to the true solution when part of the problem has been solved, and that the variance of the solution approaches zero as its size increases. The Least-squares algorithm performs nearly as well as Admixture for these theoretical scenarios. We compare least-squares, Admixture, and FRAPPE for a variety of problem sizes and difficulties. For particularly hard problems with a large number of populations, small number of samples, or greater degree of admixture, least-squares performs better than the other methods. On simulated mixtures of real population allele frequencies from the HapMap project, Admixture estimates sparsely mixed individuals better than Least-squares. The least-squares approach, however, performs within 1.5% of the Admixture error. On individual genotypes from the HapMap project, Admixture and least-squares perform qualitatively similarly and within 1.2% of each other. Significantly, the least-squares approach nearly always converges 1.5- to 6-times faster. The computational advantage of the least-squares approach along with its good estimation performance warrants further research, especially for very large datasets. As problem sizes increase, the difference in estimation performance between all algorithms decreases. In addition, when prior information is known, the least-squares approach easily incorporates the expected degree of admixture to improve the estimate.
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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.
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Ambreen, Fareeha; Ismail, Muhammad; Qureshi, Irfan Zia
2015-01-01
To study the association of serum levels of inflammatory mediators and angiogenic factors with genetic polymorphism in Pakistani age-related macular degeneration (AMD) patients. This was a cross-sectional and case-control study that included 90 AMD patients diagnosed through slit-lamp examination, fundoscopy, and ocular coherence tomography. For reference and comparison purposes, 100 healthy age-matched subjects (controls) were also recruited. IL-6, IL-8, VEGF, and CRP levels were estimated in the serum samples of patients and control subjects. Using restriction fragment length polymorphism, single nucleotide polymorphisms were studied in IL-6 (rs1800795, rs1800796, rs1800797), IL-8 (rs4073, rs2227306, rs2227543), VEGF (rs3025039, rs699947), and CRP genes (rs1205, rs1130864). Since the data were obtained from a sample population, the Box-Cox transformation algorithm was applied to reduce heterogeneity of error. Multivariate analyses of variance (M-ANOVA) were applied on the transformed data to investigate the association of serum levels of IL-6, IL-8, VEGF, and CRP with AMD. Genotype and allele frequencies were compared through χ(2) tests applying Hardy-Weinberg equilibrium. The serum concentrations of IL-6 and IL-8, VEGF, and CRP between homozygotes and heterozygotes were compared through one-way ANOVA. Significance level was p<0.05. Compared to control subjects, serum IL-6 (p<0.0001), IL-8 (p<0.0001), VEGF (p<0.0001), and CRP (p<0.0001) levels were significantly elevated in the AMD patients. For rs1800795, patients with the GG genotype showed significantly raised levels of IL-6 compared to those with GC and CC genotypes (p<0.0001). Serum IL-8 levels were significantly higher in patients with the GG genotype compared to the GC and CC genotypes for the single nucleotide polymorphism (SNP) rs2227543 (p<0.002). Similarly, significantly higher VEGF levels were detected for genotype TT for rs3025039 SNP (p<0.038). However, no significant alteration in serum CRP levels was detected in hetero- or homozygotes for rs1205 and rs1130864 SNPs. Serum IL-6, IL-8, and VEGF levels are substantially increased in AMD, and the levels coincide with polymorphism in the respective gene. No such relationship appears to exist with regard to SNPs of CRP.
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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.
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Genotype calling from next-generation sequencing data using haplotype information of reads
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
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Assumption-free estimation of the genetic contribution to refractive error across childhood.
Guggenheim, Jeremy A; St Pourcain, Beate; McMahon, George; Timpson, Nicholas J; Evans, David M; Williams, Cathy
2015-01-01
Studies in relatives have generally yielded high heritability estimates for refractive error: twins 75-90%, families 15-70%. However, because related individuals often share a common environment, these estimates are inflated (via misallocation of unique/common environment variance). We calculated a lower-bound heritability estimate for refractive error free from such bias. Between the ages 7 and 15 years, participants in the Avon Longitudinal Study of Parents and Children (ALSPAC) underwent non-cycloplegic autorefraction at regular research clinics. At each age, an estimate of the variance in refractive error explained by single nucleotide polymorphism (SNP) genetic variants was calculated using genome-wide complex trait analysis (GCTA) using high-density genome-wide SNP genotype information (minimum N at each age=3,404). The variance in refractive error explained by the SNPs ("SNP heritability") was stable over childhood: Across age 7-15 years, SNP heritability averaged 0.28 (SE=0.08, p<0.001). The genetic correlation for refractive error between visits varied from 0.77 to 1.00 (all p<0.001) demonstrating that a common set of SNPs was responsible for the genetic contribution to refractive error across this period of childhood. Simulations suggested lack of cycloplegia during autorefraction led to a small underestimation of SNP heritability (adjusted SNP heritability=0.35; SE=0.09). To put these results in context, the variance in refractive error explained (or predicted) by the time participants spent outdoors was <0.005 and by the time spent reading was <0.01, based on a parental questionnaire completed when the child was aged 8-9 years old. Genetic variation captured by common SNPs explained approximately 35% of the variation in refractive error between unrelated subjects. This value sets an upper limit for predicting refractive error using existing SNP genotyping arrays, although higher-density genotyping in larger samples and inclusion of interaction effects is expected to raise this figure toward twin- and family-based heritability estimates. The same SNPs influenced refractive error across much of childhood. Notwithstanding the strong evidence of association between time outdoors and myopia, and time reading and myopia, less than 1% of the variance in myopia at age 15 was explained by crude measures of these two risk factors, indicating that their effects may be limited, at least when averaged over the whole population.
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Janani, Madhuravasal Krishnan; Malathi, Jambulingam; Biswas, Jyothirmay; Sridharan, Sudharshan; Madhavan, Hajib Naraharirao
2015-01-01
To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples.
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Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data
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
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Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data.
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.
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AB014. Beta-ketothiolase deficiency: phenotype, genotype and outcome of 48 Vietnamese patients
Nguyen, Khanh Ngoc; Nguyen, Hoan Thi; Can, Ngoc Thi Bich; Do, Mai Thi Thanh; Bui, Thao Phuong; Fukao, Toshiyuki; Vu, Dung Chi
2017-01-01
Background Beta-ketothiolase deficiency (BKT) is an inherited metabolic disease of isoleucine and ketone body caused by mutations in the T2 gene. It is a rare disease with over 100 patients reported worldwide. We aimed to describe phenotypes and genotypes and to evaluate outcomes of Vietnamese patients with BKT. Methods Patients who were diagnosed with BKT, and followed up at National Children Hospital from January 2015 to June 2017 were enrolled. Results Forty-eight patients from 40 different and unrelated families were diagnosed through high risk screening in Vietnam. Forty-six patients (96%) presented with acute episodes of intermittent ketotic acidosis (pH <7.1, increased anion gap), and were asymptomatic between episodes. Ages of onset were between 6 and 18 months. Characteristics of metabolic chemistry revealed elevated urinary 2-methylacetoacetate, 2-methyl-3-hydroxybutyrate, tiglylglycine, and plasma C5:1 and C5:OH carnitines. We identified 8 different mutations with 9 kinds of genotypes. The common mutations of T2 gene were p.R208X and IVS10-1g>c (85%). Five novel mutations were identified (IVS10-1g>c, c.1032_1033insA, p.S284N, exon 6 -11del, and c.163_167delinsAA). Eight out of nine genotypes were null mutations. There was no correlation between genotypes and phenotypes. The outcome was good in most patients with 83% had complete recovery, 7% mental consequences, and 12% death. All patients had normal growth rate according to growth chart by World Health Organization (WHO) 2007. Conclusions BKT is a common inborn error of metabolism in Vietnam with good outcome in most patients. A newborn screening program for BKT may have a high detection rate in Vietnam.
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Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo
2016-01-01
Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.
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Mohamed Saini, Suriati; Muhamad Radzi, Azizah; Abdul Rahman, Abdul Hamid
2012-06-01
The serotonin transporter promoter (5-HTTLPR) is a potential susceptibility locus in the pathogenesis of major depressive disorder. However, data from Malaysia is lacking. The present study aimed to determine the association between the homozygous short variant of the serotonin transporter promoter gene (5-HTTLPR) with major depressive disorder. This is a candidate gene case-control association study. The sample consists of 55 major depressive disorder probands and 66 controls. They were Malaysian descents and were unrelated. The Axis I diagnosis was determined using Mini International Neuropsychiatric Interview (M.I.N.I.). The control group comprised healthy volunteers without personal psychiatric history and family history of mood disorders. Participants' blood was sent to the Institute Medical Research for genotyping. The present study failed to detect an association between 5-HTTLPR ss genotype with major depressive disorder (χ(2) = 3.67, d.f. = 1, P = 0.055, odds ratio 0.25, 95% confidence interval = 0.07-1.94). Sub-analysis revealed that the frequency of l allele in healthy controls was higher (78.0%) than that of Caucasian and East Asian population. However, in view of the small sample size this study may be prone to type II error (and type I error). This preliminary study suggests that the homozygous short variant of the 5-HTTLPR did not appear to be a risk factor for increasing susceptibility to major depressive disorder. Copyright © 2012 Blackwell Publishing Asia Pty Ltd.
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Re-Ranking Sequencing Variants in the Post-GWAS Era for Accurate Causal Variant Identification
Faye, Laura L.; Machiela, Mitchell J.; Kraft, Peter; Bull, Shelley B.; Sun, Lei
2013-01-01
Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website. PMID:23950724
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Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus.
Letellier, C; Kerkhofs, P
2003-12-01
Since two genotypes of bovine viral diarrhea viruses (BVDV) occur in Belgian herds, their differentiation is important for disease surveillance. A quantitative real-time PCR assay was developed to detect and classify bovine viral diarrhea viruses in genotype I and II. A pair of primers specific for highly conserved regions of the 5'UTR and two TaqMan probes were designed. The FAM and VIC-labeled probe sequences differed by three nucleotides, allowing the differentiation between genotype I and II. The assay detectability of genotype I and II real-time PCR assay was 1000 and 100 copies, respectively. Highly reproducible data were obtained as the coefficients of variation of threshold cycle values in inter-runs were less than 2.2%. The correct classification of genotype I and II viruses was assessed by using reference strains and characterized field isolates of both genotypes. The application to clinical diagnosis was evaluated on pooled blood samples by post run measurement of the FAM- and VIC-associated fluorescence. The 100% agreement with the conventional RT-PCR method confirmed that this new technique could be used for routine detection of persistently infected immunotolerant animals.
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Flores-Miramontes, María Guadalupe; Torres-Reyes, Luis Alberto; Alvarado-Ruíz, Liliana; Romero-Martínez, Salvador Angel; Ramírez-Rodríguez, Verenice; Balderas-Peña, Luz María Adriana; Vallejo-Ruíz, Verónica; Piña-Sánchez, Patricia; Cortés-Gutiérrez, Elva Irene; Jave-Suárez, Luis Felipe; Aguilar-Lemarroy, Adriana
2015-10-06
The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS. Two hundred thirty three cervical samples from women Without cervical lesions (WCL, n = 48), with Cervical intraepithelial neoplasia grade 1 (CIN I, n = 98), or with Cervical cancer (CC, n = 87) were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples. We identified 35 HPV genotypes, among which 30 were identified by both technologies; in addition, the HPV genotypes 32, 44, 74, 102 and 114 were detected by NGS. These latter genotypes, to our knowledge, have not been previously reported in Mexican population. Furthermore, we found that LA did not detect, in some diagnosis groups, certain HPV genotypes included in the test, such as 6, 11, 16, 26, 35, 51, 58, 68, 73, and 89, which indicates possible variations at the species level. There are HPV genotypes in Mexican population that cannot be detected by LA, which is, at present, the most complete commercial genotyping test. More studies are necessary to determine the impact of HPV-44, 74, 102 and 114 on the risk of developing CC. A greater number of samples must be analyzed by NGS for the most accurate determination of Mexican HPV variants.
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Gonzaga, Rosa Maria S.; Rodart, Itatiana F.; Reis, Mitermayer Galvão; Ramalho Neto, Cícero Eduardo; Silva, Denise Wanderlei
2008-01-01
We determined the frequency of hepatitis C virus (HCV) genotypes in anti-HCV seropositive patients in the state of Alagoas, Brazil, by means of nested-reverse transcription-polymerase chain reaction (RT-nested-PCR) followed by restriction fragment length polymorphism (RFLP) of amplified fragments of the 5´NCR. The nested-PCR with genotype-specific primers from the core region was carried out when detection was not possible by the first approach. Detectable HCV-RNA was present in 115 (74.7%) of 154 serum samples. Genotype 1 was the most frequent (77.4%), against 20.9% of genotype 3 and 0.8% of genotype 2. Subtype 1b was predominant (65.2%), followed by subtypes 1a (8.7%), and 3a (6.1%). Coinfection (1a/3a) was detected in 0.8% of the samples. Indeed, there was no significant differences in the prevalence of genotype 1 compared to what has been obtained from anti-HCV seropositive patients from other locations in Brazil. Here we report for the first time the genotype 2 in the state of Alagoas. PMID:24031281
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Santos, Paula Fernanda Gonçalves Dos; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M
2017-06-01
To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.
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Height, weight, body mass index and ocular biometry in patients with sickle cell disease.
Osuobeni, Ebi Peter; Okpala, Iheanyi; Williamson, Tom H; Thomas, Peter
2009-03-01
To investigate the effects of physical size on refractive error and the dimensions of optical components in sickle cell disease (SCD). The design was cross sectional. Height and weight of adult patients suffering from SCD were measured, and body mass index (BMI) was calculated. Anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD) and axial length (AL) were measured using A-scan ultrasonography. Corneal radius of curvature (CR) was measured using a keratometer. Non-cycloplegic refractive error was determined subjectively. Subjects with SC genotype were significantly taller than those with SS genotype. In the unadjusted data, height was correlated with VCD [p = 0.02, 0.44 mm deeper per 10 cm increase in height, 95% CI (0.65, 8.25)] and AL [p = 0.03, 0.42 mm longer for every 10 cm increase in height, 95%CI (0.49, 7.99)]. The relationship between height, VCD and AL was absent after adjustment for age, gender, genotype and weight. BMI (kg m(-2)) was correlated with AL/CR ratio in both unadjusted (p = 0.04, -0.10 decrease per 1 kg m(-2), 95% CI (-0.018, -0.001) and adjusted data (p = 0.05, -0.10 decrease per 10 kg m(-2), 95% CI (-0.0189, 0.0001). Refractive error was not related to height, weight or BMI. Physical size does not affect refractive error or optical components in adult patients with SCD.
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Konietschke, Frank; Libiger, Ondrej; Hothorn, Ludwig A
2012-01-01
Statistical association between a single nucleotide polymorphism (SNP) genotype and a quantitative trait in genome-wide association studies is usually assessed using a linear regression model, or, in the case of non-normally distributed trait values, using the Kruskal-Wallis test. While linear regression models assume an additive mode of inheritance via equi-distant genotype scores, Kruskal-Wallis test merely tests global differences in trait values associated with the three genotype groups. Both approaches thus exhibit suboptimal power when the underlying inheritance mode is dominant or recessive. Furthermore, these tests do not perform well in the common situations when only a few trait values are available in a rare genotype category (disbalance), or when the values associated with the three genotype categories exhibit unequal variance (variance heterogeneity). We propose a maximum test based on Marcus-type multiple contrast test for relative effect sizes. This test allows model-specific testing of either dominant, additive or recessive mode of inheritance, and it is robust against variance heterogeneity. We show how to obtain mode-specific simultaneous confidence intervals for the relative effect sizes to aid in interpreting the biological relevance of the results. Further, we discuss the use of a related all-pairwise comparisons contrast test with range preserving confidence intervals as an alternative to Kruskal-Wallis heterogeneity test. We applied the proposed maximum test to the Bogalusa Heart Study dataset, and gained a remarkable increase in the power to detect association, particularly for rare genotypes. Our simulation study also demonstrated that the proposed non-parametric tests control family-wise error rate in the presence of non-normality and variance heterogeneity contrary to the standard parametric approaches. We provide a publicly available R library nparcomp that can be used to estimate simultaneous confidence intervals or compatible multiplicity-adjusted p-values associated with the proposed maximum test.
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Padilla-España, Laura; Repiso-Jiménez, Juan Bosco; Fernández-Sánchez, Fernando; Pereda, Teresa; Rivas-Ruiz, Francisco; Fernández-Morano, Teresa; de la Torre-Lima, Javier; Palma, Fermín; Redondo, Maximino; de Troya-Martín, Magdalena
2016-01-01
The incidence of high-grade anal intraepithelial neoplasia (HGAIN) -with an aetiological based on high-risk types of human papillomavirus- is increasing in some high-risk groups. Screening for HGAIN includes routine anal cytology and, more recently, HPV genotyping. The main objective of this study was to determine the sensitivity and specificity of anal cytology and HPV genotyping for the detection of HGAIN. This is a study to determine the correlation of cytological and microbiological findings with anal biopsy findings in a cohort of patients at high risk of developing AIN referred to the department of sexually transmitted infections of the Hospital Costa del Sol, Spain, between January 2008 and December 2014. Of the 151 patients subjected to screening, a total of 92 patients, all of them with the result of three screening test (anal cytology, genotyping and biopsy) were included in the study. Just under two-thirds (62%) of them were HIV-positive. The sensitivity and specificity of anal cytology to detect HGAIN were 52.8 and 85.7%, respectively (k: 0.328), and 78 and 62.8% to detect two or more HPV oncogenic genotypes (k: 0.417). The detection of oncogenic HPV genotypes allowed the identification of 23 new cases of HGAIN that had been underdiagnosed with anal cytology, with 14 cases containing at least three high-risk genotypes. Anal cytology did not show enough sensitivity in HGAIN screening. HPV genotyping has shown to be a useful tool to detect HGAIN cases, although it could lead to an over-diagnosis as a solitary screening procedure. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.
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Deans, Zandra C; Tull, Justyna; Beighton, Gemma; Abbs, Stephen; Robinson, David O; Butler, Rachel
2011-11-01
Laboratories are increasingly required to perform molecular tests for the detection of mutations in the KRAS gene in metastatic colorectal cancers to allow better clinical management and more effective treatment for these patients. KRAS mutation status predicts a patient's likely response to the monoclonal antibody cetuximab. To provide a high standard of service, these laboratories require external quality assessment (EQA) to monitor the level of laboratory output and measure the performance of the laboratory against other service providers. National External Quality Assurance Services for Molecular Genetics provided a pilot EQA scheme for KRAS molecular analysis in metastatic colorectal cancers during 2009. Very few genotyping errors were reported by participating laboratories; however, the reporting nomenclature of the genotyping results varied considerably between laboratories. The pilot EQA scheme highlighted the need for continuing EQA in this field which will assess the laboratories' ability not only to obtain accurate, reliable results but also to interpret them safely and correctly ensuring that the referring clinician has the correct information to make the best clinical therapeutic decision for their patient.
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Testing Gene-Gene Interactions in the Case-Parents Design
Yu, Zhaoxia
2011-01-01
The case-parents design has been widely used to detect genetic associations as it can prevent spurious association that could occur in population-based designs. When examining the effect of an individual genetic locus on a disease, logistic regressions developed by conditioning on parental genotypes provide complete protection from spurious association caused by population stratification. However, when testing gene-gene interactions, it is unknown whether conditional logistic regressions are still robust. Here we evaluate the robustness and efficiency of several gene-gene interaction tests that are derived from conditional logistic regressions. We found that in the presence of SNP genotype correlation due to population stratification or linkage disequilibrium, tests with incorrectly specified main-genetic-effect models can lead to inflated type I error rates. We also found that a test with fully flexible main genetic effects always maintains correct test size and its robustness can be achieved with negligible sacrifice of its power. When testing gene-gene interactions is the focus, the test allowing fully flexible main effects is recommended to be used. PMID:21778736
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Bremer, James; Bertagnolio, Silvia
2012-01-01
The World Health Organization (WHO) has developed a global laboratory network to support human immunodeficiency virus drug resistance genotyping for public health surveillance in resource-limited countries. Blinded proficiency panels are an essential part of a genotyping quality-assurance program and are used to monitor the reliability of genotyping data in the WHO laboratory network. Laboratories in Europe, North America, Asia, Africa, and the Caribbean have tested panels annually since 2007; 103 of 131 submissions (79%) had >99% nucleotide sequence identity and resistance mutation concordance, compared with consensus. Most errors were associated with mixtures in the test specimen, leading to subjectivity in base-calling or amplification bias. Overall, genotyping assays used by the WHO laboratory network are reliable. PMID:22544186
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Cooper, K.; Huang, F. F.; Batista, L.; Rayo, C. D.; Bezanilla, J. C.; Toth, T. E.; Meng, X. J.
2005-01-01
Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. Increasing evidence indicates that hepatitis E is a zoonotic disease. There exist four major genotypes of HEV, and HEV isolates identified in samples from pigs belong to either genotype 3 or 4. Genotype 1 and 2 HEVs are found exclusively in humans. To determine whether genotype 1 and 2 HEVs also exist in pigs, a universal reverse transcription-PCR assay that is capable of detecting all four HEV genotypes was used to test for the presence of HEV RNA in serum and/or fecal samples from pigs in Thailand, where genotype 1 human HEV is prevalent, and from pigs in Mexico, where genotype 2 human HEV was epidemic. In Thailand, swine HEV RNA was detected in sera from 10/26 pigs of 2 to 4 months of age but not in sera from 50 pigs of other ages. In Mexico, swine HEV RNA was detected in 8/125 sera and 28/92 fecal samples from 2- to 4-month-old pigs. Antibodies to swine HEV were also detected in about 81% of the Mexican pigs. A total of 44 swine HEV isolates were sequenced for the open reading frame 2 gene region. Sequence analyses revealed that all swine HEV isolates identified in samples from pigs in Thailand and Mexico belong to genotype 3. Phylogenetic analyses revealed that minor branches associated with geographic origin exist among the swine HEV isolates. The results indicated that genotype 1 or 2 swine HEV does not exist in pigs from countries where the respective human HEV genotype 1 or 2 is prevalent. It is likely that only genotype 3 and 4 HEV strains have zoonotic potential. PMID:15814985
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Identity-by-Descent-Based Phasing and Imputation in Founder Populations Using Graphical Models
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
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Qiu, Ping; Stevens, Richard; Wei, Bo; Lahser, Fred; Howe, Anita Y. M.; Klappenbach, Joel A.; Marton, Matthew J.
2015-01-01
Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection. PMID:25830316
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dos Ramos Farías, María Sol; Picconi, María Alejandra; Garcia, María Noé; González, Joaquín V; Basiletti, Jorge; Pando, María de los Ángeles; Avila, María Mercedes
2011-06-01
Reports on the prevalence and genotypes of HPV among trans (male to female transvestites, transsexuals or transgender) sex workers (TSW) are scarce in the literature. The aim of the study was to determine the infecting HPV genotypes among TSW in Argentina. 119 TSW were recruited. Anal cells were self collected with a cytobrush. HPV DNA detection was carried out by PCR and genotyping was performed by RLB. HPV prevalence was 97.4%. 103/111 HPV positive samples were genotyped. High risk genotypes were detected in 82.5%. Two or more coinfecting HPV genotypes were found in 70.9%. One case showed up to 10 different coinfecting types. The number of genotypes was not related to condom usage. Infection rates were similar for HIV positive (100%) and HIV negative (95.8%) participants. However, 18.8% of HIV negative had 4-9 different genotypes, while among HIV positive this percentage raised to 46.2% (p=0.006). Prevalence of high risk genotypes and the frequency of each high risk type were similar between HIV positive and HIV negative groups. According to the participants' answers HIV status showed no association with condom usage. The high HPV prevalence, the coinfection with multiple genotypes and the high frequency of high risk genotypes detected, together with a situation of extreme social marginalization, discrimination and stigmatization make this population to be of extreme vulnerability. Copyright © 2011 Elsevier B.V. All rights reserved.
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Jothikumar, Narayanan; Cromeans, Theresa L; Robertson, Betty H; Meng, X J; Hill, Vincent R
2006-01-01
Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.
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Detection of G1 genotype of human cystic echinococcosis in Egypt.
Abd El Baki, Mohammad H; El Missiry, Adel M G; Abd El Aaty, Heba E M; Mohamad, Anhar A; Aminou, Heba A R
2009-12-01
The first trial to detect G1 genotype in Egyptian human isolates of hydatid cysts (HC) and serum samples to approach diagnosis of cystic echinococcosis (CE) using human sera by PCR. Using strain specific primers, 27/36 confirmed CE patients (75%) showed G1 specific band in their sera at 254 bp. Specificity was 100% without detecting bands for either other parasitosis, or mass occupying lesions. Using PCR, G1 genotype was detected in 83.3% of HC samples, without significant difference between types of human isolates (pulmonary, hepatic, or multi-organ). G1 genotype detection in human sera was in 75% of CE patients compared to 83.3% in HC samples of the same group of patients proved satisfactory, simple and safer than HCF sampling. IHAT gave sensitivity of 58.3% compared to histopathological examination of surgically removed cysts or examination of hydatid cyst fluid (HCF) for protoscolices (gold standards). The specificity was 70% with false positive reactions with other parasitic infections and mass occupying lesions. PCR detection of G1 genotype in Egyptian animal hydatid cysts showed 90% in camel isolates and 80% in sheep isolates, but pig isolates were negative. The presence of this genotype in a high percentage in camel isolates incriminated sheep strain as the source of CE camel infection. The results may give an explanation to the contradicting results of other studies that did not relay upon molecular aspects.
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Abele-Horn, Marianne; Hommers, Leif; Trabold, René; Frosch, Matthias
2006-01-01
We evaluated the ability of the new VITEK 2 version 4.01 software to identify and detect glycopeptide-resistant enterococci compared to that of the reference broth microdilution method and to classify them into the vanA, vanB, vanC1, and vanC2 genotypes. Moreover, the accuracy of antimicrobial susceptibility testing with agents with improved potencies against glycopeptide-resistant enterococci was determined. A total of 121 enterococci were investigated. The new VITEK 2 software was able to identify 114 (94.2%) enterococcal strains correctly to the species level and to classify 119 (98.3%) enterococci correctly to the glycopeptide resistance genotype level. One Enterococcus casseliflavus strain and six Enterococcus faecium vanA strains with low-level resistance to vancomycin were identified with low discrimination, requiring additional tests. One of the vanA strains was misclassified as the vanB type, and one glycopeptide-susceptible E. facium wild type was misclassified as the vanA type. The overall essential agreements for antimicrobial susceptibility testing results were 94.2% for vancomycin, 95.9% for teicoplanin, 100% for quinupristin-dalfopristin and moxifloxacin, and 97.5% for linezolid. The rates of minor errors were 9% for teicoplanin and 5% for the other antibiotic agents. The identification and susceptibility data were produced within 4 h to 6 h 30 min and 8 h 15 min to 12 h 15 min. In conclusion, use of VITEK 2 version 4.01 software appears to be a reliable method for the identification and detection of glycopeptide-resistant enterococci as well as an improvement over the use of the former VITEK 2 database. However, a significant reduction in the detection time would be desirable. PMID:16390951
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Multiple HPV genotype infection impact on invasive cervical cancer presentation and survival
Martins, Toni Ricardo; Mendoza Lopez, Rossana V.; Sadalla, José Carlos; de Carvalho, João Paulo Mancusi; Baracat, Edmund Chada
2017-01-01
Background Invasive cervical cancer (ICC) is the third most common malignant neoplasm affecting Brazilian women. Little is known about the impact of specific HPV genotypes in the prognosis of ICC. We hypothesized that HPV genotype would impact ICC clinical presentation and survival. Methods Women diagnosed with ICC at the Instituto do Câncer do Estado de São Paulo (ICESP) between May 2008 and June 2012 were included in the study and were followed until December 2015. HPV genotype was detected from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples using Onclarity™ system (BD Viper™ LT automated system). Results 292 patients aged 50±14 years were analyzed. HPVDNA was detected in 84% of patients. The HPV genotypes studied were: HPV16 (64%), HPV18 (10%), HPV33-58 (7%), HPV45 (5%), HPV31 (4%) and other high-risk HPV genotypes (11%). HPV genotypes showed different distributions regarding histological type and clinical stage. Patients were followed for 35±21 months. The overall survival at 5 years after diagnosis of cervical cancer was 54%. Age, clinical staging, histological type and multiple HPV genotypes infection detected in the same tumor specimen were associated with poorer overall survival on multivariate Cox proportional hazard analysis (p<0.05). No specific HPV genotype affected survival. Conclusion Multiple HPV genotype infection was associated with poorer ICC survival in our study, compared with single genotype infection. HPV genotyping from FFPE tumor tissue using an automated assay such as the Onclarity BD™ assay provides a simpler alternative for routine clinical use. Impact This is the largest study employing an automated HPV genotyping assay using FFPE of ICC. Multiple HPV genotype infection adversely influenced survival. PMID:28829791
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Genotypic diversity of european Phytophthora ramorum isolates based on SSR analysis
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...
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Maroso, F; Hillen, J E J; Pardo, B G; Gkagkavouzis, K; Coscia, I; Hermida, M; Franch, R; Hellemans, B; Van Houdt, J; Simionati, B; Taggart, J B; Nielsen, E E; Maes, G; Ciavaglia, S A; Webster, L M I; Volckaert, F A M; Martinez, P; Bargelloni, L; Ogden, R
2018-06-01
The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 10 12 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.
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Haplotype estimation using sequencing reads.
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.
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Aldridge, Kristina; Boyadjiev, Simeon A.; Capone, George T.; DeLeon, Valerie B.; Richtsmeier, Joan T.
2015-01-01
The genetic basis for complex phenotypes is currently of great interest for both clinical investigators and basic scientists. In order to acquire a thorough understanding of the translation from genotype to phenotype, highly precise measures of phenotypic variation are required. New technologies, such as 3D photogrammetry are being implemented in phenotypic studies due to their ability to collect data rapidly and non-invasively. Before these systems can be broadly implemented the error associated with data collected from images acquired using these technologies must be assessed. This study investigates the precision, error, and repeatability associated with anthropometric landmark coordinate data collected from 3D digital photogrammetric images acquired with the 3dMDface System. Precision, error due to the imaging system, error due to digitization of the images, and repeatability are assessed in a sample of children and adults (N=15). Results show that data collected from images with the 3dMDface System are highly repeatable and precise. The average error associated with the placement of landmarks is sub-millimeter; both the error due to digitization and to the imaging system are very low. The few measures showing a higher degree of error include those crossing the labial fissure, which are influenced by even subtle movement of the mandible. These results suggest that 3D anthropometric data collected using the 3dMDface System are highly reliable and therefore useful for evaluation of clinical dysmorphology and surgery, analyses of genotype-phenotype correlations, and inheritance of complex phenotypes. PMID:16158436
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AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING
We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...
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Detection and genotyping of human papilloma virus in cervical cancer specimens from Saudi patients.
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.
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Rodriguez-Jimenez, R; Hoenicka, J; Jimenez-Arriero, M A; Ponce, G; Bagney, A; Aragues, M; Palomo, T
2006-01-01
Previous studies have associated a decreased striatal D2 dopamine receptor (DRD2) binding with impaired performance in cognitive tasks. In vivo studies have found a lower DRD2 binding associated with the CC genotype of the C957T single nucleotide polymorphism (SNP) of the DRD2 gene. The aim of this study was to investigate the relationship between executive functions and the C957T DRD2 SNP. We hypothesized that the CC genotype would be associated with a poorer executive functioning. Our sample consisted of 83 healthy volunteers (28 males and 55 females; mean age 25.2, SD 1.7 years). To assess executive functions, the Wisconsin Card Sorting Test was used, considering the variables perseverative errors, perseverative responses, and number of categories achieved. The genotype distribution was 13 CC, 41 CT, and 29 TT, satisfying Hardy-Weinberg equilibrium. Carriers of the CC genotype, compared with carriers of the CT/TT genotypes, achieved significantly fewer categories (5.00 vs. 5.81; p = 0.004), made a greater number of perseverative errors (13.46 vs. 8.39; p = 0.018), and had a greater number of perseverative responses (14.92 vs. 8.94; p = 0.014). Our results support the hypothesis that the C957T DRD2 SNP may influence cognitive performance through its repercussions on central dopaminergic function. 2006 S. Karger AG, Basel
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Park, Subin; Kim, Jae-Won; Yang, Young-Hui; Hong, Soon-Beom; Park, Min-Hyeon; Kim, Boong-Nyun; Shin, Min-Sup; Yoo, Hee-Jeong; Cho, Soo-Churl
2012-05-16
Dysregulation of noradrenergic system may play important roles in pathophysiology of attention-deficit/hyperactivity disorder (ADHD). We examined the relationship between polymorphisms in the norepinephrine transporter SLC6A2 gene and attentional performance before and after medication in children with ADHD. Fifty-three medication-naïve children with ADHD were genotyped and evaluated using the continuous performance test (CPT). After 8-weeks of methylphenidate treatment, these children were evaluated by CPT again. We compared the baseline CPT measures and the post-treatment changes in the CPT measures based on the G1287A and the A-3081T polymorphisms of SLC6A2. There was no significant difference in the baseline CPT measures associated with the G1287A or A-3081T polymorphisms. After medication, however, ADHD subjects with the G/G genotype at the G1287A polymorphism showed a greater decrease in the mean omission error scores (p = 0.006) than subjects with the G/A or A/A genotypes, and subjects with the T allele at the A-3081T polymorphism (T/T or A/T) showed a greater decrease in the mean commission error scores (p = 0.003) than those with the A/A genotypes. Our results provide evidence for the possible role of the G1287A and A-3081T genotypes of SLC6A2 in methylphenidate-induced improvement in attentional performance and support the noradrenergic hypothesis for the pathophysiology of ADHD.
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Next-Generation Sequencing Analysis of the Diversity of Human Noroviruses in Japanese Oysters.
Imamura, Saiki; Kanezashi, Hiromi; Goshima, Tomoko; Haruna, Mika; Okada, Tsukasa; Inagaki, Nobuya; Uema, Masashi; Noda, Mamoru; Akimoto, Keiko
2017-08-01
To obtain detailed information on the diversity of infectious norovirus in oysters (Crossostrea gigas), oysters obtained from fish producers at six different sites (sites A, B, C, D, E, and F) in Japan were analyzed once a month during the period spanning October 2015-February 2016. To avoid false-positive polymerase chain reaction (PCR) results derived from noninfectious virus particles, samples were pretreated with RNase before reverse transcription-PCR (RT-PCR). RT-PCR products were subjected to next-generation sequencing to identify norovirus genotypes in oysters. As a result, all GI genotypes were detected in the investigational period. The detection rate and proportion of norovirus GI genotypes differed depending on the sampling site and month. GII.3, GII.4, GII.13, GII.16, and GII.17 were detected in this study. Both the detection rate and proportion of norovirus GII genotypes differed depending on the sampling site and month. In total, the detection rate and proportion of GII.3 were highest from October to December among all detected genotypes. In January, the detection rates of GII.4 and GII.17 reached the same level as that of GII.3. The proportion of GII.17 was relatively lower from October to December, whereas it was the highest in January. To our knowledge, this is the first investigation on noroviruses in oysters in Japan, based on a method that can distinguish their infectivity.
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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.
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García-Cervantes, Patricia Catalina; Báez-Flores, María Elena; Delgado-Vargas, Francisco; Ponce-Macotela, Martha; Nawa, Yukifumi; De-la-Cruz-Otero, María Del-Carmen; Martínez-Gordillo, Mario N; Díaz-Camacho, Sylvia Páz
2017-02-28
Giardiasis is a human health concern worldwide, especially among schoolchildren. Giardia duodenalis genotypes A and B are infective to humans, but their zoonotic potential remains controversial. In Mexico, the most prevalent genotype is A, but B was also detected in southeastern Mexico. In Sinaloa state, northwestern Mexico, giardiasis is highly prevalent, but Giardia genotypes have been poorly studied. This study aimed to investigate the distribution and clinical-epidemiological correlation of G. duodenalis genotypes in schoolchildren and their families and pets in urban and rural areas of Sinaloa state, Mexico. Among 395 schoolchildren (274 urban, 121 rural), 76 (49 urban, 27 rural) were infected with G. duodenalis. In total, 22 families (15 urban, 7 rural) of infected schoolchildren, consisting of 60 family members (41 urban, 19 rural) and 21 pet dogs (15 urban, 6 rural) were examined; 10 family members (5 urban, 5 rural) and 5 pet dogs (3 urban, 2 rural) of 10 families (6 urban, 4 rural) were infected. After PCR-RFLP analyses of vsp417 and gdh genes, genotype prevalence among infected urban schoolchildren was 79.5% AI, 12.8% AII, and 7.7% mixed AI+B. However, only AI genotype was found in family members and pets. In the rural area, only the AI genotype was detected. Genotypes were not correlated with clinical manifestations. This paper shows the presence of B genotype in northwestern Mexico for the first time. Detection of AI genotype in dogs suggested the possible role of dogs as the reservoir for human giardiasis in Sinaloa, Mexico.
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Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa
2017-01-01
Objective: The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Materials and Methods: Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. Results: The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. Conclusion: The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis. PMID:29123441
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Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa
2017-10-01
The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis.
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Genetic characterization of measles virus in the Philippines, 2008-2011.
Centeno, Rex; Fuji, Naoko; Okamoto, Michiko; Dapat, Clyde; Saito, Mariko; Tandoc, Amado; Lupisan, Socorro; Oshitani, Hitoshi
2015-06-03
Large outbreaks of measles occurred in the Philippines in 2010 and 2011. Genetic analysis was performed to identify the genotype of measles virus (MeV) that was responsible for the large outbreaks. A total of 114 representative MeVs that were detected in the Philippines from 2008 to 2011 were analyzed by sequencing the C-terminal region of nucleocapsid (N) gene and partial hemagglutinin (H) gene and by inferring the phylogenetic trees. Genetic analysis showed that genotype D9 was the predominant circulating strain during the 4-year study period. Genotype D9 was detected in 23 samples (92%) by N gene sequencing and 93 samples (94%) by H gene analysis. Sporadic cases of genotype G3 MeV were identified in 2 samples (8%) by N gene sequencing and 6 samples (6%) by H gene analysis. Genotype G3 MeV was detected mainly in Panay Island in 2009 and 2010. Molecular clock analysis of N gene showed that the recent genotype D9 viruses that caused the big outbreaks in 2010 and 2011 diverged from a common ancestor in 2005 in one of the neighboring Southeast Asian countries, where D9 was endemic. These big outbreaks of measles resulted in a spillover and were associated with genotype D9 MeV importation to Japan and the USA. Genotype D9 MeV became endemic and caused two big outbreaks in the Philippines in 2010 and 2011. Genotype G3 MeV was detected sporadically with limited geographic distribution. This study highlights the importance of genetic analysis not only in helping with the assessment of measles elimination program in the country but also in elucidating the transmission dynamics of measles virus.
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Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong
2014-09-19
The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.
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Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong
2014-01-01
The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN. PMID:25236476
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McClure, Matthew C.; Sonstegard, Tad S.; Wiggans, George R.; Van Eenennaam, Alison L.; Weber, Kristina L.; Penedo, Cecilia T.; Berry, Donagh P.; Flynn, John; Garcia, Jose F.; Carmo, Adriana S.; Regitano, Luciana C. A.; Albuquerque, Milla; Silva, Marcos V. G. B.; Machado, Marco A.; Coffey, Mike; Moore, Kirsty; Boscher, Marie-Yvonne; Genestout, Lucie; Mazza, Raffaele; Taylor, Jeremy F.; Schnabel, Robert D.; Simpson, Barry; Marques, Elisa; McEwan, John C.; Cromie, Andrew; Coutinho, Luiz L.; Kuehn, Larry A.; Keele, John W.; Piper, Emily K.; Cook, Jim; Williams, Robert; Van Tassell, Curtis P.
2013-01-01
To assist cattle producers transition from microsatellite (MS) to single nucleotide polymorphism (SNP) genotyping for parental verification we previously devised an effective and inexpensive method to impute MS alleles from SNP haplotypes. While the reported method was verified with only a limited data set (N = 479) from Brown Swiss, Guernsey, Holstein, and Jersey cattle, some of the MS-SNP haplotype associations were concordant across these phylogenetically diverse breeds. This implied that some haplotypes predate modern breed formation and remain in strong linkage disequilibrium. To expand the utility of MS allele imputation across breeds, MS and SNP data from more than 8000 animals representing 39 breeds (Bos taurus and B. indicus) were used to predict 9410 SNP haplotypes, incorporating an average of 73 SNPs per haplotype, for which alleles from 12 MS markers could be accurately be imputed. Approximately 25% of the MS-SNP haplotypes were present in multiple breeds (N = 2 to 36 breeds). These shared haplotypes allowed for MS imputation in breeds that were not represented in the reference population with only a small increase in Mendelian inheritance inconsistancies. Our reported reference haplotypes can be used for any cattle breed and the reported methods can be applied to any species to aid the transition from MS to SNP genetic markers. While ~91% of the animals with imputed alleles for 12 MS markers had ≤1 Mendelian inheritance conflicts with their parents' reported MS genotypes, this figure was 96% for our reference animals, indicating potential errors in the reported MS genotypes. The workflow we suggest autocorrects for genotyping errors and rare haplotypes, by MS genotyping animals whose imputed MS alleles fail parentage verification, and then incorporating those animals into the reference dataset. PMID:24065982
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Telomere length analysis in Down syndrome birth.
Bhaumik, Pranami; Bhattacharya, Mandar; Ghosh, Priyanka; Ghosh, Sujay; Kumar Dey, Subrata
2017-06-01
Human reproductive fitness depends upon telomere chemistry. Maternal age, meiotic nondisjunction error and telomere length of mother of trisomic child are someway associated. Reports exhibiting maternal inheritance of telomere length in Down syndrome child are very scanty. To investigate this, we collected peripheral blood from 170 mothers of Down syndrome child and 186 age matched mothers of euploid child with their newly born babies. Telomere length was measured by restriction digestion - southern blotting technique. Meiotic nondisjunction error was detected by STR genotyping. Subjects are classified by age (old >35 years and young ˂35 years) and by meiotic error (MI and MII). Linear regression was run to explore the age - telomere length relationship in each maternal groups. The study reveals that with age, telomere erodes in length. Old MII mothers carry the shortest (p˂0.001), control mothers have the longest telomere and MI lies in between. Babies from older mother have longer telomere (p˂0.001) moreover; telomeres are longer in Down syndrome babies than control babies (p˂0.001). To conclude, this study represents not only the relation between maternal aging and telomere length but also explore the maternal heritability of telomere length in families with Down syndrome child. Copyright © 2017 Elsevier B.V. All rights reserved.
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Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).
Gaydos, J K; Miller, W A; Johnson, C; Zornetzer, H; Melli, A; Packham, A; Jeffries, S J; Lance, M M; Conrad, P A
2008-12-01
Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.
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Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia.
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.
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Developing and Evaluating the HRM Technique for Identifying Cytochrome P450 2D6 Polymorphisms.
Lu, Hsiu-Chin; Chang, Ya-Sian; Chang, Chun-Chi; Lin, Ching-Hsiung; Chang, Jan-Gowth
2015-05-01
Cytochrome P450 2D6 is one of the important enzymes involved in the metabolism of many widely used drugs. Genetic polymorphisms of CYP2D6 can affect its activity. Therefore, an efficient method for identifying CYP2D6 polymorphisms is clinically important. We developed a high-resolution melting (HRM) analysis to investigate CYP2D6 polymorphisms. Genomic DNA was extracted from peripheral blood samples from 71 healthy individuals. All nine exons of the CYP2D6 gene were sequenced before screening by HRM analysis. This method can detect the most genotypes (*1, *2, *4, *10, *14, *21 *39, and *41) of CYP2D6 in Chinese. All samples were successfully genotyped. The four most common mutant CYP2D6 alleles (*1, *2, *10, and *41) can be genotyped. The single nucleotides polymorphism (SNP) frequencies of 100C > T (rs1065852), 1039C > T (rs1081003), 1661G > C (rs1058164), 2663G > A (rs28371722), 2850C > T (rs16947), 2988G > A (rs28371725), 3181A > G, and 4180G > C (rs1135840) were 58%, 61%, 73%, 1%, 13%, 3%, 1%, 73%, respectively. We identified 100% of all heterozygotes without any errors. The two homozygous genotypes (1661G > C and 4180G > C) can be distinguished by mixing with a known genotype sample to generate an artificial heterozygote for HRM analysis. Therefore, all samples could be identified using our HRM method, and the results of HRM analysis are identical to those obtained by sequencing. Our method achieved 100% sensitivity, specificity, positive prediction value and negative prediction value. HRM analysis is a nongel resolution method that is faster and less expensive than direct sequencing. Our study shows that it is an efficient tool for typing CYP2D6 polymorphisms. © 2014 Wiley Periodicals, Inc.
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Wright, David M; Allen, Adrian R; Mallon, Thomas R; McDowell, Stanley W J; Bishop, Stephen C; Glass, Elizabeth J; Bermingham, Mairead L; Woolliams, John A; Skuce, Robin A
2013-10-01
Strains of many infectious diseases differ in parameters that influence epidemic spread, for example virulence, transmissibility, detectability and host specificity. Knowledge of inter-strain variation can be exploited to improve management and decrease disease incidence. Bovine tuberculosis (bTB) is increasingly prevalent among farmed cattle in the UK, exerting a heavy economic burden on the farming industry and government. We aimed to determine whether strains of Mycobacterium bovis (the causative agent of bTB) identified and classified using genetic markers (spoligotyping and multi-locus VNTR analysis) varied in response to the tuberculin skin test; this being the primary method of bTB detection used in the UK. Inter-strain variation in detectability of M. bovis could have important implications for disease control. The skin test is based on a differential delayed type hypersensitivity (DTH) response to intradermal injections of purified protein derivative (PPD) from M. bovis (PPD-B) and Mycobacterium avium (PPD-A). We searched for an association between skin test response (PPD-B skin rise minus PPD-A skin rise) and M. bovis genotype at the disclosing test in culture-confirmed cases using a field dataset consisting of 21,000 isolates belonging to 63 genotypes of M. bovis from cattle in Northern Ireland. We found no substantial variation among genotypes (estimated responses clustered tightly around the mean) controlling for animal sex, breed and test effects. We also estimated the ratio of skin test detected to undetected cases (i.e. cases only detected at abattoir). The skin test detection ratio varied among abattoirs with some detecting a greater proportion of cases than others but this variation was unrelated to the community composition of genotypes within each abattoir catchment. These two lines of evidence indicate that M. bovis genotypes in Northern Ireland have similar detectability using the skin test. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.
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Accelerating root system phenotyping of seedlings through a computer-assisted processing pipeline.
Dupuy, Lionel X; Wright, Gladys; Thompson, Jacqueline A; Taylor, Anna; Dekeyser, Sebastien; White, Christopher P; Thomas, William T B; Nightingale, Mark; Hammond, John P; Graham, Neil S; Thomas, Catherine L; Broadley, Martin R; White, Philip J
2017-01-01
There are numerous systems and techniques to measure the growth of plant roots. However, phenotyping large numbers of plant roots for breeding and genetic analyses remains challenging. One major difficulty is to achieve high throughput and resolution at a reasonable cost per plant sample. Here we describe a cost-effective root phenotyping pipeline, on which we perform time and accuracy benchmarking to identify bottlenecks in such pipelines and strategies for their acceleration. Our root phenotyping pipeline was assembled with custom software and low cost material and equipment. Results show that sample preparation and handling of samples during screening are the most time consuming task in root phenotyping. Algorithms can be used to speed up the extraction of root traits from image data, but when applied to large numbers of images, there is a trade-off between time of processing the data and errors contained in the database. Scaling-up root phenotyping to large numbers of genotypes will require not only automation of sample preparation and sample handling, but also efficient algorithms for error detection for more reliable replacement of manual interventions.
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Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed
Guo, Yaqiong; Alderisio, Kerri A.; Yang, Wenli; Cama, Vitaliano; Xiao, Lihua
2014-01-01
To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City's source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed. PMID:24141128
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Wang, Chaolong; Schroeder, Kari B.; Rosenberg, Noah A.
2012-01-01
Allelic dropout is a commonly observed source of missing data in microsatellite genotypes, in which one or both allelic copies at a locus fail to be amplified by the polymerase chain reaction. Especially for samples with poor DNA quality, this problem causes a downward bias in estimates of observed heterozygosity and an upward bias in estimates of inbreeding, owing to mistaken classifications of heterozygotes as homozygotes when one of the two copies drops out. One general approach for avoiding allelic dropout involves repeated genotyping of homozygous loci to minimize the effects of experimental error. Existing computational alternatives often require replicate genotyping as well. These approaches, however, are costly and are suitable only when enough DNA is available for repeated genotyping. In this study, we propose a maximum-likelihood approach together with an expectation-maximization algorithm to jointly estimate allelic dropout rates and allele frequencies when only one set of nonreplicated genotypes is available. Our method considers estimates of allelic dropout caused by both sample-specific factors and locus-specific factors, and it allows for deviation from Hardy–Weinberg equilibrium owing to inbreeding. Using the estimated parameters, we correct the bias in the estimation of observed heterozygosity through the use of multiple imputations of alleles in cases where dropout might have occurred. With simulated data, we show that our method can (1) effectively reproduce patterns of missing data and heterozygosity observed in real data; (2) correctly estimate model parameters, including sample-specific dropout rates, locus-specific dropout rates, and the inbreeding coefficient; and (3) successfully correct the downward bias in estimating the observed heterozygosity. We find that our method is fairly robust to violations of model assumptions caused by population structure and by genotyping errors from sources other than allelic dropout. Because the data sets imputed under our model can be investigated in additional subsequent analyses, our method will be useful for preparing data for applications in diverse contexts in population genetics and molecular ecology. PMID:22851645
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Effects of Listening Conditions, Error Types, and Ensemble Textures on Error Detection Skills
ERIC Educational Resources Information Center
Waggoner, Dori T.
2011-01-01
This study was designed with three main purposes: (a) to investigate the effects of two listening conditions on error detection accuracy, (b) to compare error detection responses for rhythm errors and pitch errors, and (c) to examine the influences of texture on error detection accuracy. Undergraduate music education students (N = 18) listened to…
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Zhang, Y; Wang, H; Xu, S; Mao, N; Zhu, Z; Shi, J; Huang, G; Liu, C; Bo, F; Feng, D; Lu, P; Liu, Y; Wang, Y; Lei, Y; Chen, M; Chen, H; Wang, C; Fu, H; Li, C; He, J; Gao, H; Gu, S; Wang, S; Ling, H; Liu, Y; Ding, Z; Ba, Z; Feng, Y; Zheng, H; Tang, X; Lei, Y; Xiong, Y; Bellini, W J; Rota, P A; Jee, Y; Xu, W
2014-09-01
With the achievement of high coverage for routine immunization and supplementary immunization activities (SIAs), measles incidence in mainland China reached its lowest level in 2010. The proportion of measles cases in the vaccination-targeted population decreased during 2007-2010 after the SIAs. More than 60% of measles cases were in adults or infants, especially in the coastal and eastern provinces during 2009 and 2010. A total 567 isolates of measles virus were obtained from clinical specimens from 27 of 31 provinces in mainland China during 2009 and 2010. Except for two vaccine-associated cases, one genotype D4 strain, two genotype D9 strains, and four genotype D11 strains, the other 558 strains were genotype H1 cluster H1a. Genotype H1 has been the only endemic genotype detected in China since surveillance began in 1993. Only genotype H1 was found in mainland China during 1993-2008, except for one detection of genotype H2. More recently, multiple genotypes of imported measles were detected even with the background of endemic genetotype H1 viruses. Analysis of the 450-nucleotide sequencing window of the measles virus N gene showed that the overall genetic diversity of the recent geneotype H1 strains decreased between 2008 and 2010. The lower genetic diversity of H1 strains suggested that enhanced vaccination may have reduced the co-circulating lineages of endemic genotype H1 strains in mainland China. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.
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Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian
2014-01-01
Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402
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Ferragut, Fátima; Vega, Celina G; Mauroy, Axel; Conceição-Neto, Nádia; Zeller, Mark; Heylen, Elisabeth; Uriarte, Enrique Louge; Bilbao, Gladys; Bok, Marina; Matthijnssens, Jelle; Thiry, Etienne; Badaracco, Alejandra; Parreño, Viviana
2016-06-01
Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America. Copyright © 2016. Published by Elsevier B.V.
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Peng, Zhong; Wang, Haonan; Liang, Wan; Chen, Yibao; Tang, Xibiao; Chen, Huanchun; Wu, Bin
2018-01-01
Pasteurella multocida is a leading cause of respiratory disease in pigs worldwide. In this study, we determined the genetic characteristics of 115 P. multocida isolates from the lungs of pigs with respiratory disease in China in 2015 using capsular typing, lipopolysaccharide (LPS) genotyping, and virulence genotyping based on the detection of virulence-associated genes. The results showed that the isolates belonged to three capsular types: A (49.6%), D (46.1%), and nontypable (4.3%); and two LPS genotypes: L3 (22.6%) and L6 (77.4%). When combining the capsular types with the LPS genotypes, a genotype group D: L6 (46.1%) was the most prevalent among the strains. Among the 23 virulence-associated genes detected in this study, a small number of them displayed a certain level of "genotype-preference". We found that pfhA, hgbA, and hgbB had a close association with P. multocida LPS genotypes, while tadD was more associated with P. multocida capsular types. In addition, multilocus sequence typing (MLST) on 40 P. multocida isolates identified four sequence types: ST3, ST10, ST11, and ST16, and the distribution of ST11 was significantly higher than the other MLST genotypes. Interestingly, all of the ST11 isolates detected in this study were genotype D: L6 strains and they were 100% positive for hgbB. Our data suggest that a capsule/LPS/MLST genotype D/L6/ST11 is likely to be strongly associated with respiratory clinical manifestation of the disease in pigs.
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Genetic diversity of Phytophthora ramorum in nursery trade and managed environment in Scotland
Alexandra Schlenzig; David Cook
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...
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Precise genotyping and recombination detection of Enterovirus
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
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Ding, Qianlan; Wu, Xi; Lu, Yeling; Chen, Changming; Shen, Rui; Zhang, Xi; Jiang, Zhengwen; Wang, Xuefeng
2016-07-01
To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection. Copyright © 2016 Elsevier B.V. All rights reserved.
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Fairfield, Beth; Mammarella, Nicola; Di Domenico, Alberto; D'Aurora, Marco; Stuppia, Liborio; Gatta, Valentina
2017-08-30
False memories are common memory distortions in everyday life and seem to increase with affectively connoted complex information. In line with recent studies showing a significant interaction between the noradrenergic system and emotional memory, we investigated whether healthy volunteer carriers of the deletion variant of the ADRA2B gene that codes for the α2b-adrenergic receptor are more prone to false memories than non-carriers. In this study, we collected genotype data from 212 healthy female volunteers; 91 ADRA2B carriers and 121 non-carriers. To assess gene effects on false memories for affective information, factorial mixed model analysis of variances (ANOVAs) were conducted with genotype as the between-subjects factor and type of memory error as the within-subjects factor. We found that although carriers and non-carriers made comparable numbers of false memory errors, they showed differences in the direction of valence biases, especially for inferential causal errors. Specifically, carriers produced fewer causal false memory errors for scripts with a negative outcome, whereas non-carriers showed a more general emotional effect and made fewer causal errors with both positive and negative outcomes. These findings suggest that putatively higher levels of noradrenaline in deletion carriers may enhance short-term consolidation of negative information and lead to fewer memory distortions when facing negative events. Copyright © 2017 Elsevier B.V. All rights reserved.
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Local neutral networks help maintain inaccurately replicating ribozymes.
Szilágyi, András; Kun, Ádám; Szathmáry, Eörs
2014-01-01
The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.
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Simulundu, E; Chambaro, H M; Sinkala, Y; Kajihara, M; Ogawa, H; Mori, A; Ndebe, J; Dautu, G; Mataa, L; Lubaba, C H; Simuntala, C; Fandamu, P; Simuunza, M; Pandey, G S; Samui, K L; Misinzo, G; Takada, A; Mweene, A S
2018-02-01
During 2013-2015, several and severe outbreaks of African swine fever (ASF) affected domestic pigs in six provinces of Zambia. Genetic characterization of ASF viruses (ASFVs) using standardized genotyping procedures revealed that genotypes I, II and XIV were associated with these outbreaks. Molecular and epidemiological data suggest that genotype II ASFV (Georgia 2007/1-like) detected in Northern Province of Zambia may have been introduced from neighbouring Tanzania. Also, a genotype II virus detected in Eastern Province of Zambia showed a p54 phylogenetic relationship that was inconsistent with that of p72, underscoring the genetic variability of ASFVs. While it appears genotype II viruses detected in Zambia arose from a domestic pig cycle, genotypes I and XIV possibly emerged from a sylvatic cycle. Overall, this study demonstrates the co-circulation of multiple genotypes of ASFVs, involvement of both the sylvatic and domestic pig cycle in ASF outbreaks in Zambia and possible trans-boundary spread of the disease in south-eastern Africa. Indeed, while there is need for regional or international concerted efforts in the control of ASF, understanding pig marketing practices, pig population dynamics, pig housing and rearing systems and community engagement will be important considerations when designing future prevention and control strategies of this disease in Zambia. © 2017 Blackwell Verlag GmbH.
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Cloete, Kevin Wesley; Ristow, Peter Gustav; Kasu, Mohaimin; D'Amato, Maria Eugenia
2017-03-01
CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Gifford, Robert J.; Rhee, Soo-Yon; Eriksson, Nicolas; Liu, Tommy F.; Kiuchi, Mark; Das, Amar K.; Shafer, Robert W.
2008-01-01
Design Promiscuous guanine (G) to adenine (A) substitutions catalysed by apolipoprotein B RNA-editing catalytic component (APOBEC) enzymes are observed in a proportion of HIV-1 sequences in vivo and can introduce artifacts into some genetic analyses. The potential impact of undetected lethal editing on genotypic estimation of transmitted drug resistance was assessed. Methods Classifiers of lethal, APOBEC-mediated editing were developed by analysis of lentiviral pol gene sequence variation and evaluated using control sets of HIV-1 sequences. The potential impact of sequence editing on genotypic estimation of drug resistance was assessed in sets of sequences obtained from 77 studies of 25 or more therapy-naive individuals, using mixture modelling approaches to determine the maximum likelihood classification of sequences as lethally edited as opposed to viable. Results Analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals using a novel classifier of lethal, APOBEC3G-mediated sequence editing, the polypeptide-like 3G (APOBEC3G)-mediated defectives (A3GD) index’, detected lethal editing in association with spurious ‘transmitted drug resistance’ in nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma. Conclusion Screening for lethally edited sequences in datasets containing a proportion of proviral DNA, such as those likely to be obtained for epidemiological surveillance of transmitted drug resistance in the developing world, can eliminate rare but potentially significant errors in genotypic estimation of transmitted drug resistance. PMID:18356601
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Tapia, Lorena I; Shaw, Chad A; Aideyan, Letisha O; Jewell, Alan M; Dawson, Brian C; Haq, Taha R; Piedra, Pedro A
2014-01-01
Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.
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Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.
2014-01-01
Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544
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Genotype 3 is the predominant hepatitis C genotype in a multi-ethnic Asian population in Malaysia.
Ho, Shiaw-Hooi; Ng, Kee-Peng; Kaur, Harvinder; Goh, Khean-Lee
2015-06-01
Genotypes of hepatitis C virus (HCV) are distributed differently across the world. There is a paucity of such data in a multi-ethnic Asian population like Malaysia. The objectives of this study were to determine the distribution of HCV genotypes between major ethnic groups and to ascertain their association with basic demographic variables like age and gender. This was a cross-sectional prospective study conducted from September 2007 to September 2013. Consecutive patients who were detected to have anti-HCV antibodies in the University of Malaya Medical Centre were included and tested for the presence of HCV RNA using Roche Cobas Amplicor Analyzer and HCV genotype using Roche single Linear Array HCV Genotyping strip. Five hundred and ninety-six subjects were found to have positive anti-HCV antibodies during this period of time. However, only 396 (66.4%) were HCV RNA positive and included in the final analysis. Our results showed that HCV genotype 3 was the predominant genotype with overall frequency of 61.9% followed by genotypes 1 (35.9%), 2 (1.8%) and 6 (0.5%). There was a slightly higher prevalence of HCV genotype 3 among the Malays when compared to the Chinese (P=0.043). No other statistical significant differences were observed in the distribution of HCV genotypes among the major ethnic groups. There was also no association between the predominant genotypes and basic demographic variables. In a multi-ethnic Asian society in Malaysia, genotype 3 is the predominant genotype among all the major ethnic groups with genotype 1 as the second commonest genotype. Both genotypes 2 and 6 are uncommon. Neither genotype 4 nor 5 was detected. There is no identification of HCV genotype according to ethnic origin, age and gender.
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Molecular Epidemiology of Rhinovirus Detections in Young Children
Howard, Leigh M.; Johnson, Monika; Gil, Ana I.; Griffin, Marie R.; Edwards, Kathryn M.; Lanata, Claudio F.; Williams, John V.; Grijalva, Carlos G.
2016-01-01
Background. Human rhinoviruses (HRVs) are frequently detected in children with acute respiratory illnesses (ARIs) but also in asymptomatic children. We compared features of ARI with HRV species (A, B, C) and determined genotypes associated with repeated HRV detections within individuals. Methods. We used clinical data and respiratory samples obtained from children <3 years old during weekly active household-based surveillance. A random subset of samples in which HRV was detected from individuals during both ARI and an asymptomatic period within 120 days of the ARI were genotyped. Features of ARI were compared among HRV species. Concordance of genotype among repeated HRV detections within individuals was assessed. Results. Among 207 ARI samples sequenced, HRV-A, HRV-B, and HRV-C were detected in 104 (50%), 20 (10%), and 83 (40%), respectively. Presence of fever, decreased appetite, and malaise were significantly higher in children with HRV-B. When codetections with other viruses were excluded (n = 155), these trends persisted, but some did not reach statistical significance. When 58 paired sequential HRV detections during asymptomatic and ARI episodes were sequenced, only 9 (16%) were identical genotypes of HRV. Conclusions. Clinical features may differ among HRV species. Repeated HRV detections in young children frequently represented acquisition of new HRV strains. PMID:26900577
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Tembuyser, Lien; Ligtenberg, Marjolijn J L; Normanno, Nicola; Delen, Sofie; van Krieken, J Han; Dequeker, Elisabeth M C
2014-05-01
Precision medicine is now a key element in clinical oncology. RAS mutational status is a crucial predictor of responsiveness to anti-epidermal growth factor receptor agents in metastatic colorectal cancer. In an effort to guarantee high-quality testing services in molecular pathology, the European Society of Pathology has been organizing an annual KRAS external quality assessment program since 2009. In 2012, 10 formalin-fixed, paraffin-embedded samples, of which 8 from invasive metastatic colorectal cancer tissue and 2 artificial samples of cell line material, were sent to more than 100 laboratories from 26 countries with a request for routine KRAS testing. Both genotyping and clinical reports were assessed independently. Twenty-seven percent of the participants genotyped at least 1 of 10 samples incorrectly. In total, less than 5% of the distributed specimens were genotyped incorrectly. Genotyping errors consisted of false negatives, false positives, and incorrectly genotyped mutations. Twenty percent of the laboratories reported a technical error for one or more samples. A review of the written reports showed that several essential elements were missing, most notably a clinical interpretation of the test result, the method sensitivity, and the use of a reference sequence. External quality assessment serves as a valuable educational tool in assessing and improving molecular testing quality and is an important asset for monitoring quality assurance upon incorporation of new biomarkers in diagnostic services. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Kerr, Shona M; Campbell, Archie; Murphy, Lee; Hayward, Caroline; Jackson, Cathy; Wain, Louise V; Tobin, Martin D; Dominiczak, Anna; Morris, Andrew; Smith, Blair H; Porteous, David J
2013-03-22
Generation Scotland: Scottish Family Health Study (GS:SFHS) is a family-based biobank of 24,000 participants with rich phenotype and DNA available for genetic research. This paper describes the laboratory results from genotyping 32 single nucleotide polymorphisms (SNPs) on DNA from over 10,000 participants who attended GS:SFHS research clinics. The analysis described here was undertaken to test the quality of genetic information available to researchers. The success rate of each marker genotyped (call rate), minor allele frequency and adherence to Mendelian inheritance are presented. The few deviations in marker transmission in the 925 parent-child trios analysed were assessed as to whether they were likely to be miscalled genotypes, data or sample handling errors, or pedigree inaccuracies including non-paternity. The first 10,450 GS:SFHS clinic participants who had spirometry and smoking data available and DNA extracted were selected. 32 SNPs were assayed, chosen as part of a replication experiment from a Genome-Wide Association Study meta-analysis of lung function. In total 325,336 genotypes were returned. The overall project pass rate (32 SNPs on 10,450 samples) was 97.29%. A total of 925 parent-child trios were assessed for transmission of the SNP markers, with 16 trios indicating evidence of inconsistency in the recorded pedigrees. The Generation Scotland: Scottish Family Health Study used well-validated study methods and can produce good quality genetic data, with a low error rate. The GS:SFHS DNA samples are of high quality and the family groups were recorded and processed with accuracy during collection of the cohort.
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Qiu, Xianbo; Song, Liuwei; Yang, Shuo; Guo, Meng; Yuan, Quan; Ge, Shengxiang; Min, Xiaoping; Xia, Ningshao
2016-01-01
A fast and low-cost method for HBV genotyping especially for genotypes A, B, C and D was developed and tested. A classifier was used to detect and analyze a one-step immunoassay lateral flow strip functionalized with genotype-specific monoclonal antibodies (mAbs) on multiple capture lines in the form of pattern recognition for point-of-care (POC) diagnostics. The fluorescent signals from the capture lines and the background of the strip were collected via multiple optical channels in parallel. A digital HBV genotyping model, whose inputs are the fluorescent signals and outputs are a group of genotype-specific digital binary codes (0/1), was developed based on the HBV genotyping strategy. Meanwhile, a companion decoding table was established to cover all possible pairing cases between the states of a group of genotype-specific digital binary codes and the HBV genotyping results. A logical analyzing module was constructed to process the detected signals in parallel without program control, and its outputs were used to drive a set of LED indicators, which determine the HBV genotype. Comparing to the nucleic acid analysis to HBV viruses, much faster HBV genotyping with significantly lower cost can be obtained with the developed method. PMID:27306485
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Murillo, Gabriel H; You, Na; Su, Xiaoquan; Cui, Wei; Reilly, Muredach P; Li, Mingyao; Ning, Kang; Cui, Xinping
2016-05-15
Single nucleotide variant (SNV) detection procedures are being utilized as never before to analyze the recent abundance of high-throughput DNA sequencing data, both on single and multiple sample datasets. Building on previously published work with the single sample SNV caller genotype model selection (GeMS), a multiple sample version of GeMS (MultiGeMS) is introduced. Unlike other popular multiple sample SNV callers, the MultiGeMS statistical model accounts for enzymatic substitution sequencing errors. It also addresses the multiple testing problem endemic to multiple sample SNV calling and utilizes high performance computing (HPC) techniques. A simulation study demonstrates that MultiGeMS ranks highest in precision among a selection of popular multiple sample SNV callers, while showing exceptional recall in calling common SNVs. Further, both simulation studies and real data analyses indicate that MultiGeMS is robust to low-quality data. We also demonstrate that accounting for enzymatic substitution sequencing errors not only improves SNV call precision at low mapping quality regions, but also improves recall at reference allele-dominated sites with high mapping quality. The MultiGeMS package can be downloaded from https://github.com/cui-lab/multigems xinping.cui@ucr.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Assessing the Relationship of Ancient and Modern Populations
Schraiber, Joshua G.
2018-01-01
Genetic material sequenced from ancient samples is revolutionizing our understanding of the recent evolutionary past. However, ancient DNA is often degraded, resulting in low coverage, error-prone sequencing. Several solutions exist to this problem, ranging from simple approach, such as selecting a read at random for each site, to more complicated approaches involving genotype likelihoods. In this work, we present a novel method for assessing the relationship of an ancient sample with a modern population, while accounting for sequencing error and postmortem damage by analyzing raw reads from multiple ancient individuals simultaneously. We show that, when analyzing SNP data, it is better to sequence more ancient samples to low coverage: two samples sequenced to 0.5× coverage provide better resolution than a single sample sequenced to 2× coverage. We also examined the power to detect whether an ancient sample is directly ancestral to a modern population, finding that, with even a few high coverage individuals, even ancient samples that are very slightly diverged from the modern population can be detected with ease. When we applied our approach to European samples, we found that no ancient samples represent direct ancestors of modern Europeans. We also found that, as shown previously, the most ancient Europeans appear to have had the smallest effective population sizes, indicating a role for agriculture in modern population growth. PMID:29167200
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Hidden Markov Model-Based CNV Detection Algorithms for Illumina Genotyping Microarrays.
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.
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Kulik, Tomasz; Jestoi, Marika; Okorski, Adam
2011-01-01
Fungi of the genus Fusarium are important plant pathogens and contaminants of cereal grains producing different types of mycotoxins. Enniatins are a group of mycotoxins with ionophoric properties frequently detected in North European grains. Within the Fusarium complex responsible for grain infection, Fusarium avenaceum, Fusarium poae and Fusarium tricinctum are the most potential enniatins producers. This study presents the development of two quantitative TaqMan MGB (Minor Groove Binder) assays for the specific quantification of F. avenaceum/F. tricinctum and F. poae esyn1 genotypes, respectively. Two sets of genotype-specific primers/probes were designed on the basis of esyn1 gene homologues encoding multifunctional enzyme enniatin synthetase. The specificity of the assays developed has been tested successfully on 111 Fusarium isolates from different geographical origins. The detection limits for F. avenaceum/F. tricinctum esyn1 genotype and F. poae genotype were 19 and 0.3 pg, respectively. The application of the assays developed on asymptomatic wheat grain samples revealed significant positive correlations between the enniatins levels and the amount of F. avenaceum/F. tricinctum esyn1 genotype (R=0.61) and F. poae esyn1 genotype (R=0.42). © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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Vidal, Adriana C; Smith, Jennifer S; Valea, Fidel; Bentley, Rex; Gradison, Maggie; Yarnall, Kimberly S H; Ford, Anne; Overcash, Francine; Grant, Kathy; Murphy, Susan K; Hoyo, Cathrine
2014-08-01
For poorly understood reasons, invasive cervical cancer (ICC) incidence and mortality rates are higher in women of African descent. Oncogenic human papillomavirus (HPV) genotypes distribution may vary between European American (EA) and African-American (AA) women and may contribute to differences in ICC incidence. The current study aimed at disentangling differences in HPV distribution among AA and EA women. Five-hundred and seventy-two women were enrolled at the time of colposcopic evaluation following an abnormal liquid-based cytology screen. HPV infections were detected using HPV linear array, and chi-squared tests and linear regression models were used to compare HPV genotypes across racial/ethnic groups by CIN status. Of the 572 participants, 494 (86 %) had detectable HPV; 245 (43 %) had no CIN lesion, 239 (42 %) had CIN1, and 88 (15 %) had CIN2/3. Seventy-three percent of all women were infected with multiple HPV genotypes. After adjusting for race, age, parity, income, oral contraception use, and current smoking, AAs were two times less likely to harbor HPV 16/18 (OR 0.48, 95 % CI 0.21-0.94, p = 0.03) when all women were considered. This association remained unchanged when only women with CIN2/3 lesions were examined (OR 0.22, 95 % CI 0.05-0.95, p = 0.04). The most frequent high-risk HPV genotypes detected among EAs were 16, 18, 56, 39, and 66, while HPV genotypes 33, 35, 45, 58, and 68 were the most frequent ones detected in AAs. Our data suggest that while HPV 16/18 are the most common genotypes among EA women with CIN, AAs may harbor different genotypes.
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Heikrujam, Monika; Kumar, Jatin; Agrawal, Veena
2015-09-01
To detect genetic variations among different Simmondsia chinensis genotypes, two gene targeted markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) were employed in terms of their informativeness and efficiency in analyzing genetic relationships among different genotypes. A total of 15 SCoT and 17 CBDP primers detected genetic polymorphism among 39 Jojoba genotypes (22 females and 17 males). Comparatively, CBDP markers proved to be more effective than SCoT markers in terms of percentage polymorphism as the former detecting an average of 53.4% and the latter as 49.4%. The Polymorphic information content (PIC) value and marker index (MI) of CBPD were 0.43 and 1.10, respectively which were higher than those of SCoT where the respective values of PIC and MI were 0.38 and 1.09. While comparing male and female genotype populations, the former showed higher variation in respect of polymorphic percentage and PIC, MI and Rp values over female populations. Nei's diversity (h) and Shannon index (I) were calculated for each genotype and found that the genotype "MS F" (in both markers) was highly diverse and genotypes "Q104 F" (SCoT) and "82-18 F" (CBDP) were least diverse among the female genotype populations. Among male genotypes, "32 M" (CBDP) and "MS M" (SCoT) revealed highest h and I values while "58-5 M" (both markers) was the least diverse. Jaccard's similarity co-efficient of SCoT markers ranged from 0.733 to 0.922 in female genotypes and 0.941 to 0.746 in male genotype population. Likewise, CBDP data analysis also revealed similarity ranging from 0.751 to 0.958 within female genotypes and 0.754 to 0.976 within male genotype populations thereby, indicating genetically diverse Jojoba population. Employing the NTSYS (Numerical taxonomy and multivariate analysis system) Version 2.1 software, both the markers generated dendrograms which revealed that all the Jojoba genotypes were clustered into two major groups, one group consisting of all female genotypes and another group comprising of all male genotypes. During the present investigation, CBDP markers proved more informative in studying genetic diversity among Jojoba. Such genetically diverse genotypes would thus be of great significance for breeding, management and conservation of elite (high yielding) Jojoba germplasm.
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Payne, M S; Furfaro, L L; Tucker, R; Tan, L Y; Mokany, E
2017-08-01
Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10 -5 ng DNA (SV3) and 0.4 × 10 -6 ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. © 2017 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.
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Milekovic, Tomislav; Ball, Tonio; Schulze-Bonhage, Andreas; Aertsen, Ad; Mehring, Carsten
2013-01-01
Background Brain-machine interfaces (BMIs) can translate the neuronal activity underlying a user’s movement intention into movements of an artificial effector. In spite of continuous improvements, errors in movement decoding are still a major problem of current BMI systems. If the difference between the decoded and intended movements becomes noticeable, it may lead to an execution error. Outcome errors, where subjects fail to reach a certain movement goal, are also present during online BMI operation. Detecting such errors can be beneficial for BMI operation: (i) errors can be corrected online after being detected and (ii) adaptive BMI decoding algorithm can be updated to make fewer errors in the future. Methodology/Principal Findings Here, we show that error events can be detected from human electrocorticography (ECoG) during a continuous task with high precision, given a temporal tolerance of 300–400 milliseconds. We quantified the error detection accuracy and showed that, using only a small subset of 2×2 ECoG electrodes, 82% of detection information for outcome error and 74% of detection information for execution error available from all ECoG electrodes could be retained. Conclusions/Significance The error detection method presented here could be used to correct errors made during BMI operation or to adapt a BMI algorithm to make fewer errors in the future. Furthermore, our results indicate that smaller ECoG implant could be used for error detection. Reducing the size of an ECoG electrode implant used for BMI decoding and error detection could significantly reduce the medical risk of implantation. PMID:23383315
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Ware, M W; Keely, S P; Villegas, E N
2013-07-01
This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions. Journal of Applied Microbiology Published [2013]. This article is a U.S. Government work and is in the public domain in the USA.
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Power analysis of QTL detection in half-sib families using selective DNA pooling
Baro, Jesús Á; Carleos, Carlos; Corral, Norberto; López, Teresa; Cañón, Javier
2001-01-01
Individual loci of economic importance (QTL) can be detected by comparing the inheritance of a trait and the inheritance of loci with alleles readily identifiable by laboratory methods (genetic markers). Data on allele segregation at the individual level are costly and alternatives have been proposed that make use of allele frequencies among progeny, rather than individual genotypes. Among the factors that may affect the power of the set up, the most important are those intrinsic to the QTL: the additive effect of the QTL, and its dominance, and distance between markers and QTL. Other factors are relative to the choice of animals and markers, such as the frequency of the QTL and marker alleles among dams and sires. Data collection may affect the detection power through the size of half-sib families, selection rate within families, and the technical error incurred when estimating genetic frequencies. We present results for a sensitivity analysis for QTL detection using pools of DNA from selected half-sibs. Simulations showed that conclusive detection may be achieved with families of at least 500 half-sibs if sires are chosen on the criteria that most of their marker alleles are either both missing, or one is fixed, among dams. PMID:11403746
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Rapid typing of Mannheimia haemolytica major genotypes 1 and 2 using MALDI-TOF mass spectrometry
USDA-ARS?s Scientific Manuscript database
Genotype 2 M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate ge...
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Digital Droplet PCR: CNV Analysis and Other Applications.
Mazaika, Erica; Homsy, Jason
2014-07-14
Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. Copyright © 2014 John Wiley & Sons, Inc.
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Symptomatic and Subclinical Infection with Rotavirus P[8]G9, Rural Ecuador
Endara, Pablo; Trueba, Gabriel; Solberg, Owen D.; Bates, Sarah J.; Ponce, Karina; Cevallos, William; Matthijnssens, Jelle
2007-01-01
During the past decade, rotavirus genotype G9 has spread throughout the world, adding to and sometimes supplanting the common genotypes G1–G4. We report evidence of this spread in a population sample within rural Ecuador. A total of 1,656 stool samples were collected from both patients with diarrhea and asymptomatic residents in 22 remote communities in northwestern Ecuador from August 2003 through February 2006. Rotavirus was detected in 23.4% of case-patients and 3.2% of controls. From these 136 rotavirus-positive samples, a subset of 47 were genotyped; 72% were of genotype G9, and 62% were genotype P[8]G9. As a comparison, 29 rotavirus-positive stool samples were collected from a hospital in Quito during March 2006 and genotyped; 86% were of genotype P[8]G9. Few countries have reported P[8]G9 rotavirus detection rates as high as those of the current study. This growing prevalence may require changes to current vaccination programs to include coverage for this genotype. PMID:17553272
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Michelli, Elvia; Téllez, Luis; Mendoza, José-Andrés; Noguera, María-Eugenia; Milano, Melisse; Vera, Reauben; Callejas, Diana
2013-12-01
Genotyping of human papillomavirus (HPV) by molecular methods may enhance assessment information for screening and following of cervical infection. In this study, cervical samples were obtained from 250 women, along with colposcopic and cytological evaluations. A Nested-PCR-Multiplex assay was used for HPV detection and genotyping for HPV E6/E7 early regions. Infection with HPV was detected in 26.0% of the samples, with 98.46% positive for at least one genotype. High-risk HPVs were identified in 98.44%. HPV18 infection was detected in 76.92% of samples and HPV16 in 36.92%, whether as individual or as multiple infections. These infections were seen more frequently in women under 35 years of age (64.7%). The Pap-smear examination showed that 16.92% (11/65) of the samples had cervical changes suggesting HPV infection, whereas the colposcopic evaluation was suggestive of HPV infection in 47.69% (31/65) of DNA-HPV positive samples. There was a high frequency of high-risk HPV genotypes, particularly HPV18, alone or in multiple-type infections. Colposcopy findings showed to have a high predictive value for the diagnosis of HPV infection. The results reflect that over 50% of HPV-positive patients had a normal colposcopy and/or cytology, highlighting the importance of including HPV testing along with genotype identification in routine gynecological evaluations.
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Lukášová, Radka; Marková, Jiřina; Bártová, Eva; Murat, Jean-Benjamin; Sedlák, Kamil
2018-05-07
Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi are important infectious agents, with T. gondii and E. cuniculi having zoonotic potential. There are two main clonal lineages (types I and II) of T. gondii in Europe, but little is known about genotypes of T. gondii in wild animals. The aim of our study was molecular detection of these three pathogens in tissues of wild red foxes ( Vulpes vulpes) from the Czech Republic. Using PCR (B1 gene), we detected T. gondii in 10% of the animals that we tested ( n=100); N. caninum and E. cuniculi were not detected. The T. gondii samples were genotyped by single multiplex PCR assay with 15 microsatellite markers. Five samples were successfully genotyped as genotype II, a unique finding for T. gondii isolated from red foxes from the Czech Republic.
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Linkage disequilibrium among commonly genotyped SNP and variants detected from bull sequence
USDA-ARS?s Scientific Manuscript database
Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNP genotyped by commercial assays. A number of variants detected from sequencing influential sires are likely to be causal, but noticable improvements in prediction accuracy using imputed sequen...
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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.
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Melo, Thaise P; Takada, Luciana; Baldi, Fernando; Oliveira, Henrique N; Dias, Marina M; Neves, Haroldo H R; Schenkel, Flavio S; Albuquerque, Lucia G; Carvalheiro, Roberto
2016-06-21
QTL mapping through genome-wide association studies (GWAS) is challenging, especially in the case of low heritability complex traits and when few animals possess genotypic and phenotypic information. When most of the phenotypic information is from non-genotyped animals, GWAS can be performed using the weighted single-step GBLUP (WssGBLUP) method, which permits to combine all available information, even that of non-genotyped animals. However, it is not clear to what extent phenotypic information from non-genotyped animals increases the power of QTL detection, and whether factors such as the extent of linkage disequilibrium (LD) in the population and weighting SNPs in WssGBLUP affect the importance of using information from non-genotyped animals in GWAS. These questions were investigated in this study using real and simulated data. Analysis of real data showed that the use of phenotypes of non-genotyped animals affected SNP effect estimates and, consequently, QTL mapping. Despite some coincidence, the most important genomic regions identified by the analyses, either using or ignoring phenotypes of non-genotyped animals, were not the same. The simulation results indicated that the inclusion of all available phenotypic information, even that of non-genotyped animals, tends to improve QTL detection for low heritability complex traits. For populations with low levels of LD, this trend of improvement was less pronounced. Stronger shrinkage on SNPs explaining lower variance was not necessarily associated with better QTL mapping. The use of phenotypic information from non-genotyped animals in GWAS may improve the ability to detect QTL for low heritability complex traits, especially in populations in which the level of LD is high.
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Reemergence of enterovirus 71 epidemic in northern Taiwan, 2012.
Luo, Shu-Ting; Chiang, Pai-Shan; Chung, Wan-Yu; Chia, Min-Yuan; Tsao, Kuo-Chien; Wang, Ying-Hsiang; Lin, Tzou-Yien; Lee, Min-Shi
2015-01-01
Enterovirus 71 (EV71) belongs to picornavirus family and could be classified phylogenetically into three major genogroups (A, B and C) including 11 genotypes (A, B1-B5 and C1-C5). Since 1997, EV71 has caused large-scale of epidemics with neurological complications in Asian children. In Taiwan, nationwide EV71 epidemics with different predominant genotypes have occurred cyclically since 1998. A nationwide EV71 epidemic occurred again in 2012. We conducted genetic and antigenic characterizations of the 2012 epidemic. Chang Gung Memorial Hospital (CGMH) is a medical center in northern Taiwan. In CGMH, specimens were collected from pediatric inpatients with suspected enterovirus infections for virus isolation. Enterovirus isolates were serotyped and genotyped and sera from EV71 inpatients were collected for measuring neutralizing antibody titers. There were 10, 16 and 99 EV71 inpatients identified in 2010, 2011 and 2012, respectively. There were 82 EV71 isolates genotyped, which identified 17 genotype C4a viruses and 65 genotype B5 viruses. The genotype B5 viruses were not detected until November 2011 and caused epidemics in 2012. Interestingly, the B5-2011 viruses were genetically distinguishable from the B5 viruses causing the 2008 epidemic and are likely introduced from China or Southeastern Asia. Based on antigenic analysis, minor antigenic variations were detected among the B5-2008, B5-2011, C4a-2008 and C4a-2012 viruses but these viruses antigenically differed from genotype A. Genotype B5 and C4a viruses antigenically differ from genotype A viruses which have disappeared globally for 30 years but have been detected in China since 2008. Enterovirus surveillance should monitor genetic and antigenic variations of EV71.
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Polymorphism analysis of the prion gene in BSE-affected and unaffected cattle.
Neibergs, H L; Ryan, A M; Womack, J E; Spooner, R L; Williams, J L
1994-10-01
Polymerase chain reaction (PCR) primers designed to amplify the octapeptide repeat region of the bovine prion gene were used to test the association of genotypes with bovine spongiform encephalitis (BSE) in 56 BSE-affected and 177 unaffected animals. Three alleles (A,B,C) were detected as single-strand conformation polymorphisms (SSCPs) and two alleles (1,2--representing six or five copies of the octapeptide repeat respectively) were detected as amplified double-strand fragment length polymorphisms (AMFLPs). Observed genotypes of SSCPs and AMFLPs were analysed by chi-square. The SSCP genotypes of nuclear family members of animals with BSE and BSE-affected animals were different (P < 0.001, P < 0.01) from unrelated animals of the same breed without BSE. No genotypic differences were found between the BSE-affected animals and their relatives (P > 0.469). No AMFLP genotypic differences were detected between BSE-affected animals, their relatives, unrelated animals of the same breed or animals of different breeds (P > 0.05). These data suggest that BSE-affected animals and their relatives are more likely to have the AA SSCP genotype than unrelated animals of the same breed or animals of different breeds.
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Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique.
Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Shaw, Philip J; Ukosakit, Kittipat; Tragoonrung, Somvong; Tongsima, Sissades
2015-01-01
DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. This work presents an automated genotyping tool from DNA gel electrophoresis images, called GELect, which was written in Java and made available through the imageJ framework. With a novel automated image processing workflow, the tool can accurately segment lanes from a gel matrix, intelligently extract distorted and even doublet bands that are difficult to identify by existing image processing tools. Consequently, genotyping from DNA gel electrophoresis can be performed automatically allowing users to efficiently conduct large scale DNA fingerprinting via DNA gel electrophoresis. The software is freely available from http://www.biotec.or.th/gi/tools/gelect.
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Geographical distribution of HCV genotypes in Mexico.
Sánchez-Avila, Juan Francisco; González, Elizabeth; Vázquez, Victoria; Suárez, Susana; Uribe, Misael
2007-01-01
Chronic hepatitis C (CHC) is the second cause of endstage liver disease in our country and one of the main indications of liver transplantation. Hepatitis C virus (HCV) genotype is the principal prognostic factor and the determinant of the therapeutic scheme. In our country few data exist regarding the prevalence of HCV infection and genotype distribution in the Mexican Republic has not been determined. The aim of this study was to characterize the prevalence of the different HCV genotypes and to explore their geographical distribution. Mexican patients with hepatitis C infection, detected throughout the country between 2003 and 2006, were included. All samples were analyzed by a central laboratory and Hepatitis C genotype was identified by Line Immuno Probe Assay in PCR positive samples (Versant Line Probe Assay Quest Diagnostics Nichols Institute, San Juan Capistrano CA). Data were analyzed according to the four geographical areas in Mexico. One thousand three hundred and ninety CHC patients were included. The most frequent genotype detected was genotype 1 (69%) followed by genotype 2 (21.4%) and genotype 3 (9.2%). Genotype 4 and 5 were infrequent. There was no subject infected with genotype 6. Genotype 1 and 2 exhibit very similar distribution in all geographical areas. Genotype 3 infected patients were more frequent in the North region (52%) compared with other areas:center-western (30%), center (17%), South-South east (1%) (p < 0.001). The most prevalent HCV genotype in Mexico is genotype 1. Geographical distribution of HCV genotypes in the four geographical areas in Mexico is not homogenous with a greater frequency of genotype3 in the north region. This difference could be related to the global changes of risk factors for HCV infection.
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Genetic Characterization of Rubella Virus Strains Detected in Spain, 1998-2014.
Martínez-Torres, Alex O; Mosquera, María M; De Ory, Fernando; González-Praetorius, Alejandro; Echevarría, Juan E
2016-01-01
The National Plan for the Elimination of Rubella was implemented in Spain in 2008 using the logistics of the National Plan for the Elimination of Measles that have been employed since year 2000. Molecular characterization of rubella virus (RUBV) is important for disease surveillance and for monitoring elimination of the disease throughout the world. We describe the first complete series of data regarding the circulation of RUBV genotypes in Spain. The 739-nucleotide fragment designated by the WHO for RUBV genotyping was sequenced in 88 selected cases collected from 1998 to 2014. Five genotypes were identified: 1E, 2B, 1J, 1I, and 1a. Genotype 1E was predominant between 1998 and 2003 but was replaced by genotype 2B, which was detected in sporadic cases in 2004, 2006, 2008, 2012, 2013 and 2014. There was an outbreak of genotype 2B in Algeciras (Andalusia) in 2008. Genotype 1J caused an outbreak in Madrid in 2004/2005 and sporadic cases in 2005 and 2007. Genotype 1I was found to have infected an immune-suppressed patient with neurological symptoms in 2008. Finally, vaccine strain RA 27/3 was detected in three sporadic cases, two of them immune-suppressed and without a recent history of vaccination. This suggests that during these years there were a series of imported sporadic cases and outbreaks, confirming the findings of epidemiological data analysis. The importation sources were generally consistent with our geographic and cultural ties, mainly with Europe (genotypes 1E, 2B, 1I) and Latin America (1J).
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Genetic Characterization of Rubella Virus Strains Detected in Spain, 1998-2014
Martínez-Torres, Alex O.; Mosquera, María M.; De Ory, Fernando; González-Praetorius, Alejandro; Echevarría, Juan E.
2016-01-01
The National Plan for the Elimination of Rubella was implemented in Spain in 2008 using the logistics of the National Plan for the Elimination of Measles that have been employed since year 2000. Molecular characterization of rubella virus (RUBV) is important for disease surveillance and for monitoring elimination of the disease throughout the world. We describe the first complete series of data regarding the circulation of RUBV genotypes in Spain. The 739-nucleotide fragment designated by the WHO for RUBV genotyping was sequenced in 88 selected cases collected from 1998 to 2014. Five genotypes were identified: 1E, 2B, 1J, 1I, and 1a. Genotype 1E was predominant between 1998 and 2003 but was replaced by genotype 2B, which was detected in sporadic cases in 2004, 2006, 2008, 2012, 2013 and 2014. There was an outbreak of genotype 2B in Algeciras (Andalusia) in 2008. Genotype 1J caused an outbreak in Madrid in 2004/2005 and sporadic cases in 2005 and 2007. Genotype 1I was found to have infected an immune-suppressed patient with neurological symptoms in 2008. Finally, vaccine strain RA 27/3 was detected in three sporadic cases, two of them immune-suppressed and without a recent history of vaccination. This suggests that during these years there were a series of imported sporadic cases and outbreaks, confirming the findings of epidemiological data analysis. The importation sources were generally consistent with our geographic and cultural ties, mainly with Europe (genotypes 1E, 2B, 1I) and Latin America (1J). PMID:27622271
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Ahlen, Catrine; Aas, Marianne; Krusnell, Jadwiga; Iversen, Ole-Jan
2016-01-01
Recurrent legionella outbreaks at one and the same location are common. We have identified a single Legionella pneumophila genotype associated with recurrent Legionella outbreaks over 6 years. Field emergency surveys following Legionella outbreaks were performed on a vessel in 2008, 2009 and 2013. Water samples from both the distribution and technical parts of the potable water system were analyzed with respect to L. pneumophila [Real-Time PCR, cultivation, serotyping and genotyping (PFGE)] and free-living amoebae, (FLA). Legionella pneumophila serogroup 1 was present in the ship's potable water system during every outbreak. Genotyping of the 2008 survey material showed two separate PFGE genotypes while those in 2009 and 2013 demonstrated the presence of only one of the two genotypes. FLA with intracellular L. pneumophila of the same genotype were also detected. Analyses of the freshwater system on a ship following three separate Legionella outbreaks, for L. pneumophila and FLAs, revealed a single L. pneumophila genotype and FLA (Hartmanella). It is reasonable to assume that the L. pneumophila genotype detected in the freshwater system was the causal agent in the outbreaks onboard. Persistence of an apparently low-pathogenic L. pneumophila genotype and FLA in a potable water system represent a potential risk for recurrent outbreaks.
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Dynamics of HCV epidemiology in Aydin province of Turkey and the associated factors.
Kirdar, Sevin; Aydin, Neriman; Tiryaki, Yasin; Ertugrul, Bulent; Coskun, Adil; Bilgen, Mehmet
2018-02-01
This paper gives an update on the local distributions of HCV genotypes in Aydin province of Turkey, provides a comparison with the previous records, and discusses the potential causal reasons shaping the evolving genotype profiles. Patient files from 2011 to 2016 were retrospectively analyzed, and newly detected cases were documented. Out of 286 patients, male and female ratios were determined to remain nearly the same (~50%). Genotype 1 was still the most common (90.2%), followed by genotype 3 (5.9%), genotype 2 (2.1%), and genotype 4 (1.4%) in frequency. There were international patients (4.50%). One patient had genotyped 2+3 together. Genotypes 4 and 2+3 were detected for the first time, and the patients with genotype 4 were interestingly all male and also domestic individuals. However, these patients traveled or lived abroad in the past due to occupational reasons, thereby likely acquired the infection while abroad. HCV surveillance system is currently inadequate and some infected patients may go undetected in the province. Remapping the regional distribution of HCV genotypes from time-to-time is required for identifying the local dynamics and causes leading to it. This process enhances the clinical preparation and readiness for the better management of the disease. © 2017 APMIS. Published by John Wiley & Sons Ltd.
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DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER
Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) base...
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Ocular manifestations of sickle cell disease and genetic susceptibility for refractive errors
Shukla, Palak; Verma, Henu; Patel, Santosh; Patra, P. K.; Bhaskar, L. V. K. S.
2017-01-01
PURPOSE: Sickle cell disease (SCD) is the most common and serious form of an inherited blood disorder that lead to higher risk of early mortality. SCD patients are at high risk for developing multiorgan acute and chronic complications linked with significant morbidity and mortality. Some of the ophthalmological complications of SCD include retinal changes, refractive errors, vitreous hemorrhage, and abnormalities of the cornea. MATERIALS AND METHODS: The present study includes 96 SCD patients. A dilated comprehensive eye examination was performed to know the status of retinopathy. Refractive errors were measured in all patients. In patients with >10 years of age, cycloplegia was not performed before autorefractometry. A subset of fifty patients’ genotyping was done for NOS3 27-base pair (bp) variable number of tandem repeat (VNTR) and IL4 intron-3 VNTR polymorphisms using polymerase chain reaction-electrophoresis. Chi-square test was performed to test the association between the polymorphisms and refractive errors. RESULTS: The results of the present study revealed that 63.5% of patients have myopia followed by 19.8% hyperopia. NOS3 27-bp VNTR genotypes significantly deviated from Hardy–Weinberg equilibrium (P < 0.0001). Although IL4 70-bp VNTR increased the risk of developing refractive errors, it is not statistically significant. However, NOS3 27-bp VNTR significantly reduced the risk of development of myopia. CONCLUSION: In summary, our study documents the prevalence of refractive errors along with some retinal changes in Indian SCD patients. Further, this study demonstrates that the NOS3 VNTR contributes to the susceptibility to development of myopia in SCD cases. PMID:29018763
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Ocular manifestations of sickle cell disease and genetic susceptibility for refractive errors.
Shukla, Palak; Verma, Henu; Patel, Santosh; Patra, P K; Bhaskar, L V K S
2017-01-01
Sickle cell disease (SCD) is the most common and serious form of an inherited blood disorder that lead to higher risk of early mortality. SCD patients are at high risk for developing multiorgan acute and chronic complications linked with significant morbidity and mortality. Some of the ophthalmological complications of SCD include retinal changes, refractive errors, vitreous hemorrhage, and abnormalities of the cornea. The present study includes 96 SCD patients. A dilated comprehensive eye examination was performed to know the status of retinopathy. Refractive errors were measured in all patients. In patients with >10 years of age, cycloplegia was not performed before autorefractometry. A subset of fifty patients' genotyping was done for NOS3 27-base pair (bp) variable number of tandem repeat (VNTR) and IL4 intron-3 VNTR polymorphisms using polymerase chain reaction-electrophoresis. Chi-square test was performed to test the association between the polymorphisms and refractive errors. The results of the present study revealed that 63.5% of patients have myopia followed by 19.8% hyperopia. NOS3 27-bp VNTR genotypes significantly deviated from Hardy-Weinberg equilibrium ( P < 0.0001). Although IL4 70-bp VNTR increased the risk of developing refractive errors, it is not statistically significant. However, NOS3 27-bp VNTR significantly reduced the risk of development of myopia. In summary, our study documents the prevalence of refractive errors along with some retinal changes in Indian SCD patients. Further, this study demonstrates that the NOS3 VNTR contributes to the susceptibility to development of myopia in SCD cases.
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Genotype harmonizer: automatic strand alignment and format conversion for genotype data integration.
Deelen, Patrick; Bonder, Marc Jan; van der Velde, K Joeri; Westra, Harm-Jan; Winder, Erwin; Hendriksen, Dennis; Franke, Lude; Swertz, Morris A
2014-12-11
To gain statistical power or to allow fine mapping, researchers typically want to pool data before meta-analyses or genotype imputation. However, the necessary harmonization of genetic datasets is currently error-prone because of many different file formats and lack of clarity about which genomic strand is used as reference. Genotype Harmonizer (GH) is a command-line tool to harmonize genetic datasets by automatically solving issues concerning genomic strand and file format. GH solves the unknown strand issue by aligning ambiguous A/T and G/C SNPs to a specified reference, using linkage disequilibrium patterns without prior knowledge of the used strands. GH supports many common GWAS/NGS genotype formats including PLINK, binary PLINK, VCF, SHAPEIT2 & Oxford GEN. GH is implemented in Java and a large part of the functionality can also be used as Java 'Genotype-IO' API. All software is open source under license LGPLv3 and available from http://www.molgenis.org/systemsgenetics. GH can be used to harmonize genetic datasets across different file formats and can be easily integrated as a step in routine meta-analysis and imputation pipelines.
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Takahashi, Masaharu; Nishizawa, Tsutomu; Gotanda, Yuhko; Tsuda, Fumio; Komatsu, Fumio; Kawabata, Terue; Hasegawa, Kyoko; Altankhuu, Murdorjyn; Chimedregzen, Ulziiburen; Narantuya, Luvsanbasaryn; Hoshino, Hiromi; Hino, Kunihiko; Kagawa, Yasuo; Okamoto, Hiroaki
2004-03-01
The prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.
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Selection of core animals in the Algorithm for Proven and Young using a simulation model.
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.
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van Hal, S. J.; Stark, D.; Lockwood, B.; Marriott, D.; Harkness, J.
2007-01-01
Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-β-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct. PMID:17537949
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Prabdial-Sing, N; Chirwa, T; Thaver, J; Smuts, H; Vermeulen, M; Suchard, M; Puren, A J
2016-11-01
There are limited molecular epidemiological studies of hepatitis C at a national level in South Africa. The introduction of newer treatment modalities for hepatitis C requires knowledge of the genotypes as these may have different prognostic and therapeutic implications. This retrospective study describes genotype distributions of patients attending specialist clinics and a blood donor group studied during the period 2008-2012 in South Africa. Residual samples from diagnostic viral load testing from specialist clinics in South Africa (n=941) and from the South African National Blood Service (n=294) were analysed quantitatively by real-time PCR and genotyped using the Versant line probe assay or sequencing. Genotype 1 was predominant in blood donors (34%), whilst genotype 5a was prevalent in patients (36%). In the blood donor group, genotype 4 was detected for the first time. Genotype 2 was rare in the patient group and not detected in blood donors. Genotype 1 was the predominant genotype in the younger age groups (less than 30 years), whereas genotype 5a was found at higher proportions in the older age groups for both the patient and blood donor groups, comprising more than 60% of genotypes in those older than 50 years. Genotypes 1 and 5 were at highest proportions across all provinces compared to other genotypes. In blood donors, genotype 1 was predominant among Caucasians (43%) and genotype 5a among Blacks (54%). Such information is required for planning the impact on the health sector with regard to newly emerging therapies for hepatitis C and burden of disease. © 2016 John Wiley & Sons Ltd.
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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.
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[Hepatitis B virus genotype E infection in Turkey: the detection of the first case].
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.
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Human Papillomavirus (HPV) Genotyping: Automation and Application in Routine Laboratory Testing
Torres, M; Fraile, L; Echevarria, JM; Hernandez Novoa, B; Ortiz, M
2012-01-01
A large number of assays designed for genotyping human papillomaviruses (HPV) have been developed in the last years. They perform within a wide range of analytical sensitivity and specificity values for the different viral types, and are used either for diagnosis, epidemiological studies, evaluation of vaccines and implementing and monitoring of vaccination programs. Methods for specific genotyping of HPV-16 and HPV-18 are also useful for the prevention of cervical cancer in screening programs. Some commercial tests are, in addition, fully or partially automated. Automation of HPV genotyping presents advantages such as the simplicity of the testing procedure for the operator, the ability to process a large number of samples in a short time, and the reduction of human errors from manual operations, allowing a better quality assurance and a reduction of cost. The present review collects information about the current HPV genotyping tests, with special attention to practical aspects influencing their use in clinical laboratories. PMID:23248734
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2013-01-01
Background We studied anal specimens to determine the distribution of human papillomavirus (HPV) genotypes and co-infection occurrence. This information will contribute to the knowledge of HPV genotype distributions and provide an estimate of the prevalence of different oncogenic HPV genotypes found in patients in Madrid (Spain). Methods We studied a total of 82 anal biopsies from the Hospital General Universitario Gregorio Marañón of Madrid. These included 4 specimens with benign lesions, 52 specimens with low-grade anal squamous intraepithelial lesion, 24 specimens with high-grade anal squamous intraepithelial lesions and 2 specimens with invasive anal carcinoma. HPV genotyping was performed with PCR amplification and reverse dot blot hybridization. Results We detected 33 different HPV genotypes, including 16 HPVs associated with a high risk of carcinogenesis, 3 HPVs associated with a highly likely risk of carcinogenesis and 14 HPVs associated with a low-risk of carcinogenesis. In two specimens, an uncharacterized HPV genotype was detected. The most frequent HPV genotypes found were HPV-16 (10.3%; 95% CI: 6.6%-15.1%), HPV-52 (8.5%; 95% CI: 5.2%-13%) and HPV-43/44 (7.6%; 95% CI: 4.5%-11.9%). HPV-18 was only detected in 0.9% (95% CI: 0.1%-3.2%) of the total viruses detected in all lesions. HPV co-infections were found in 83.9% of all types of lesions. The majority of cases (90.2%) were concomitantly infected with the human immunodeficiency virus (HIV). Conclusion The prevalence of high-risk carcinogenic genotypes in anal pathological samples was remarkable. Therefore, further studies that include a greater number of samples, particularly invasive carcinoma cases are needed to evaluate the potential influence of these HPV genotypes in the appearance of anal carcinomas. Also, the influence of other accompanying infections should be evaluated clarify the appearance of this type of carcinoma. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2075238024106058. PMID:24325764
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Comparison of Three Different Commercial Kits for the Human Papilloma Virus Genotyping.
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.
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Nozari, Nazbanou; Dell, Gary S.; Schwartz, Myrna F.
2011-01-01
Despite the existence of speech errors, verbal communication is successful because speakers can detect (and correct) their errors. The standard theory of speech-error detection, the perceptual-loop account, posits that the comprehension system monitors production output for errors. Such a comprehension-based monitor, however, cannot explain the double dissociation between comprehension and error-detection ability observed in the aphasic patients. We propose a new theory of speech-error detection which is instead based on the production process itself. The theory borrows from studies of forced-choice-response tasks the notion that error detection is accomplished by monitoring response conflict via a frontal brain structure, such as the anterior cingulate cortex. We adapt this idea to the two-step model of word production, and test the model-derived predictions on a sample of aphasic patients. Our results show a strong correlation between patients’ error-detection ability and the model’s characterization of their production skills, and no significant correlation between error detection and comprehension measures, thus supporting a production-based monitor, generally, and the implemented conflict-based monitor in particular. The successful application of the conflict-based theory to error-detection in linguistic, as well as non-linguistic domains points to a domain-general monitoring system. PMID:21652015
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Detection of drought tolerant genes within seedling apple rootstocks in Syria
USDA-ARS?s Scientific Manuscript database
This investigation was conducted to detect the drought tolerant genes (four genes) within seedling apple rootstocks derived from five apple genotypes, including Syrian apple cultivars. The results showed that the gene MdPepPro (a cyclophilin) was found in all studied genotypes and their progenies e...
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SNP discovery and genotyping using Genotyping-by-Sequencing in Pekin ducks.
Zhu, Feng; Cui, Qian-Qian; Hou, Zhuo-Cheng
2016-11-15
Genomic selection and genome-wide association studies need thousands to millions of SNPs. However, many non-model species do not have reference chips for detecting variation. Our goal was to develop and validate an inexpensive but effective method for detecting SNP variation. Genotyping by sequencing (GBS) can be a highly efficient strategy for genome-wide SNP detection, as an alternative to microarray chips. Here, we developed a GBS protocol for ducks and tested it to genotype 49 Pekin ducks. A total of 169,209 SNPs were identified from all animals, with a mean of 55,920 SNPs per individual. The average SNP density reached 1156 SNPs/MB. In this study, the first application of GBS to ducks, we demonstrate the power and simplicity of this method. GBS can be used for genetic studies in to provide an effective method for genome-wide SNP discovery.
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ERIC Educational Resources Information Center
Hallin, Anna Eva; Reuterskiöld, Christina
2017-01-01
Purpose: The first aim of this study was to investigate if Swedish-speaking school-age children with language impairment (LI) show specific morphosyntactic vulnerabilities in error detection. The second aim was to investigate the effects of lexical frequency on error detection, an overlooked aspect of previous error detection studies. Method:…
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Kempf, Marie; Abdissa, Alemseged; Diatta, Georges; Trape, Jean-François; Angelakis, Emmanouil; Mediannikov, Oleg; La Scola, Bernard; Raoult, Didier
2012-09-01
Acinetobacter baumannii has previously been detected and genotyped in human body lice. The objectives of this study were to determine the presence of this bacterium in head and body lice collected from healthy individuals in Ethiopia by molecular methods and to characterize the genotype. Human lice from locations at different altitudes in Ethiopia were screened for the presence of Acinetobacter sp by targeting the rpoB gene. Acinetobacter baumannii was detected and genotyped using recA PCR amplification. A total of 115 head and 109 body lice were collected from 134 healthy individuals. Acinetobacter sp were found in 54 head (47%) and 77 body (71%) lice. The recA gene was sequenced for 60 of the Acinetobacter sp and 67% were positive for A. baumannii; genotype 1 was retrieved the most frequently. Our study is the first to show the presence of A. baumannii in human body lice, and also in head lice, in Ethiopia. Copyright © 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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Analysis of the impact of error detection on computer performance
NASA Technical Reports Server (NTRS)
Shin, K. C.; Lee, Y. H.
1983-01-01
Conventionally, reliability analyses either assume that a fault/error is detected immediately following its occurrence, or neglect damages caused by latent errors. Though unrealistic, this assumption was imposed in order to avoid the difficulty of determining the respective probabilities that a fault induces an error and the error is then detected in a random amount of time after its occurrence. As a remedy for this problem a model is proposed to analyze the impact of error detection on computer performance under moderate assumptions. Error latency, the time interval between occurrence and the moment of detection, is used to measure the effectiveness of a detection mechanism. This model is used to: (1) predict the probability of producing an unreliable result, and (2) estimate the loss of computation due to fault and/or error.
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Richman, Susan D; Fairley, Jennifer; Butler, Rachel; Deans, Zandra C
2017-12-01
Evidence strongly indicates that extended RAS testing should be undertaken in mCRC patients, prior to prescribing anti-EGFR therapies. With more laboratories implementing testing, the requirement for External Quality Assurance schemes increases, thus ensuring high standards of molecular analysis. Data was analysed from 15 United Kingdom National External Quality Assessment Service (UK NEQAS) for Molecular Genetics Colorectal cancer external quality assurance (EQA) schemes, delivered between 2009 and 2016. Laboratories were provided annually with nine colorectal tumour samples for genotyping. Information on methodology and extent of testing coverage was requested, and scores given for genotyping, interpretation and clerical accuracy. There has been a sixfold increase in laboratory participation (18 in 2009 to 108 in 2016). For RAS genotyping, fewer laboratories now use Roche cobas®, pyrosequencing and Sanger sequencing, with more moving to next generation sequencing (NGS). NGS is the most commonly employed technology for BRAF and PIK3CA mutation screening. KRAS genotyping errors were seen in ≤10% laboratories, until the 2014-2015 scheme, when there was an increase to 16.7%, corresponding to a large increase in scheme participants. NRAS genotyping errors peaked at 25.6% in the first 2015-2016 scheme but subsequently dropped to below 5%. Interpretation and clerical accuracy scores have been consistently good throughout. Within this EQA scheme, we have observed that the quality of molecular analysis for colorectal cancer has continued to improve, despite changes in the required targets, the volume of testing and the technologies employed. It is reassuring to know that laboratories clearly recognise the importance of participating in EQA schemes.
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Automatic-repeat-request error control schemes
NASA Technical Reports Server (NTRS)
Lin, S.; Costello, D. J., Jr.; Miller, M. J.
1983-01-01
Error detection incorporated with automatic-repeat-request (ARQ) is widely used for error control in data communication systems. This method of error control is simple and provides high system reliability. If a properly chosen code is used for error detection, virtually error-free data transmission can be attained. Various types of ARQ and hybrid ARQ schemes, and error detection using linear block codes are surveyed.
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Heikrujam, Monika; Kumar, Jatin; Agrawal, Veena
2015-01-01
To detect genetic variations among different Simmondsia chinensis genotypes, two gene targeted markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) were employed in terms of their informativeness and efficiency in analyzing genetic relationships among different genotypes. A total of 15 SCoT and 17 CBDP primers detected genetic polymorphism among 39 Jojoba genotypes (22 females and 17 males). Comparatively, CBDP markers proved to be more effective than SCoT markers in terms of percentage polymorphism as the former detecting an average of 53.4% and the latter as 49.4%. The Polymorphic information content (PIC) value and marker index (MI) of CBPD were 0.43 and 1.10, respectively which were higher than those of SCoT where the respective values of PIC and MI were 0.38 and 1.09. While comparing male and female genotype populations, the former showed higher variation in respect of polymorphic percentage and PIC, MI and Rp values over female populations. Nei's diversity (h) and Shannon index (I) were calculated for each genotype and found that the genotype “MS F” (in both markers) was highly diverse and genotypes “Q104 F” (SCoT) and “82–18 F” (CBDP) were least diverse among the female genotype populations. Among male genotypes, “32 M” (CBDP) and “MS M” (SCoT) revealed highest h and I values while “58-5 M” (both markers) was the least diverse. Jaccard's similarity co-efficient of SCoT markers ranged from 0.733 to 0.922 in female genotypes and 0.941 to 0.746 in male genotype population. Likewise, CBDP data analysis also revealed similarity ranging from 0.751 to 0.958 within female genotypes and 0.754 to 0.976 within male genotype populations thereby, indicating genetically diverse Jojoba population. Employing the NTSYS (Numerical taxonomy and multivariate analysis system) Version 2.1 software, both the markers generated dendrograms which revealed that all the Jojoba genotypes were clustered into two major groups, one group consisting of all female genotypes and another group comprising of all male genotypes. During the present investigation, CBDP markers proved more informative in studying genetic diversity among Jojoba. Such genetically diverse genotypes would thus be of great significance for breeding, management and conservation of elite (high yielding) Jojoba germplasm. PMID:26110116
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What errors do peer reviewers detect, and does training improve their ability to detect them?
Schroter, Sara; Black, Nick; Evans, Stephen; Godlee, Fiona; Osorio, Lyda; Smith, Richard
2008-10-01
To analyse data from a trial and report the frequencies with which major and minor errors are detected at a general medical journal, the types of errors missed and the impact of training on error detection. 607 peer reviewers at the BMJ were randomized to two intervention groups receiving different types of training (face-to-face training or a self-taught package) and a control group. Each reviewer was sent the same three test papers over the study period, each of which had nine major and five minor methodological errors inserted. BMJ peer reviewers. The quality of review, assessed using a validated instrument, and the number and type of errors detected before and after training. The number of major errors detected varied over the three papers. The interventions had small effects. At baseline (Paper 1) reviewers found an average of 2.58 of the nine major errors, with no notable difference between the groups. The mean number of errors reported was similar for the second and third papers, 2.71 and 3.0, respectively. Biased randomization was the error detected most frequently in all three papers, with over 60% of reviewers rejecting the papers identifying this error. Reviewers who did not reject the papers found fewer errors and the proportion finding biased randomization was less than 40% for each paper. Editors should not assume that reviewers will detect most major errors, particularly those concerned with the context of study. Short training packages have only a slight impact on improving error detection.
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[Detection and genotyping of human papillomavirus in biopsies of uterine cervical adenocarcinoma].
Brebi M, Priscilla; Ili G, Carmen Gloria; López M, Jaime; García M, Patricia; Melo A, Angélica; Montenegro H, Sonia; Leal R, Pamela; Guzmán G, Pablo; Roa S, Juan Carlos
2009-03-01
The genotyping of Human Papillomavirus (HPV) will improve knowledge about the local epidemiological association of this virus with adenocarcinoma. To determine the frequency of HPV genotypes in biopsies of women with uterine cervical adenocarcinoma in a geographic region of Chile. Forty-one cervical biopsies with a pathological diagnosis of adenocarcinoma, corresponding to all women diagnosed with this cancer between 2002 and 2004, were analyzed. Viral gene Ll was amplified by PCRfor viral detection. HPV genotyping was carried out by a Reverse Line Blot technique. Seventy one percent of biopsies were positive for HPV. The most common genotypes found were HPV 16 (61%), followed by HPV 18 (19.5%). Eighty seven percent of biopsies had a single HPV infection. Three patients had a multiple HPV infection. All of the latter were infected by HPV 16, associated with other three viral genotypes (45, 52 and 66). No low-risk HPV genotypes were found. In this sample of biopsies, there was a high prevelence of HPV 16 and a low prevalence of HPV 18, which historically has been related to adenocarcinoma. The genotypes found correspond to those described in South America.
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Liefeldt, Lutz; Plentz, Annelie; Klempa, Boris; Kershaw, Olivia; Endres, Anne-Sophie; Raab, Ulla; Neumayer, Hans-H; Meisel, Helga; Modrow, Susanne
2005-01-01
Organ transplant recipients infected with parvovirus B19 frequently develop persistent viremia associated with chronic anemia and pure red cell aplasia. In this study, a male renal transplant recipient who had been infected with parvovirus B19/genotype 2 after renal transplantation at the age of 34 years is described. The patient was repeatedly treated with high dose intravenous immunoglobulin (IVIG) that resulted in the resolvement of symptoms but not in virus eradication. During an observation period of 33 months after transplantation three phases associated with high parvovirus B19 viremia were observed. Both the first and the second viremic phases were combined with severe anemia. Parvovirus B19 specific IgM-antibodies were initially detected at the beginning of the second phase in continually rising concentrations. Initially eradication of the virus by immunoglobulin therapy was reported after the first viremic phase [Liefeldt et al. (2002): Nephrol Dial Transplant 17:1840-1842]. Retrospectively this statement has to be corrected. It was based on the use of a qualitative PCR assay specific for parvovirus B19 genotype 1 associated with reduced sensitivity for detection of genotype 2. After sequence analysis of the viral DNA and adjustment of a real-time PCR assay (TaqMan) for quantitative detection of all three B19 virus genotypes analysis of consecutive serum samples allowed the demonstration of long lasting phases with reduced viral loads following IVIG-treatment. These results demonstrate that IVIG treatment of parvovirus B19-triggered anemia in transplant recipients offers an opportunity to resolve symptoms, but does not guarantee eradication of the virus. Since reactivation of parvovirus B19 infection can result in high virus load associated with the recurrence of symptoms repeated screening for viral DNA is recommended using the TaqMan system established for quantitative detection of all three genotypes of parvovirus B19. Copyright 2005 Wiley-Liss, Inc.
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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.
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Multi-criteria decision making approaches for quality control of genome-wide association studies.
Malovini, Alberto; Rognoni, Carla; Puca, Annibale; Bellazzi, Riccardo
2009-03-01
Experimental errors in the genotyping phases of a Genome-Wide Association Study (GWAS) can lead to false positive findings and to spurious associations. An appropriate quality control phase could minimize the effects of this kind of errors. Several filtering criteria can be used to perform quality control. Currently, no formal methods have been proposed for taking into account at the same time these criteria and the experimenter's preferences. In this paper we propose two strategies for setting appropriate genotyping rate thresholds for GWAS quality control. These two approaches are based on the Multi-Criteria Decision Making theory. We have applied our method on a real dataset composed by 734 individuals affected by Arterial Hypertension (AH) and 486 nonagenarians without history of AH. The proposed strategies appear to deal with GWAS quality control in a sound way, as they lead to rationalize and make explicit the experimenter's choices thus providing more reproducible results.
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Bontempi, Iván A; Bizai, María L; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana; Solari, Aldo; Diez, Cristina
2016-09-01
Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa=0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed. Copyright © 2016 Elsevier B.V. All rights reserved.
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da Silva, Marcelle Figueira Marques; Fumian, Tulio Machado; de Assis, Rosane Maria Santos; Fialho, Alexandre Madi; Carvalho-Costa, Filipe Anibal; da Silva Ribeiro de Andrade, Juliana; Leite, José Paulo Gagliardi
2017-01-01
Group A rotavirus (RVA) genotype G12 is habitually associated with diarrhea disease (DD) in African children and recently its detection has increased worldwide. A total of 970 stool samples collected from individuals with DD in the Northeastern, Southeastern, and Southern Brazilian regions, Eastern coast, were analyzed and 321 (33%) were positive for RVA and of these, 241 (75%) genotyped as G12P[8]. The rate of RVA positivity was higher among children aged 5-10 years old (60%). All RVA infections observed in adults aged >21 years were G12P[8] (n = 27) showing that this genotype affected older age groups during the year of 2014 in Brazil. Phylogenetic analysis of VP7 and VP8* G12P[8] strains demonstrated an elevated similarity among Brazilian and G12-III prototypes strains circulating worldwide recently, suggesting that this lineage is associated with the global spread of the G12 genotype, considered as the 6th most prevalent human RVA genotype nowadays; while other G12 lineages remain sporadically detected and usually detected in association with other P genotypes. VP8* analysis revealed that Brazilian strains belong to P[8]-3 lineage, the single P[8] lineage presently detected in the country. No major nucleotide/amino acid disparities were observed among strains recovered from children and adults for VP7 and VP8* genes. These data are essential to support the surveillance studies, particularly in countries where the RVA vaccine was introduced in their National Immunization Program enabling identification of potential alterations in the epidemiological profile that can impact its efficacy in vaccination programs. J. Med. Virol. 89:64-70, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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Paweletz, Cloud P; Sacher, Adrian G; Raymond, Chris K; Alden, Ryan S; O'Connell, Allison; Mach, Stacy L; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M; Lim, Lee P; Jänne, Pasi A; Oxnard, Geoffrey R
2016-02-15
Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. ©2015 American Association for Cancer Research.
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Paweletz, Cloud P.; Sacher, Adrian G.; Raymond, Chris K.; Alden, Ryan S.; O'Connell, Allison; Mach, Stacy L.; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M.; Lim, Lee P.; Jänne, Pasi A.; Oxnard, Geoffrey R.
2015-01-01
Purpose Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care, however comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusion Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. PMID:26459174
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Genotyping of polyomavirus BK by Real Time PCR for VP1 gene.
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.
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Johansson, Ewa; Welinder-Olsson, Christina; Gilljam, Marita
2014-02-01
Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. © 2013 APMIS. Published by John Wiley & Sons Ltd.
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Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H
2015-08-01
HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.
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Denaturing high-performance liquid chromatography for mutation detection and genotyping.
Fackenthal, Donna Lee; Chen, Pei Xian; Howe, Ted; Das, Soma
2013-01-01
Denaturing high-performance liquid chromatography (DHPLC) is an accurate and efficient screening technique used for detecting DNA sequence changes by heteroduplex analysis. It can also be used for genotyping of single nucleotide polymorphisms (SNPs). The high sensitivity of DHPLC has made this technique one of the most reliable approaches to mutation analysis and, therefore, used in various areas of genetics, both in the research and clinical arena. This chapter describes the methods used for mutation detection analysis and the genotyping of SNPs by DHPLC on the WAVE™ system from Transgenomic Inc. ("WAVE" and "DNASep" are registered trademarks, and "Navigator" is a trademark, of Transgenomic, used with permission. All other trademarks are property of the respective owners).
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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.
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Stanton, Jeffrey J; Zong, Jian-Chao; Eng, Crystal; Howard, Lauren; Flanagan, Joe; Stevens, Martina; Schmitt, Dennis; Wiedner, Ellen; Graham, Danielle; Junge, Randall E; Weber, Martha A; Fischer, Martha; Mejia, Alicia; Tan, Jie; Latimer, Erin; Herron, Alan; Hayward, Gary S; Ling, Paul D
2013-03-01
Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.
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A real time genotyping PCR assay for polyomavirus BK.
Gard, Lilli; Niesters, Hubert G M; Riezebos-Brilman, Annelies
2015-09-01
Polyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I-IV). On the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630-1956). For every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed. This study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype. Copyright © 2015 Elsevier B.V. All rights reserved.
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[Detection and application of PIS genetic deficiency gene in dairy goat].
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.
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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
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Randhawa, P; Pastrana, D V; Zeng, G; Huang, Y; Shapiro, R; Sood, P; Puttarajappa, C; Berger, M; Hariharan, S; Buck, C B
2015-04-01
Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype-specificity, and mechanism of action of anti-polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co-transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon-modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype-specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti-viral effects, until effective small molecule inhibitors of BKV replication can be developed. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
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Auguste, Albert J.; Liria, Jonathan; Forrester, Naomi L.; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C.; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B.; Halsey, Eric S.; Kochel, Tadeusz J.; Hernandez, Rosa; Navarro, Juan-Carlos
2015-01-01
In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions. PMID:26401714
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Auguste, Albert J; Liria, Jonathan; Forrester, Naomi L; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B; Halsey, Eric S; Kochel, Tadeusz J; Hernandez, Rosa; Navarro, Juan-Carlos; Weaver, Scott C
2015-10-01
In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.
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Hepatitis A and E Viruses in Wastewaters, in River Waters, and in Bivalve Molluscs in Italy.
Iaconelli, M; Purpari, G; Della Libera, S; Petricca, S; Guercio, A; Ciccaglione, A R; Bruni, R; Taffon, S; Equestre, M; Fratini, M; Muscillo, M; La Rosa, Giuseppina
2015-12-01
Several studies have reported the detection of hepatitis A (HAV) and E (HEV) virus in sewage waters, indicating a possibility of contamination of aquatic environments. The objective of the present study was to assess the occurrence of HAV and HEV in different water environments, following the route of contamination from raw sewage through treated effluent to the surface waters receiving wastewater discharges . Bivalve molluscan shellfish samples were also analyzed, as sentinel of marine pollution. Samples were tested by RT-PCR nested type in the VP1/2A junction for HAV, and in the ORF1 and ORF2 regions for HEV. Hepatitis A RNA was detected in 12 water samples: 7/21 (33.3%) raw sewage samples, 3/21 (14.3%) treated sewage samples, and 2/27 (7.4%) river water samples. Five sequences were classified as genotype IA, while the remaining 7 sequences belonged to genotype IB. In bivalves, HAV was detected in 13/56 samples (23.2%), 12 genotype IB and one genotype IA. Whether the presence of HAV in the matrices tested indicates the potential for waterborne and foodborne transmission is unknown, since infectivity of the virus was not demonstrated. HEV was detected in one raw sewage sample and in one river sample, both belonging to genotype 3. Sequences were similar to sequences detected previously in Italy in patients with autochthonous HEV (no travel history) and in animals (swine). To our knowledge, this is the first detection of HEV in river waters in Italy, suggesting that surface water can be a potential source for exposure .
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Díaz, Luis Adrian; Albrieu Llinás, Guillermo; Vázquez, Ana; Tenorio, Antonio; Contigiani, Marta Silvia
2012-01-01
St. Louis encephalitis virus is a complex zoonoses. In 2005, 47 laboratory-confirmed and probable clinical cases of SLEV infection were reported in Córdoba, Argentina. Although the causes of 2005 outbreak remain unknown, they might be related not only to virological factors, but also to ecological and environmental conditions. We hypothesized that one of the factors for SLE reemergence in Córdoba, Argentina, was the introduction of a new SLEV genotype (SLEV genotype III), with no previous activity in the area. In order to evaluate this hypothesis we carried out a molecular characterization of SLEV detections from mosquitoes collected between 2001 and 2004 in Córdoba city. A total of 315 mosquito pools (11,002 individuals) including 12 mosquitoes species were analyzed. Overall, 20 pools (8 mosquitoes species) were positive for SLEV. During this study, genotypes II, V and VII were detected. No mosquito pool infected with genotype III was detected before the 2005 outbreak. Genotype V was found every year and in the 8 sampled sites. Genotypes II and VII showed limited temporal and spatial activities. We cannot dismiss the association of genotype II and V as etiological agents during the outbreak. However, the silent circulation of other SLEV strains in Córdoba city before the 2005 outbreak suggests that the introduction of genotype III was an important factor associated to this event. Not mutually exclusive, other factors such as changes in avian hosts and mosquitoes vectors communities, driven by climatic and environmental modifications, should also be taken into consideration in further studies. PMID:22303490
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Chorny, Joseph A; Frye, Teresa C; Fisher, Beth L; Remmers, Carol L
2018-03-23
The primary high-risk human papillomavirus (hrHPV) assays in the United States are the cobas (Roche) and the Aptima (Hologic). The cobas assay detects hrHPV by DNA analysis while the Aptima detects messenger RNA (mRNA) oncogenic transcripts. As the Aptima assay identifies oncogenic expression, it should have a lower rate of hrHPV and genotype detection. The Kaiser Permanente Regional Reference Laboratory in Denver, Colorado changed its hrHPV assay from the cobas to the Aptima assay. The rates of hrHPV detection and genotyping were compared over successive six-month periods. The overall hrHPV detection rates by the two platforms were similar (9.5% versus 9.1%) and not statistically different. For genotyping, the HPV 16 rate by the cobas was 1.6% and by the Aptima it was 1.1%. These differences were statistically different with the Aptima detecting nearly one-third less HPV 16 infections. With the HPV 18 and HPV 18/45, there was a slightly higher detection rate of HPV 18/45 by the Aptima platform (0.5% versus 0.9%) and this was statistically significant. While HPV 16 represents a low percentage of hrHPV infections, it was detected significantly less by the Aptima assay compared to the cobas assay. This has been previously reported, although not highlighted. Given the test methodologies, one would expect the Aptima to detect less HPV 16. This difference appears to be mainly due to a significantly increased number of non-oncogenic HPV 16 infections detected by the cobas test as there were no differences in HPV 16 detection rates in the high-grade squamous intraepithelial lesions indicating that the two tests have similar sensitivities for oncogenic HPV 16. © 2018 Wiley Periodicals, Inc.
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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.
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Sugimoto, Daichi; Nakano, Mamoru; Inada, Machi; Fujitani, Misako; Chiba, Shoko; Sakai, Takeshi
2017-09-25
This study was conducted to investigate the distribution of rotavirus genotypes in Nara Prefecture, Japan before and after the introduction of rotavirus vaccination in 2011. Since the 2011/2012 season, DS-1-like G1P[8] strains have been detected in Nara Prefecture, accounting for about half of all strains in the 2014/2015 season. During the 2015/2016 season, no DS-1-like G1P[8] strains were detected; G2P[4] was the predominant genotype.
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Meaza, Abyot; Kebede, Abebaw; Yaregal, Zelalem; Dagne, Zekarias; Moga, Shewki; Yenew, Bazezew; Diriba, Getu; Molalign, Helina; Tadesse, Mengistu; Adisse, Desalegn; Getahun, Muluwork; Desta, Kassu
2017-04-17
Multi drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples. A cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant. The sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples. The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance. The diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.
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Córcoles, A.D.; Magesan, Easwar; Srinivasan, Srikanth J.; Cross, Andrew W.; Steffen, M.; Gambetta, Jay M.; Chow, Jerry M.
2015-01-01
The ability to detect and deal with errors when manipulating quantum systems is a fundamental requirement for fault-tolerant quantum computing. Unlike classical bits that are subject to only digital bit-flip errors, quantum bits are susceptible to a much larger spectrum of errors, for which any complete quantum error-correcting code must account. Whilst classical bit-flip detection can be realized via a linear array of qubits, a general fault-tolerant quantum error-correcting code requires extending into a higher-dimensional lattice. Here we present a quantum error detection protocol on a two-by-two planar lattice of superconducting qubits. The protocol detects an arbitrary quantum error on an encoded two-qubit entangled state via quantum non-demolition parity measurements on another pair of error syndrome qubits. This result represents a building block towards larger lattices amenable to fault-tolerant quantum error correction architectures such as the surface code. PMID:25923200
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Córcoles, A D; Magesan, Easwar; Srinivasan, Srikanth J; Cross, Andrew W; Steffen, M; Gambetta, Jay M; Chow, Jerry M
2015-04-29
The ability to detect and deal with errors when manipulating quantum systems is a fundamental requirement for fault-tolerant quantum computing. Unlike classical bits that are subject to only digital bit-flip errors, quantum bits are susceptible to a much larger spectrum of errors, for which any complete quantum error-correcting code must account. Whilst classical bit-flip detection can be realized via a linear array of qubits, a general fault-tolerant quantum error-correcting code requires extending into a higher-dimensional lattice. Here we present a quantum error detection protocol on a two-by-two planar lattice of superconducting qubits. The protocol detects an arbitrary quantum error on an encoded two-qubit entangled state via quantum non-demolition parity measurements on another pair of error syndrome qubits. This result represents a building block towards larger lattices amenable to fault-tolerant quantum error correction architectures such as the surface code.
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Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui
2012-01-13
Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.
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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...
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Integrated analysis of error detection and recovery
NASA Technical Reports Server (NTRS)
Shin, K. G.; Lee, Y. H.
1985-01-01
An integrated modeling and analysis of error detection and recovery is presented. When fault latency and/or error latency exist, the system may suffer from multiple faults or error propagations which seriously deteriorate the fault-tolerant capability. Several detection models that enable analysis of the effect of detection mechanisms on the subsequent error handling operations and the overall system reliability were developed. Following detection of the faulty unit and reconfiguration of the system, the contaminated processes or tasks have to be recovered. The strategies of error recovery employed depend on the detection mechanisms and the available redundancy. Several recovery methods including the rollback recovery are considered. The recovery overhead is evaluated as an index of the capabilities of the detection and reconfiguration mechanisms.
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Failed triple therapy in a treatment-experienced patient with genotype 6 hepatitis C infection.
Gammal, Roseann S; Spooner, Linda M; Abraham, George M
2014-02-01
The first published report of the use of triple therapy in a patient with hepatitis C virus (HCV) genotype 6 infection-a treatment that was prescribed due to incorrect HCV genotyping and which ultimately failed-is presented. A 70-year-old male U.S. resident of Vietnamese descent requested treatment for chronic HCV infection acquired decades earlier. He reported experiencing hepatitis C treatment failures twice before-13 years prior (interferon alfa monotherapy for six months) and 7 years prior (standard dual therapy with pegylated interferon alfa-2b and ribavirin for nine months). Initial viral genotyping indicated infection with HCV genotypes 1a and 6c (a form of mixed HCV disease amenable to triple therapy), and treatment with pegylated interferon alfa-2a, ribavirin, and boceprevir was initiated. By week 8 of triple therapy, the patient's viral load had decreased from 15,700,000 (7.20 log) to 462,882 (5.67 log) IU/mL, but the viral load subsequently rebounded to baseline levels, and treatment was discontinued at week 16. When repeat HCV genotyping was performed, it was discovered that initial genotyping was incorrect and that the man's infection involved not mixed genotypes but only genotype 6; he was not an appropriate candidate for triple therapy. The case emphasizes the need for clinicians to be cognizant of potential HCV genotyping errors, particularly with regard to patients of Southeast Asian descent. Three courses of interferon-based treatment, including triple therapy with boceprevir, failed to produce a sustained therapeutic response in a 70-year-old ethnic Vietnamese man with genotype 6 HCV infection.
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Barstad, Bjørn; Quarsten, Hanne; Tveitnes, Dag; Noraas, Sølvi; Ask, Ingvild S; Saeed, Maryam; Bosse, Franziskus; Vigemyr, Grete; Huber, Ilka; Øymar, Knut
2018-05-01
The current diagnostic marker of Lyme neuroborreliosis (LNB), the Borrelia burgdorferi sensu lato antibody index (AI) in the cerebrospinal fluid (CSF), has insufficient sensitivity in the early phase of LNB. We aimed to elucidate the diagnostic value of PCR for B. burgdorferi sensu lato in CSF from children with symptoms suggestive of LNB and to explore B. burgdorferi sensu lato genotypes associated with LNB in children. Children were prospectively included in predefined groups with a high or low likelihood of LNB based on diagnostic guidelines (LNB symptoms, CSF pleocytosis, and B. burgdorferi sensu lato antibodies) or the detection of other causative agents. CSF samples were analyzed by two B. burgdorferi sensu lato -specific real-time PCR assays and, if B. burgdorferi sensu lato DNA was detected, were further analyzed by five singleplex real-time PCR assays for genotype determination. For children diagnosed as LNB patients (58 confirmed and 18 probable) ( n = 76) or non-LNB controls ( n = 28), the sensitivity and specificity of PCR for B. burgdorferi sensu lato in CSF were 46% and 100%, respectively. B. burgdorferi sensu lato DNA was detected in 26/58 (45%) children with AI-positive LNB and in 7/12 (58%) children with AI-negative LNB and symptoms of short duration. Among 36 children with detectable B. burgdorferi sensu lato DNA, genotyping indicated Borrelia garinii ( n = 27) and non- B. garinii ( n = 1) genotypes, while 8 samples remained untyped. Children with LNB caused by B. garinii did not have a distinct clinical picture. The rate of detection of B. burgdorferi sensu lato DNA in the CSF of children with LNB was higher than that reported previously. PCR for B. burgdorferi sensu lato could be a useful supplemental diagnostic tool in unconfirmed LNB cases with symptoms of short duration. B. garinii was the predominant genotype in children with LNB. Copyright © 2018 American Society for Microbiology.
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Rotavirus Strain Trends During the Postlicensure Vaccine Era: United States, 2008–2013
Bowen, Michael D.; Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Teel, Elizabeth N.; Gautam, Rashi; Sturgeon, Michele; Azimi, Parvin H.; Baker, Carol J.; Bernstein, David I.; Boom, Julie A.; Chappell, James; Donauer, Stephanie; Edwards, Kathryn M.; Englund, Janet A.; Halasa, Natasha B.; Harrison, Christopher J.; Johnston, Samantha H.; Klein, Eileen J.; McNeal, Monica M.; Moffatt, Mary E.; Rench, Marcia A.; Sahni, Leila C.; Selvarangan, Rangaraj; Staat, Mary A.; Szilagyi, Peter G.; Weinberg, Geoffrey A.; Wikswo, Mary E.; Parashar, Umesh D.; Payne, Daniel C.
2016-01-01
Background Group A rotaviruses (RVA) are a significant cause of pediatric gastroenteritis worldwide. The New Vaccine Surveillance Network (NVSN) has conducted active surveillance for RVA at pediatric hospitals and emergency departments at 3–7 geographically diverse sites in the United States since 2006. Methods Over 6 consecutive years, from 2008 to 2013, 1523 samples from NVSN sites that were tested positive by a Rotaclone enzyme immunoassay were submitted to the Centers for Disease Control and Prevention for genotyping. Results In the 2009, 2010, and 2011 seasons, genotype G3P[8] was the predominant genotype throughout the network, with a 46%–84% prevalence. In the 2012 season, G12P[8] replaced G3P[8] as the most common genotype, with a 70% prevalence, and this trend persisted in 2013 (68.0% prevalence). Vaccine (RotaTeq; Rotarix) strains were detected in 0.6%–3.4% of genotyped samples each season. Uncommon and unusual strains (eg, G8P[4], G3P[24], G2P[8], G3P[4], G3P[6], G24P[14], G4P[6], and G9P[4]) were detected sporadically over the study period. Year, study site, and race were found to be significant predictors of genotype. Conclusions Continued active surveillance is needed to monitor RVA genotypes in the United States and to detect potential changes since vaccine licensure. PMID:27302190
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Bulk Genotyping of Biopsies Can Create Spurious Evidence for Hetereogeneity in Mutation Content.
Kostadinov, Rumen; Maley, Carlo C; Kuhner, Mary K
2016-04-01
When multiple samples are taken from the neoplastic tissues of a single patient, it is natural to compare their mutation content. This is often done by bulk genotyping of whole biopsies, but the chance that a mutation will be detected in bulk genotyping depends on its local frequency in the sample. When the underlying mutation count per cell is equal, homogenous biopsies will have more high-frequency mutations, and thus more detectable mutations, than heterogeneous ones. Using simulations, we show that bulk genotyping of data simulated under a neutral model of somatic evolution generates strong spurious evidence for non-neutrality, because the pattern of tissue growth systematically generates differences in biopsy heterogeneity. Any experiment which compares mutation content across bulk-genotyped biopsies may therefore suggest mutation rate or selection intensity variation even when these forces are absent. We discuss computational and experimental approaches for resolving this problem.
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Ogunsemowo, Olukunle; Olaleye, David O; Odaibo, Georgina N
2018-01-01
Human respiratory syncytial virus (HRSV) is the most common viral cause of acute lower respiratory tract infections (LRTIs) in infants and young children however, without an effective vaccine licensed for human use till date. Information on the circulating genotypes of HRSV from regions with high-burden of infection is vital in the global efforts towards the development of protective vaccine. We report here the genotypes of HRSV circulating among children in Ibadan, the first of such from Nigeria.Nasopharyngeal and oropharyngeal swabs collected from 231 children presenting with respiratory infections in some health facilities for care as well as those attending immunization centers for routine vaccination in Ibadan, Nigeria were used for the study. The 2nd hypervariable (HVR2) region of the glycoprotein (G) gene of HRSV was amplified and sequenced using HRSV group specific primers. HRSV was detected in 41 out of the 231 samples. Thirty-three of the isolates were successfully subtyped(22 subtype A and 11 subtype B). Fourteen of the subtype A and all the subtype B were successfully sequenced and genotyped. Phylogenetic analysis showed that genotype ON1 with 72 nucleotide (nt) duplication was the major subgroup A virus (11 of 14) detected together with genotype NA2. All the HRSV subtype B detected belong to the BA genotype with characteristic 60nt duplication. The ON1 genotypes vary considerably from the prototype strain due to amino acid substitutions including T292I which has not been reported elsewhere. The NA2 genotypes have mutations on four antigenic sites within the HVR2relative to the prototype A2. In conclusion, three genotypes of HRSV were found circulating in Ibadan, Nigeria. Additional study that will include isolates from other parts of the country will be done to determine the extent of genotype diversity of HRSV circulating in Nigeria.
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Sensitivity in error detection of patient specific QA tools for IMRT plans
NASA Astrophysics Data System (ADS)
Lat, S. Z.; Suriyapee, S.; Sanghangthum, T.
2016-03-01
The high complexity of dose calculation in treatment planning and accurate delivery of IMRT plan need high precision of verification method. The purpose of this study is to investigate error detection capability of patient specific QA tools for IMRT plans. The two H&N and two prostate IMRT plans with MapCHECK2 and portal dosimetry QA tools were studied. Measurements were undertaken for original and modified plans with errors introduced. The intentional errors composed of prescribed dose (±2 to ±6%) and position shifting in X-axis and Y-axis (±1 to ±5mm). After measurement, gamma pass between original and modified plans were compared. The average gamma pass for original H&N and prostate plans were 98.3% and 100% for MapCHECK2 and 95.9% and 99.8% for portal dosimetry, respectively. In H&N plan, MapCHECK2 can detect position shift errors starting from 3mm while portal dosimetry can detect errors started from 2mm. Both devices showed similar sensitivity in detection of position shift error in prostate plan. For H&N plan, MapCHECK2 can detect dose errors starting at ±4%, whereas portal dosimetry can detect from ±2%. For prostate plan, both devices can identify dose errors starting from ±4%. Sensitivity of error detection depends on type of errors and plan complexity.
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Molecular Characterization of Chikungunya Virus, Philippines, 2011-2013.
Sy, Ava Kristy; Saito-Obata, Mariko; Medado, Inez Andrea; Tohma, Kentaro; Dapat, Clyde; Segubre-Mercado, Edelwisa; Tandoc, Amado; Lupisan, Socorro; Oshitani, Hitoshi
2016-05-01
During 2011-2013, a nationwide outbreak of chikungunya virus infection occurred in the Philippines. The Asian genotype was identified as the predominant genotype; sporadic cases of the East/Central/South African genotype were detected in Mindanao. Further monitoring is needed to define the transmission pattern of this virus in the Philippines.
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Microsatellite markers associated with resistance to Marek's disease in commercial layer chickens.
McElroy, J P; Dekkers, J C M; Fulton, J E; O'Sullivan, N P; Soller, M; Lipkin, E; Zhang, W; Koehler, K J; Lamont, S J; Cheng, H H
2005-11-01
The objective of the current study was to identify QTL conferring resistance to Marek's disease (MD) in commercial layer chickens. To generate the resource population, 2 partially inbred lines that differed in MD-caused mortality were intermated to produce 5 backcross families. Vaccinated chicks were challenged with very virulent plus (vv+) MD virus strain 648A at 6 d and monitored for MD symptoms. A recent field isolate of the MD virus was used because the lines were resistant to commonly used older laboratory strains. Selective genotyping was employed using 81 microsatellites selected based on prior results with selective DNA pooling. Linear regression and Cox proportional hazard models were used to detect associations between marker genotypes and survival. Significance thresholds were validated by simulation. Seven and 6 markers were significant based on proportion of false positive and false discovery rate thresholds less than 0.2, respectively. Seventeen markers were associated with MD survival considering a comparison-wise error rate of 0.10, which is about twice the number expected by chance, indicating that at least some of the associations represent true effects. Thus, the present study shows that loci affecting MD resistance can be mapped in commercial layer lines. More comprehensive studies are under way to confirm and extend these results.
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A Mechanism for Error Detection in Speeded Response Time Tasks
ERIC Educational Resources Information Center
Holroyd, Clay B.; Yeung, Nick; Coles, Michael G. H.; Cohen, Jonathan D.
2005-01-01
The concept of error detection plays a central role in theories of executive control. In this article, the authors present a mechanism that can rapidly detect errors in speeded response time tasks. This error monitor assigns values to the output of cognitive processes involved in stimulus categorization and response generation and detects errors…
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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.
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Procedural error monitoring and smart checklists
NASA Technical Reports Server (NTRS)
Palmer, Everett
1990-01-01
Human beings make and usually detect errors routinely. The same mental processes that allow humans to cope with novel problems can also lead to error. Bill Rouse has argued that errors are not inherently bad but their consequences may be. He proposes the development of error-tolerant systems that detect errors and take steps to prevent the consequences of the error from occurring. Research should be done on self and automatic detection of random and unanticipated errors. For self detection, displays should be developed that make the consequences of errors immediately apparent. For example, electronic map displays graphically show the consequences of horizontal flight plan entry errors. Vertical profile displays should be developed to make apparent vertical flight planning errors. Other concepts such as energy circles could also help the crew detect gross flight planning errors. For automatic detection, systems should be developed that can track pilot activity, infer pilot intent and inform the crew of potential errors before their consequences are realized. Systems that perform a reasonableness check on flight plan modifications by checking route length and magnitude of course changes are simple examples. Another example would be a system that checked the aircraft's planned altitude against a data base of world terrain elevations. Information is given in viewgraph form.
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Molecular surveillance of Dengue in Sukabumi, West Java province, Indonesia.
Nusa, Roy; Prasetyowati, Heni; Meutiawati, Febrina; Yohan, Benediktus; Trimarsanto, Hidayat; Setianingsih, Tri Yuli; Sasmono, R Tedjo
2014-06-11
Dengue is endemic and affects people in all Indonesian provinces. Increasing dengue cases have been observed every year in Sukabumi in West Java province. Despite the endemicity, limited data is available on the genetic of dengue viruses (DENV) circulating in the country. To understand the dynamics of dengue disease, we performed molecular and serological surveillance of dengue in Sukabumi. A total of 113 patients were recruited for this study. Serological data were obtained using anti-dengue IgM and IgG tests plus dengue NS1 antigen detection. Dengue detection and serotyping were performed using real-time RT-PCR. Viruses were isolated and the envelope genes were sequenced. Phylogenetic and evolutionary analyses were performed to determine the genotype of the viruses and their evolutionary rates. Real-time RT-PCR detected DENV in 25 (22%) of 113 samples. Serotyping revealed the predominance of DENV-2 (16 isolates, 64%), followed by DENV-1 (5 isolates, 20%), and DENV-4 (4 isolates, 16%). No DENV-3 was detected in the samples. Co-circulation of genotype I and IV of DENV-1 was observed. The DENV-2 isolates all belonged to the Cosmopolitan genotype, while DENV-4 isolates were grouped into genotype II. Overall, their evolutionary rates were similar to DENV from other countries. We revealed the distribution of DENV serotypes and genotypes in Sukabumi. Compared to data obtained from other cities in Indonesia, we observed the differing predominance of DENV serotypes but similar genotype distribution, where the infecting viruses were closely related with Indonesian endemic viruses isolated previously.
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ERIC Educational Resources Information Center
Nozari, Nazbanou; Dell, Gary S.; Schwartz, Myrna F.
2011-01-01
Despite the existence of speech errors, verbal communication is successful because speakers can detect (and correct) their errors. The standard theory of speech-error detection, the perceptual-loop account, posits that the comprehension system monitors production output for errors. Such a comprehension-based monitor, however, cannot explain the…
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The case-only test for gene-environment interaction is not uniformly powerful: an empirical example
Wu, Chen; Chang, Jiang; Ma, Baoshan; Miao, Xiaoping; Zhou, Yifeng; Liu, Yu; Li, Yun; Wu, Tangchun; Hu, Zhibin; Shen, Hongbing; Jia, Weihua; Zeng, Yixin; Lin, Dongxin; Kraft, Peter
2016-01-01
The case-only test has been proposed as a more powerful approach to detect gene-environment (G×E) interactions. This approach assumes that the genetic and environmental factors are independent. While it is well known that Type I error rate will increase if this assumption is violated, it is less widely appreciated that gene-environment correlation can also lead to power loss. We illustrate this phenomenon by comparing the performance of the case-only test to other approaches to detect G×E interactions in a genome-wide association study of esophageal squamous carcinoma (ESCC) in Chinese populations. Some of these approaches do not use information on the correlation between exposure and genotype (standard logistic regression), while others seek to use this information in a robust fashion to boost power without increasing Type I error (two-step, empirical Bayes and cocktail methods). G×E interactions were identified involving drinking status and two regions containing genes in the alcohol metabolism pathway, 4q23 and 12q24. Although the case-only test yielded the most significant tests of G×E interaction in the 4q23 region, the case-only test failed to identify significant interactions in the 12q24 region which were readily identified using other approaches. The low power of the case-only test in the 12q24 region is likely due to the strong inverse association between the SNPs in this region and drinking status. This example underscores the need to consider multiple approaches to detect gene-environment interactions, as different tests are more or less sensitive to different alternative hypotheses and violations of the gene-environment independence assumption. PMID:23595356
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Guibert, N; Hu, Y; Feeney, N; Kuang, Y; Plagnol, V; Jones, G; Howarth, K; Beeler, J F; Paweletz, C P; Oxnard, G R
2018-04-01
Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.
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Molecular appraisal of intestinal parasitic infection in transplant recipients
Yadav, Pooja; Khalil, Shehla; Mirdha, Bijay Ranjan
2016-01-01
Background & objectives: Diarrhoea is the main clinical manifestation caused by intestinal parasitic infections in patients, with special reference to transplant recipients who require careful consideration to reduce morbidity and mortality. Further, molecular characterization of some important parasites is necessary to delineate the different modes of transmission to consider appropriate management strategies. We undertook this study to investigate the intestinal parasitic infections in transplant recipients with or without diarrhoea, and the genotypes of the isolated parasites were also determined. Methods: Stool samples from 38 transplant recipients comprising 29 post-renal, two liver and seven bone marrow transplant (BMT) recipients presenting with diarrhoea and 50 transplant recipients (42 post-renal transplant, eight BMT) without diarrhoea were examined for the presence of intestinal parasites by light microscopy using wet mount, modified Ziehl–Neelsen staining for intestinal coccidia and modified trichrome staining for microsporidia. Genotypes of Cryptosporidium species were determined by multilocus genotyping using small subunit ribosomal (SSUrRNA), Cryptosporidium oocyst wall protein (COWP) and dihydrofolate reductase (DHFR) as the target genes. Assemblage study for Giardia lamblia was performed using triose phosphate isomerase (TPI) as the target gene. Samples were also screened for bacterial, fungal and viral pathogens. Results: The parasites that were detected included Cryptosporidium species (21%, 8/38), Cystoisospora (Isospora) belli (8%, 3), Cyclospora cayetanensis (5%, 2), G. lamblia (11%, 4), Hymenolepis nana (11%, 4), Strongyloides stercoralis (3%, 1) and Blastocystis hominis (3%, 1). Multilocus genotyping of Cryptosporidium species at SSUrRNA, COWP and DHFR loci could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. All the C. hominis isolates were detected in adult post-renal transplant (PRT) recipients, whereas the C. parvum isolates included a child with BMT and an adult with PRT. Clostridium difficle, cytomegalovirus and Candida albicans were found in 2, 3 and 2 patients, respectively. Interpretation & conclusions: In the present study, C. hominis was observed as an important parasite causing intestinal infections in transplant recipients. Multilocus genotyping of Cryptosporidium species could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. Genotyping of G. lamblia revealed that assemblage B was most common. PMID:27934806
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Clover: Compiler directed lightweight soft error resilience
Liu, Qingrui; Lee, Dongyoon; Jung, Changhee; ...
2015-05-01
This paper presents Clover, a compiler directed soft error detection and recovery scheme for lightweight soft error resilience. The compiler carefully generates soft error tolerant code based on idem-potent processing without explicit checkpoint. During program execution, Clover relies on a small number of acoustic wave detectors deployed in the processor to identify soft errors by sensing the wave made by a particle strike. To cope with DUE (detected unrecoverable errors) caused by the sensing latency of error detection, Clover leverages a novel selective instruction duplication technique called tail-DMR (dual modular redundancy). Once a soft error is detected by either themore » sensor or the tail-DMR, Clover takes care of the error as in the case of exception handling. To recover from the error, Clover simply redirects program control to the beginning of the code region where the error is detected. Lastly, the experiment results demonstrate that the average runtime overhead is only 26%, which is a 75% reduction compared to that of the state-of-the-art soft error resilience technique.« less
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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.
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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.
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The molecular and clinical features of dengue during outbreak in Jambi, Indonesia in 2015
Haryanto, Sotianingsih; Hayati, Rahma F.; Yohan, Benediktus; Sijabat, Lanceria; Sihite, Ifo F.; Fahri, Sukmal; Meutiawati, Febrina; Halim, Jonathan A. N.; Halim, Stefanie N.; Soebandrio, Amin
2016-01-01
Dengue is hyperendemic in Indonesia. In 2015, reported cases of dengue fever doubled those of 2014 in the Jambi municipality of Sumatra. We examined viral aetiology and its relationship with disease outcome in Jambi. Dengue-suspected patients’ sera were collected and NS1 detection and IgM/IgG serology were performed. Dengue virus (DENV) serotyping was performed using real-time RT-PCR. Envelope genes were sequenced to determine the genotypes of DENV. Clinical, haematologic, and demographic data were recorded. Of 210 dengue-suspected patients, 107 were confirmed. The disease manifested as Dengue Fever (62%), Dengue Haemorrhagic Fever (36%), and Dengue Shock Syndrome (2%). The serotypes of 94 DENV were determined. All DENV serotypes were detected with DENV-1 as the predominant serotype (66%). Genotypically, the DENV-1 viruses belong to Genotype I, DENV-2 was of Cosmopolitan genotype, DENV-3 as Genotype I, and DENV-4 belonged to Genotype II. Comparison with historical data revealed serotype predominance switched from DENV-3 to DENV-1, and the replacement of Genotype IV of DENV-1 with Genotype I. In summary, DENV-1 predominated during the 2015 dengue outbreak in Jambi. The full spectrum of dengue disease occurred and was characterized by a switch in predominant serotypes. PMID:27215933
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García-Hoyos, María; Cortón, Marta; Ávila-Fernández, Almudena; Riveiro-Álvarez, Rosa; Giménez, Ascensión; Hernan, Inma; Carballo, Miguel; Ayuso, Carmen
2012-01-01
Purpose Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients. Methods We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray. Results The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%. Conclusions The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP. PMID:22736939
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Menage, Lucy; Yodmeeklin, Arpaporn; Khamrin, Pattara; Kumthip, Kattareeya; Maneekarn, Niwat
2017-09-01
Human cosavirus and saffold virus are both newly discovered members of the Picornaviridae family. It has been suggested that these viruses may be the causative agents of acute gastroenteritis. In this study, 1093 stool samples collected from patients with acute gastroenteritis between January 2014 and December 2016, were screened for cosavirus and saffold virus using reverse transcription-polymerase chain reaction. The viral genotypes were then established via nucleotide sequencing. Here, cosavirus was detected in 16 of 1093 stool samples (1.5%) and saffold virus was detected in 18 of 1093 stool samples (1.6%). The saffold virus genotypes 1 (16.7%), 2 (50%) and 6 (33.3%), and the cosavirus genetic groups A (87.5%), C (6.25%) and D (6.25%), were all identified across the three-year study period. Interestingly, saffold virus genotype 6 has now been detected for the first time in Thailand. The present study provides the prevalence of cosavirus and saffold virus with the emergence of saffold virus genotype 6 in Thailand. Copyright © 2017 Elsevier B.V. All rights reserved.
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Wakeley, P. R.; Johnson, N.; McElhinney, L. M.; Marston, D.; Sawyer, J.; Fooks, A. R.
2005-01-01
Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus. PMID:15956398
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Aichi Virus in Sewage and Surface Water, the Netherlands
Rutjes, Saskia A.; Takumi, Katsuhisa; Husman, Ana Maria de Roda
2013-01-01
Detection of Aichi virus in humans was initially reported in Japan in 1989. To establish a timeline for the prevalence of Aichi virus infection among humans in the Netherlands, we conducted molecular analysis of archival water samples from 1987–2000 and 2009–2012. Aichi virus RNA was detected in 100% (8/8) of sewage samples and 100% (7/7) of surface water samples collected during 1987–2000 and 100% (8/8) of sewage samples and 71% (5/7) of surface water samples collected during 2009–2012. Several genotype A and B Aichi virus lineages were observed over the 25-year period studied, but the time course of viral genetic diversity showed recent expansion of the genotype B population over genotype A. Our results show that Aichi virus has been circulating among the human population in the Netherlands since before its initial detection in humans was reported and that genotype B now predominates in this country. PMID:23876456
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D'Angelino, Rubens Henrique Ramos; Pituco, Edviges Maristela; Villalobos, Eliana Monteforte Cassaro; Harakava, Ricardo; Gregori, Fábio
2013-01-01
Bovine leukemia virus (BLV) was investigated in the central nervous system (CNS) of cattle with neurological syndrome. A total of 269 CNS samples were submitted to nested-PCR (BLV env gene gp51), and the viral genotypes were identified. The nested-PCR was positive in 4.8% (13/269) CNS samples, with 2.7% (2/74) presenting at histological examination lesions of nonpurulent meningoencephalitis (NPME), whereas 5.6% (11/195) not presenting NPME (P > 0.05). No samples presented lymphosarcoma. The PCR products (437 bp) were sequenced and submitted to phylogenetic analysis by neighbor-joining and maximum composite likelihood methods, and genotypes 1, 5, and 6 were detected, corroborating other South American studies. The genotype 6 barely described in Brazil and Argentina was more frequently detected in this study. The identity matrices showed maximum similarity (100%) among some samples of this study and one from Argentina (FJ808582), recovered from GenBank. There was no association among the genotypes and NPME lesions. PMID:23710448
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Avadhanula, Vasanthi; Chemaly, Roy F; Shah, Dimpy P; Ghantoji, Shashank S; Azzi, Jacques M; Aideyan, Letisha O; Mei, Minghua; Piedra, Pedro A
2015-02-15
Respiratory syncytial virus (RSV) can cause severe respiratory disease in adult hematopoietic cell transplant (HCT) recipients. RSV subgroups A and B have evolved into multiple genotypes. We report on a recently described RSV genotype (ON1) in a cohort of adult HCT recipients in Texas. Twenty adult HCT recipients were enrolled as a part of an efficacy trial of ribavirin therapy. RSV identification and genotyping was performed using molecular techniques. RSV-specific neutralizing antibody (NAb) responses were measured. ON1 genotype was detected in 3 of 6 patients in the 2011-2012 season and in 8 of 14 patients in 2012-2013 season. Other genotypes detected were NA1 and BA. NAb levels were low at enrollment. Eight of 9 patients who cleared the RSV infection within 2 weeks mounted a ≥4-fold NAb response, compared with 2 of 8 who shed the virus for >2 weeks. The clinical course of those infected with ON1 was comparable to the course for individuals infected with other genotypes. This is the first report of RSV ON1 genotype in the United States, and ON1 genotype was dominant genotype in adult HCT recipients. Interestingly, faster viral clearance was associated with a ≥4-fold NAb response, likely indicating a reconstituted immune system. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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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
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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
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Land, Sally; Zhou, Julian; Cunningham, Philip; Sohn, Annette H; Singtoroj, Thida; Katzenstein, David; Mann, Marita; Sayer, David; Kantor, Rami
2013-01-01
Background The TREAT Asia Quality Assessment Scheme (TAQAS) was developed as a quality assessment programme through expert education and training, for laboratories in the Asia-Pacific and Africa that perform HIV drug-resistance (HIVDR) genotyping. We evaluated the programme performance and factors associated with high-quality HIVDR genotyping. Methods Laboratories used their standard protocols to test panels of human immunodeficiency virus (HIV)-positive plasma samples or electropherograms. Protocols were documented and performance was evaluated according to a newly developed scoring system, agreement with panel-specific consensus sequence, and detection of drug-resistance mutations (DRMs) and mixtures of wild-type and resistant virus (mixtures). High-quality performance was defined as detection of ≥95% DRMs. Results Over 4.5 years, 23 participating laboratories in 13 countries tested 45 samples (30 HIV-1 subtype B; 15 non-B subtypes) in nine panels. Median detection of DRMs was 88–98% in plasma panels and 90–97% in electropherogram panels. Laboratories were supported to amend and improve their test outcomes as appropriate. Three laboratories that detected <80% DRMs in early panels demonstrated subsequent improvement. Sample complexity factors – number of DRMs (p<0.001) and number of DRMs as mixtures (p<0.001); and laboratory performance factors – detection of mixtures (p<0.001) and agreement with consensus sequence (p<0.001), were associated with high performance; sample format (plasma or electropherogram), subtype and genotyping protocol were not. Conclusion High-quality HIVDR genotyping was achieved in the TAQAS collaborative laboratory network. Sample complexity and detection of mixtures were associated with performance quality. Laboratories conducting HIVDR genotyping are encouraged to participate in quality assessment programmes. PMID:23845227
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Genotyping of Coxiella burnetii from domestic ruminants in northern Spain.
Astobiza, Ianire; Tilburg, Jeroen J H C; Piñero, Alvaro; Hurtado, Ana; García-Pérez, Ana L; Nabuurs-Franssen, Marrigje H; Klaassen, Corné H W
2012-12-10
Information on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST). A total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but some of the MLVA genotypes are described here for the first time. Genotyping revealed a substantial genetic diversity among domestic ruminants from Northern Spain.
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Baroudi, Djamel; Zhang, Hongwei; Amer, Said; Khelef, Djamel; Roellig, Dawn M; Wang, Yuanfei; Feng, Yaoyu; Xiao, Lihua
2018-03-01
Little information is available on the occurrence of the zoonotic protists Cryptosporidium spp. and none on Enterocytozoon bieneusi in camels. This preliminary study was conducted to examine the identity of Cryptosporidium subtypes and E. bieneusi genotypes in dromedary camels in Algeria. A total of 39 fecal specimens were collected from young camels. PCR-sequence analysis of the small subunit rRNA was used to detect and genotype Cryptosporidium spp. Cryptosporidium parvum present was further subtyped by sequence analysis of the 60 kDa glycoprotein gene. PCR-sequence analysis of the ribosomal internal transcribed spacer gene was used to detect and genotype E. bieneusi. Altogether, two and eight of the specimens analyzed were positive for C. parvum and E. bieneusi, respectively. The former was identified as a new subtype that is genetically related to the C. hominis If subtype family, whereas the latter was identified as two related genotypes (Macaque1 and a novel genotype) in the newly assigned E. bieneusi genotype group 8. Although they are not known hosts for C. parvum and E. bieneusi, camels are apparently infected with genetically distinct variants of these pathogens.
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Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays
DOE Office of Scientific and Technical Information (OSTI.GOV)
Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.
2000-12-01
Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less
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Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays
DOE Office of Scientific and Technical Information (OSTI.GOV)
Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.
2001-07-05
Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less
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Impact of Measurement Error on Synchrophasor Applications
DOE Office of Scientific and Technical Information (OSTI.GOV)
Liu, Yilu; Gracia, Jose R.; Ewing, Paul D.
2015-07-01
Phasor measurement units (PMUs), a type of synchrophasor, are powerful diagnostic tools that can help avert catastrophic failures in the power grid. Because of this, PMU measurement errors are particularly worrisome. This report examines the internal and external factors contributing to PMU phase angle and frequency measurement errors and gives a reasonable explanation for them. It also analyzes the impact of those measurement errors on several synchrophasor applications: event location detection, oscillation detection, islanding detection, and dynamic line rating. The primary finding is that dynamic line rating is more likely to be influenced by measurement error. Other findings include themore » possibility of reporting nonoscillatory activity as an oscillation as the result of error, failing to detect oscillations submerged by error, and the unlikely impact of error on event location and islanding detection.« less
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Jääskeläinen, Anne J; Kallio-Kokko, Hannimari; Ozkul, Aykut; Bodur, Hurrem; Korukruoglu, Gulay; Mousavi, Mehrdad; Pranav, Patel; Vaheri, Antti; Mirazimi, Ali; Vapalahti, Olli
2014-12-01
Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.
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Yi, Ping; Chen, Zhuqin; Zhao, Yan; Guo, Jianxin; Fu, Huabin; Zhou, Yuanguo; Yu, Lili; Li, Li
2009-03-01
The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive diagnosis. We have now assessed the possibility of detecting single-nucleotide differences between fetal and maternal DNA in maternal plasma by polymerase chain reaction (PCR)/ligase detection reaction((LDR)/capillary electrophoresis. PCR/LDR/capillary electrophoresis was applied to detect the genotype of c.454-397T>gene (ESR1) from experimental DNA models of maternal plasma at different sensitivity levels and 13 maternal plasma samples.alphaC in estrogen receptor. (1) Our results demonstrated that the technique could discriminate low abundance single-nucleotide mutation with a mutant/normal allele ratio up to 1:10 000. (2) Examination of ESR1 c.454-397T>C genotypes by using the method of restriction fragment length analysis was performed in 25 pregnant women, of whom 13 pregnant women had homozygous genotypes. The c.454-397T>C genotypes of paternally inherited fetal DNA in maternal plasma of these 13 women were detected by PCR/LDR/capillary electrophoresis, which were accordant with the results of umbilical cord blood. PCR/LDR/capillary electrophoresis has very high sensitivity to distinguish low abundance single nucleotide differences and can discriminate point mutations and single-nucleotide polymorphisms(SNPs) of paternally inherited fetal DNA in maternal plasma.
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Taira, Chiaki; Matsuda, Kazuyuki; Yamaguchi, Akemi; Sueki, Akane; Koeda, Hiroshi; Takagi, Fumio; Kobayashi, Yukihiro; Sugano, Mitsutoshi; Honda, Takayuki
2013-09-23
Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations. Copyright © 2013 Elsevier B.V. All rights reserved.
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Land, Sally; Cunningham, Philip; Zhou, Jialun; Frost, Kevin; Katzenstein, David; Kantor, Rami; Chen, Yi-Ming Arthur; Oka, Shinichi; DeLong, Allison; Sayer, David; Smith, Jeffery; Dax, Elizabeth M.; Law, Matthew
2010-01-01
The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme – designated TAQAS – is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratory’s result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0–83%) and a significant correlation with the detection of DRMs (p < 0.01). Interpretation of antiretroviral resistance showed ~70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories. PMID:19490972
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Ferro, Marta; Macher, Hada C; Noguerol, Pilar; Jimenez-Arriscado, Pilar; Molinero, Patrocinio; Guerrero, Juan M; Rubio, Amalia
2016-01-01
Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.
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LaMere, Brandon J.; Kornegay, Janet; Fetterman, Barbara; Sadorra, Mark; Shieh, Jen; Castle, Philip E.
2009-01-01
Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV Persistence and Progression cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized specimens from 100 women with low-grade intraepithelial lesion cytology. Test aliquots were left in denaturant for 10 or 18 hours at 4°C and then neutralized; comparison aliquots were not denatured but diluted to match the timing, temperature, concentration and salt conditions of the treated specimens. The masked aliquots were tested using a commercialized PCR-based assay that detects of 37 HPV genotypes. There was no overall effect of treatment on test positivity or number of types. HPV16 was marginally more likely to be detected in untreated versus treated aliquots (P = 0.09) but HPV45 was marginally more likely to be detected in treated than untreated aliquots (P = 0.07), suggesting that these differences represented chance (intra-test variability). It can be concluded that residual Hybrid Capture-positive specimens can be accurately genotyped by PCR after Hybrid Capture 2 processing. PMID:17673302
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Transmission and molecular characterisation of wild measles virus in Romania, 2008 to 2012.
Necula, G; Lazar, M; Stanescu, A; Pistol, A; Santibanez, S; Mankertz, A; Lupulescu, E
2013-12-12
Molecular characterisation of measles virus is a powerful tool for tracing transmission. Genotyping may prove the absence of endemic circulation of measles virus, i.e. transmission for more than 12 months, which is one of the criteria for verifying elimination of the disease. We have genetically characterised measles viruses detected in Romania from 2008 to 2012, focusing on the recent outbreaks from 2010 to 2012 that affected mainly groups with limited access to healthcare and schools. The findings emphasise the importance of genotyping during the different phases of an outbreak. A total of 8,170 cases were notified, and 5,093 (62%) of the 7,559 possible cases were serologically confirmed. RT-PCR was performed for 104 samples: from the 101 positive samples obtained from sporadic measles cases or clusters from different counties, 73 were genotyped. Sporadic measles cases associated with D4 and D5 viruses were observed from2008 to 2009. Genotype D4-Manchester was predominant in 2011 and 2012. In addition, the related variant D4-Maramures and MVs/Limoges.FRA/17.10[D4] and a few D4-Hamburg strains were detected. The detection of several distinct MV-D4 genotypes suggests multiple virus importations to Romania. The outbreak associated with D4 genotype is the second largest outbreak in Romania in less than 10 years.
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Development of a QTL-environment-based predictive model for node addition rate in common bean.
Zhang, Li; Gezan, Salvador A; Eduardo Vallejos, C; Jones, James W; Boote, Kenneth J; Clavijo-Michelangeli, Jose A; Bhakta, Mehul; Osorno, Juan M; Rao, Idupulapati; Beebe, Stephen; Roman-Paoli, Elvin; Gonzalez, Abiezer; Beaver, James; Ricaurte, Jaumer; Colbert, Raphael; Correll, Melanie J
2017-05-01
This work reports the effects of the genetic makeup, the environment and the genotype by environment interactions for node addition rate in an RIL population of common bean. This information was used to build a predictive model for node addition rate. To select a plant genotype that will thrive in targeted environments it is critical to understand the genotype by environment interaction (GEI). In this study, multi-environment QTL analysis was used to characterize node addition rate (NAR, node day - 1 ) on the main stem of the common bean (Phaseolus vulgaris L). This analysis was carried out with field data of 171 recombinant inbred lines that were grown at five sites (Florida, Puerto Rico, 2 sites in Colombia, and North Dakota). Four QTLs (Nar1, Nar2, Nar3 and Nar4) were identified, one of which had significant QTL by environment interactions (QEI), that is, Nar2 with temperature. Temperature was identified as the main environmental factor affecting NAR while day length and solar radiation played a minor role. Integration of sites as covariates into a QTL mixed site-effect model, and further replacing the site component with explanatory environmental covariates (i.e., temperature, day length and solar radiation) yielded a model that explained 73% of the phenotypic variation for NAR with root mean square error of 16.25% of the mean. The QTL consistency and stability was examined through a tenfold cross validation with different sets of genotypes and these four QTLs were always detected with 50-90% probability. The final model was evaluated using leave-one-site-out method to assess the influence of site on node addition rate. These analyses provided a quantitative measure of the effects on NAR of common beans exerted by the genetic makeup, the environment and their interactions.
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Identifying and mitigating batch effects in whole genome sequencing data.
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.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Almasi, Gheorghe; Blumrich, Matthias Augustin; Chen, Dong
Methods and apparatus perform fault isolation in multiple node computing systems using commutative error detection values for--example, checksums--to identify and to isolate faulty nodes. When information associated with a reproducible portion of a computer program is injected into a network by a node, a commutative error detection value is calculated. At intervals, node fault detection apparatus associated with the multiple node computer system retrieve commutative error detection values associated with the node and stores them in memory. When the computer program is executed again by the multiple node computer system, new commutative error detection values are created and stored inmore » memory. The node fault detection apparatus identifies faulty nodes by comparing commutative error detection values associated with reproducible portions of the application program generated by a particular node from different runs of the application program. Differences in values indicate a possible faulty node.« less
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Effect of Linkage Disequilibrium on the Identification of Functional Variants
Thomas, Alun; Abel, Haley J; Di, Yanming; Faye, Laura L; Jin, Jing; Liu, Jin; Wu, Zheyan; Paterson, Andrew D
2011-01-01
We summarize the contributions of Group 9 of Genetic Analysis Workshop 17. This group addressed the problems of linkage disequilibrium and other longer range forms of allelic association when evaluating the effects of genotypes on phenotypes. Issues raised by long-range associations, whether a result of selection, stratification, possible technical errors, or chance, were less expected but proved to be important. Most contributors focused on regression methods of various types to illustrate problematic issues or to develop adaptations for dealing with high-density genotype assays. Study design was also considered, as was graphical modeling. Although no method emerged as uniformly successful, most succeeded in reducing false-positive results either by considering clusters of loci within genes or by applying smoothing metrics that required results from adjacent loci to be similar. Two unexpected results that questioned our assumptions of what is required to model linkage disequilibrium were observed. The first was that correlations between loci separated by large genetic distances can greatly inflate single-locus test statistics, and, whether the result of selection, stratification, possible technical errors, or chance, these correlations seem overabundant. The second unexpected result was that applying principal components analysis to genome-wide genotype data can apparently control not only for population structure but also for linkage disequilibrium. PMID:22128051
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Luo, Zhao-Yun; Chen, Qiang; Yang, Hui; Lin, Min; Chen, Chan-Yu; Yang, Chun; Yang, Li-Ye
2015-01-01
Low-risk human papillomavirus (LR-HPV) infection is the main cause of genital warts. LR- HPV genotypes 6 and 11 are associated with genital warts, but there have only been a few published studies about the genotype-specific prevalence of HPV in genital warts in China. The objective of our study was to assess the prevalence of HPV genotypes for clinical cases involving both men and women and to evaluate the potential benefit of a quadrivalent (genotypes 6, 11, 16, and 18) HPV vaccine in eastern Guangdong province of China. A total of 696 eligible patients with genital warts were enrolled during the period Aug 2009 through Oct 2014. Specimens were collected from genital warts, the HPV GenoArray test was used for HPV detection and genotyping, which could detect 21 HPV genotypes, including genotypes 6, 11, 16, and 18. Among the 696 cases, 675 samples were successfully genotyped. The median age of patients was 32.1 years (range, 16-67 years). The most prevalent genotypes were HPV-6 (285/675, 42.2%), HPV-11 (265/675, 39.3%), HPV-52 (52/675, 7.7%), HPV-16 (51/675, 7.56%), HPV-81 (50/675, 7.40%) and HPV-58 (37/675, 5.48%). Low-risk genotypes predominated, with a prevalence of 96.59%. The cumulative prevalence of genotypes 6 and 11 was 78.7% (531/675), the cumulative prevalence of genotypes 16 and 18 was 11.6% (78/675), and the cumulative prevalence of genotypes 6, 11, 16, and 18 was 82.5% (557/675). Our results provide strong evidence that, in eastern Guangdong, different from Western countries, the most prevalent low risk HPV genotypes in patients with genital warts are 6, 11 and 81. The quadrivalent HPV vaccine could prevent 82.5% of genital warts in eastern Guangdong.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gopan, O; Kalet, A; Smith, W
2016-06-15
Purpose: A standard tool for ensuring the quality of radiation therapy treatments is the initial physics plan review. However, little is known about its performance in practice. The goal of this study is to measure the effectiveness of physics plan review by introducing simulated errors into “mock” treatment plans and measuring the performance of plan review by physicists. Methods: We generated six mock treatment plans containing multiple errors. These errors were based on incident learning system data both within the department and internationally (SAFRON). These errors were scored for severity and frequency. Those with the highest scores were included inmore » the simulations (13 errors total). Observer bias was minimized using a multiple co-correlated distractor approach. Eight physicists reviewed these plans for errors, with each physicist reviewing, on average, 3/6 plans. The confidence interval for the proportion of errors detected was computed using the Wilson score interval. Results: Simulated errors were detected in 65% of reviews [51–75%] (95% confidence interval [CI] in brackets). The following error scenarios had the highest detection rates: incorrect isocenter in DRRs/CBCT (91% [73–98%]) and a planned dose different from the prescribed dose (100% [61–100%]). Errors with low detection rates involved incorrect field parameters in record and verify system (38%, [18–61%]) and incorrect isocenter localization in planning system (29% [8–64%]). Though pre-treatment QA failure was reliably identified (100%), less than 20% of participants reported the error that caused the failure. Conclusion: This is one of the first quantitative studies of error detection. Although physics plan review is a key safety measure and can identify some errors with high fidelity, others errors are more challenging to detect. This data will guide future work on standardization and automation. Creating new checks or improving existing ones (i.e., via automation) will help in detecting those errors with low detection rates.« less
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[Analysis of allele dropout at TH01 locus in paternity testing].
Lai, Li; Shen, Xiao-li; Xue, Shi-jie; Hu, Jie
2013-10-01
To analyze allele dropout at TH01 locus in paternity testing in order to determine the accurate genotype. To use a two STR loci genotyping system to verify an abnormal genotype for the TH01 locus with PCR using specific primers, cloning and DNA sequencing. A rare allele at TH01 locus named 5.2, which was undetectable with PowerPlex 21 system, was detected with an Identifiler system. Genetic variations may result in rare alleles and loci loss. To avoid misjudgment, laboratories should have a variety of methods for detecting loci loss.
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Palmer, Tom M; Holmes, Michael V; Keating, Brendan J; Sheehan, Nuala A
2017-01-01
Abstract Mendelian randomization studies use genotypes as instrumental variables to test for and estimate the causal effects of modifiable risk factors on outcomes. Two-stage residual inclusion (TSRI) estimators have been used when researchers are willing to make parametric assumptions. However, researchers are currently reporting uncorrected or heteroscedasticity-robust standard errors for these estimates. We compared several different forms of the standard error for linear and logistic TSRI estimates in simulations and in real-data examples. Among others, we consider standard errors modified from the approach of Newey (1987), Terza (2016), and bootstrapping. In our simulations Newey, Terza, bootstrap, and corrected 2-stage least squares (in the linear case) standard errors gave the best results in terms of coverage and type I error. In the real-data examples, the Newey standard errors were 0.5% and 2% larger than the unadjusted standard errors for the linear and logistic TSRI estimators, respectively. We show that TSRI estimators with modified standard errors have correct type I error under the null. Researchers should report TSRI estimates with modified standard errors instead of reporting unadjusted or heteroscedasticity-robust standard errors. PMID:29106476
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Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993-2007.
Seme, Katja; Vrhovac, Maja; Mocilnik, Tina; Maticic, Mojca; Lesnicar, Gorazd; Baklan, Zvonko; Volkar, Jelka Meglic; Rajter, Mojca; Stepec, Srecko; Lunar, Maja; Poljak, Mario
2009-04-01
In order to identify the main routes of hepatitis C (HCV) transmission and to determine the HCV genotype distribution and its dynamics during a 15-year period in Slovenia, HCV genotypes were detected using the INNO-LiPA HCV II (Innogenetics) test for serum samples obtained from 1,504 patients representing 72.6% of all patients with chronic hepatitis C diagnosed from 1993 to 2007. HCV genotype 1 was predominant (56%), followed by genotypes 3, 2, and 4, with a prevalence of 37.8%, 5%, and 1.2%, respectively. HCV genotypes 5 and 6 were not detected in any patient. Patients infected with HCV genotype 3 were significantly younger (mean age 28.9 +/- 8.5 years) than those infected with genotype 1 (mean age 38.9 +/- 14.8 years; P < 0.0001) and those infected with HCV genotype 2 (mean age 50.3 +/- 18.2 years; P < 0.0001). Intravenous drug use was identified as the most frequent possible HCV transmission route (34.3%), followed by medical-related transmission such as transfusion of HCV-contaminated blood or blood products, and hemodialysis (12.5%). Being an intravenous drug user was found to be strongly associated with HCV genotype 3 (OR, 3.71 [95% CI, 2.97-4.65]; P < 0.0001) and reporting infection by transfusion of blood or blood products was found to be strongly associated with HCV genotype 1 (OR, 3.28 [95% CI, 2.18-4.95]; P < 0.0001). During the 15-year period, the proportion of genotype 3 increased substantially, reflecting the fact that the HCV epidemic in Slovenia is driven mostly by intravenous drug use. (c) 2009 Wiley-Liss, Inc.
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Molecular Characterization of Chikungunya Virus, Philippines, 2011–2013
Sy, Ava Kristy; Saito-Obata, Mariko; Medado, Inez Andrea; Tohma, Kentaro; Dapat, Clyde; Segubre-Mercado, Edelwisa; Tandoc, Amado; Lupisan, Socorro
2016-01-01
During 2011–2013, a nationwide outbreak of chikungunya virus infection occurred in the Philippines. The Asian genotype was identified as the predominant genotype; sporadic cases of the East/Central/South African genotype were detected in Mindanao. Further monitoring is needed to define the transmission pattern of this virus in the Philippines. PMID:27088593
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NASA Technical Reports Server (NTRS)
Buechler, W.; Tucker, A. G.
1981-01-01
Several methods were employed to detect both the occurrence and source of errors in the operational software of the AN/SLQ-32. A large embedded real time electronic warfare command and control system for the ROLM 1606 computer are presented. The ROLM computer provides information about invalid addressing, improper use of privileged instructions, stack overflows, and unimplemented instructions. Additionally, software techniques were developed to detect invalid jumps, indices out of range, infinte loops, stack underflows, and field size errors. Finally, data are saved to provide information about the status of the system when an error is detected. This information includes I/O buffers, interrupt counts, stack contents, and recently passed locations. The various errors detected, techniques to assist in debugging problems, and segment simulation on a nontarget computer are discussed. These error detection techniques were a major factor in the success of finding the primary cause of error in 98% of over 500 system dumps.
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Wang, S C; Ding, M M; Wei, X L; Zhang, T; Yao, F
2016-06-01
To recognize the possibility of Y fragment deletion of Amelogenin gene intuitively and simply according to the genotyping graphs. By calculating the ratio of total peak height of genotyping graphs, the statistics of equilibrium distribution between Amelogenin and D3S1358 loci, Amelogenin X-gene and Amelogenin Y-gene, and different alleles of D3S1358 loci from 1 968 individuals was analyzed after amplified by PowerPlex ® 21 detection kit. Sum of peak height of Amelogenin X allele was not less than 60% that of D3S1358 loci alleles in 90.8% female samples, and sum of peak height of Amelogenin X allele was not higher than 70% that of D3S1358 loci alleles in 94.9% male samples. The result of genotyping after amplified by PowerPlex ® 21 detection kit shows that the possibility of Y fragment deletion should be considered when only Amelogenin X-gene of Amelogenin is detected and the peak height of Amelogenin X-gene is not higher than 70% of the total peak height of D3S1358 loci. Copyright© by the Editorial Department of Journal of Forensic Medicine
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Permanence analysis of a concatenated coding scheme for error control
NASA Technical Reports Server (NTRS)
Costello, D. J., Jr.; Lin, S.; Kasami, T.
1983-01-01
A concatenated coding scheme for error control in data communications is analyzed. In this scheme, the inner code is used for both error correction and detection, however, the outer code is used only for error detection. A retransmission is requested if the outer code detects the presence of errors after the inner code decoding. Probability of undetected error is derived and bounded. A particular example, proposed for the planetary program, is analyzed.
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Probability of undetected error after decoding for a concatenated coding scheme
NASA Technical Reports Server (NTRS)
Costello, D. J., Jr.; Lin, S.
1984-01-01
A concatenated coding scheme for error control in data communications is analyzed. In this scheme, the inner code is used for both error correction and detection, however the outer code is used only for error detection. A retransmission is requested if the outer code detects the presence of errors after the inner code decoding. Probability of undetected error is derived and bounded. A particular example, proposed for NASA telecommand system is analyzed.
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Feltus, Dawn C; Giddings, Catherine W; Khaitsa, Margaret L; McEvoy, John M
2008-02-14
Recent studies have identified the novel, host adapted Cryptosporidium bovis and the deer-like genotype in dairy cattle from farms in the United States, China, India and Europe. This novel species and genotype appear to be more prevalent in older, post-weaned dairy cattle than previously thought. However, little information is available on their prevalence in beef cow-calf operations. In the present study, we determined the prevalence of Cryptosporidium species in 98 calves (6-8 months old) and 114 cows (>2 years old) in seven beef cow-calf herds in western North Dakota. DNA was extracted from fecal samples and Cryptosporidium spp. were identified by amplification of the 18S rRNA gene followed by sequencing or RFLP analysis. All seven herds tested positive for Cryptosporidium. Overall, 43/212 (20.3%) animals were positive. Only five of these positives were from cows. C. bovis, the deer-like genotype and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. C. parvum was not identified in any of the positive samples. C. bovis, the deer-like genotype and C. andersoni were detected in 6/7, 5/7 and 2/7 herds, respectively. C. bovis and the deer-like genotype were primarily detected in calves, while C. andersoni was only detected in cows. Six isolates could not be typed. These results show a relatively high prevalence of C. bovis and the deer-like genotype in 6-8-month-old beef calves compared to cows older than 2 years in the seven herds studied.
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Rotavirus Strain Trends During the Postlicensure Vaccine Era: United States, 2008-2013.
Bowen, Michael D; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Teel, Elizabeth N; Gautam, Rashi; Sturgeon, Michele; Azimi, Parvin H; Baker, Carol J; Bernstein, David I; Boom, Julie A; Chappell, James; Donauer, Stephanie; Edwards, Kathryn M; Englund, Janet A; Halasa, Natasha B; Harrison, Christopher J; Johnston, Samantha H; Klein, Eileen J; McNeal, Monica M; Moffatt, Mary E; Rench, Marcia A; Sahni, Leila C; Selvarangan, Rangaraj; Staat, Mary A; Szilagyi, Peter G; Weinberg, Geoffrey A; Wikswo, Mary E; Parashar, Umesh D; Payne, Daniel C
2016-09-01
Group A rotaviruses (RVA) are a significant cause of pediatric gastroenteritis worldwide. The New Vaccine Surveillance Network (NVSN) has conducted active surveillance for RVA at pediatric hospitals and emergency departments at 3-7 geographically diverse sites in the United States since 2006. Over 6 consecutive years, from 2008 to 2013, 1523 samples from NVSN sites that were tested positive by a Rotaclone enzyme immunoassay were submitted to the Centers for Disease Control and Prevention for genotyping. In the 2009, 2010, and 2011 seasons, genotype G3P[8] was the predominant genotype throughout the network, with a 46%-84% prevalence. In the 2012 season, G12P[8] replaced G3P[8] as the most common genotype, with a 70% prevalence, and this trend persisted in 2013 (68.0% prevalence). Vaccine (RotaTeq; Rotarix) strains were detected in 0.6%-3.4% of genotyped samples each season. Uncommon and unusual strains (eg, G8P[4], G3P[24], G2P[8], G3P[4], G3P[6], G24P[14], G4P[6], and G9P[4]) were detected sporadically over the study period. Year, study site, and race were found to be significant predictors of genotype. Continued active surveillance is needed to monitor RVA genotypes in the United States and to detect potential changes since vaccine licensure. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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Tracing Males From Different Continents by Genotyping JC Polyomavirus in DNA From Semen Samples.
Rotondo, John Charles; Candian, Tommaso; Selvatici, Rita; Mazzoni, Elisa; Bonaccorsi, Gloria; Greco, Pantaleo; Tognon, Mauro; Martini, Fernanda
2017-05-01
The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%), and 3A (2.9%). Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected, suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of for instance sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. J. Cell. Physiol. 232: 982-985, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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calculate eigenvectors to adjust for population stratification in association analyses. SNP filters are developed including missing data filters, duplicate and Mendelian errors, minor allele frequency and Hardy genotype and phenotype datasets and apply the filters correctly by repeating the pre-compute results. A QC
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Paternity testing in an autotetraploid alfalfa breeding polycross
USDA-ARS?s Scientific Manuscript database
Determining unknown parentage in autotetraploid alfalfa (Medicago sativa L.) (2n = 4x = 32) can improve breeding gains. Exclusion analysis based paternity testing SAS code is presented, amenable to genotyping errors, for autotetraploid species utilizing co-dominant molecular markers with ambiguous d...
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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.
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Endicott, Phillip; Metspalu, Mait; Stringer, Chris; Macaulay, Vincent; Cooper, Alan; Sanchez, Juan J
2006-12-20
The issue of errors in genetic data sets is of growing concern, particularly in population genetics where whole genome mtDNA sequence data is coming under increased scrutiny. Multiplexed PCR reactions, combined with SNP typing, are currently under-exploited in this context, but have the potential to genotype whole populations rapidly and accurately, significantly reducing the amount of errors appearing in published data sets. To show the sensitivity of this technique for screening mtDNA genomic sequence data, 20 historic samples of the enigmatic Andaman Islanders and 12 modern samples from three Indian tribal populations (Chenchu, Lambadi and Lodha) were genotyped for 20 coding region sites after provisional haplogroup assignment with control region sequences. The genotype data from the historic samples significantly revise the topologies for the Andaman M31 and M32 mtDNA lineages by rectifying conflicts in published data sets. The new Indian data extend the distribution of the M31a lineage to South Asia, challenging previous interpretations of mtDNA phylogeography. This genetic connection between the ancestors of the Andamanese and South Asian tribal groups approximately 30 kya has important implications for the debate concerning migration routes and settlement patterns of humans leaving Africa during the late Pleistocene, and indicates the need for more detailed genotyping strategies. The methodology serves as a low-cost, high-throughput model for the production and authentication of data from modern or ancient DNA, and demonstrates the value of museum collections as important records of human genetic diversity.
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Endicott, Phillip; Metspalu, Mait; Stringer, Chris; Macaulay, Vincent; Cooper, Alan; Sanchez, Juan J.
2006-01-01
The issue of errors in genetic data sets is of growing concern, particularly in population genetics where whole genome mtDNA sequence data is coming under increased scrutiny. Multiplexed PCR reactions, combined with SNP typing, are currently under-exploited in this context, but have the potential to genotype whole populations rapidly and accurately, significantly reducing the amount of errors appearing in published data sets. To show the sensitivity of this technique for screening mtDNA genomic sequence data, 20 historic samples of the enigmatic Andaman Islanders and 12 modern samples from three Indian tribal populations (Chenchu, Lambadi and Lodha) were genotyped for 20 coding region sites after provisional haplogroup assignment with control region sequences. The genotype data from the historic samples significantly revise the topologies for the Andaman M31 and M32 mtDNA lineages by rectifying conflicts in published data sets. The new Indian data extend the distribution of the M31a lineage to South Asia, challenging previous interpretations of mtDNA phylogeography. This genetic connection between the ancestors of the Andamanese and South Asian tribal groups ∼30 kya has important implications for the debate concerning migration routes and settlement patterns of humans leaving Africa during the late Pleistocene, and indicates the need for more detailed genotyping strategies. The methodology serves as a low-cost, high-throughput model for the production and authentication of data from modern or ancient DNA, and demonstrates the value of museum collections as important records of human genetic diversity. PMID:17218991
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Porter, Danielle P; Toma, Jonathan; Tan, Yuping; Solberg, Owen; Cai, Suqin; Kulkarni, Rima; Andreatta, Kristen; Lie, Yolanda; Chuck, Susan K; Palella, Frank; Miller, Michael D; White, Kirsten L
2016-02-01
Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.
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Yang, Song; Xing, Huichun; Feng, Shenghu; Ju, Wei; Liu, Shunai; Wang, Xiaomei; Ou, Weini; Cheng, Jun; Pan, Calvin Q
2018-02-01
There is little information on the association between baseline non-structural protein (NS) 5b resistance-associated variants (RAVs) and treatment failure in hepatitis C patients. This study examined the frequencies of natural hepatitis C virus (HCV) NS5B resistance-associated variants (RAVs) in an Asian cohort. Samples from Asian HCV patients enrolled between October 2009 and September 2014 were analyzed for NS5B RAVs within the region from amino acid 230 to 371. Serum samples were tested by PCR genotyping, with sequence alignment performed using the neighbor-joining method. NS5B was detected by Sanger sequencing followed by Geno2pheno analysis. NS5B RAVs were detected in 80.52% (1199/1489) of patients; 68.4% (1019/1489) and 79.7% (1186/1489) were associated with resistance to sofosbuvir (SOF) and dasabuvir (DSV), respectively. These RAVs were present in 95% (1004/1058) of genotype 1b patients. When genotypes 1b and 2a were compared, SOF-associated RAVs were detected at a higher frequency in genotype 1b (94.8% [1004/1058] vs. 2.9% [9/309]; χ 2 = 1054.433, P < 0.001), C316H/N was more common in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ 2 = 1096.014, P < 0.001), M289F/L/I/W/V had a higher frequency in genotype 2a (0.7% [7/309] vs. 2.3% [7/1058]; χ 2 = 4.589, P = 0.032), DSV-associated RAVs were most often found in genotype 1b (95.0% [1005/1058] vs. 40.1% 124/309]; χ 2 = 500.577, P < 0.001), and frequency of C316Y/H/N/W was higher in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ 2 = 1096.014, P < 0.001). In conclusion, baseline SOF and DSV RAVs are common in Asian HCV patients and predominantly occur in genotype 1b.
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Jönsson, E G; Nöthen, M M; Gustavsson, J P; Neidt, H; Brené, S; Tylec, A; Propping, P; Sedvall, G C
1997-05-01
Personality traits in human subjects have shown considerable heritable components. Recently, two research groups reported associations between dopamine D4 receptor genotypes and the personality trait known as novelty seeking. This study was an attempt to replicate these findings. Three different exonic dopamine D4 receptor polymorphisms were genotyped in 126 healthy Swedish subjects. Personality traits of the subjects were assessed with the Karolinska Scales of Personality. Although there was a tendency in the direction hypothesized, no significant association between genotype constellations and personality traits was found. The previously reported association between dopamine D4 receptor alleles and novelty seeking was not replicated. Possible reasons for this include differences in personality inventories, ethnicity, and type I or type II errors.
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Yilmaz, Huseyin; Karakullukcu, Asiye; Turan, Nuri; Cizmecigil, Utku Y; Yilmaz, Aysun; Ozkul, Ayse A; Aydin, Ozge; Gunduz, Alper; Mete, Mahmut; Zeyrek, Fadile Y; Kirazoglu, Taner T; Richt, Juergen A; Kocazeybek, Bekir
2017-08-11
Hepatitis A virus (HAV) is a food and water-borne virus causing clinical (mainly hepatitis) and subclinical disease in humans. It is important to characterize circulating strains of HAV in order to prevent HAV infections using efficacious vaccines. The aim of this study was the detection and characterization of the circulating strains of HAV in Turkey by performing serology, RT-PCR, sequencing and phylogenetic analysis. In this study, 355 HAV suspected cases were analysed by ELISA for the presence of antibodies to HAV. RNA was extracted from 54 HAV IgM positive human sera. None of the suspect cases were vaccinated against HAV and they never received blood transfusions. Samples found positive by RT-PCR using primers targeting the VP1/VP2A junction and VP1/VP3 capsid region of HAV, were subjected to sequencing and phylogenetic analyses. IgM type antibodies to HAV were detected in 54 patients. Twenty one of them were students. The age of IgM positive cases was between 3 and 60 years. IgM positivity differed in age groups and was higher in the age group 3 to 10 years. Phylogenetic analysis showed that the majority of HAV strains detected in this study belong to the "HAV 1B" cluster. In addition, the HAV sub-genotypes IA (KT874461.1) and IIIA (KT222963.1) were found in 2 children. These sub-genotypes were not previously reported in Turkey. The child who carried sub-genotype IIIA travelled to Afghanistan and presented with abdominal pain, icterus and vomitus. He was positive for anti-HAV IgM and IgG but negative for hepatitis B and C. Liver enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and lactate dehydrogenase were severely elevated. Bilirubin levels were also increased. White blood cells, neutrophils and hemoglobin were decreased while lymphocytes and monocytes were increased. Similar clinical signs and laboratory findings were reported for the child infected with sub-genotype IA but aspartate aminotransferase and alanine aminotransferase were not severely elevated. The results indicate that molecular studies determining the HAV genotype variation in Turkey are timely and warranted. The majority of IgM positive cases in 3-10 year old patients indicate that childhood vaccination is important. Sub-genotype IB is the most prevalant genotype in Turkey. Surprisingly, sub-genotype IA and IIIA are also present in Turkey; future diagnostic efforts need to include diagnostic methods which can identify this emerging HAV genotypes. Our results also show that one important risk factor for contracting hepatitis A virus is international travel since genotype IIIA was detected in a child who had travelled to Afghanistan.
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Rosenberg, Gillian K; Lattimore, Sam; Brailsford, Susan R; Hewitt, Patricia E; Tettmar, Kate I; Kitchen, Alan D; Ijaz, Samreen; Tedder, Richard S
2013-10-01
In 2010 hepatitis B virus (HBV) was the most frequently detected infection in UK blood donation screening, typically found in first-time, male, chronically infected donors born abroad. To date there has been no comprehensive characterization of the virologic profile of these infections. Epidemiologic and serologic data were collected retrospectively for 344 chronically HBV-infected blood donors identified from July 2005 to June 2010. Additional laboratory testing was carried out to determine the HBV genotype, viral load, and prevalence of clinically significant mutations and to detect hepatitis delta virus (HDV) coinfection. Five HBV genotypes (A-E) were found, Genotypes D (45%), A (20%), and E (20%) were the most prevalent. A strong association was seen between genotype and donor ethnicity (p < 0.001) and between genotype and place of residence (p = 0.006). Clinically significant mutations were observed across hepatitis B surface antigen (17%), basal core promoter (25%) and precore (78%) regions. An antiviral resistance profile was identified in one donor. Evidence of HDV coinfection was found in 2% of donors. The data show the diversity of HBV in asymptomatic chronic infections detected in blood donors in England and North Wales and demonstrates the presence of mutations which may impact on disease. The global nature of these infections and the inability to identify chronically infected donors before donation highlights the importance of using screening assays capable of detecting a broad range of genotypes and mutations. Furthermore, the integration of the virologic and demographic data allows us to more accurately construct a profile of our chronically HBV-infected blood donors. © 2012 American Association of Blood Banks.
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Wang, Jiao; Qi, Xiaoming; Zhang, Xiaozhen; Yan, Wen; You, Chongge
2017-12-01
Objective To establish the methods for detecting single nucleotide polymorphisms (SNPs) of ADP-ribosylation factor-like GTPase 15 (ARL15), major histocompatibility complex class II-DM alpha (HLA-DMA ) and nuclear factor kappa B subunit 2 (NFKB2) genes using high resolution melting (HRM) technology, and to explore the association of those SNPs with the susceptibility of rheumatoid arthritis (RA) in northwestern Han Chinese population. Methods The PCR-HRM detection system for four SNPs (rs255758, rs1063478, rs397514331 and rs397514332) was established for genotyping, and gene sequencing was performed to validate the genotyping ability of the system. 588 RA cases and 200 controls were enrolled in a case-control study to analyze the associations of ARL15 and HLA-DMA gene polymorphisms with RA risk. Results The direct sequencing validated that the established PCR-HRM detection system could be used for genotyping clinical samples correctly. The mutated genotype of rs397514331 and rs397514332 from NFKB2 gene are not found in this study. The genotype frequencies of rs255758 and rs1063478 had statistical difference between the cases and controls, but no statistical difference in allelic frequencies. Under the dominant model (AA vs AC/CC), the AA genotype of rs255758 decreases the RA risk (OR=0.666, 95%CI=0.478-0.927, P=0.016). Conclusion The method of PCR-HRM we established can be applied to the routine detection of rs255758, rs1063478, rs397514331 and rs397514332. The ARL15 and HLA-DMA gene polymorphisms are associated with RA risk in Northwestern Han Chinese population.
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Stanton, Jeffrey J.; Zong, Jian-Chao; Eng, Crystal; Howard, Lauren; Flanagan, Joe; Stevens, Martina; Schmitt, Dennis; Wiedner, Ellen; Graham, Danielle; Junge, Randall E.; Weber, Martha A.; Fischer, Martha; Mejia, Alicia; Tan, Jie; Latimer, Erin; Herron, Alan; Hayward, Gary S.; Ling, Paul D.
2013-01-01
Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elephas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3–8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7–21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants. PMID:23505702
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Passarge, Michelle; Fix, Michael K; Manser, Peter; Stampanoni, Marco F M; Siebers, Jeffrey V
2017-04-01
To develop a robust and efficient process that detects relevant dose errors (dose errors of ≥5%) in external beam radiation therapy and directly indicates the origin of the error. The process is illustrated in the context of electronic portal imaging device (EPID)-based angle-resolved volumetric-modulated arc therapy (VMAT) quality assurance (QA), particularly as would be implemented in a real-time monitoring program. A Swiss cheese error detection (SCED) method was created as a paradigm for a cine EPID-based during-treatment QA. For VMAT, the method compares a treatment plan-based reference set of EPID images with images acquired over each 2° gantry angle interval. The process utilizes a sequence of independent consecutively executed error detection tests: an aperture check that verifies in-field radiation delivery and ensures no out-of-field radiation; output normalization checks at two different stages; global image alignment check to examine if rotation, scaling, and translation are within tolerances; pixel intensity check containing the standard gamma evaluation (3%, 3 mm) and pixel intensity deviation checks including and excluding high dose gradient regions. Tolerances for each check were determined. To test the SCED method, 12 different types of errors were selected to modify the original plan. A series of angle-resolved predicted EPID images were artificially generated for each test case, resulting in a sequence of precalculated frames for each modified treatment plan. The SCED method was applied multiple times for each test case to assess the ability to detect introduced plan variations. To compare the performance of the SCED process with that of a standard gamma analysis, both error detection methods were applied to the generated test cases with realistic noise variations. Averaged over ten test runs, 95.1% of all plan variations that resulted in relevant patient dose errors were detected within 2° and 100% within 14° (<4% of patient dose delivery). Including cases that led to slightly modified but clinically equivalent plans, 89.1% were detected by the SCED method within 2°. Based on the type of check that detected the error, determination of error sources was achieved. With noise ranging from no random noise to four times the established noise value, the averaged relevant dose error detection rate of the SCED method was between 94.0% and 95.8% and that of gamma between 82.8% and 89.8%. An EPID-frame-based error detection process for VMAT deliveries was successfully designed and tested via simulations. The SCED method was inspected for robustness with realistic noise variations, demonstrating that it has the potential to detect a large majority of relevant dose errors. Compared to a typical (3%, 3 mm) gamma analysis, the SCED method produced a higher detection rate for all introduced dose errors, identified errors in an earlier stage, displayed a higher robustness to noise variations, and indicated the error source. © 2017 American Association of Physicists in Medicine.
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Abdel-Moneim, Ahmed S; Soliman, May S; Kamel, Mahmoud M; El-Kholy, Amani A
2018-03-01
Human respiratory syncytial virus causes severe lower respiratory tract infection in neonates and children. Genotype ON1, with duplication of 72-nt in the G gene, was first detected in Canada and then recorded in other countries. In the current study, we describe the first detection of the ON1 genotype among children in Egypt in 2014/2015. Sequence analysis of the full-attachment G gene revealed that the majority of the strains examined were related to the ON1 genotype and only one sample related to N1 genotype. The Egyptian ON1 strains showed unique non-silent mutations in addition to variable mutations near the antigenic sites in comparison to the original ON1 ancestor strain. Continuous surveillance of hRSV regionally and globally is needed to understand the evolutionary mechanisms and strategies adopted by hRSV and their inducers for better adaption to the host.
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Isolation and RFLP genotyping of Toxoplasma gondii from the gray wolf (Canis lupus).
Dubey, J P; Choudhary, S; Ferreira, L R; Kwok, O C H; Butler, E; Carstensen, M; Yu, L; Su, C
2013-11-08
Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study feral gray wolves (Canis lupus) from Minnesota were examined for T. gondii infection. Antibodies to T. gondii were detected in 130 (52.4%) of 248 wolves tested by the modified agglutination test (cut-off titer of 25). Tissues (hearts, brains or both) of 109 wolves were bioassayed in mice for protozoal isolation. Viable T. gondii was isolated from 25 and the isolates were further propagated in cell culture. T. gondii DNA from these isolates was characterized using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Four genotypes were detected. Twenty-one isolates were Type 12 (ToxoDB PCR-RFLP genotype #5), 2 were Type II clonal (ToxoDB #1), 1 was Type II variant (ToxoDB #3), and 1 was a new genotype designated as ToxoDB genotype #219. Published by Elsevier B.V.
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Tabrizi, Sepehr N; Law, Irwin; Buadromo, Eka; Stevens, Matthew P; Fong, James; Samuela, Josaia; Patel, Mahomed; Mulholland, E Kim; Russell, Fiona M; Garland, Suzanne M
2011-09-01
There is currently limited information about human papillomavirus (HPV) genotype distribution in women in the South Pacific region. This study's objective was to determine HPV genotypes present in cervical cancer (CC) and precancers (cervical intraepithelial lesion (CIN) 3) in Fiji. Cross-sectional analysis evaluated archival CC and CIN3 biopsy samples from 296 women of Melanesian Fijian ethnicity (n=182, 61.5%) and Indo-Fijian ethnicity (n=114, 38.5%). HPV genotypes were evaluated using the INNO-LiPA assay in archival samples from CC (n=174) and CIN3 (n=122) among women in Fiji over a 5-year period from 2003 to 2007. Overall, 99% of the specimens tested were HPV DNA-positive for high-risk genotypes, with detection rates of 100%, 97.4% and 100% in CIN3, squamous cell carcinoma (SCC) and adenosquamous carcinoma biopsies, respectively. Genotypes 16 and 18 were the most common (77%), followed by HPV 31 (4.3%). Genotype HPV 16 was the most common identified (59%) in CIN3 specimens, followed by HPV 31 (9%) and HPV 52 (6.6%). Multiple genotypes were detected in 12.5-33.3% of specimens, depending on the pathology. These results indicated that the two most prevalent CC-associated HPV genotypes in Fiji parallel those described in other regions worldwide, with genotype variations thereafter. These data suggest that the currently available bivalent and quadrivalent HPV vaccines could potentially reduce cervical cancers in Fiji by over 80% and reduce precancers by at least 60%.
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Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines.
Chung, Ju-Young; Kim, Min-Sung; Jung, Tae Woong; Kim, Seong Joon; Kang, Jin-Han; Han, Seung Beom; Kim, Sang Yong; Rhim, Jung Woo; Kim, Hwang-Min; Park, Jae Hong; Jo, Dae Sun; Ma, Sang Hyuk; Jeong, Hye-Sook; Cheon, Doo-Sung; Kim, Jong-Hyun
2015-10-01
Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.
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Genotypic Diversity of Escherichia coli in the Water and Soil of Tropical Watersheds in Hawaii ▿
Goto, Dustin K.; Yan, Tao
2011-01-01
High levels of Escherichia coli were frequently detected in tropical soils in Hawaii, which present important environmental sources of E. coli to water bodies. This study systematically examined E. coli isolates from water and soil of several watersheds in Hawaii and observed high overall genotypic diversity (35.5% unique genotypes). In the Manoa watershed, fewer than 9.3% of the observed E. coli genotypes in water and 6.6% in soil were shared between different sampling sites, suggesting the lack of dominant fecal sources in the watershed. High temporal variability of E. coli genotypes in soil was also observed, which suggests a dynamic E. coli population corresponding with the frequently observed high concentrations in tropical soils. When E. coli genotypes detected from the same sampling events were compared, limited sharing between the soil and water samples was observed in the majority of comparisons (73.5%). However, several comparisons reported up to 33.3% overlap of E. coli genotypes between soil and water, illustrating the potential for soil-water interactions under favorable environmental conditions. In addition, genotype accumulation curves for E. coli from water and soil indicated that the sampling efforts in the Manoa watershed could not exhaust the overall genotypic diversity. Comparisons of E. coli genotypes from other watersheds on Oahu, Hawaii, identified no apparent grouping according to sampling locations. The results of the present study demonstrate the complexity of using E. coli as a fecal indicator bacterium in tropical watersheds and highlight the need to differentiate environmental sources of E. coli from fecal sources in water quality monitoring. PMID:21515724
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Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping
2017-06-01
The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.
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Human Bocavirus in Korean Children with Gastroenteritis and Respiratory Tract Infections.
Lee, Eun Jin; Kim, Han-Sung; Kim, Hyun Soo; Kim, Jae-Seok; Song, Wonkeun; Kim, MiYoung; Lee, Young Kyung; Kang, Hee Jung
2016-01-01
Human bocaviruses (HBoVs) are suggested to be etiologic agents of childhood respiratory and gastrointestinal infections. There are four main recognized genotypes of HBoVs (HBoV1-4); the HBoV-1 genotype is considered to be the primary etiologic agent in respiratory infections, whereas the HBoV2-4 genotypes have been mainly associated with gastrointestinal infections. The aim of the present study was to determine the distribution of HBoV genotypes in children with respiratory or gastrointestinal infections in a hospital in Korea. A total of 662 nasopharyngeal swabs (NPSs) and 155 fecal specimens were collected from children aged 5 years or less. Polymerase chain reaction (PCR) was conducted to detect the NS1 HBoV gene. The VP1 gene of HBoV was further amplified in samples that were positive for the NS1 gene. The PCR products of VP1 gene amplification were genotyped by sequence analysis. HBoV was detected in 69 (14.5%) of 662 NPSs and in 10 (6.5%) of 155 fecal specimens. Thirty-three isolates from NPSs and five isolates from fecal specimens were genotyped, and all 38 sequenced isolates were identified as the HBoV-1 genotype. HBoV-1 is the most prevalent genotype in children with respiratory or gastrointestinal HBoV infections in a hospital in Korea.
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Human Bocavirus in Korean Children with Gastroenteritis and Respiratory Tract Infections
Lee, Eun Jin; Kim, Jae-Seok; Song, Wonkeun; Kim, MiYoung; Lee, Young Kyung
2016-01-01
Human bocaviruses (HBoVs) are suggested to be etiologic agents of childhood respiratory and gastrointestinal infections. There are four main recognized genotypes of HBoVs (HBoV1–4); the HBoV-1 genotype is considered to be the primary etiologic agent in respiratory infections, whereas the HBoV2–4 genotypes have been mainly associated with gastrointestinal infections. The aim of the present study was to determine the distribution of HBoV genotypes in children with respiratory or gastrointestinal infections in a hospital in Korea. A total of 662 nasopharyngeal swabs (NPSs) and 155 fecal specimens were collected from children aged 5 years or less. Polymerase chain reaction (PCR) was conducted to detect the NS1 HBoV gene. The VP1 gene of HBoV was further amplified in samples that were positive for the NS1 gene. The PCR products of VP1 gene amplification were genotyped by sequence analysis. HBoV was detected in 69 (14.5%) of 662 NPSs and in 10 (6.5%) of 155 fecal specimens. Thirty-three isolates from NPSs and five isolates from fecal specimens were genotyped, and all 38 sequenced isolates were identified as the HBoV-1 genotype. HBoV-1 is the most prevalent genotype in children with respiratory or gastrointestinal HBoV infections in a hospital in Korea. PMID:27990436
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Isolation and molecular identification of Acanthamoeba spp from oasis water in Tunisia.
Dendana, F; Trabelsi, H; Neiji, S; Sellami, H; Kammoun, S; Makni, F; Feki, J; Cheikhrouhou, F; Ayadi, A
2018-04-01
In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health. Copyright © 2018. Published by Elsevier Inc.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hoffman, D; Dyer, B; Kumaran Nair, C
Purpose: The Integral Quality Monitor (IQM), developed by iRT Systems GmbH (Koblenz, Germany) is a large-area, linac-mounted ion chamber used to monitor photon fluence during patient treatment. Our previous work evaluated the change of the ion chamber’s response to deviations from static 1×1 cm2 and 10×10 cm2 photon beams and other characteristics integral to use in external beam detection. The aim of this work is to simulate two external beam radiation delivery errors, quantify the detection of simulated errors and evaluate the reduction in patient harm resulting from detection. Methods: Two well documented radiation oncology delivery errors were selected formore » simulation. The first error was recreated by modifying a wedged whole breast treatment, removing the physical wedge and calculating the planned dose with Pinnacle TPS (Philips Radiation Oncology Systems, Fitchburg, WI). The second error was recreated by modifying a static-gantry IMRT pharyngeal tonsil plan to be delivered in 3 unmodulated fractions. A radiation oncologist evaluated the dose for simulated errors and predicted morbidity and mortality commiserate with the original reported toxicity, indicating that reported errors were approximately simulated. The ion chamber signal of unmodified treatments was compared to the simulated error signal and evaluated in Pinnacle TPS again with radiation oncologist prediction of simulated patient harm. Results: Previous work established that transmission detector system measurements are stable within 0.5% standard deviation (SD). Errors causing signal change greater than 20 SD (10%) were considered detected. The whole breast and pharyngeal tonsil IMRT simulated error increased signal by 215% and 969%, respectively, indicating error detection after the first fraction and IMRT segment, respectively. Conclusion: The transmission detector system demonstrated utility in detecting clinically significant errors and reducing patient toxicity/harm in simulated external beam delivery. Future work will evaluate detection of other smaller magnitude delivery errors.« less
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Lupini, Caterina; Giovanardi, Davide; Pesente, Patrizia; Bonci, Michela; Felice, Viviana; Rossi, Giulia; Morandini, Emilio; Cecchinato, Mattia; Catelli, Elena
2016-08-01
A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.
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Iwai, Masae; Hasegawa, Sumiyo; Obara, Mayumi; Nakamura, Kazuya; Horimoto, Eiji; Takizawa, Takenori; Kurata, Takeshi; Sogen, Shun-ichi; Shiraki, Kimiyasu
2009-01-01
Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis. PMID:19124591
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[The virological and epidemiological aspects of human adenoviral conjunctivitis in Tunisia].
Fedaoui, N; Ben Ayed, N; Ben Yahia, A; Matri, L; Nacef, L; Triki, H
2017-01-01
Human adenoviruses (HAdV) are the main cause of viral conjunctivitis. In Tunisia and North Africa more generally, there is no regular nationwide surveillance program that monitors viruses causing conjunctivitis and keratoconjunctivitis. In this study, we report the results of HAdV screening in conjunctival samples collected for over 14 years in Tunisia. A total of 282 conjunctival samples received between 2000 and 2013 were investigated. Detection and identification of genotype were performed by PCR-sequencing at the hexon gene; 64.5% of samples (n=182) revealed positive by PCR detection without correlation noted between infection, age, sex, social class or clinical manifestations of viral conjunctivitis. HAdV-D8 was the largely predominant genotype in Tunisia, representing 81.3% of all isolates, and was detected continuously from 2000 to 2013. Minor co-circulating genotypes were also identified - HAdV-E4, HAdV-B3, B55 and HAdV-B7 - accounting for 10.7%, 4.9%, 1.9% and 0.9% of isolates, respectively. In conclusion, this work reports epidemiological data on adenoviral conjunctivitis from a region where such information is very scarce and contributes to a better knowledge of the worldwide distribution of causative genotypes. It also presents an approach for the identification of circulating HAdV in the country and demonstrates the importance of molecular tools for both detection and identification of genotypes, which allow rapid virological investigation, especially during epidemics. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
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Zhao, Wei; Wang, Jianguang; Yang, Ziyin; Liu, Aiqin
2017-01-01
Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans and is also found in a wide range of animals. It is considered to be an important but neglected zoonotic pathogen. With the development of deer bred in captivity, the number of deer has been increasing in recent years in China and there are more people involved in this work. The aims of this study were to determine prevalence and genotypes of E. bieneusi in red deer (Cervus elaphus) and Siberian roe deer (Capreolus pygargus), and to assess their potential zoonotic transmission. A total of 122 fecal specimens were collected from 104 red deer and 18 roe deer from three deer farms in Heilongjiang and Jilin Provinces, China. Enterocytozoon bieneusi was detected and genotyped by PCR and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. The average infection rate was 8.2% (10/122), with 7.7% (8/104) for red deer and 11.1% (2/18) for roe deer. Two genotypes were identified: a known genotype BEB6 (n = 9) and a novel genotype named HLJD-VI (n = 1). This is the first report of E. bieneusi infection in Siberian roe deer. The fact that genotype BEB6 was detected previously in one human case of microsporidiosis, and that genotype HLJD-VI fell into zoonotic group 1, suggest the possibility of transmission to humans. A brief review of E. bieneusi genotypes in deer worldwide shows that 40 genotypes have been found in seven deer species, with genotype BEB6 being predominant. PMID:29267159
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Dou, Xin-Man; Cheng, Hui-Juan; Meng, Ling; Zhou, Lin-Lin; Ke, Yi-Hong; Liu, Li-Ping; Li, Yu-Min
2017-04-30
The aim of the present study is to investigate association between septic shock (SS) and angiotensin I-converting enzyme ( ACE ) single nucleotide polymorphisms (SNPs). From October 2009 to December 2016, 238 SS patients and 242 healthy individuals were selected for our study. ACE activity was detected, ACE rs4291 and rs4646994 polymorphisms were detected using PCR-restriction fragment length polymorphism (PCR-RFLP). The Kaplan-Meier survival curve was employed to evaluate the association between ACE SNPs and patients' survival and univariate and multivariate analyses to estimate risk factors for SS. ACE activity in the case group was increased in comparison with the control group. Allele and genotype frequencies of rs4291 and rs4646994 were different between the case and control groups. The TT genotype frequency of the rs4291 polymorphisms and the DD genotype of the rs4646994 polymorphisms of the case group were higher than those in the control group. The AT and TT genotypes indicated a significant elevation of ACE activity than the AA genotype, while a significant decline was found in the DI and II genotypes in comparison with the DI genotype. Patients with TT or DD genotypes had increased fatality rate within 7 and 30 days when compared with those with non-TT or non-DD genotypes. Lower sepsis-related organ failure assessment (SOFA) scores, rs4291, serum ACE and rs4646994 were all considered as risky factors for SS patients. The study demonstrates that TT genotype of rs4291 or DD genotype of rs4646994 may be indicative of a higher risk of SS and a poorer prognosis in SS patients. © 2017 The Author(s).
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Perry, Brea L.; Pescosolido, Bernice A.; Bucholz, Kathleen; Edenberg, Howard; Kramer, John; Kuperman, Samuel; Schuckit, Marc Alan; Nurnberger, John I.
2015-01-01
Gender-moderated gene–environment interactions are rarely explored, raising concerns about inaccurate specification of etiological models and inferential errors. The current study examined the influence of gender, negative and positive daily life events, and GABRA2 genotype (SNP rs279871) on alcohol dependence, testing two- and three-way interactions between these variables using multilevel regression models fit to data from 2,281 White participants in the Collaborative Study on the Genetics of Alcoholism. Significant direct effects of variables of interest were identified, as well as gender-specific moderation of genetic risk on this SNP by social experiences. Higher levels of positive life events were protective for men with the high-risk genotype, but not among men with the low-risk genotype or women, regardless of genotype. Our findings support the disinhibition theory of alcohol dependence, suggesting that gender differences in social norms, constraints and opportunities, and behavioral undercontrol may explain men and women’s distinct patterns of association. PMID:23974430
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Zhou, Zhiyong; Wagar, Nick; DeVos, Joshua R.; Rottinghaus, Erin; Diallo, Karidia; Nguyen, Duc B.; Bassey, Orji; Ugbena, Richard; Wadonda-Kabondo, Nellie; McConnell, Michelle S.; Zulu, Isaac; Chilima, Benson; Nkengasong, John; Yang, Chunfu
2011-01-01
Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX. Conclusions The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR. PMID:22132237
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Multi-Criteria Decision Making Approaches for Quality Control of Genome-Wide Association Studies
Malovini, Alberto; Rognoni, Carla; Puca, Annibale; Bellazzi, Riccardo
2009-01-01
Experimental errors in the genotyping phases of a Genome-Wide Association Study (GWAS) can lead to false positive findings and to spurious associations. An appropriate quality control phase could minimize the effects of this kind of errors. Several filtering criteria can be used to perform quality control. Currently, no formal methods have been proposed for taking into account at the same time these criteria and the experimenter’s preferences. In this paper we propose two strategies for setting appropriate genotyping rate thresholds for GWAS quality control. These two approaches are based on the Multi-Criteria Decision Making theory. We have applied our method on a real dataset composed by 734 individuals affected by Arterial Hypertension (AH) and 486 nonagenarians without history of AH. The proposed strategies appear to deal with GWAS quality control in a sound way, as they lead to rationalize and make explicit the experimenter’s choices thus providing more reproducible results. PMID:21347174
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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
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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
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[Study on the relationship of MTHFR polymorphisms with unexplained recurrent spontaneous abortion].
Li, Xiao-mei; Zhang, You-zhong; Xu, Yan-xue; Jiang, Sen
2004-02-01
To assess the relationship of methylenetetrahydrofolate reductase (MTHFR) C677T genotypes to unexplained recurrent spontaneous abortion (URSA). This study included two groups:57 currently non-pregnant women with a history of URSA (URSA group), and 50 currently non-pregnant women with a history of having given birth to at least one live baby and without any history of spontaneous abortion, still-born fetus, placental thrombosis and intrauterine growth retardation(IUGR)(control group). The fasting serum-Hcy was measured with high pressure liquid chromatography. Folic acid and vitamin B(12) were detected by radioimmune assay; antiphospholipid antibody (ACA) was detected by ELISA. MTHFR C677T gene polymorphisms were detected by the technique of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). C/C genotype in URSA group was significantly lower than that in control group, the total mutant T allele frequency was significantly higher than that in control group. There was no significant difference in respect of "age, rural area/city, period, primary/secondary abortion" between the genotype distributions of MTHFR C677T. The T/T genotype and C/T+T/T genotypes frequencies for "abortion times>or=3" were higher than those for "abortion time <3". MTHFR C677T gene polymorphism is a genetic risk factor for URSA.
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Detection and sequencing of rotavirus among sudanese children.
Magzoub, Magzoub Abbas; Bilal, Naser Eldin; Bilal, Jalal Ali; Alzohairy, Mohammad Abdulrahman; Elamin, Bahaeldin Khalid; Gasim, Gasim Ibrahim
2017-01-01
Diarrheal diseases are a big public health problem worldwide, particularly among developing countries. The current study was conducted to detect and characterize group A rotavirus among admitted children with gastroenteritis to the pediatric hospitals, Sudan. A total of 755 stool samples were collected from Sudanese children with less than 5 years of age presenting with acute gastroenteritis during the period from April to September 2010. Enzyme-linked immunosorbent assay (ELISA) was used to Detection of Rotavirus antigens. Ribonucleic acid (RNAs) were extracted from rotavirus-positive stool samples using (QIAamp ® Viral RNA Mini Kit). (Omniscript ® Reverse Transcription kit) was used to convert RNA to complementary Deoxyribonucleic acid (cDNA). The cDNAs were used as template for detection of VP4-P (P for Protease-sensitive) and VP7-G (G for Glycoprotein) genotyping of Rotavirus using nested PCR and sequencing. Out of the 755 stool samples from children with acute gastroenteritis, 121 were positive for rotavirus A. Among 24 samples that were sequenced; the VP7 predominant G type was G1 (83.3%), followed by G9 (16.7%). Out of these samples, only one VP4 P[8] genotype was detected. As a conclusion the VP7 predominant G type was G1, followed by G9 whereas only one VP4 genotype was detected and showed similarity to P[8] GenBank strain. It appears that the recently approved rotavirus vaccines in Sudan are well matched to the rotavirus genotypes identified in this study, though more studies are needed.
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Rew, Mary Beth; Robbins, Jooke; Mattila, David; Palsbøll, Per J; Bérube, Martine
2011-04-01
Genetic identification of individuals is now commonplace, enabling the application of tagging methods to elusive species or species that cannot be tagged by traditional methods. A key aspect is determining the number of loci required to ensure that different individuals have non-matching multi-locus genotypes. Closely related individuals are of particular concern because of elevated matching probabilities caused by their recent co-ancestry. This issue may be addressed by increasing the number of loci to a level where full siblings (the relatedness category with the highest matching probability) are expected to have non-matching multi-locus genotypes. However, increasing the number of loci to meet this "full-sib criterion" greatly increases the laboratory effort, which in turn may increase the genotyping error rate resulting in an upward-biased mark-recapture estimate of abundance as recaptures are missed due to genotyping errors. We assessed the contribution of false matches from close relatives among 425 maternally related humpback whales, each genotyped at 20 microsatellite loci. We observed a very low (0.5-4%) contribution to falsely matching samples from pairs of first-order relatives (i.e., parent and offspring or full siblings). The main contribution to falsely matching individuals from close relatives originated from second-order relatives (e.g., half siblings), which was estimated at 9%. In our study, the total number of observed matches agreed well with expectations based upon the matching probability estimated for unrelated individuals, suggesting that the full-sib criterion is overly conservative, and would have required a 280% relative increase in effort. We suggest that, under most circumstances, the overall contribution to falsely matching samples from close relatives is likely to be low, and hence applying the full-sib criterion is unnecessary. In those cases where close relatives may present a significant issue, such as unrepresentative sampling, we propose three different genotyping strategies requiring only a modest increase in effort, which will greatly reduce the number of false matches due to the presence of related individuals.
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Effects of Contextual Sight-Singing and Aural Skills Training on Error-Detection Abilities.
ERIC Educational Resources Information Center
Sheldon, Deborah A.
1998-01-01
Examines the effects of contextual sight-singing and ear training on pitch and rhythm error detection abilities among undergraduate instrumental music education majors. Shows that additional training produced better error detection, particularly with rhythm errors and in one-part examples. Maintains that differences attributable to texture were…
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A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms
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
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Distribution of Porphyromonas gingivalis fimA genotypes in primary endodontic infections.
Rôças, Isabela N; Siqueira, José F
2010-03-01
Long fimbriae (FimA) are important virulence factors of Porphyromonas gingivalis. Based on the diversity of the fimA gene, this species is classified into 6 genotypes. This study surveyed samples from primary endodontic infections for the presence of these P. gingivalis fimA variants. Genomic DNA isolated from samples taken from 25 root canals of teeth with chronic apical periodontitis and 25 aspirates from acute apical abscess was used as template in polymerase chain reaction (PCR) assays directed toward the detection of the different P. gingivalis fimA genotypes. Porphyromonas gingivalis was detected by a 16S rRNA gene-based PCR in 36% of the total number of cases sampled (44% of chronic apical periodontitis and 28% of abscess aspirates). In cases of chronic apical periodontitis, P. gingivalis variant type IV was the most prevalent (24%), followed by types I (20%), II (16%), and III (8%). In acute abscess samples, variant type II was the most prevalent (12%), followed by types III and IV (8% of each) and type I (4%). Combinations of up to 3 different genotypes were detected in a few cases. No single fimA genotype variant or combination thereof was significantly associated with symptoms. Overall, fimA types IV (16%), II (14%), and I (12%) were the most prevalent. Findings demonstrated that different P. gingivalis fimA genotypes can be present in primary endodontic infections. Copyright 2010 Mosby, Inc. All rights reserved.
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Szuhai, Károly; Sandhaus, Emily; Kolkman-Uljee, Sandra M.; Lemaître, Marc; Truffert, Jean-Christophe; Dirks, Roeland W.; Tanke, Hans J.; Fleuren, Gert Jan; Schuuring, Ed; Raap, Anton K.
2001-01-01
Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogenous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology. PMID:11696426
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Influence of COMT genotype and affective distractors on the processing of self-generated thought.
Kilford, Emma J; Dumontheil, Iroise; Wood, Nicholas W; Blakemore, Sarah-Jayne
2015-06-01
The catechol-O-methyltransferase (COMT) enzyme is a major determinant of prefrontal dopamine levels. The Val(158)Met polymorphism affects COMT enzymatic activity and has been associated with variation in executive function and affective processing. This study investigated the effect of COMT genotype on the flexible modulation of the balance between processing self-generated and processing stimulus-oriented information, in the presence or absence of affective distractors. Analyses included 124 healthy adult participants, who were also assessed on standard working memory (WM) tasks. Relative to Val carriers, Met homozygotes made fewer errors when selecting and manipulating self-generated thoughts. This effect was partly accounted for by an association between COMT genotype and visuospatial WM performance. We also observed a complex interaction between the influence of affective distractors, COMT genotype and sex on task accuracy: male, but not female, participants showed a sensitivity to the affective distractors that was dependent on COMT genotype. This was not accounted for by WM performance. This study provides novel evidence of the role of dopaminergic genetic variation on the ability to select and manipulate self-generated thoughts. The results also suggest sexually dimorphic effects of COMT genotype on the influence of affective distractors on executive function. © The Author (2014). Published by Oxford University Press.
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Gish, R G; Gutierrez, J A; Navarro-Cazarez, N; Giang, K; Adler, D; Tran, B; Locarnini, S; Hammond, R; Bowden, S
2014-12-01
Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource-limited settings and among marginalized populations. High-quality point-of-care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign(®) HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the 'a' determinant. Thirty-seven serum samples from patients with HBV infection, covering HBV genotypes A-G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV 'a' determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource-limited countries due to its low cost (0.50$) and immediately available results. © 2014 John Wiley & Sons Ltd.
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Bledsoe, Sarah; Van Buskirk, Alex; Falconer, R James; Hollon, Andrew; Hoebing, Wendy; Jokic, Sladan
2018-02-01
The effectiveness of barcode-assisted medication preparation (BCMP) technology on detecting oral liquid dose preparation errors. From June 1, 2013, through May 31, 2014, a total of 178,344 oral doses were processed at Children's Mercy, a 301-bed pediatric hospital, through an automated workflow management system. Doses containing errors detected by the system's barcode scanning system or classified as rejected by the pharmacist were further reviewed. Errors intercepted by the barcode-scanning system were classified as (1) expired product, (2) incorrect drug, (3) incorrect concentration, and (4) technological error. Pharmacist-rejected doses were categorized into 6 categories based on the root cause of the preparation error: (1) expired product, (2) incorrect concentration, (3) incorrect drug, (4) incorrect volume, (5) preparation error, and (6) other. Of the 178,344 doses examined, 3,812 (2.1%) errors were detected by either the barcode-assisted scanning system (1.8%, n = 3,291) or a pharmacist (0.3%, n = 521). The 3,291 errors prevented by the barcode-assisted system were classified most commonly as technological error and incorrect drug, followed by incorrect concentration and expired product. Errors detected by pharmacists were also analyzed. These 521 errors were most often classified as incorrect volume, preparation error, expired product, other, incorrect drug, and incorrect concentration. BCMP technology detected errors in 1.8% of pediatric oral liquid medication doses prepared in an automated workflow management system, with errors being most commonly attributed to technological problems or incorrect drugs. Pharmacists rejected an additional 0.3% of studied doses. Copyright © 2018 by the American Society of Health-System Pharmacists, Inc. All rights reserved.
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Spatial effects, sampling errors, and task specialization in the honey bee.
Johnson, B R
2010-05-01
Task allocation patterns should depend on the spatial distribution of work within the nest, variation in task demand, and the movement patterns of workers, however, relatively little research has focused on these topics. This study uses a spatially explicit agent based model to determine whether such factors alone can generate biases in task performance at the individual level in the honey bees, Apis mellifera. Specialization (bias in task performance) is shown to result from strong sampling error due to localized task demand, relatively slow moving workers relative to nest size, and strong spatial variation in task demand. To date, specialization has been primarily interpreted with the response threshold concept, which is focused on intrinsic (typically genotypic) differences between workers. Response threshold variation and sampling error due to spatial effects are not mutually exclusive, however, and this study suggests that both contribute to patterns of task bias at the individual level. While spatial effects are strong enough to explain some documented cases of specialization; they are relatively short term and not explanatory for long term cases of specialization. In general, this study suggests that the spatial layout of tasks and fluctuations in their demand must be explicitly controlled for in studies focused on identifying genotypic specialists.
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Gregory, Lilian; Carrillo Gaeta, Natália; Araújo, Jansen; Matsumiya Thomazelli, Luciano; Harakawa, Ricardo; Ikuno, Alice A; Hiromi Okuda, Liria; de Stefano, Eliana; Pituco, Edviges Maristela
2017-12-01
Enzootic bovine leucosis (EBL) is a silent disease caused by a retrovirus [bovine leukaemia virus (BLV)]. BLV is classified into almost 10 genotypes that are distributed in several countries. The present research aimed to describe two BLV gp51 env sequences of strains detected in the state of São Paulo, Brazil and perform a phylogenetic analysis to compare them to other BLV gp51 env sequences of strains around the world. Two bovines from different herds were admitted to the Bovine and Small Ruminant Hospital, School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil. In both, lymphosarcoma was detected and the presence of BLV was confirmed by nested PCR. The neighbour-joining algorithm distance method was used to genotype the BLV sequences by phylogenetic reconstruction, and the maximum likelihood method was used for the phylogenetic reconstruction. The phylogeny estimates were calculated by performing 1000 bootstrap replicates. Analysis of the partial envelope glycoprotein (env) gene sequences from two isolates (25 and 31) revealed two different genotypes of BLV. Isolate 25 clustered with ten genotype 6 isolates from Brazil, Argentina, Thailand and Paraguay. On the other hand, isolate 31 clustered with two genotype 5 isolates (one was also from São Paulo and one was from Costa Rica). The detected genotypes corroborate the results of previous studies conducted in the state of São Paulo, Brazil. The prediction of amino acids showed substitutions, particularly between positions 136 and 150 in 11 out of 13 sequences analysed, including sequences from GenBank. BLV is still important in Brazil and this research should be continued.
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Molecular epidemiology of hepatitis A virus infection in Northeast India.
Bose, Moumita; Bose, Sujoy; Saikia, Anjan; Medhi, Subhash; Deka, Manab
2015-07-01
The present study was undertaken to screen the molecular epidemiology of Hepatitis A virus (HAV) in Northeast India (NEI) who are ethnically distinct, tribal dominated and of lower socio-economic status with almost no information available from NEI on these aspects. Briefly, 3 ml blood was collected from 324 random liver disease cases with jaundice, receiving care at Central Hospital, N.F. Railway, Guwahati, Assam with informed consent. The patients detected with HAV-IgM positive status were included and were stratified as acute viral hepatitis (AVH) and fulminant hepatitis (FHF) based on clinical profile. Viral RNA was isolated and HAV-RNA was detected by Real-time PCR using primers for the VP3-VP1 region. HAV genotyping was studied by PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of HAV isolates. Statistical analysis was performed using SPSS13.0 software. A total of 69 cases were HAV infected with two HBV co-infected cases (n = 69 + 2 = 71), 62 cases and two co-infected cases were AVH and others were FHF cases. HAV infection was predominant in especially in the young and adult age group. HAV-RNA was detected in 28 cases, out of which 19 cases could be genotyped (12 AVH, 7 FHF); which showed the prevalence of genotype IIIA or IA only. Although HAV genotype IIIA was the major genotype in both the AVH (10/12, 83.33%) and FHF (5/7, 71.43%) group, but the difference in distribution of genotypes in AVH and FHF cases was statistically non-significant (P = 0.550). HAV genotype IIIA is associated with the majority of HAV infected cases and severity in NEI. © 2015 Wiley Periodicals, Inc.
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Distribution of HPV genotypes in cervical cancer in multi- ethnic Malaysia.
Hamzi Abdul Raub, Sayyidi; Isa, Nurismah Md; Zailani, Hatta Ahmad; Omar, Baharudin; Abdullah, Mohamad Farouk; Mohd Amin, Wan Anna; Noor, Rushdan Md; Ayub, Mukarramah Che; Abidin, Zainal; Kassim, Fauziah; Vicknesh, Visvalingam; Zakaria, Zubaidah; Kamaluddin, Muhammad Amir; Tan, Geok Chin; Syed Husain, Sharifah Noor Akmal
2014-01-01
Cervical cancer is the third commonest type of cancer among women in Malaysia. Our aim was to determine the distribution of human papilloma virus (HPV) genotypes in cervical cancer in our multi-ethnic population. This was a multicentre study with a total of 280 cases of cervical cancer from 4 referral centres in Malaysia, studied using real-time polymerase chain reaction (qPCR) detection of 12 high risk-HPV genotypes. Overall HPV was detected in 92.5% of cases, in 95.9% of squamous cell carcinomas and 84.3%of adenocarcinomas. The five most prevalent high-risk HPV genotypes were HPV 16 (68.2%), 18 (40%), 58 (10.7%), 33 (10.4%) and 52 (10.4%). Multiple HPV infections were more prevalent (55.7%) than single HPV infections (36.8%). The percentage of HPV positive cases in Chinese, Malays and Indians were 95.5%, 91.9% and 80.0%, respectively. HPV 16 and 18 genotypes were the commonest in all ethnic groups. We found that the percentage of HPV 16 infection was significantly higher in Chinese (75.9%) compared to Malays (63.7%) and Indians (52.0%) (p<0.05), while HPV 18 was significantly higher in Malays (52.6%) compared to Chinese (25.0%) and Indians (28%) (p<0.05). Meanwhile, HPV 33 (17.9%) and 52 (15.2%) were also more commonly detected in the Chinese (p<0.05). This study showed that the distribution of HPV genotype in Malaysia is similar to other Asian countries. Importantly, we found that different ethnic groups in Malaysia have different HPV genotype infection rates, which is a point to consider during the implementation of HPV vaccination.
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A concatenated coding scheme for error control
NASA Technical Reports Server (NTRS)
Lin, S.
1985-01-01
A concatenated coding scheme for error contol in data communications was analyzed. The inner code is used for both error correction and detection, however the outer code is used only for error detection. A retransmission is requested if either the inner code decoder fails to make a successful decoding or the outer code decoder detects the presence of errors after the inner code decoding. Probability of undetected error of the proposed scheme is derived. An efficient method for computing this probability is presented. Throughout efficiency of the proposed error control scheme incorporated with a selective repeat ARQ retransmission strategy is analyzed.
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Form Overrides Meaning When Bilinguals Monitor for Errors
Ivanova, Iva; Ferreira, Victor S.; Gollan, Tamar H.
2016-01-01
Bilinguals rarely produce unintended language switches, which may in part be because switches are detected and corrected by an internal monitor. But are language switches easier or harder to detect than within-language semantic errors? To approximate internal monitoring, bilinguals listened (Experiment 1) or read aloud (Experiment 2) stories, and detected language switches (translation equivalents or semantically unrelated to expected words) and within-language errors (semantically related or unrelated to expected words). Bilinguals detected semantically related within-language errors most slowly and least accurately, language switches more quickly and accurately than within-language errors, and (in Experiment 2), translation equivalents as quickly and accurately as unrelated language switches. These results suggest that internal monitoring of form (which can detect mismatches in language membership) completes earlier than, and is independent of, monitoring of meaning. However, analysis of reading times prior to error detection revealed meaning violations to be more disruptive for processing than language violations. PMID:28649169
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Liquid biopsy genotyping in lung cancer: ready for clinical utility?
Huang, Wei-Lun; Chen, Yi-Lin; Yang, Szu-Chun; Ho, Chung-Liang; Wei, Fang; Wong, David T; Su, Wu-Chou; Lin, Chien-Chung
2017-03-14
Liquid biopsy is a blood test that detects evidence of cancer cells or tumor DNA in the circulation. Despite complicated collection methods and the requirement for technique-dependent platforms, it has generated substantial interest due, in part, to its potential to detect driver oncogenes such as epidermal growth factor receptor (EGFR) mutants in lung cancer. This technology is advancing rapidly and is being incorporated into numerous EGFR tyrosine kinase inhibitor (EGFR-TKI) development programs. It appears ready for integration into clinical care. Recent studies have demonstrated that biological fluids such as saliva and urine can also be used for detecting EGFR mutant DNA through application other user-friendly techniques. This review focuses on the clinical application of liquid biopsies to lung cancer genotyping, including EGFR and other targets of genotype-directed therapy and compares multiple platforms used for liquid biopsy.
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Molecular Epidemiology of Measles Viruses in the United States, 1997–2001
Liffick, Stephanie L.; Rota, Jennifer S.; Katz, Russell S.; Redd, Susan; Papania, Mark; Bellini, William J.
2002-01-01
From 1997 to 2001, sequence data from 55 clinical specimens were obtained from confirmed measles cases in the United States, representing 21 outbreaks and 34 sporadic cases. Sequence analysis indicated the presence of 11 of the recognized genotypes. The most common genotypes detected were genotype D6, usually identified from imported cases from Europe, and genotype D5, associated with importations from Japan. A number of viruses belonging to genotype D4 were imported from India and Pakistan. Overall, viral genotypes were determined for 13 chains of transmission with an unknown source of virus, and seven different genotypes were identified. Therefore, the diversity of Measles virus genotypes observed in the United States from 1997 to 2001 reflected multiple imported sources of virus and indicated that no strain of measles is endemic in the United States. PMID:12194764
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Nowak, Rebecca G; Ambulos, Nicholas P; Schumaker, Lisa M; Mathias, Trevor J; White, Ruth A; Troyer, Jennifer; Wells, David; Charurat, Manhattan E; Bentzen, Søren M; Cullen, Kevin J
2017-06-13
Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it's diagnostic performance has not been tested in cases with multiple concurrent HPV infections. We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections. In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2-91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5-96.2; sensitivity: 93.1%, 95% CI: 83.3-98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1-87.7; sensitivity: 61.1, 95% CI: 43.5-76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4-95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114). The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.
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Moltke, Ida; Albrechtsen, Anders; Hansen, Thomas v.O.; Nielsen, Finn C.; Nielsen, Rasmus
2011-01-01
All individuals in a finite population are related if traced back long enough and will, therefore, share regions of their genomes identical by descent (IBD). Detection of such regions has several important applications—from answering questions about human evolution to locating regions in the human genome containing disease-causing variants. However, IBD regions can be difficult to detect, especially in the common case where no pedigree information is available. In particular, all existing non-pedigree based methods can only infer IBD sharing between two individuals. Here, we present a new Markov Chain Monte Carlo method for detection of IBD regions, which does not rely on any pedigree information. It is based on a probabilistic model applicable to unphased SNP data. It can take inbreeding, allele frequencies, genotyping errors, and genomic distances into account. And most importantly, it can simultaneously infer IBD sharing among multiple individuals. Through simulations, we show that the simultaneous modeling of multiple individuals makes the method more powerful and accurate than several other non-pedigree based methods. We illustrate the potential of the method by applying it to data from individuals with breast and/or ovarian cancer, and show that a known disease-causing mutation can be mapped to a 2.2-Mb region using SNP data from only five seemingly unrelated affected individuals. This would not be possible using classical linkage mapping or association mapping. PMID:21493780
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Human papilloma virus (HPV) genotypes prevalence in a region of South Italy (Apulia).
Coscia, Maria Franca; Monno, Rosa; Ballini, Andrea; Mirgaldi, Rosanna; Dipalma, Gianna; Pettini, Francesco; Cristallo, Vincenzo; Inchingolo, Francesco; Foti, Caterina; de Vito, Danila
2015-01-01
Since human papillomavirus (HPV) is the central casual factor in cervical cancer, understanding the epidemiology and geographical area distribution of the most prevalent HPV genotypes constitutes an important step towards development of strategies of prevention. The aim of this study was to investigate the prevalence of HPV infection and to determine HPV types distribution among 822 HPV positive women and some sexual male partners in Apulia (Italy). HPV DNA detection and genotyping was performed by nested-PCR for the L1 region and reverse line blot hybridization allowing the specific detection of 24 HPV genotyping both high risk (HR) and low risk (LR). The most prevalent HPV genotypes were HPV 16 (35%), HPV 31 (16%) HPV 6 (9%), HPV 58 and 66 (7%), followed by HPV 33 (6%), HPV 18 and 56 (4%), HPV 70 and 45 (3%), HPV 53 and 11 (2%). Currently 1.5% of tested specimens remained unclassified. Multiple infections with at last two different high- risk HPV genotypes were observed in 10% of specimens. This finding adds knowledge to HPV epidemiological investigation, and addresses further studies aimed to consider public health for identifying groups at risk for cervical cancer.
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NASA Astrophysics Data System (ADS)
Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.
2017-02-01
In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.
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Palmer, Tom M; Holmes, Michael V; Keating, Brendan J; Sheehan, Nuala A
2017-11-01
Mendelian randomization studies use genotypes as instrumental variables to test for and estimate the causal effects of modifiable risk factors on outcomes. Two-stage residual inclusion (TSRI) estimators have been used when researchers are willing to make parametric assumptions. However, researchers are currently reporting uncorrected or heteroscedasticity-robust standard errors for these estimates. We compared several different forms of the standard error for linear and logistic TSRI estimates in simulations and in real-data examples. Among others, we consider standard errors modified from the approach of Newey (1987), Terza (2016), and bootstrapping. In our simulations Newey, Terza, bootstrap, and corrected 2-stage least squares (in the linear case) standard errors gave the best results in terms of coverage and type I error. In the real-data examples, the Newey standard errors were 0.5% and 2% larger than the unadjusted standard errors for the linear and logistic TSRI estimators, respectively. We show that TSRI estimators with modified standard errors have correct type I error under the null. Researchers should report TSRI estimates with modified standard errors instead of reporting unadjusted or heteroscedasticity-robust standard errors. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health.
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[Detection of UGT1A1*28 Polymorphism Using Fragment Analysis].
Huang, Ying; Su, Jian; Huang, Xiaosui; Lu, Danxia; Xie, Zhi; Yang, Suqing; Guo, Weibang; Lv, Zhiyi; Wu, Hongsui; Zhang, Xuchao
2017-12-20
Uridine-diphosphoglucuronosyl transferase 1A1 (UGT1A1), UGT1A1*28 polymorphism can reduce UGT1A1 enzymatic activity, which may lead to severe toxicities in patients who receive irinotecan. This study tries to build a fragment analysis method to detect UGT1A1*28 polymorphism. A total of 286 blood specimens from the lung cancer patients who were hospitalized in Guangdong General Hospital between April 2014 to May 2015 were detected UGT1A1*28 polymorphism by fragment analysis method. Comparing with Sanger sequencing, precision and accuracy of the fragment analysis method were 100%. Of the 286 patients, 236 (82.5% harbored TA6/6 genotype, 48 (16.8%) TA 6/7 genotype and 2 (0.7%) TA7/7 genotype. Our data suggest hat the fragment analysis method is robust for detecting UGT1A1*28 polymorphism in clinical practice. It's simple, time-saving, and easy-to-carry.
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Scott, John D; Lee, Min-Kuang; Fernando, Keerthi; Durden, Lance A; Jorgensen, Danielle R; Mak, Sunny; Morshed, Muhammad G
2010-06-01
Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three-year, bird-tick-pathogen study (2004-2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada-wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf-rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete-infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first-time records for: ticks in the Yukon (north of 60 degrees latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird-derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden-crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir-competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.-infected ticks into Canada. Bird-feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.
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Wang, X T; Wang, R J; Ren, G J; Yu, Z Q; Zhang, L X; Zhang, S Y; Lu, H; Peng, X Q; Zhao, G H
2016-03-01
Giardia duodenalis and Enterocytozoon bieneusi are two common protozoa that parasitize the intestinal epithelium of animals and humans. Calves have been identified as important reservoirs of these two pathogens, but limited data is available for these two pathogens in calves in China. In the present study, the prevalence and assemblages/genotypes of both parasites in calves of dairy and native beef (Qinchuan) cattle in Shaanxi province, northwestern China, were analyzed using multilocus genotyping (MLST). Of 371 fecal samples collected from calves (including 198 dairy calves and 173 Qinchuan calves), the respective overall prevalence of G. duodenalis and E. bieneusi was 18.87 (70 of 371) and 19.68 % (73 of 371). Both the zoonotic G. duodenalis assemblage A and animal adapted assemblage E were found in dairy and Qinchuan calves. Seventeen, eight, five, and two G. duodenalis subtypes were detected at the triose phosphate isomerase (tpi), β-giardin (bg), glutamate dehydrogenase (gdh), and small subunit ribosomal RNA (SSU-rRNA) loci, with five and two novel subtypes detected at the tpi and bg loci, forming 25 multiple genotypes (MLGs) (15 and 11 in dairy and Qinchuan calves, respectively). Of 73 samples that were positive for E. bieneusi at the ribosomal RNA internal transcribed spacer (ITS) locus, five ITS genotypes were found, including three known zoonotic genotypes (I, J, CHN1) and two novel genotypes (CSX1 and CSX2). MLST analysis of three microsatellite loci (MS1, MS3, MS7) and one minisatellite locus (MS4) detected six, two, two, and two genotypes at the MS1, MS3, MS4, and MS7 loci, respectively, forming ten MLGs (seven and four in dairy and Qinchuan calves, respectively). These results indicate complex population structures of G. duodenalis and E. bieneusi in calves in Shaanxi province and the zoonotic potential of these two pathogens in calves in this province.
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Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A
2017-04-01
Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.
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Do, Loan Phuong; Bui, Trang Minh; Hasebe, Futoshi; Morita, Kouichi; Phan, Nga Thi
2015-04-01
Japanese encephalitis virus (JEV) is an arthropod-borne virus causing serious public health issues in Asia. JEV consists of five genotypes and recent studies have shown the emergence of JEV genotype I (GI) and its replacement of genotype III (GIII). Using an archival JEV collection, we investigated the molecular evolution of JEV in Vietnam over the last 48 years (1964-2012) in humans, mosquitoes, and pigs, within the global context. The nine JEV isolates from humans, pigs, and mosquitoes sequenced in this study and 29 sequences available in GenBank were used to analyze the envelope (E) protein of the Vietnamese JEVs. A collection of 225 cerebrospinal fluid specimens from patients with suspected Japanese encephalitis (JE) was also tested and genotyped with real-time RT-PCR. The 38 E genes identified with sequencing and nine Vietnamese JEV strains genotyped with real-time RT-PCR, belonging to two lineages, evolved in accordance with those in the rest of the world. The first GIII strain was detected in humans in Vietnam in 1964, and in mosquitoes in 1979, whereas GI strains were first detected in humans and mosquitoes in 1990 and 1994, respectively. After 2004, GI was the only genotype detected in Vietnam, demonstrating that the GIIII strains had been displaced by GI strains. Five haplotypes were identified in the Vietnamese JEVs, with SKSS predominant. The S123N and S123R substitutions in the E protein were already present in the Vietnamese JEVs. This study describes the long evolutionary history of JEV in Vietnam over 34 years, which correlates well with the global evolution of JEV. The Vietnamese GIII strains have been replaced by GI strains in mosquitoes, pigs, and humans. The predominant haplotypes of the Vietnamese strains support this genotype displacement in Vietnam. Further surveillance is required to confirm the disappearance of the GIII strains in nature and the emergence of new pathogens causing encephalitis in Vietnam, after the long-term use of JEV vaccines in that country.
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Zhao, Jian-Jun; Yan, Xi-Jun; Chai, Xiu-Li; Martella, Vito; Luo, Guo-Liang; Zhang, Hai-Ling; Gao, Han; Liu, Ying-Xue; Bai, Xue; Zhang, Lei; Chen, Tao; Xu, Lei; Zhao, Chun-Fei; Wang, Feng-Xue; Shao, Xi-Qun; Wu, Wei; Cheng, Shi-Peng
2010-01-06
Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. Genetic/antigenic heterogeneity has been observed among the various CDV strains, notably in the haemagglutinin (H) gene, that appears as a good target to gather epidemiological information. Based on sequence analysis of the H gene, wild-type CDV strains cluster into distinct geographic lineages (genotypes), irrespective of the species of isolation. The sequence of the H gene of 28 CDV strains detected from both vaccinated and non-vaccinated breeding foxes, raccoon dogs and minks from different geographical areas of China during the years 2004-2008 was determined. All the CDV strains but two (strains HL and HLJ2) were characterized as Asia-1 genotype and were highly similar to each other (96.2-99.7% at the amino acid [aa] level) and to other Asia-1 strains (96.1-99.5% aa) previously detected in China. The CDV strains HL and HLJ2 were both collected from foxes in Heilongjiang province in 2005. Strain HL resembled CDVs of the Arctic genotype (GR88-like) and displayed high aa identity (98.0%) to the Chinese canine strain Liu. By converse, strain HLJ2 was barely related to CDVs of the Asia-2 genotype (88.7-90.3% aa identity), and could represent a novel CDV genotype, tentatively proposed as Asia-3. These results suggest that at least three different CDV genotypes, distantly related (81.8-91.6% aa identity) to the vaccine strains, Onderstepoort-like (America-1 genotype), are currently circulating in breeding foxes, raccoon dogs and minks in China, and that the genotype Asia-1 is predominant. Whether the diversity between wild-type CDVs and the vaccine strains may affect, to some extent, the efficacy of the vaccines deserves further investigations.
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Román-Román, Adolfo; Giono-Cerezo, Silvia; Camorlinga-Ponce, Margarita; Martínez-Carrillo, Dinorah Nashely; Loaiza-Loeza, Salome; Fernández-Tilapa, Gloria
2013-03-01
Helicobacter pylori adheres to various components of the human saliva. Therefore, the objective of this research was to simultaneously detect H. pylori in saliva and in gastric biopsy, and to determine the agreement between the vacA genotypes in both saliva and gastric biopsy. A total of 162 patients with chronic gastritis and 34 with gastric ulcer were studied, and saliva and biopsy samples were collected from each patient. H. pylori DNA was detected by conventional PCR and nested PCR was used for vacA genotyping. In 24% of the patients (47/196) H. pylori DNA was found in saliva and in biopsy; 52.5% (103/196) were saliva(negative)/biopsy(positive) and 6.6% (13/196) were saliva(positive)/biopsy(negative). In either or both H. pylori vacAs1m1 or s1m2 genotypes were detected in saliva in 41.5% of the patients with chronic gastritis. Forty-seven percent had >1 genotype, and the s1m1/s1m2 combination was found in 36% of them. H. pylori vacAs1m1 and s1m2 were also found in the saliva and biopsy of patients with gastric ulcer. The genotypes found in saliva and biopsy of the same patient had 51.1% agreement. In 27.6% of the 47 patients saliva(positive)/biopsy(positive) two genotypes were found in saliva, and one or both in the stomach. The s1m1/s1m2 genotypes, alone or together, are found simultaneously in saliva and gastric biopsy of the same patient. These results suggest that H. pylori reaches the oral cavity by various ways, and that saliva can be the transmitting and re-infecting vector. Copyright © 2012 Elsevier España, S.L. All rights reserved.
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Westbrook, Johanna I.; Li, Ling; Lehnbom, Elin C.; Baysari, Melissa T.; Braithwaite, Jeffrey; Burke, Rosemary; Conn, Chris; Day, Richard O.
2015-01-01
Objectives To (i) compare medication errors identified at audit and observation with medication incident reports; (ii) identify differences between two hospitals in incident report frequency and medication error rates; (iii) identify prescribing error detection rates by staff. Design Audit of 3291patient records at two hospitals to identify prescribing errors and evidence of their detection by staff. Medication administration errors were identified from a direct observational study of 180 nurses administering 7451 medications. Severity of errors was classified. Those likely to lead to patient harm were categorized as ‘clinically important’. Setting Two major academic teaching hospitals in Sydney, Australia. Main Outcome Measures Rates of medication errors identified from audit and from direct observation were compared with reported medication incident reports. Results A total of 12 567 prescribing errors were identified at audit. Of these 1.2/1000 errors (95% CI: 0.6–1.8) had incident reports. Clinically important prescribing errors (n = 539) were detected by staff at a rate of 218.9/1000 (95% CI: 184.0–253.8), but only 13.0/1000 (95% CI: 3.4–22.5) were reported. 78.1% (n = 421) of clinically important prescribing errors were not detected. A total of 2043 drug administrations (27.4%; 95% CI: 26.4–28.4%) contained ≥1 errors; none had an incident report. Hospital A had a higher frequency of incident reports than Hospital B, but a lower rate of errors at audit. Conclusions Prescribing errors with the potential to cause harm frequently go undetected. Reported incidents do not reflect the profile of medication errors which occur in hospitals or the underlying rates. This demonstrates the inaccuracy of using incident frequency to compare patient risk or quality performance within or across hospitals. New approaches including data mining of electronic clinical information systems are required to support more effective medication error detection and mitigation. PMID:25583702
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Polymorphism of angiotensin-converting enzyme gene in sarcoidosis.
Arbustini, E; Grasso, M; Leo, G; Tinelli, C; Fasani, R; Diegoli, M; Banchieri, N; Cipriani, A; Gorrini, M; Semenzato, G; Luisetti, M
1996-02-01
Sarcoidosis is the disease in which increased levels of serum Angiotensin-converting enzyme (sACE) are most often detected. It has recently been shown that the deletion (D) or the insertion (I) of a 250bp-DNA fragment in the ACE gene accounts for three main ACE genotypes (i.e., II, ID, and DD) and for 47% of total phenotypic variance in sACE level. The aim of our work was to investigate whether or not patients with sarcoidosis have an increased incidence of those ACE genotypes coding for highest sACE levels and to investigate whether or not sACE level in sarcoidosis is related to ACE genotypes. We studied 61 unrelated patients with sarcoidosis (test group) and 80 unrelated healthy control subjects (control group). The ACE I and D alleles were detected with polymerase chain reaction on genomic DNA. In the control group we found an ACE genotype distribution that agreed with the Hardy-Weinberg proportion. The ACE genotype distribution was not significantly different in the test group. There was no correlation between ACE genotype and roentgenologic stage of sarcoidosis. Plotting the sACE level in the control group against ACE genotype, we found a trend of increasing mean sACE value according to the order II < ID < DD. The same trend for ACE genotype was found in the test group, in which it also paralleled the trend of sACE values plotted against roentgenologic stage, according to the order Stage I < Stage II < Stage III. We conclude that in sarcoidosis the ACE genotype distribution is not altered. The trends for increasing sACE values in sarcoidosis according to both ACE genotype and roentgenologic stage would suggest that both mechanisms play a role in determining sACE level.
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Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013.
Malasao, Rungnapa; Okamoto, Michiko; Chaimongkol, Natthawan; Imamura, Tadatsugu; Tohma, Kentaro; Dapat, Isolde; Dapat, Clyde; Suzuki, Akira; Saito, Mayuko; Saito, Mariko; Tamaki, Raita; Pedrera-Rico, Gay Anne Granada; Aniceto, Rapunzel; Quicho, Reynaldo Frederick Negosa; Segubre-Mercado, Edelwisa; Lupisan, Socorro; Oshitani, Hitoshi
2015-01-01
Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.
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Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013
Malasao, Rungnapa; Okamoto, Michiko; Chaimongkol, Natthawan; Imamura, Tadatsugu; Tohma, Kentaro; Dapat, Isolde; Dapat, Clyde; Suzuki, Akira; Saito, Mayuko; Saito, Mariko; Tamaki, Raita; Pedrera-Rico, Gay Anne Granada; Aniceto, Rapunzel; Quicho, Reynaldo Frederick Negosa; Segubre-Mercado, Edelwisa; Lupisan, Socorro; Oshitani, Hitoshi
2015-01-01
Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines. PMID:26540236
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Verbeke, Joren; Van Poucke, Mario; Peelman, Luc; Piepers, Sofie; De Vliegher, Sarne
2014-12-01
The CXCR1 gene plays an important role in the innate immunity of the bovine mammary gland. Associations between single nucleotide polymorphisms (SNP) CXCR1c.735C>G and c.980A>G and udder health have been identified before in small populations. A fluorescent multiprobe PCR assay was designed specifically and validated to genotype both SNP simultaneously in a reliable and cost-effective manner. In total, 3,106 cows from 50 commercial Flemish dairy herds were genotyped using this assay. Associations between genotype and detailed phenotypic data, including pathogen-specific incidence rate of clinical mastitis (IRCM), test-day somatic cell count, and test-day milk yield (MY) were analyzed. Staphylococcus aureus IRCM tended to associate with SNP c.735C>G. Cows with genotype c.735GG had lower Staph. aureus IRCM compared with cows with genotype c.735CC (rate ratio = 0.35, 95% confidence interval = 0.14–0.90). Additionally, a parity-specific association between Staph. aureus IRCM and SNP c.980A>G was detected. Heifers with genotype c.980GG had a lower Staph. aureus IRCM compared with heifers with genotype c.980AG (rate ratio = 0.15, 95% confidence interval = 0.04–0.56). Differences were less pronounced in multiparous cows. Associations between CXCR1 genotype and somatic cell count were not detected. However, MY was associated with SNP c.735C>G. Cows with genotype c.735GG out-produced cows with genotype c.735CC by 0.8 kg of milk/d. Results provide a basis for further research on the relation between CXCR1 polymorphism and pathogen-specific mastitis resistance and MY.
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Local concurrent error detection and correction in data structures using virtual backpointers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, C.C.J.; Chen, P.P.; Fuchs, W.K.
1989-11-01
A new technique, based on virtual backpointers, is presented in this paper for local concurrent error detection and correction in linked data structures. Two new data structures utilizing virtual backpointers, the Virtual Double-Linked List and the B-Tree and Virtual Backpointers, are described. For these structures, double errors within a fixed-size checking window can be detected in constant time and single errors detected during forward moves can be corrected in constant time.
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Laqua, C; Zill, P; Koller, G; Preuss, U; Soyka, M
2015-03-01
We have analysed the MAOA-uVNTR polymorphism in the promoter region of the X-chromosomal monoamine oxidase A (MAOA) gene. The first aim was to examine the association between the MAOA genotype and the alcoholic phenotype. In the second part of the paper we have analysed the association of the MAOA genotype with impulsive and aggressive behaviour. Genotypes with 3 or 5-repeat alleles (MAOA-L-genotype) were reported to be associated with impulsive and aggressive traits. The MAOA genotype was determined in 371 male alcohol-dependent subjects and 236 male controls all of German descent. Behavioural and personality traits were evaluated using the self-report questionnaires Barratt Impulsiveness Scale (BIS), Buss Durkee Hostility Inventory (BDHI), Temperament and Character Inventory (TCI) and NEO-Five Factor Inventory (NEO-FFI). A median split in BIS, Buss Durkee Physical Assault, Buss Durkee Irritability, TCI and NEO-FFI was conducted. No association could be detected between the MAOA genotype and the alcoholic phenotype. Based on the results of the BIS questionnaire, we were able to make out an association between the MAOA-L genotype and higher levels of impulsivity (p = 0.043). Furthermore - without reaching statistical significance - we detected a very slight association between the MAOA-L genotype and higher scores in the BDHI subcategory physical aggression (p = 0.058). Taken together, these findings suggest that the MAOA-L genotype is to some extent associated with impulsive and antisocial personality traits in alcoholic men. Further studies on that question are needed. © Georg Thieme Verlag KG Stuttgart · New York.
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Tannerella forsythensis prtH genotype and association with periodontal status.
Hamlet, Stephen M; Taiyeb-Ali, Tara B; Cullinan, Mary P; Westerman, Bill; Palmer, Janet E; Seymour, Gregory J
2007-02-01
The prtH gene of Tannerella forsythensis encodes for a cysteine protease possessing virulent properties. Subgingival colonization by T. forsythensis with this genotype has been suggested to be a discriminator between periodontal health and disease. This study examined the prevalence of T. forsythensis prtH genotype in subgingival plaque and its association with periodontal disease progression and current disease status. Subjects harboring T. forsythensis in their subgingival plaque were identified using real-time polymerase chain reaction (PCR). The presence or absence of the prtH genotype was assessed by conventional PCR. Probing depths and relative attachment levels were also assessed. The prtH genotype was detected in 13 of 56 (23.2%) subjects harboring T. forsythensis in their subgingival plaque. Periodontal disease progression was defined as two or more sites with > or = 2 mm attachment loss in the previous 2-year period; current disease was defined as four or more sites with probing depths > or = 4 mm. The odds of periodontal disease (progression and/or current disease) were 1.55 times greater in subjects harboring prtH genotype T. forsythensis than in subjects in whom prtH was not detected. The prtH genotype was associated with higher numbers of T. forsythensis. In subjects with high levels of T. forsythensis, prtH genotype was associated with an increased extent of periodontal disease 2 years subsequently. These results show that T. forsythensis prtH genotype is associated with high levels of T. forsythensis. However, further work is needed to determine whether it also is a useful marker of periodontal disease progression in T. forsythensis-infected subjects.
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Kreilinger, Alex; Hiebel, Hannah; Müller-Putz, Gernot R
2016-03-01
This work aimed to find and evaluate a new method for detecting errors in continuous brain-computer interface (BCI) applications. Instead of classifying errors on a single-trial basis, the new method was based on multiple events (MEs) analysis to increase the accuracy of error detection. In a BCI-driven car game, based on motor imagery (MI), discrete events were triggered whenever subjects collided with coins and/or barriers. Coins counted as correct events, whereas barriers were errors. This new method, termed ME method, combined and averaged the classification results of single events (SEs) and determined the correctness of MI trials, which consisted of event sequences instead of SEs. The benefit of this method was evaluated in an offline simulation. In an online experiment, the new method was used to detect erroneous MI trials. Such MI trials were discarded and could be repeated by the users. We found that, even with low SE error potential (ErrP) detection rates, feasible accuracies can be achieved when combining MEs to distinguish erroneous from correct MI trials. Online, all subjects reached higher scores with error detection than without, at the cost of longer times needed for completing the game. Findings suggest that ErrP detection may become a reliable tool for monitoring continuous states in BCI applications when combining MEs. This paper demonstrates a novel technique for detecting errors in online continuous BCI applications, which yields promising results even with low single-trial detection rates.
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Lee-Montero, I; Navarro, A; Borrell, Y; García-Celdrán, M; Martín, N; Negrín-Báez, D; Blanco, G; Armero, E; Berbel, C; Zamorano, M J; Sánchez, J J; Estévez, A; Ramis, G; Manchado, M; Afonso, J M
2013-08-01
The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Passarge, M; Fix, M K; Manser, P
Purpose: To create and test an accurate EPID-frame-based VMAT QA metric to detect gross dose errors in real-time and to provide information about the source of error. Methods: A Swiss cheese model was created for an EPID-based real-time QA process. The system compares a treatmentplan- based reference set of EPID images with images acquired over each 2° gantry angle interval. The metric utilizes a sequence of independent consecutively executed error detection Methods: a masking technique that verifies infield radiation delivery and ensures no out-of-field radiation; output normalization checks at two different stages; global image alignment to quantify rotation, scaling andmore » translation; standard gamma evaluation (3%, 3 mm) and pixel intensity deviation checks including and excluding high dose gradient regions. Tolerances for each test were determined. For algorithm testing, twelve different types of errors were selected to modify the original plan. Corresponding predictions for each test case were generated, which included measurement-based noise. Each test case was run multiple times (with different noise per run) to assess the ability to detect introduced errors. Results: Averaged over five test runs, 99.1% of all plan variations that resulted in patient dose errors were detected within 2° and 100% within 4° (∼1% of patient dose delivery). Including cases that led to slightly modified but clinically equivalent plans, 91.5% were detected by the system within 2°. Based on the type of method that detected the error, determination of error sources was achieved. Conclusion: An EPID-based during-treatment error detection system for VMAT deliveries was successfully designed and tested. The system utilizes a sequence of methods to identify and prevent gross treatment delivery errors. The system was inspected for robustness with realistic noise variations, demonstrating that it has the potential to detect a large majority of errors in real-time and indicate the error source. J. V. Siebers receives funding support from Varian Medical Systems.« less
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Human papillomavirus detection in paraffin-embedded colorectal cancer tissues.
Tanzi, Elisabetta; Bianchi, Silvia; Frati, Elena R; Amicizia, Daniela; Martinelli, Marianna; Bragazzi, Nicola L; Brisigotti, Maria Pia; Colzani, Daniela; Fasoli, Ester; Zehender, Gianguglielmo; Panatto, Donatella; Gasparini, Roberto
2015-01-01
Human papillomavirus (HPV) has a well-recognized aetiological role in the development of cervical cancer and other anogenital tumours. Recently, an association between colorectal cancer and HPV infection has been suggested, although this is still controversial. This study aimed at detecting and characterizing HPV infection in 57 paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3 %. In particular, 15.8 % of tumour tissues and 8.8 % of non-cancerous tissue samples were HPV DNA-positive. Of these samples, 85.7 % were genotyped successfully, with 41.7 % of sequences identifying four genotypes of the HR (high oncogenic risk) clade Group 1; the remaining 58.3 % of HPV-genotyped specimens had an unclassified β-HPV. Examining additional cases and analysing whole genomes will help to outline the significance of these findings.
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A prevalence-based association test for case-control studies.
Ryckman, Kelli K; Jiang, Lan; Li, Chun; Bartlett, Jacquelaine; Haines, Jonathan L; Williams, Scott M
2008-11-01
Genetic association is often determined in case-control studies by the differential distribution of alleles or genotypes. Recent work has demonstrated that association can also be assessed by deviations from the expected distributions of alleles or genotypes. Specifically, multiple methods motivated by the principles of Hardy-Weinberg equilibrium (HWE) have been developed. However, these methods do not take into account many of the assumptions of HWE. Therefore, we have developed a prevalence-based association test (PRAT) as an alternative method for detecting association in case-control studies. This method, also motivated by the principles of HWE, uses an estimated population allele frequency to generate expected genotype frequencies instead of using the case and control frequencies separately. Our method often has greater power, under a wide variety of genetic models, to detect association than genotypic, allelic or Cochran-Armitage trend association tests. Therefore, we propose PRAT as a powerful alternative method of testing for association.
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Kim, Hee Sup; Jeong, Sook Hyang; Jang, Je Hyuck; Myung, Hyung Joon; Kim, Jin Wook; Bang, Soo Mee; Song, Sang Hoon; Kim, Haeryoung; Yun, Hae Sun
2011-12-01
A 37-year-old male presented with fever and jaundice was diagnosed as hepatitis A complicated with progressive cholestasis and severe autoimmune hemolytic anemia. He was treated with high-dose prednisolone (1.5 mg/kg), and eventually recovered. His initial serum contained genotype IA hepatitis A virus (HAV), which was subsequently replaced by genotype IIIA HAV. Moreover, at the time of development of hemolytic anemia, he became positive for immunoglobulin M (IgM) anti-hepatitis E virus (HEV). We detected HAV antigens in the liver biopsy specimen, while we detected neither HEV antigen in the liver nor HEV RNA in his serum. This is the first report of hepatitis A coinfected with two different genotypes manifesting with autoimmune hemolytic anemia, prolonged cholestasis, and false-positive IgM anti-HEV.
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Kim, Hee-Sup; Jang, Je-Hyuck; Myung, Hyung-Joon; Kim, Jin-Wook; Bang, Soo-Mee; Song, Sang Hoon; Kim, Haeryoung; Yun, Hae Sun
2011-01-01
A 37-year-old male presented with fever and jaundice was diagnosed as hepatitis A complicated with progressive cholestasis and severe autoimmune hemolytic anemia. He was treated with high-dose prednisolone (1.5 mg/kg), and eventually recovered. His initial serum contained genotype IA hepatitis A virus (HAV), which was subsequently replaced by genotype IIIA HAV. Moreover, at the time of development of hemolytic anemia, he became positive for immunoglobulin M (IgM) anti-hepatitis E virus (HEV). We detected HAV antigens in the liver biopsy specimen, while we detected neither HEV antigen in the liver nor HEV RNA in his serum. This is the first report of hepatitis A coinfected with two different genotypes manifesting with autoimmune hemolytic anemia, prolonged cholestasis, and false-positive IgM anti-HEV. PMID:22310798
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Damato, AL; Bhagwat, MS; Buzurovic, I
Purpose: To investigate the use of a system using EM tracking, postprocessing and error-detection algorithms for measuring brachytherapy catheter locations and for detecting errors and resolving uncertainties in treatment-planning catheter digitization. Methods: An EM tracker was used to localize 13 catheters in a clinical surface applicator (A) and 15 catheters inserted into a phantom (B). Two pairs of catheters in (B) crossed paths at a distance <2 mm, producing an undistinguishable catheter artifact in that location. EM data was post-processed for noise reduction and reformatted to provide the dwell location configuration. CT-based digitization was automatically extracted from the brachytherapy planmore » DICOM files (CT). EM dwell digitization error was characterized in terms of the average and maximum distance between corresponding EM and CT dwells per catheter. The error detection rate (detected errors / all errors) was calculated for 3 types of errors: swap of two catheter numbers; incorrect catheter number identification superior to the closest position between two catheters (mix); and catheter-tip shift. Results: The averages ± 1 standard deviation of the average and maximum registration error per catheter were 1.9±0.7 mm and 3.0±1.1 mm for (A) and 1.6±0.6 mm and 2.7±0.8 mm for (B). The error detection rate was 100% (A and B) for swap errors, mix errors, and shift >4.5 mm (A) and >5.5 mm (B); errors were detected for shifts on average >2.0 mm (A) and >2.4 mm (B). Both mix errors associated with undistinguishable catheter artifacts were detected and at least one of the involved catheters was identified. Conclusion: We demonstrated the use of an EM tracking system for localization of brachytherapy catheters, detection of digitization errors and resolution of undistinguishable catheter artifacts. Automatic digitization may be possible with a registration between the imaging and the EM frame of reference. Research funded by the Kaye Family Award 2012.« less
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Bogema, D. R.; Deutscher, A. T.; Fell, S.; Collins, D.; Eamens, G. J.
2015-01-01
Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease. PMID:25588653
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A novel dNTP-limited PCR and HRM assay to detect Williams-Beuren syndrome.
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.
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Muver, a computational framework for accurately calling accumulated mutations.
Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A; Grimm, Sara A; Kunkel, Thomas A; Fargo, David C
2018-05-09
Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.
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An improved 10-SSR pyrus fingerprinting DNA test to confirm parentage [abstract
USDA-ARS?s Scientific Manuscript database
Pedigree confirmation is a critical part of breeding, managing genetic resources, and developing genetic mapping populations for out-crossing plants, such as the genus Pyrus. Individuals which are not progeny of a biparental cross can cause mapping errors or result in the costly genotyping of indivi...
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Ayala-Valdovinos, Miguel Angel; Galindo-García, Jorge; Sánchez-Chiprés, David; Duifhuis-Rivera, Theodor
2017-04-01
Hydrocephalus in Friesian horses is an autosomal recessive hereditary disease that can result in an abortion, a stillbirth, or euthanization of a newborn foal. Here, the hydrocephalus-associated c.1423C > T mutation in B3GALNT2 gene was detected with PCR-RFLP and PCR-PIRA methods for horse genotyping. A preliminary genotyping survey was performed on 83 randomly selected Friesian stallion horses to determine the current allele frequency in Mexico. The frequency of the mutant T allele was 9.6%. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Errors, error detection, error correction and hippocampal-region damage: data and theories.
MacKay, Donald G; Johnson, Laura W
2013-11-01
This review and perspective article outlines 15 observational constraints on theories of errors, error detection, and error correction, and their relation to hippocampal-region (HR) damage. The core observations come from 10 studies with H.M., an amnesic with cerebellar and HR damage but virtually no neocortical damage. Three studies examined the detection of errors planted in visual scenes (e.g., a bird flying in a fish bowl in a school classroom) and sentences (e.g., I helped themselves to the birthday cake). In all three experiments, H.M. detected reliably fewer errors than carefully matched memory-normal controls. Other studies examined the detection and correction of self-produced errors, with controls for comprehension of the instructions, impaired visual acuity, temporal factors, motoric slowing, forgetting, excessive memory load, lack of motivation, and deficits in visual scanning or attention. In these studies, H.M. corrected reliably fewer errors than memory-normal and cerebellar controls, and his uncorrected errors in speech, object naming, and reading aloud exhibited two consistent features: omission and anomaly. For example, in sentence production tasks, H.M. omitted one or more words in uncorrected encoding errors that rendered his sentences anomalous (incoherent, incomplete, or ungrammatical) reliably more often than controls. Besides explaining these core findings, the theoretical principles discussed here explain H.M.'s retrograde amnesia for once familiar episodic and semantic information; his anterograde amnesia for novel information; his deficits in visual cognition, sentence comprehension, sentence production, sentence reading, and object naming; and effects of aging on his ability to read isolated low frequency words aloud. These theoretical principles also explain a wide range of other data on error detection and correction and generate new predictions for future test. Copyright © 2013 Elsevier Ltd. All rights reserved.
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McClintock, Brett T.; Bailey, Larissa L.; Pollock, Kenneth H.; Simons, Theodore R.
2010-01-01
The recent surge in the development and application of species occurrence models has been associated with an acknowledgment among ecologists that species are detected imperfectly due to observation error. Standard models now allow unbiased estimation of occupancy probability when false negative detections occur, but this is conditional on no false positive detections and sufficient incorporation of explanatory variables for the false negative detection process. These assumptions are likely reasonable in many circumstances, but there is mounting evidence that false positive errors and detection probability heterogeneity may be much more prevalent in studies relying on auditory cues for species detection (e.g., songbird or calling amphibian surveys). We used field survey data from a simulated calling anuran system of known occupancy state to investigate the biases induced by these errors in dynamic models of species occurrence. Despite the participation of expert observers in simplified field conditions, both false positive errors and site detection probability heterogeneity were extensive for most species in the survey. We found that even low levels of false positive errors, constituting as little as 1% of all detections, can cause severe overestimation of site occupancy, colonization, and local extinction probabilities. Further, unmodeled detection probability heterogeneity induced substantial underestimation of occupancy and overestimation of colonization and local extinction probabilities. Completely spurious relationships between species occurrence and explanatory variables were also found. Such misleading inferences would likely have deleterious implications for conservation and management programs. We contend that all forms of observation error, including false positive errors and heterogeneous detection probabilities, must be incorporated into the estimation framework to facilitate reliable inferences about occupancy and its associated vital rate parameters.
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PRESAGE: Protecting Structured Address Generation against Soft Errors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sharma, Vishal C.; Gopalakrishnan, Ganesh; Krishnamoorthy, Sriram
Modern computer scaling trends in pursuit of larger component counts and power efficiency have, unfortunately, lead to less reliable hardware and consequently soft errors escaping into application data ("silent data corruptions"). Techniques to enhance system resilience hinge on the availability of efficient error detectors that have high detection rates, low false positive rates, and lower computational overhead. Unfortunately, efficient detectors to detect faults during address generation (to index large arrays) have not been widely researched. We present a novel lightweight compiler-driven technique called PRESAGE for detecting bit-flips affecting structured address computations. A key insight underlying PRESAGE is that any addressmore » computation scheme that flows an already incurred error is better than a scheme that corrupts one particular array access but otherwise (falsely) appears to compute perfectly. Enabling the flow of errors allows one to situate detectors at loop exit points, and helps turn silent corruptions into easily detectable error situations. Our experiments using PolyBench benchmark suite indicate that PRESAGE-based error detectors have a high error-detection rate while incurring low overheads.« less
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PRESAGE: Protecting Structured Address Generation against Soft Errors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sharma, Vishal C.; Gopalakrishnan, Ganesh; Krishnamoorthy, Sriram
Modern computer scaling trends in pursuit of larger component counts and power efficiency have, unfortunately, lead to less reliable hardware and consequently soft errors escaping into application data ("silent data corruptions"). Techniques to enhance system resilience hinge on the availability of efficient error detectors that have high detection rates, low false positive rates, and lower computational overhead. Unfortunately, efficient detectors to detect faults during address generation have not been widely researched (especially in the context of indexing large arrays). We present a novel lightweight compiler-driven technique called PRESAGE for detecting bit-flips affecting structured address computations. A key insight underlying PRESAGEmore » is that any address computation scheme that propagates an already incurred error is better than a scheme that corrupts one particular array access but otherwise (falsely) appears to compute perfectly. Ensuring the propagation of errors allows one to place detectors at loop exit points and helps turn silent corruptions into easily detectable error situations. Our experiments using the PolyBench benchmark suite indicate that PRESAGE-based error detectors have a high error-detection rate while incurring low overheads.« less
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Schönherr, Sebastian; Neuner, Mathias; Forer, Lukas; Specht, Günther; Kloss-Brandstätter, Anita; Kronenberg, Florian; Coassin, Stefan
2013-01-01
Single nucleotide polymorphisms (SNPs) play a prominent role in modern genetics. Current genotyping technologies such as Sequenom iPLEX, ABI TaqMan and KBioscience KASPar made the genotyping of huge SNP sets in large populations straightforward and allow the generation of hundreds of thousands of genotypes even in medium sized labs. While data generation is straightforward, the subsequent data conversion, storage and quality control steps are time-consuming, error-prone and require extensive bioinformatic support. In order to ease this tedious process, we developed SNPflow. SNPflow is a lightweight, intuitive and easily deployable application, which processes genotype data from Sequenom MassARRAY (iPLEX) and ABI 7900HT (TaqMan, KASPar) systems and is extendible to other genotyping methods as well. SNPflow automatically converts the raw output files to ready-to-use genotype lists, calculates all standard quality control values such as call rate, expected and real amount of replicates, minor allele frequency, absolute number of discordant replicates, discordance rate and the p-value of the HWE test, checks the plausibility of the observed genotype frequencies by comparing them to HapMap/1000-Genomes, provides a module for the processing of SNPs, which allow sex determination for DNA quality control purposes and, finally, stores all data in a relational database. SNPflow runs on all common operating systems and comes as both stand-alone version and multi-user version for laboratory-wide use. The software, a user manual, screenshots and a screencast illustrating the main features are available at http://genepi-snpflow.i-med.ac.at. PMID:23527209
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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.
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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
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A Dual Frequency Carrier Phase Error Difference Checking Algorithm for the GNSS Compass.
Liu, Shuo; Zhang, Lei; Li, Jian
2016-11-24
The performance of the Global Navigation Satellite System (GNSS) compass is related to the quality of carrier phase measurement. How to process the carrier phase error properly is important to improve the GNSS compass accuracy. In this work, we propose a dual frequency carrier phase error difference checking algorithm for the GNSS compass. The algorithm aims at eliminating large carrier phase error in dual frequency double differenced carrier phase measurement according to the error difference between two frequencies. The advantage of the proposed algorithm is that it does not need additional environment information and has a good performance on multiple large errors compared with previous research. The core of the proposed algorithm is removing the geographical distance from the dual frequency carrier phase measurement, then the carrier phase error is separated and detectable. We generate the Double Differenced Geometry-Free (DDGF) measurement according to the characteristic that the different frequency carrier phase measurements contain the same geometrical distance. Then, we propose the DDGF detection to detect the large carrier phase error difference between two frequencies. The theoretical performance of the proposed DDGF detection is analyzed. An open sky test, a manmade multipath test and an urban vehicle test were carried out to evaluate the performance of the proposed algorithm. The result shows that the proposed DDGF detection is able to detect large error in dual frequency carrier phase measurement by checking the error difference between two frequencies. After the DDGF detection, the accuracy of the baseline vector is improved in the GNSS compass.
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A concatenated coding scheme for error control
NASA Technical Reports Server (NTRS)
Kasami, T.; Fujiwara, T.; Lin, S.
1986-01-01
In this paper, a concatenated coding scheme for error control in data communications is presented and analyzed. In this scheme, the inner code is used for both error correction and detection; however, the outer code is used only for error detection. A retransmission is requested if either the inner code decoder fails to make a successful decoding or the outer code decoder detects the presence of errors after the inner code decoding. Probability of undetected error (or decoding error) of the proposed scheme is derived. An efficient method for computing this probability is presented. Throughput efficiency of the proposed error control scheme incorporated with a selective-repeat ARQ retransmission strategy is also analyzed. Three specific examples are presented. One of the examples is proposed for error control in the NASA Telecommand System.
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Self-checking self-repairing computer nodes using the mirror processor
NASA Technical Reports Server (NTRS)
Tamir, Yuval
1992-01-01
Circuitry added to fault-tolerant systems for concurrent error deduction usually reduces performance. Using a technique called micro rollback, it is possible to eliminate most of the performance penalty of concurrent error detection. Error detection is performed in parallel with intermodule communication, and erroneous state changes are later undone. The author reports on the design and implementation of a VLSI RISC microprocessor, called the Mirror Processor (MP), which is capable of micro rollback. In order to achieve concurrent error detection, two MP chips operate in lockstep, comparing external signals and a signature of internal signals every clock cycle. If a mismatch is detected, both processors roll back to the beginning of the cycle when the error occurred. In some cases the erroneous state is corrected by copying a value from the fault-free processor to the faulty processor. The architecture, microarchitecture, and VLSI implementation of the MP, emphasizing its error-detection, error-recovery, and self-diagnosis capabilities, are described.
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Differential detection in quadrature-quadrature phase shift keying (Q2PSK) systems
NASA Astrophysics Data System (ADS)
El-Ghandour, Osama M.; Saha, Debabrata
1991-05-01
A generalized quadrature-quadrature phase shift keying (Q2PSK) signaling format is considered for differential encoding and differential detection. Performance in the presence of additive white Gaussian noise (AWGN) is analyzed. Symbol error rate is found to be approximately twice the symbol error rate in a quaternary DPSK system operating at the same Eb/N0. However, the bandwidth efficiency of differential Q2PSK is substantially higher than that of quaternary DPSK. When the error is due to AWGN, the ratio of double error rate to single error rate can be very high, and the ratio may approach zero at high SNR. To improve error rate, differential detection through maximum-likelihood decoding based on multiple or N symbol observations is considered. If N and SNR are large this decoding gives a 3-dB advantage in error rate over conventional N = 2 differential detection, fully recovering the energy loss (as compared to coherent detection) if the observation is extended to a large number of symbol durations.
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Kirdar, Sevİn; Sener, Asli Gamze; Cengİz, Merve; Aydin, Nerİman
2016-11-01
The prevalence of autoantibody in the patients with chronic hepatitis C infection, and the relationship between the autoantibodies and HCV genotypes were investigated in this study. One hundred and eight anti-HCV positive and 86 anti-HCV negative patients were included in the study. Anti-HCV were studied by enzyme immunassay (EIA). HCV RNA was determined by real time polymerase chain reaction (PCR) and HCV genotypes were determined by a reverse-line blot hybridization. Anti-nuclear antibodies (ANA), anti-smooth muscle antibodies (ASMA), Anti-mitochondrial antibodies (AMA), liver kidney microsomal antibodies (LKM) were detected by indirect immunofluorescence assay. Among patients, 13 (12.03%) of 108 were positive for at least one autoantibody. The positivity was not observed in control group. The most prevalent autoantibody in anti-HCV positive group was ANA. ANA was positive in six HCV patients with genotype 1. In HCV patients with genotype 1, the frequencies of ANA, ASMA, AMA and LKM1 were six, two, three and one, respectively. In HCV patients with genotype 2, ANA was positive one patient and ASMA, AMA and LKM1 were not detected in HCV patients with genotype 2. In conclusion, the autoantibodies in patients with chronic hepatitis C in the study were low as compared to those reported in previous studies. © 2016 APMIS. Published by John Wiley & Sons Ltd.
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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.
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Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman
2014-10-01
We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.
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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.
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He, Fei; Zhou, Wanjun; Cai, Ren; Yan, Tizhen; Xu, Xiangmin
2018-04-01
In this study, we aimed to assess the performance of two whole-genome amplification methods, multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycle (MALBAC), for β-thalassemia genotyping and single-nucleotide polymorphism (SNP)/copy-number variant (CNV) detection using two DNA sequencing assays. We collected peripheral blood, cell lines, and discarded embryos, and carried out MALBAC and MDA on single-cell and five-cell samples. We detected and statistically analyzed differences in the amplification efficiency, positive predictive value, sensitivity, allele dropout (ADO) rate, SNPs, and CV values between the two methods. Through Sanger sequencing at the single-cell and five-cell levels, we showed that both the amplification rate and ADO rate of MDA were better than those using MALBAC, and the sensitivity and positive predictive value obtained from MDA were higher than those from MALBAC for β-thalassemia genotyping. Using next-generation sequencing (NGS) at the single-cell level, we confirmed that MDA has better properties than MALBAC for SNP detection. However, MALBAC was more stable and homogeneous than MDA using low-depth NGS at the single-cell level for CNV detection. We conclude that MALBAC is the better option for CNV detection, while MDA is better suited for SNV detection.
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Chen, Jian; Luo, Bi; Qi, Zhu; Huo, Pei-Dan; Zhang, Quan-Sheng; Wang, Hong
2010-06-01
This study was aimed to establish a method of PCR combination with PCR-RFLP for detecting the South-East Asian (SEA) deletion type alpha-thalassemia 1 and non-deletion mutation of Hb Constant Spring (CS), and to investigate the application value of this method. For the members of the families with alpha-thalassemia, SEA deletion mutation was detected by PCR, then the HbCS point mutation was screened by PCR-RFLP. The results indicated that 15 carriers with alpha-thalassemia (--(SEA)/) were found in 19 members from 7 families, and 2 families with genotype of --(SEA)/alpha(CS)alpha were screened out successfully. It is concluded that the PCR combination with PCR-RFLP is a simple, rapid, and reliable method for screening HbH disease with genotype of --(SEA)/alpha(CS)alpha.
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Liquid biopsy genotyping in lung cancer: ready for clinical utility?
Ho, Chung-Liang; Wei, Fang; Wong, David T.; Su, Wu-Chou; Lin, Chien-Chung
2017-01-01
Liquid biopsy is a blood test that detects evidence of cancer cells or tumor DNA in the circulation. Despite complicated collection methods and the requirement for technique-dependent platforms, it has generated substantial interest due, in part, to its potential to detect driver oncogenes such as epidermal growth factor receptor (EGFR) mutants in lung cancer. This technology is advancing rapidly and is being incorporated into numerous EGFR tyrosine kinase inhibitor (EGFR-TKI) development programs. It appears ready for integration into clinical care. Recent studies have demonstrated that biological fluids such as saliva and urine can also be used for detecting EGFR mutant DNA through application other user-friendly techniques. This review focuses on the clinical application of liquid biopsies to lung cancer genotyping, including EGFR and other targets of genotype-directed therapy and compares multiple platforms used for liquid biopsy. PMID:28099915
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Mancianti, Francesca; Nardoni, Simona; Papini, Roberto; Mugnaini, Linda; Martini, Mina; Altomonte, Iolanda; Salari, Federica; D'Ascenzi, Carlo; Dubey, Jitender P
2014-04-03
Toxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys. Antibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe. The occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection.
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Local concurrent error detection and correction in data structures using virtual backpointers
NASA Technical Reports Server (NTRS)
Li, C. C.; Chen, P. P.; Fuchs, W. K.
1987-01-01
A new technique, based on virtual backpointers, for local concurrent error detection and correction in linked data structures is presented. Two new data structures, the Virtual Double Linked List, and the B-tree with Virtual Backpointers, are described. For these structures, double errors can be detected in 0(1) time and errors detected during forward moves can be corrected in 0(1) time. The application of a concurrent auditor process to data structure error detection and correction is analyzed, and an implementation is described, to determine the effect on mean time to failure of a multi-user shared database system. The implementation utilizes a Sequent shared memory multiprocessor system operating on a shared databased of Virtual Double Linked Lists.
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Local concurrent error detection and correction in data structures using virtual backpointers
NASA Technical Reports Server (NTRS)
Li, Chung-Chi Jim; Chen, Paul Peichuan; Fuchs, W. Kent
1989-01-01
A new technique, based on virtual backpointers, for local concurrent error detection and correction in linked data strutures is presented. Two new data structures, the Virtual Double Linked List, and the B-tree with Virtual Backpointers, are described. For these structures, double errors can be detected in 0(1) time and errors detected during forward moves can be corrected in 0(1) time. The application of a concurrent auditor process to data structure error detection and correction is analyzed, and an implementation is described, to determine the effect on mean time to failure of a multi-user shared database system. The implementation utilizes a Sequent shared memory multiprocessor system operating on a shared database of Virtual Double Linked Lists.
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Mans, Ben J; Pienaar, Ronel; Latif, Abdalla A; Potgieter, Fred T
2011-05-01
Sequence variation within the 18S SSU rRNA V4 hyper-variable region can affect the accuracy of real-time hybridization probe-based diagnostics for the detection of Theileria spp. infections. This is relevant for assays that use non-specific primers, such as the real-time hybridization assay for T. parva (Sibeko et al. 2008). To assess the effect of sequence variation on this test, the Theileria 18S gene from 62 buffalo and 49 cattle samples was cloned and ∼1000 clones sequenced. Twenty-six genotypes were detected which included known and novel genotypes for the T. buffeli, T. mutans, T. taurotragi and T. velifera clades. A novel genotype related to T. sp. (sable) was also detected in 1 bovine sample. Theileria genotypic diversity was higher in buffalo compared to cattle. Polymorphism within the T. parva hyper-variable region was confirmed by aberrant real-time melting peaks and supported by sequencing of the S5 ribosomal gene. Analysis of the S5 gene suggests that this gene can be a marker for species differentiation. T. parva, T. sp. (buffalo) and T. sp. (bougasvlei) remain the only genotypes amplified by the primer set of the hybridization assay. Therefore, the 18S sequence diversity observed does not seem to affect the current real-time hybridization assay for T. parva.
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Maver, Polona J; Poljak, Mario; Seme, Katja; Kocjan, Bostjan J
2010-10-01
A novel PCR-restriction fragment length polymorphism assay (PCR-RFLP) was developed for sensitive detection and reliable differentiation of five low-risk human papillomavirus (lr-HPV) genotypes: HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44, as well as differentiation of prototypic and non-prototypic HPV 6 genomic variants. The assay is based on the amplification of a 320-bp fragment of the HPV E1 gene and subsequent analysis of PCR-products with BsaJI and HinFI. Testing on plasmid standards showed that PCR-RFLP enabled simple and reliable identification and differentiation of five targeted lr-HPV genotypes and could detect reproducibly down to 10 copies of viral genome equivalents per PCR. The PCR-RFLP showed almost complete agreement with previously obtained genotyping results on 42 HPV-DNA negative samples and 223 HPV-DNA positive samples (45 HPV 6, 34 HPV 11, 35 HPV 42, 10 HPV 43, 24 HPV 44 positive samples and 75 samples containing 28 non-targeted HPV genotypes). The novel assay is simple and robust, does not require any sophisticated equipment and can be of great value for epidemiological studies, particularly in settings in which financial resources are limited. Copyright (c) 2010 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Novak, A; Nyflot, M; Sponseller, P
2014-06-01
Purpose: Radiation treatment planning involves a complex workflow that can make safety improvement efforts challenging. This study utilizes an incident reporting system to identify detection points of near-miss errors, in order to guide our departmental safety improvement efforts. Previous studies have examined where errors arise, but not where they are detected or their patterns. Methods: 1377 incidents were analyzed from a departmental nearmiss error reporting system from 3/2012–10/2013. All incidents were prospectively reviewed weekly by a multi-disciplinary team, and assigned a near-miss severity score ranging from 0–4 reflecting potential harm (no harm to critical). A 98-step consensus workflow was usedmore » to determine origination and detection points of near-miss errors, categorized into 7 major steps (patient assessment/orders, simulation, contouring/treatment planning, pre-treatment plan checks, therapist/on-treatment review, post-treatment checks, and equipment issues). Categories were compared using ANOVA. Results: In the 7-step workflow, 23% of near-miss errors were detected within the same step in the workflow, while an additional 37% were detected by the next step in the workflow, and 23% were detected two steps downstream. Errors detected further from origination were more severe (p<.001; Figure 1). The most common source of near-miss errors was treatment planning/contouring, with 476 near misses (35%). Of those 476, only 72(15%) were found before leaving treatment planning, 213(45%) were found at physics plan checks, and 191(40%) were caught at the therapist pre-treatment chart review or on portal imaging. Errors that passed through physics plan checks and were detected by therapists were more severe than other errors originating in contouring/treatment planning (1.81 vs 1.33, p<0.001). Conclusion: Errors caught by radiation treatment therapists tend to be more severe than errors caught earlier in the workflow, highlighting the importance of safety checks in dosimetry and physics. We are utilizing our findings to improve manual and automated checklists for dosimetry and physics.« less
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Error-Related Psychophysiology and Negative Affect
ERIC Educational Resources Information Center
Hajcak, G.; McDonald, N.; Simons, R.F.
2004-01-01
The error-related negativity (ERN/Ne) and error positivity (Pe) have been associated with error detection and response monitoring. More recently, heart rate (HR) and skin conductance (SC) have also been shown to be sensitive to the internal detection of errors. An enhanced ERN has consistently been observed in anxious subjects and there is some…
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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.
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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.
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Simulating and Detecting Radiation-Induced Errors for Onboard Machine Learning
NASA Technical Reports Server (NTRS)
Wagstaff, Kiri L.; Bornstein, Benjamin; Granat, Robert; Tang, Benyang; Turmon, Michael
2009-01-01
Spacecraft processors and memory are subjected to high radiation doses and therefore employ radiation-hardened components. However, these components are orders of magnitude more expensive than typical desktop components, and they lag years behind in terms of speed and size. We have integrated algorithm-based fault tolerance (ABFT) methods into onboard data analysis algorithms to detect radiation-induced errors, which ultimately may permit the use of spacecraft memory that need not be fully hardened, reducing cost and increasing capability at the same time. We have also developed a lightweight software radiation simulator, BITFLIPS, that permits evaluation of error detection strategies in a controlled fashion, including the specification of the radiation rate and selective exposure of individual data structures. Using BITFLIPS, we evaluated our error detection methods when using a support vector machine to analyze data collected by the Mars Odyssey spacecraft. We found ABFT error detection for matrix multiplication is very successful, while error detection for Gaussian kernel computation still has room for improvement.
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An advanced SEU tolerant latch based on error detection
NASA Astrophysics Data System (ADS)
Xu, Hui; Zhu, Jianwei; Lu, Xiaoping; Li, Jingzhao
2018-05-01
This paper proposes a latch that can mitigate SEUs via an error detection circuit. The error detection circuit is hardened by a C-element and a stacked PMOS. In the hold state, a particle strikes the latch or the error detection circuit may cause a fault logic state of the circuit. The error detection circuit can detect the upset node in the latch and the fault output will be corrected. The upset node in the error detection circuit can be corrected by the C-element. The power dissipation and propagation delay of the proposed latch are analyzed by HSPICE simulations. The proposed latch consumes about 77.5% less energy and 33.1% less propagation delay than the triple modular redundancy (TMR) latch. Simulation results demonstrate that the proposed latch can mitigate SEU effectively. Project supported by the National Natural Science Foundation of China (Nos. 61404001, 61306046), the Anhui Province University Natural Science Research Major Project (No. KJ2014ZD12), the Huainan Science and Technology Program (No. 2013A4011), and the National Natural Science Foundation of China (No. 61371025).
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[Detection and classification of medication errors at Joan XXIII University Hospital].
Jornet Montaña, S; Canadell Vilarrasa, L; Calabuig Mũoz, M; Riera Sendra, G; Vuelta Arce, M; Bardají Ruiz, A; Gallart Mora, M J
2004-01-01
Medication errors are multifactorial and multidisciplinary, and may originate in processes such as drug prescription, transcription, dispensation, preparation and administration. The goal of this work was to measure the incidence of detectable medication errors that arise within a unit dose drug distribution and control system, from drug prescription to drug administration, by means of an observational method confined to the Pharmacy Department, as well as a voluntary, anonymous report system. The acceptance of this voluntary report system's implementation was also assessed. A prospective descriptive study was conducted. Data collection was performed at the Pharmacy Department from a review of prescribed medical orders, a review of pharmaceutical transcriptions, a review of dispensed medication and a review of medication returned in unit dose medication carts. A voluntary, anonymous report system centralized in the Pharmacy Department was also set up to detect medication errors. Prescription errors were the most frequent (1.12%), closely followed by dispensation errors (1.04%). Transcription errors (0.42%) and administration errors (0.69%) had the lowest overall incidence. Voluntary report involved only 4.25% of all detected errors, whereas unit dose medication cart review contributed the most to error detection. Recognizing the incidence and types of medication errors that occur in a health-care setting allows us to analyze their causes and effect changes in different stages of the process in order to ensure maximal patient safety.
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Bianchi, Silvia; Frati, Elena Rosanna; Panatto, Donatella; Martinelli, Marianna; Amicizia, Daniela; Zotti, Carla Maria; Martinese, Morena; Bonanni, Paolo; Boccalini, Sara; Coppola, Rosa Cristina; Masia, Giuseppina; Meloni, Angelo; Castiglia, Paolo; Piana, Andrea; Gasparini, Roberto; Tanzi, Elisabetta
2013-01-01
The introduction of vaccination against Human Papillomavirus (HPV) in adolescent girls in 2006 has focused virological surveillance on this age group. As few studies have evaluated HPV infections in young populations, further data are needed in order to improve and extend prophylactic policy and to monitor epidemiological changes. The present study aimed at evaluating overall and type-specific HPV prevalence in both female and male adolescents in Italy. HPV DNA detection and genotyping was performed on urine samples collected from 870 unvaccinated adolescents (369 females, 501 males, 11-18 years of age) in five cities in Italy. Following DNA extraction by means of a commercial kit (NucliSENS®-miniMAG®, bioMérieux), the L1 gene fragment was PCR amplified and genotyped by restriction fragment length polymorphism analysis. HPV DNA was detected in 1.5% of all samples, and in 3% and 0.4% of samples from females and males, respectively. In approximately 70% of HPV DNA positive adolescents, the infection was due to a single genotype, with 88.9% of genotypes belonging to the HR-clade. The only two HPV-positive boys (14 and 18 years old) had HPV-70 genotype. Only one of the 11 HPV-infected girls was in the 11-14 age-group. HPV prevalence was 4.2% in girls aged 15-18 years and 60% of infections were due to vaccine types HPV-16 or HPV-6/-11. This is one of the few studies, the first conducted in Italy, on HPV infection in adolescents. Urine testing is the easier way of detecting HPV infection in younger populations. Our data revealed a very low HPV prevalence, and no infections were observed in the 12-year-old vaccine target population. The majority of infections were seen in females aged 15-18 years. Overall, more than 50% and 30% of the potentially persistent HPV infections detected in this group could have been prevented by the quadrivalent and the bivalent vaccines, respectively. PMID:24255711
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Bianchi, Silvia; Frati, Elena Rosanna; Panatto, Donatella; Martinelli, Marianna; Amicizia, Daniela; Zotti, Carla Maria; Martinese, Morena; Bonanni, Paolo; Boccalini, Sara; Coppola, Rosa Cristina; Masia, Giuseppina; Meloni, Angelo; Castiglia, Paolo; Piana, Andrea; Gasparini, Roberto; Tanzi, Elisabetta
2013-01-01
The introduction of vaccination against Human Papillomavirus (HPV) in adolescent girls in 2006 has focused virological surveillance on this age group. As few studies have evaluated HPV infections in young populations, further data are needed in order to improve and extend prophylactic policy and to monitor epidemiological changes. The present study aimed at evaluating overall and type-specific HPV prevalence in both female and male adolescents in Italy. HPV DNA detection and genotyping was performed on urine samples collected from 870 unvaccinated adolescents (369 females, 501 males, 11-18 years of age) in five cities in Italy. Following DNA extraction by means of a commercial kit (NucliSENS(®)-miniMAG(®), bioMérieux), the L1 gene fragment was PCR amplified and genotyped by restriction fragment length polymorphism analysis. HPV DNA was detected in 1.5% of all samples, and in 3% and 0.4% of samples from females and males, respectively. In approximately 70% of HPV DNA positive adolescents, the infection was due to a single genotype, with 88.9% of genotypes belonging to the HR-clade. The only two HPV-positive boys (14 and 18 years old) had HPV-70 genotype. Only one of the 11 HPV-infected girls was in the 11-14 age-group. HPV prevalence was 4.2% in girls aged 15-18 years and 60% of infections were due to vaccine types HPV-16 or HPV-6/-11. This is one of the few studies, the first conducted in Italy, on HPV infection in adolescents. Urine testing is the easier way of detecting HPV infection in younger populations. Our data revealed a very low HPV prevalence, and no infections were observed in the 12-year-old vaccine target population. The majority of infections were seen in females aged 15-18 years. Overall, more than 50% and 30% of the potentially persistent HPV infections detected in this group could have been prevented by the quadrivalent and the bivalent vaccines, respectively.
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[RHD variant in RhD/-/ mother with anti-D makes noninvasive fetal RHD genotyping impossible].
Orzińska, Agnieszka; Engel, Karina; Łakomy, Magdalena; Smolarczyk-Wodzyńska, Justyna; Lipińska, Anna; Pelc-Kłopotowska, Monika; Brojer, Ewa
2009-10-01
Noninvasive fetal RHD genotyping from maternal plasma of RhD(/-) pregnant women of Caucasian race may be used for predicting the risk of hemolytic disease because the RHD gene is usually absent in such populations. If detected in plasma of such women, the RHD gene originates from the RhD(+) fetus. The number of fetal copies of the gene in maternal plasma is extremely small. In the presented case of the RhD(/-) pregnant woman with anti-D it was impossible to give a fetal RHD result due to mother's RHD(+) genotype. The fetal RHD was determined from amniocytes. to present the difficulties related to the interpretation of results of invasive and noninvasive procedures. whole blood, plasma and amniotic fluid of the RhD(-) woman with anti-D (14 week of pregnancy) as well as whole blood of the newborn. RHD and RHCE*c were genotyped by real-time PCR in DNA isolated from maternal plasma and amniocytes and the RHD and d-genotypes were tested by SSP methods in DNA isolated from whole blood and amniocytes. RHD and RHCE*c were detected in DNA isolated from plasma. The high level of RHD suggested its origin from the mother's DNA therefore it was impossible to determine the fetal RHD. The d-little test identified a RHD(IVS3+ 1G>A) variant in the mother's genome. A weak signal of real-time PCR for the RHD was obtained in amniocytes but the RHD was not detected by SSP. The RHCE*c was detected by both methods. Results were inconclusive; the fetal RHD status remained unknown. The child was RhD(-) with RHD in its DNA undetected by either method. 1/The RHD(IVS3+ 1G>A) variant in the RhD(-) mother precluded formal noninvasive fetal RHD genotyping. 2/Real-time PCR is too sensitive for amniocyte testing and may lead to false results as it detects trace maternal DNA in amniotic fluid. 3/The frequency of RHD(IVS3+1G>A) occurrence in Poland requires further studies.
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Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J
2009-06-29
It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available.
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Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B.; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L.; Dieckhaus, Kevin; Rosen, Marc I.; Kozal, Michael J.
2009-01-01
Background It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Methodology/Principal Findings Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004–2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85–5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5–74.3, p = 0.0016). Conclusions/Significance Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available. PMID:19562031
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Fault-tolerant quantum error detection.
Linke, Norbert M; Gutierrez, Mauricio; Landsman, Kevin A; Figgatt, Caroline; Debnath, Shantanu; Brown, Kenneth R; Monroe, Christopher
2017-10-01
Quantum computers will eventually reach a size at which quantum error correction becomes imperative. Quantum information can be protected from qubit imperfections and flawed control operations by encoding a single logical qubit in multiple physical qubits. This redundancy allows the extraction of error syndromes and the subsequent detection or correction of errors without destroying the logical state itself through direct measurement. We show the encoding and syndrome measurement of a fault-tolerantly prepared logical qubit via an error detection protocol on four physical qubits, represented by trapped atomic ions. This demonstrates the robustness of a logical qubit to imperfections in the very operations used to encode it. The advantage persists in the face of large added error rates and experimental calibration errors.
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Human papillomavirus detection in head and neck squamous cell carcinoma
Vietía, Dayahindara; Liuzzi, Juan; Ávila, Maira; De Guglielmo, Zoraya; Prado, Yrneh; Correnti, María
2014-01-01
Introduction Human Papillomavirus (HPV) has been associated with benign and malignant lesions in different epitheliums. The relationship between specific genotypes of high-risk HPV and some human cancers is well established. The aim of this work was to detect the HPV genotypes present in head and neck squamous cell carcinoma (HNSCC). Methods We evaluated 71 samples of patients with histopathological diagnosis of HNSCC. The DNA extraction was conducted with the QIAGEN commercial kit. HPV detection and genotyping were performed by reverse hybridisation (INNO-LiPA) following the commercial specifications. Results The mean age of the patients evaluated was 60.7 ± 13.11 years. The distribution of the lesions included 25 (35.20%) cases of squamous cell carcinoma (SCC) of the oral cavity, 23 (32.39%) of larynx, 16 (22.50%) of the oropharynx, 4 (5.63%) of paranasal sinus, and 2 (2. 80%) cases of SCC of the nostril. Of the patients, 78.9% were males, and of these 76% were tobacco users and 67.6% were alcohol consumers. The viral DNA was detected in 67.6% of the samples. The oral cavity and the larynx were the highest HPV-positivity sites with 35.40% and 29.10% respectively. The most frequent genotype was 16 as single infection (18.70%), or in combination with another HPV types. In the oral cavity and larynx the genotypes 16 or the combination 6 and 51 were present in 11.76% and 14.28%, respectively; and in the oropharynx the most frequent genotype was 16 in 22.50% of the cases, and in the paranasal sinus 50% presented infection with HPV-6. We observed that tumours with most advanced size and stage presented greater HPV positivity. Conclusions This study shows a high percentage of HPV positivity in SCC is mainly associated with high-risk HPV. It is important to highlight that viral infection, especially HPV-16, could be a risk factor in HNSCC progression. PMID:25374623
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Fedaoui, Nadia; Ben Ayed, Narjess; Ben Yahia, Ahlem; Hammami, Walid; Matri, Leila; Nacef, Leila; Triki, Henda
2017-02-01
Human adenoviruses (HAdVs) are common causes of conjunctivitis. This study describes the epidemiological features and characterizes by phylogenetic analysis HAdVs isolated from patients with conjunctivitis in Tunisia, North Africa. Data on out-patients presenting with conjunctivitis during 2 years (2012-2013) were analyzed. Conjunctival swabs obtained from 240 patients were assessed for the presence of HAdVs by PCR amplification on the fiber and hexon genes. Positive PCR products, together with those of nine viral isolates from previous years, were sequenced and analyzed phylogenetically. Conjunctivitis represented 11.5% of all reasons of consultations with a slight increase between mid-March and mid-June. Sixty-five percent of samples (n = 156) revealed positive by at least one PCR test. PCR amplification in the hexon gene was slightly more sensitive as compared to the fiber gene. Genotyping in the two genomic regions gave concordant results for almost all isolates. HAdV-D8 was the most predominant genotype (87.6%) and was detected continuously from 2000 to 2013. Minor co-circulating genotypes including HAdV-E4, HAdV-B3, HAdV-B55, and HAdV-D37 were identified; most of them were detected by amplification in the hexon gene. In conclusion, this work reports molecular data on adenoviral conjunctivitis from a region where such information is scarce and contributes to a better knowledge of the worldwide distribution of causative genotypes. It revealed a predominance and endemic circulation of HAdV-D8, a genotype that was mainly reported from epidemic keratoconjunctivitis. It shows that PCR amplification in two different genomic regions enhances the sensitivity of HAdV detection in clinical samples and the identification of minor genotypes. J. Med. Virol. 89:304-312, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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Design of the Detector II: A CMOS Gate Array for the Study of Concurrent Error Detection Techniques.
1987-07-01
detection schemes and temporary failures. The circuit consists- or of six different adders with concurrent error detection schemes . The error detection... schemes are - simple duplication, duplication with functional dual implementation, duplication with different &I [] .6implementations, two-rail encoding...THE SYSTEM. .. .... ...... ...... ...... 5 7. DESIGN OF CED SCHEMES .. ... ...... ...... ........ 7 7.1 Simple Duplication
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Horowitz-Kraus, Tzipi
2016-05-01
The error-detection mechanism aids in preventing error repetition during a given task. Electroencephalography demonstrates that error detection involves two event-related potential components: error-related and correct-response negativities (ERN and CRN, respectively). Dyslexia is characterized by slow, inaccurate reading. In particular, individuals with dyslexia have a less active error-detection mechanism during reading than typical readers. In the current study, we examined whether a reading training programme could improve the ability to recognize words automatically (lexical representations) in adults with dyslexia, thereby resulting in more efficient error detection during reading. Behavioural and electrophysiological measures were obtained using a lexical decision task before and after participants trained with the reading acceleration programme. ERN amplitudes were smaller in individuals with dyslexia than in typical readers before training but increased following training, as did behavioural reading scores. Differences between the pre-training and post-training ERN and CRN components were larger in individuals with dyslexia than in typical readers. Also, the error-detection mechanism as represented by the ERN/CRN complex might serve as a biomarker for dyslexia and be used to evaluate the effectiveness of reading intervention programmes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
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2011-01-01
Background Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. Results We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. Conclusions Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers. PMID:21767361
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Hwang, Kyu-Baek; Lee, In-Hee; Park, Jin-Ho; Hambuch, Tina; Choe, Yongjoon; Kim, MinHyeok; Lee, Kyungjoon; Song, Taemin; Neu, Matthew B; Gupta, Neha; Kohane, Isaac S; Green, Robert C; Kong, Sek Won
2014-08-01
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 single nucleotide variants (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 in NA12878, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and an ensemble genotyping would be essential to minimize false-positive DNM candidates. © 2014 WILEY PERIODICALS, INC.
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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
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Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.
Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing
2015-11-01
Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia.
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Olson, Eric J.
2013-06-11
An apparatus, program product, and method that run an algorithm on a hardware based processor, generate a hardware error as a result of running the algorithm, generate an algorithm output for the algorithm, compare the algorithm output to another output for the algorithm, and detect the hardware error from the comparison. The algorithm is designed to cause the hardware based processor to heat to a degree that increases the likelihood of hardware errors to manifest, and the hardware error is observable in the algorithm output. As such, electronic components may be sufficiently heated and/or sufficiently stressed to create better conditions for generating hardware errors, and the output of the algorithm may be compared at the end of the run to detect a hardware error that occurred anywhere during the run that may otherwise not be detected by traditional methodologies (e.g., due to cooling, insufficient heat and/or stress, etc.).
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Distribution and Biocontrol Potential of phlD(+) Pseudomonads in Corn and Soybean Fields.
McSpadden Gardener, Brian B; Gutierrez, Laura J; Joshi, Raghavendra; Edema, Richard; Lutton, Elizabeth
2005-06-01
ABSTRACT The abundance and diversity of phlD(+) Pseudomonas spp. colonizing the rhizospheres of young, field-grown corn and soybean plants were assayed over a 3-year period. Populations of these bacteria were detected on the large majority of plants sampled in the state of Ohio, but colonization was greater on corn. Although significant variation in the incidence of rhizosphere colonization was observed from site to site and year to year on both crops, the magnitude of the variation was greatest for soybean. The D genotype was detected on plants collected from all 15 counties examined, and it represented the most abundant subpopulation on both crops. Additionally, six other genotypes (A, C, F, I, R, and S) were found to predominate in the rhizosphere of some plants. The most frequently observed of these were the A genotype and a newly discovered S genotype, both of which were found on corn and soybean roots obtained from multiple locations. Multiple isolates of the most abundant genotypes were recovered and characterized. The S genotype was found to be phylogenetically and phenotypically similar to the D genotype. In addition, the novel R genotype was found to be most similar to the A genotype. All of the isolates displayed significant capacities to inhibit the growth of an oomycete pathogen in vitro, but such phenotypes were highly dependent on media used. When tested against multiple oomycete pathogens isolated from soybean, the A genotype was significantly more inhibitory than the D genotype when incubated on 1/10x tryptic soy agar and 1/5x corn meal agar. Seed inoculation with different isolates of the A, D, and S genotypes indicated that significant root colonization, generally in excess of log 5 cells per gram of root, could be attained on both crops. Field trials of the A genotype isolate Wayne1R indicated the capacity of inoculant populations to supplement the activities of native populations so as to increase soybean stands and yields. The relevance of these findings to natural and augmentative biocontrol of root pathogens by these bacteria is discussed.
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Chang, Wen-Shin; Tsai, Chia-Wen; Wang, Ju-Yu; Ying, Tsung-Ho; Hsiao, Tsan-Seng; Chuang, Chin-Liang; Yueh, Te-Cheng; Liao, Cheng-Hsi; Hsu, Chin-Mu; Liu, Shih-Ping; Gong, Chi-Li; Tsai, Chang-Hai; Bau, Da-Tian
2015-09-01
The present study aimed at investigating whether X-ray repair cross complementing protein 3 (XRCC3) genotype may serve as a useful marker for detecting leiomyoma and predicting risk. A total of 640 women (166 patients with leiomyoma and 474 healthy controls) were examined for their XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotype. The distributions of genotypic and allelic frequencies between the two groups were compared. The results showed that the CT and TT genotypes of XRCC3 rs861539 were associated with increased leiomyoma risk (odds ratio=2.19, 95% confidence interval=1.23-3.90; odds ratio=3.72, 95% confidence interval=1.23-11.26, respectively). On allelic frequency analysis, we found a significant difference in the distribution of the T allelic frequency of the XRCC3 rs861539 (p=5.88 × 10(-5)). None of the other six single nucleotide polymorphisms were associated with altered leiomyoma susceptibility. The T allele (CT and TT genotypes) of XRCC3 rs861539 contributes to increased risk of leiomyoma among Taiwanese women and may serve as a early detection and predictive marker. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Quambusch, Mona; Pirttilä, Anna Maria; Tejesvi, Mysore V; Winkelmann, Traud; Bartsch, Melanie
2014-05-01
The endophytic bacterial communities of six Prunus avium L. genotypes differing in their growth patterns during in vitro propagation were identified by culture-dependent and culture-independent methods. Five morphologically distinct isolates from tissue culture material were identified by 16S rDNA sequence analysis. To detect and analyze the uncultivable fraction of endophytic bacteria, a clone library was established from the amplified 16S rDNA of total plant extract. Bacterial diversity within the clone libraries was analyzed by amplified ribosomal rDNA restriction analysis and by sequencing a clone for each identified operational taxonomic unit. The most abundant bacterial group was Mycobacterium sp., which was identified in the clone libraries of all analyzed Prunus genotypes. Other dominant bacterial genera identified in the easy-to-propagate genotypes were Rhodopseudomonas sp. and Microbacterium sp. Thus, the community structures in the easy- and difficult-to-propagate cherry genotypes differed significantly. The bacterial genera, which were previously reported to have plant growth-promoting effects, were detected only in genotypes with high propagation success, indicating a possible positive impact of these bacteria on in vitro propagation of P. avium, which was proven in an inoculation experiment. © The Author 2014. Published by Oxford University Press. All rights reserved.
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VacA and cagA genotypes of Helicobacter pylori isolated from raw meat in Isfahan province, Iran
Gilani, Ali; Razavilar, Vadood; Rokni, Nordahr; Rahimi, Ebrahim
2017-01-01
Foods with animal origins play a substantial role in the transmission of Helicobacter pylori. The present investigation was carried out to study the vacA and cagA genotypes status of H. pylori isolated from various types of meat samples. Two hundred and twenty meat samples were collected and cultured. H. pylori-positive strains were analyzed for the presence of vacA and cagA genotypes. Eleven out of 220 (5.00%) samples were positive for H. pylori. Findings were confirmed by nested PCR. Prevalence of H. pylori in the meat samples of slaughterhouses and butcheries were 72.20% and 27.70%, respectively. The most commonly detected genotypes in the meat samples of slaughterhouses and butcheries were vacA m1a (66.66%) and vacA s1a (37.50%), respectively. The S1am1a was the most commonly detected genotype. Meat sampled from butcheries had the higher prevalence of H. pylori and its genotypes than those of slaughterhouses (p < 0.05). Results showed that meat samples could be the potential sources of virulent strains of H. pylori. Application of sanitary measures in the storage, transportation and sale of meat is essential for reducing the levels of H. pylori cross contamination. PMID:28473901
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VacA and cagA genotypes of Helicobacter pylori isolated from raw meat in Isfahan province, Iran.
Gilani, Ali; Razavilar, Vadood; Rokni, Nordahr; Rahimi, Ebrahim
2017-01-01
Foods with animal origins play a substantial role in the transmission of Helicobacter pylori . The present investigation was carried out to study the vacA and cagA genotypes status of H. pylori isolated from various types of meat samples. Two hundred and twenty meat samples were collected and cultured. H. pylori -positive strains were analyzed for the presence of vacA and cagA genotypes. Eleven out of 220 (5.00%) samples were positive for H. pylori . Findings were confirmed by nested PCR. Prevalence of H. pylori in the meat samples of slaughterhouses and butcheries were 72.20% and 27.70%, respectively. The most commonly detected genotypes in the meat samples of slaughterhouses and butcheries were vacA m1a (66.66%) and vacA s1a (37.50%), respectively. The S1am1a was the most commonly detected genotype. Meat sampled from butcheries had the higher prevalence of H. pylori and its genotypes than those of slaughterhouses ( p < 0.05). Results showed that meat samples could be the potential sources of virulent strains of H. pylori . Application of sanitary measures in the storage, transportation and sale of meat is essential for reducing the levels of H. pylori cross contamination.
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Meise, Philipp; Jozefowicz, Anna Maria; Uptmoor, Ralf; Mock, Hans-Peter; Ordon, Frank; Schum, Annegret
2017-08-23
Aiming at a better understanding of the physiological and biochemical background of nitrogen use efficiency, alterations in the shoot proteome under N-deficiency were investigated in two contrasting potato genotypes grown in vitro with 60 and 7.5mM N, respectively. A gel based proteomic approach was applied to identify candidate proteins associated with genotype specific responses to N-deficiency. 21% of the detected proteins differed in abundance between the two genotypes. Between control and N-deficiency conditions 19.5% were differentially accumulated in the sensitive and 15% in the tolerant genotype. 93% of the highly N-deficiency responsive proteins were identified by MALDI TOF/TOF mass spectrometry. The major part was associated with photosynthesis, carbohydrate metabolism, stress response and regulation. Differential accumulation of enzymes involved in the Calvin cycle and glycolysis suggest activation of alternative carbohydrate pathways. In the tolerant genotype, increased abundance under N-deficiency was also found for enzymes involved in chlorophyll synthesis and stability of enzymes, which increase photosynthetic carbon fixation efficiency. Out of a total of 106 differentially abundant proteins, only eight were detected in both genotypes. Our findings suggest that mutually responsive proteins reflect universal stress responses while adaptation to N-deficiency in metabolic pathways is more genotype specific. Nitrogen losses from arable farm land considerably contribute to environmental pollution. In potato, this is a special problem due cultivation on light soils, irrigation and the shallow root system. Therefore, breeding of cultivars with improved nitrogen use efficiency and stable yields under reduced N fertilization is an important issue. Knowledge of genotype dependent adaptation to N-deficiency at the proteome level can help to understand regulation of N efficiency and development of N-efficient cultivars. Copyright © 2017 Elsevier B.V. All rights reserved.
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Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants.
Paradowska, Edyta; Jabłońska, Agnieszka; Studzińska, Mirosława; Suski, Patrycja; Kasztelewicz, Beata; Zawilińska, Barbara; Wiśniewska-Ligier, Małgorzata; Dzierżanowska-Fangrat, Katarzyna; Woźniakowska-Gęsicka, Teresa; Czech-Kowalska, Justyna; Lipka, Bożena; Kornacka, Maria; Pawlik, Dorota; Tomasik, Tomasz; Kosz-Vnenchak, Magdalena; Leśnikowski, Zbigniew J
2013-09-01
Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns. Copyright © 2013 Elsevier B.V. All rights reserved.
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Walker, Andreas; Siemann, Holger; Groten, Svenja; Ross, R Stefan; Scherbaum, Norbert; Timm, Jörg
2015-09-01
People who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking. The aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naïve PWID living in Germany. The full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol. With the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%). A protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID. Copyright © 2015 Elsevier B.V. All rights reserved.
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González-Hernández, Luz A; Flores-Miramontes, María G; Aguilar-Lemarroy, Adriana; Quintanilla-Peña, Krissya S; Martin-Amaya-Barajas, Fabiola L; Ramos-Solano, Moisés; Enciso Gómez, Luis F; Andrade-Villanueva, Jaime F; Jave-Suárez, Luis F
2018-04-01
The incidence of anal cancer has been rising, especially in HIV+ patients and has been associated with HPV infection. HIV+ patients are more at risk of HPV coinfection and are seven times more likely to have persistent HPV infection; moreover, HIV+ men have an increased risk of developing anal cancer compared to HIV+ women. The development of screening strategies for the detection of HPV in HIV+ men is of major importance; however, there is not enough information about the HPV genotypes and variants that are colonizing the anal epithelia of HIV+ men in diverse geographical regions. Therefore, this work was aimed at identifying HPV genotypes present in the anal epithelium of HIV+ men who have sex with men (MSM), with or without anal lesions (n = 75). For HPV genotyping, two approaches were performed: Linear Array HPV Genotyping Test and next-generation sequencing (NGS). In general, the six most frequent HPV genotypes found by Linear Array were HPV6, 62, 61, 81, 16 and 51. On the other hand, employing NGS, a total of 36 HPV genotypes belonging to both alpha and beta genera were found. The genotypes with the greatest number of reads, according to the diagnostic group, were: HPV81, 45, 6, 51 and 61 in MSM without anal lesions (WAIN); HPV6, 61, 70, 62 and 66 in MSM with atypical lesions (AAL); HPV6, 11, 66, 81 and 61 in MSM with anal intraepithelial neoplasia grade I (AIN I); and HPV16, 81, 58, 61 and 52 with AIN III. Additionally, a great diversity of L1 variants was observed, especially in genotypes HPV16, 58, 61, 52, 45 and 59.
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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
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Evidence for two koi herpesvirus (KHV) genotypes in South Korea.
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.
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Westbrook, Johanna I; Li, Ling; Lehnbom, Elin C; Baysari, Melissa T; Braithwaite, Jeffrey; Burke, Rosemary; Conn, Chris; Day, Richard O
2015-02-01
To (i) compare medication errors identified at audit and observation with medication incident reports; (ii) identify differences between two hospitals in incident report frequency and medication error rates; (iii) identify prescribing error detection rates by staff. Audit of 3291 patient records at two hospitals to identify prescribing errors and evidence of their detection by staff. Medication administration errors were identified from a direct observational study of 180 nurses administering 7451 medications. Severity of errors was classified. Those likely to lead to patient harm were categorized as 'clinically important'. Two major academic teaching hospitals in Sydney, Australia. Rates of medication errors identified from audit and from direct observation were compared with reported medication incident reports. A total of 12 567 prescribing errors were identified at audit. Of these 1.2/1000 errors (95% CI: 0.6-1.8) had incident reports. Clinically important prescribing errors (n = 539) were detected by staff at a rate of 218.9/1000 (95% CI: 184.0-253.8), but only 13.0/1000 (95% CI: 3.4-22.5) were reported. 78.1% (n = 421) of clinically important prescribing errors were not detected. A total of 2043 drug administrations (27.4%; 95% CI: 26.4-28.4%) contained ≥ 1 errors; none had an incident report. Hospital A had a higher frequency of incident reports than Hospital B, but a lower rate of errors at audit. Prescribing errors with the potential to cause harm frequently go undetected. Reported incidents do not reflect the profile of medication errors which occur in hospitals or the underlying rates. This demonstrates the inaccuracy of using incident frequency to compare patient risk or quality performance within or across hospitals. New approaches including data mining of electronic clinical information systems are required to support more effective medication error detection and mitigation. © The Author 2015. Published by Oxford University Press in association with the International Society for Quality in Health Care.
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Double ErrP Detection for Automatic Error Correction in an ERP-Based BCI Speller.
Cruz, Aniana; Pires, Gabriel; Nunes, Urbano J
2018-01-01
Brain-computer interface (BCI) is a useful device for people with severe motor disabilities. However, due to its low speed and low reliability, BCI still has a very limited application in daily real-world tasks. This paper proposes a P300-based BCI speller combined with a double error-related potential (ErrP) detection to automatically correct erroneous decisions. This novel approach introduces a second error detection to infer whether wrong automatic correction also elicits a second ErrP. Thus, two single-trial responses, instead of one, contribute to the final selection, improving the reliability of error detection. Moreover, to increase error detection, the evoked potential detected as target by the P300 classifier is combined with the evoked error potential at a feature-level. Discriminable error and positive potentials (response to correct feedback) were clearly identified. The proposed approach was tested on nine healthy participants and one tetraplegic participant. The online average accuracy for the first and second ErrPs were 88.4% and 84.8%, respectively. With automatic correction, we achieved an improvement around 5% achieving 89.9% in spelling accuracy for an effective 2.92 symbols/min. The proposed approach revealed that double ErrP detection can improve the reliability and speed of BCI systems.
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Bellenguez, Céline; Strange, Amy; Freeman, Colin; Donnelly, Peter; Spencer, Chris C A
2012-01-01
High-throughput genotyping arrays provide an efficient way to survey single nucleotide polymorphisms (SNPs) across the genome in large numbers of individuals. Downstream analysis of the data, for example in genome-wide association studies (GWAS), often involves statistical models of genotype frequencies across individuals. The complexities of the sample collection process and the potential for errors in the experimental assay can lead to biases and artefacts in an individual's inferred genotypes. Rather than attempting to model these complications, it has become a standard practice to remove individuals whose genome-wide data differ from the sample at large. Here we describe a simple, but robust, statistical algorithm to identify samples with atypical summaries of genome-wide variation. Its use as a semi-automated quality control tool is demonstrated using several summary statistics, selected to identify different potential problems, and it is applied to two different genotyping platforms and sample collections. The algorithm is written in R and is freely available at www.well.ox.ac.uk/chris-spencer chris.spencer@well.ox.ac.uk Supplementary data are available at Bioinformatics online.
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Prevalence and genotype identification of Toxoplasma gondii in wild animals from southwestern Spain.
Calero-Bernal, Rafael; Saugar, José M; Frontera, Eva; Pérez-Martín, Juan E; Habela, Miguel A; Serrano, Francisco J; Reina, David; Fuentes, Isabel
2015-01-01
We used PCR to detect Toxoplasma gondii in the principal game species in southwestern Spain. We detected T. gondii in 32.2% of animals tested. Prevalences varied from 14.7% in wild boar (Sus scrofa) to 51.2% in red fox (Vulpes vulpes). The most prevalent genotype was type II (50.0%), followed by type III (20.6%) and type I (5.9%). Mixed infections (11.8%) were detected in wild boar (types I+III) and red fox (types II+III). Polymorphic strains (11.8%) were detected in several species. The high prevalence and the genetic variability shown could have implications for infection of farm animals and humans.
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Human papilloma virus prevalence in laryngeal squamous cell carcinoma.
Gungor, A; Cincik, H; Baloglu, H; Cekin, E; Dogru, S; Dursun, E
2007-08-01
To determine the prevalence and type of human papilloma virus deoxyribonucleic acid (DNA) in cases of laryngeal squamous cell carcinoma. We analysed the prevalence of human papilloma virus infection in archived paraffin block specimens taken from 99 cases of laryngeal squamous cell carcinoma between 1990 and 2005, using polymerase chain reaction techniques. Biopsy specimens from five proven verrucous skin lesions were used as positive controls, and peripheral blood samples from five healthy volunteers were used as negative controls. Four test samples were found to have inadequate deoxyribonucleic acid purity and were therefore excluded from the study. Human papilloma virus deoxyribonucleic acid was detected in seven of 95 cases of laryngeal squamous cell carcinoma (7.36 per cent). Human papilloma virus genotyping revealed double human papilloma virus infection in three cases and single human papilloma virus infection in the remaining four cases. The human papilloma virus genotypes detected were 6, 11 and 16 (the latter detected in only one case). In our series, a very low human papilloma virus prevalence was found among laryngeal squamous cell carcinoma cases. The human papilloma virus genotypes detected were mostly 6 and/or 11, and 16 in only one case. To the best of our knowledge, this is the first report of human papilloma virus prevalence in laryngeal squamous cell carcinoma, based on polymerase chain reaction genotyping in a Turkish population.
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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.
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Experimental investigation of observation error in anuran call surveys
McClintock, B.T.; Bailey, L.L.; Pollock, K.H.; Simons, T.R.
2010-01-01
Occupancy models that account for imperfect detection are often used to monitor anuran and songbird species occurrence. However, presenceabsence data arising from auditory detections may be more prone to observation error (e.g., false-positive detections) than are sampling approaches utilizing physical captures or sightings of individuals. We conducted realistic, replicated field experiments using a remote broadcasting system to simulate simple anuran call surveys and to investigate potential factors affecting observation error in these studies. Distance, time, ambient noise, and observer abilities were the most important factors explaining false-negative detections. Distance and observer ability were the best overall predictors of false-positive errors, but ambient noise and competing species also affected error rates for some species. False-positive errors made up 5 of all positive detections, with individual observers exhibiting false-positive rates between 0.5 and 14. Previous research suggests false-positive errors of these magnitudes would induce substantial positive biases in standard estimators of species occurrence, and we recommend practices to mitigate for false positives when developing occupancy monitoring protocols that rely on auditory detections. These recommendations include additional observer training, limiting the number of target species, and establishing distance and ambient noise thresholds during surveys. ?? 2010 The Wildlife Society.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Damato, Antonio L., E-mail: adamato@lroc.harvard.edu; Viswanathan, Akila N.; Don, Sarah M.
2014-10-15
Purpose: To investigate the use of a system using electromagnetic tracking (EMT), post-processing and an error-detection algorithm for detecting errors and resolving uncertainties in high-dose-rate brachytherapy catheter digitization for treatment planning. Methods: EMT was used to localize 15 catheters inserted into a phantom using a stepwise acquisition technique. Five distinct acquisition experiments were performed. Noise associated with the acquisition was calculated. The dwell location configuration was extracted from the EMT data. A CT scan of the phantom was performed, and five distinct catheter digitization sessions were performed. No a priori registration of the CT scan coordinate system with the EMTmore » coordinate system was performed. CT-based digitization was automatically extracted from the brachytherapy plan DICOM files (CT), and rigid registration was performed between EMT and CT dwell positions. EMT registration error was characterized in terms of the mean and maximum distance between corresponding EMT and CT dwell positions per catheter. An algorithm for error detection and identification was presented. Three types of errors were systematically simulated: swap of two catheter numbers, partial swap of catheter number identification for parts of the catheters (mix), and catheter-tip shift. Error-detection sensitivity (number of simulated scenarios correctly identified as containing an error/number of simulated scenarios containing an error) and specificity (number of scenarios correctly identified as not containing errors/number of correct scenarios) were calculated. Catheter identification sensitivity (number of catheters correctly identified as erroneous across all scenarios/number of erroneous catheters across all scenarios) and specificity (number of catheters correctly identified as correct across all scenarios/number of correct catheters across all scenarios) were calculated. The mean detected and identified shift was calculated. Results: The maximum noise ±1 standard deviation associated with the EMT acquisitions was 1.0 ± 0.1 mm, and the mean noise was 0.6 ± 0.1 mm. Registration of all the EMT and CT dwell positions was associated with a mean catheter error of 0.6 ± 0.2 mm, a maximum catheter error of 0.9 ± 0.4 mm, a mean dwell error of 1.0 ± 0.3 mm, and a maximum dwell error of 1.3 ± 0.7 mm. Error detection and catheter identification sensitivity and specificity of 100% were observed for swap, mix and shift (≥2.6 mm for error detection; ≥2.7 mm for catheter identification) errors. A mean detected shift of 1.8 ± 0.4 mm and a mean identified shift of 1.9 ± 0.4 mm were observed. Conclusions: Registration of the EMT dwell positions to the CT dwell positions was possible with a residual mean error per catheter of 0.6 ± 0.2 mm and a maximum error for any dwell of 1.3 ± 0.7 mm. These low residual registration errors show that quality assurance of the general characteristics of the catheters and of possible errors affecting one specific dwell position is possible. The sensitivity and specificity of the catheter digitization verification algorithm was 100% for swap and mix errors and for shifts ≥2.6 mm. On average, shifts ≥1.8 mm were detected, and shifts ≥1.9 mm were detected and identified.« less
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Zhu, Yumin; Yu, Xiaoming; Huang, Fenfen; Yu, Ruisong; Dong, Shijuan; Si, Fusheng; Zhang, Yuanshu; Li, Zhen
2012-11-08
Four major genotypes of hepatitis E virus (HEV), the causative agent of hepatitis E, have so far been recognized. While genotypes 3 and 4 are both zoonotic, the disease symptoms caused by the latter tend to be more severe. To examine if specific nucleotide/amino acid variations between genotypes 3 and 4 play a role in determining the severity of hepatitis E disease, the complete genome of one swine HEV genotype 4 isolate, SAAS-FX17, was determined and compared with other genotype 4 and genotype 3 genomes to identify putative HEV genotype 4 virulence determinants. A total of 42 conformable nt/aa variations between genotype 3 and 4 HEVs were detected, of which 19 were proposed to be potential disease severity determinants for genotype 4 strains. One potential determinant was located in each of the 5'-UTR and 3'-UTR, 3 and 12 within ORF1 and ORF2 respectively, and 2 in the junction region.
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Register file soft error recovery
Fleischer, Bruce M.; Fox, Thomas W.; Wait, Charles D.; Muff, Adam J.; Watson, III, Alfred T.
2013-10-15
Register file soft error recovery including a system that includes a first register file and a second register file that mirrors the first register file. The system also includes an arithmetic pipeline for receiving data read from the first register file, and error detection circuitry to detect whether the data read from the first register file includes corrupted data. The system further includes error recovery circuitry to insert an error recovery instruction into the arithmetic pipeline in response to detecting the corrupted data. The inserted error recovery instruction replaces the corrupted data in the first register file with a copy of the data from the second register file.
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NASA Technical Reports Server (NTRS)
Bernacki, Bruce E.; Mansuripur, M.
1992-01-01
A commonly used tracking method on pre-grooved magneto-optical (MO) media is the push-pull technique, and the astigmatic method is a popular focus-error detection approach. These two methods are analyzed using DIFFRACT, a general-purpose scalar diffraction modeling program, to observe the effects on the error signals due to focusing lens misalignment, Seidel aberrations, and optical crosstalk (feedthrough) between the focusing and tracking servos. Using the results of the astigmatic/push-pull system as a basis for comparison, a novel focus/track-error detection technique that utilizes a ring toric lens is evaluated as well as the obscuration method (focus error detection only).
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Error detection and correction unit with built-in self-test capability for spacecraft applications
NASA Technical Reports Server (NTRS)
Timoc, Constantin
1990-01-01
The objective of this project was to research and develop a 32-bit single chip Error Detection and Correction unit capable of correcting all single bit errors and detecting all double bit errors in the memory systems of a spacecraft. We designed the 32-bit EDAC (Error Detection and Correction unit) based on a modified Hamming code and according to the design specifications and performance requirements. We constructed a laboratory prototype (breadboard) which was converted into a fault simulator. The correctness of the design was verified on the breadboard using an exhaustive set of test cases. A logic diagram of the EDAC was delivered to JPL Section 514 on 4 Oct. 1988.
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Legione, Alistair R; Patterson, Jade L S; Whiteley, Pam L; Amery-Gale, Jemima; Lynch, Michael; Haynes, Leesa; Gilkerson, James R; Polkinghorne, Adam; Devlin, Joanne M; Sansom, Fiona M
2016-05-01
Chlamydia pecorum infection is a threat to the health of free-ranging koalas (Phascolarctos cinereus) in Australia. Utilizing an extensive sample archive we determined the prevalence of C. pecorum in koalas within six regions of Victoria, Australia. The ompA genotypes of the detected C. pecorum were characterized to better understand the epidemiology of this pathogen in Victorian koalas. Despite many studies in northern Australia (i.e. Queensland and New South Wales), prior Chlamydia studies in Victorian koalas are limited. We detected C. pecorum in 125/820 (15 %) urogenital swabs, but in only one ocular swab. Nucleotide sequencing of the molecular marker C. pecorum ompA revealed that the majority (90/114) of C. pecorum samples typed were genotype B. This genotype has not been reported in northern koalas. In general, Chlamydia infection in Victorian koalas is associated with milder clinical signs compared with infection in koalas in northern populations. Although disease pathogenesis is likely to be multifactorial, the high prevalence of genotype B in Victoria may suggest it is less pathogenic. All but three koalas had C. pecorum genotypes unique to southern koala populations (i.e. Victoria and South Australia). These included a novel C. pecorum ompA genotype and two genotypes associated with livestock. Regression analysis determined that significant factors for the presence of C. pecorum infection were sex and geographical location. The presence of 'wet bottom' in males and the presence of reproductive tract pathology in females were significantly associated with C. pecorum infection, suggesting variation in clinical disease manifestations between sexes.
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Abdel Rahman, Mohamed F.; Hashad, Ingy M.; Abdel-Maksoud, Sahar M.; Farag, Nabil M.; Abou-Aisha, Khaled
2012-01-01
Aim: The aim of this study was to detect endothelial nitric oxide synthase (eNOS) Glu298Asp gene variants in a random sample of the Egyptian population, compare it with those from other populations, and attempt to correlate these variants with serum levels of nitric oxide (NO). The association of eNOS genotypes or serum NO levels with the incidence of acute myocardial infarction (AMI) was also examined. Methods: One hundred one unrelated healthy subjects and 104 unrelated AMI patients were recruited randomly from the 57357 Hospital and intensive care units of El Demerdash Hospital and National Heart Institute, Cairo, Egypt. eNOS genotypes were determined by polymerase chain reaction–restriction fragment length polymorphism. Serum NO was determined spectrophotometrically. Results: The genotype distribution of eNOS Glu298Asp polymorphism determined for our sample was 58.42% GG (wild type), 33.66% GT, and 7.92% TT genotypes while allele frequencies were 75.25% and 24.75% for G and T alleles, respectively. No significant association between serum NO and specific eNOS genotype could be detected. No significant correlation between eNOS genotype distribution or allele frequencies and the incidence of AMI was observed. Conclusion: The present study demonstrated the predominance of the homozygous genotype GG over the heterozygous GT and homozygous TT in random samples of Egyptian population. It also showed the lack of association between eNOS genotypes and mean serum levels of NO, as well as the incidence of AMI. PMID:22731641
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Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin
2014-10-01
Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.
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Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan
2013-01-01
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula. PMID:23825624
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Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan
2013-01-01
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
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Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S
2016-05-01
Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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Legione, Alistair R; Patterson, Jade L S; Whiteley, Pam; Firestone, Simon M; Curnick, Megan; Bodley, Kate; Lynch, Michael; Gilkerson, James R; Sansom, Fiona M; Devlin, Joanne M
2017-02-01
Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas. Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status. The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and 'wet bottom' (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98). Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.
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Marked Genomic Diversity of Norovirus Genogroup I Strains in a Waterborne Outbreak
Hannoun, Charles; Larsson, Charlotte U.; Bergström, Tomas
2012-01-01
Marked norovirus (NoV) diversity was detected in patient samples from a large community outbreak of gastroenteritis with waterborne epidemiology affecting approximately 2,400 people. NoV was detected in 33 of 50 patient samples examined by group-specific real-time reverse transcription-PCR. NoV genotype I (GI) strains predominated in 31 patients, with mixed GI infections occurring in 5 of these patients. Sequence analysis of RNA-dependent polymerase-N/S capsid-coding regions (∼900 nucleotides in length) confirmed the dominance of the GI strains (n = 36). Strains of NoV GI.4 (n = 21) and GI.7 (n = 9) were identified, but six strains required full capsid amino acid analyses (530 to 550 amino acids) based on control sequencing of cloned amplicons before the virus genotype could be determined. Three strains were assigned to a new NoV GI genotype, proposed as GI.9, based on capsid amino acid analyses showing 26% dissimilarity from the established genotypes GI.1 to GI.8. Three other strains grouped in a sub-branch of GI.3 with 13 to 15% amino acid dissimilarity to GI.3 GenBank reference strains. Phylogenetic analysis (2.1 kb) of 10 representative strains confirmed these genotype clusters. Strains of NoV GII.4 (n = 1), NoV GII.6 (n = 2), sapovirus GII.2 (n = 1), rotavirus (n = 3), adenovirus (n = 1), and Campylobacter spp. (n = 2) were detected as single infections or as mixtures with NoV GI. Marked NoV GI diversity detected in patients was consistent with epidemiologic evidence of waterborne NoV infections, suggesting human fecal contamination of the water supply. Recognition of NoV diversity in a cluster of patients provided a useful warning marker of waterborne contamination in the Lilla Edet outbreak. PMID:22247153
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The 2010 Global Proficiency Study of Human Papillomavirus Genotyping in Vaccinology
Eklund, Carina; Forslund, Ola; Wallin, Keng-Ling; Zhou, Tiequn
2012-01-01
Accurate and internationally comparable human papillomavirus (HPV) DNA genotyping is essential both for evaluation of HPV vaccines and for effective monitoring and implementation of vaccination programs. The World Health Organization (WHO) HPV Laboratory Network (LabNet) regularly issues international proficiency studies. The 2010 HPV genotyping proficiency panel for HPV vaccinology contained 43 coded samples composed of purified plasmids of 16 HPV types (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a and 68b) and 3 coded extraction controls. Proficient typing was defined as detection in both single and multiple infections of 50 international units (IU) of HPV type 16 (HPV-16) and HPV-18 DNA and 500 genome equivalents (GE) for the other 14 HPV types. Ninety-eight laboratories worldwide submitted a total of 132 data sets. Twenty-four different HPV genotyping assay methods were used, with Linear Array being the most commonly used. Other major assays used were a line blot assay (Inno-LiPa), CLART, type-specific real-time PCR, PCR Luminex, and different microarray assays. Altogether, 72 data sets were proficient for detection of more than 1 type, and only 26 data sets proficiently detected all 16 HPV types. The major oncogenic HPV types, 16 and 18, were proficiently detected in 95.0% (114/120) and 87.0% (94/108) of data sets, respectively. Forty-six data sets reported multiple false-positive results and were considered nonproficient. A trend toward increased sensitivity of assays was seen for the 41 laboratories that participated in both 2008 and 2010. In conclusion, continued global proficiency studies will be required for establishing comparable and reliable HPV genotyping services for vaccinology worldwide. PMID:22535980
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Fellahi, Siham; Ducatez, Mariette; El Harrak, Mehdi; Guérin, Jean-Luc; Touil, Nadia; Sebbar, Ghizlane; Bouaiti, El Arbi; Khataby, Khadija; Ennaji, My Mustapha; El-Houadfi, Mohammed
2015-01-01
The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705-1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.
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Cosgun, Yasemin; Guldemir, Dilek; Coskun, Aslihan; Yolbakan, Sultan; Kalaycioglu, Atila Taner; Korukluoglu, Gulay; Durmaz, Riza
2018-04-01
Measles is an important childhood infection targeted to be eliminated by the World Health Organization (WHO). Virus circulation has not been interrupted in the European Region because high vaccination rates could not be achieved among some countries of the WHO European Region including Turkey. The purpose of this study was to evaluate the laboratory findings of measles cases confirmed in the last nine years, to assess the epidemiological data of the cases, to determine the molecular genotyping studies and to emphasise the importance of laboratory-based surveillance in measles. From 2007 to 2010, only 18 imported cases were detected in Turkey. However, this number increased with a local outbreak of 111 cases in 2011, followed by another outbreak in 2012 in Istanbul that spread countrywide in the following two years; a total of 8661 laboratory-confirmed measles cases were reported from 2012 to 2015. After ELISA detection of a measles IgM-positive result in serum samples of potential measles cases, RT-PCR was performed with urine or nasopharyngeal swab samples of patients, and amplicons were subjected to sequencing. In the samples of 2010 and 2011, D4 and D9 genotypes were mainly detected; as of 2012, the D8 genotype has gained importance. Although D8 was also identified in 2014, in the same year genotype H1 viruses were detected in Turkey for the first time. Therefore, it is important to perform a genotypic analysis of the virus causing the outbreak, analyse epidemiological connections of the contact, determine the source of the outbreak and plan measures based on this information.
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Circulating Cell-free DNA for Metastatic Cervical Cancer Detection, Genotyping, and Monitoring.
Kang, Zhigang; Stevanović, Sanja; Hinrichs, Christian S; Cao, Liang
2017-11-15
Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making. Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome. Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy. Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856-62. ©2017 AACR . ©2017 American Association for Cancer Research.
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HCV avidity as a tool for detection of recent HCV infection: Sensitivity depends on HCV genotype.
Shepherd, Samantha J; McDonald, Scott A; Palmateer, Norah E; Gunson, Rory N; Aitken, Celia; Dore, Gregory J; Goldberg, David J; Applegate, Tanya L; Lloyd, Andrew R; Hajarizadeh, Behzad; Grebely, Jason; Hutchinson, Sharon J
2018-01-01
Accurate detection of incident hepatitis C virus (HCV) infection is required to target and evaluate public health interventions, but acute infection is largely asymptomatic and difficult to detect using traditional methods. Our aim was to evaluate a previously developed HCV avidity assay to distinguish acute from chronic HCV infection. Plasma samples collected from recent seroconversion subjects in two large Australian cohorts were tested using the avidity assay, and the avidity index (AI) was calculated. Demographic and clinical characteristics of patients with low/high AI were compared via logistic regression. Sensitivity and specificity of the assay for recent infection and the mean duration of recent infection (MDRI) were estimated stratified by HCV genotype. Avidity was assessed in 567 samples (from 215 participants), including 304 with viraemia (defined as ≥250 IU/mL). An inverse relationship between AI and infection duration was found in viraemic samples only. The adjusted odds of a low AI (<30%) decreased with infection duration (odds ratio [OR] per week of 0.93; 95% CI:0.89-0.97), and were lower for G1 compared with G3 samples (OR = 0.14; 95% CI:0.05-0.39). Defining recent infection as <26 weeks, sensitivity (at AI cut-off of 20%) was estimated at 48% (95% CI:39-56%), 36% (95% CI:20-52%), and 65% (95% CI:54-75%) and MDRI was 116, 83, and 152 days for all genotypes, G1, and G3, respectively. Specificity (≥52 weeks infection duration, all genotypes) was 96% (95% CI:90-98%). HCV avidity testing has utility for detecting recent HCV infection in patients, and for assessing progress in reaching incidence targets for eliminating transmission, but variation in assay performance across genotype should be recognized. © 2017 Wiley Periodicals, Inc.
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A single-sampling hair trap for mesocarnivores
Jonathan N. Pauli; Matthew B. Hamilton; Edward B. Crain; Steven W. Buskirk
2007-01-01
Although techniques to analyze and quantifY DNA-based data have progressed, methods to noninvasively collect samples lag behind. Samples are generally collected from devices that permit coincident sampling of multiple individuals. Because of cross-contamination, substantive genotyping errors can arise. We developed a cost-effective (US$4.60/trap) single-capture hair...
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Cost-efficient selection of a marker panel in genetic studies
Jamie S. Sanderlin; Nicole Lazar; Michael J. Conroy; Jaxk Reeves
2012-01-01
Genetic techniques are frequently used to sample and monitor wildlife populations. The goal of these studies is to maximize the ability to distinguish individuals for various genetic inference applications, a process which is often complicated by genotyping error. However, wildlife studies usually have fixed budgets, which limit the number of geneticmarkers available...
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USDA-ARS?s Scientific Manuscript database
Microsatellite markers (MS) have traditionally been used for parental verification and are still the international standard in spite of their higher cost, error rate, and turnaround time compared with Single Nucleotide Polymorphisms (SNP) -based assays. Despite domestic and international demands fr...
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USDA-ARS?s Scientific Manuscript database
We proposed a method to estimate the error variance among non-replicated genotypes, thus to estimate the genetic parameters by using replicated controls. We derived formulas to estimate sampling variances of the genetic parameters. Computer simulation indicated that the proposed methods of estimatin...
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ERIC Educational Resources Information Center
Abedi, Razie; Latifi, Mehdi; Moinzadeh, Ahmad
2010-01-01
This study tries to answer some ever-existent questions in writing fields regarding approaching the most effective ways to give feedback to students' errors in writing by comparing the effect of error correction and error detection on the improvement of students' writing ability. In order to achieve this goal, 60 pre-intermediate English learners…
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Fault-tolerant quantum error detection
Linke, Norbert M.; Gutierrez, Mauricio; Landsman, Kevin A.; Figgatt, Caroline; Debnath, Shantanu; Brown, Kenneth R.; Monroe, Christopher
2017-01-01
Quantum computers will eventually reach a size at which quantum error correction becomes imperative. Quantum information can be protected from qubit imperfections and flawed control operations by encoding a single logical qubit in multiple physical qubits. This redundancy allows the extraction of error syndromes and the subsequent detection or correction of errors without destroying the logical state itself through direct measurement. We show the encoding and syndrome measurement of a fault-tolerantly prepared logical qubit via an error detection protocol on four physical qubits, represented by trapped atomic ions. This demonstrates the robustness of a logical qubit to imperfections in the very operations used to encode it. The advantage persists in the face of large added error rates and experimental calibration errors. PMID:29062889
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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
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Carrier screening in the era of expanding genetic technology.
Arjunan, Aishwarya; Litwack, Karen; Collins, Nick; Charrow, Joel
2016-12-01
The Center for Jewish Genetics provides genetic education and carrier screening to individuals of Jewish descent. Carrier screening has traditionally been performed by targeted mutation analysis for founder mutations with an enzyme assay for Tay-Sachs carrier detection. The development of next-generation sequencing (NGS) allows for higher detection rates regardless of ethnicity. Here, we explore differences in carrier detection rates between genotyping and NGS in a primarily Jewish population. Peripheral blood samples or saliva samples were obtained from 506 individuals. All samples were analyzed by sequencing, targeted genotyping, triplet-repeat detection, and copy-number analysis; the analyses were carried out at Counsyl. Of 506 individuals screened, 288 were identified as carriers of at least 1 condition and 8 couples were carriers for the same disorder. A total of 434 pathogenic variants were identified. Three hundred twelve variants would have been detected via genotyping alone. Although no additional mutations were detected by NGS in diseases routinely screened for in the Ashkenazi Jewish population, 26.5% of carrier results and 2 carrier couples would have been missed without NGS in the larger panel. In a primarily Jewish population, NGS reveals a larger number of pathogenic variants and provides individuals with valuable information for family planning.Genet Med 18 12, 1214-1217.
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Rabodoarivelo, Marie Sylvianne; Imperiale, Bélen; andrianiavomikotroka, Rina; Brandao, Angela; Kumar, Parveen; Singh, Sarman; Ferrazoli, Lucilaine; Morcillo, Nora; Rasolofo, Voahangy; Palomino, Juan Carlos; Vandamme, Peter; Martin, Anandi
2015-01-01
Background Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. Methods Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. Results For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. Conclusion The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings. PMID:26431352
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del Puerto, Florencia; Sánchez, Zunilda; Nara, Eva; Meza, Graciela; Paredes, Berta; Ferreira, Elizabeth; Russomando, Graciela
2010-01-01
Trypanosoma cruzi II is associated with Chagas disease in the southern part of South America. We analyzed T. cruzi variants in field-collected triatomines and congenitally infected infants living in the same disease-endemic region in Paraguay. Results of polymerase chain reactions for T. cruzi kinetoplast DNA and satellite DNA were positive in 83 triatomine feces samples and 58 infant blood samples. However, lineages were detected in 33 and 38 samples, respectively. Trypanosoma cruzi genotypes were determined in 56 (97%) blood samples after hybridization by using specific probes. The Tc I genotype was not detected. The prevalent sublineage was Tc IId in triatomines (27 of 33) and infant blood (36 of 58) as assessed by amplification of the 24Sα ribosomal RNA and the mini-exon region genes. The Tc IIc genotype was detected in 20 infant blood samples and in 1 triatomine. This study shows T. cruzi II is the predominant lineage circulating in triatomines and humans in endemic areas of eastern region of Paraguay. PMID:20207861
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Development of an improved RT-LAMP assay for detection of currently circulating rubella viruses.
Abo, H; Okamoto, K; Anraku, M; Otsuki, N; Sakata, M; Icenogle, J; Zheng, Q; Kurata, T; Kase, T; Komase, K; Takeda, M; Mori, Y
2014-10-01
Rubella virus is the causative agent of rubella. The symptoms are usually mild, and characterized by a maculopapular rash and fever. However, rubella infection in pregnant women sometimes can result in the birth of infants with congenital rubella syndrome (CRS). Global efforts have been made to reduce and eliminate CRS. Although a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of rubella virus has been reported, the primers contained several mismatched nucleotides with the genomes of currently circulating rubella virus strains. In the present study, a new RT-LAMP assay was established. The detection limit of this assay was 100-1000PFU/reaction of viruses for all rubella genotypes, except for genotype 2C, which is not commonly found in the current era. Therefore, the new RT-LAMP assay can successfully detect all current rubella virus genotypes, and does not require sophisticated devices like TaqMan real-time PCR systems. This assay should be a useful assay for laboratory diagnosis of rubella and CRS. Copyright © 2014 Elsevier B.V. All rights reserved.
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del Puerto, Florencia; Sánchez, Zunilda; Nara, Eva; Meza, Graciela; Paredes, Berta; Ferreira, Elizabeth; Russomando, Graciela
2010-03-01
Trypanosoma cruzi II is associated with Chagas disease in the southern part of South America. We analyzed T. cruzi variants in field-collected triatomines and congenitally infected infants living in the same disease-endemic region in Paraguay. Results of polymerase chain reactions for T. cruzi kinetoplast DNA and satellite DNA were positive in 83 triatomine feces samples and 58 infant blood samples. However, lineages were detected in 33 and 38 samples, respectively. Trypanosoma cruzi genotypes were determined in 56 (97%) blood samples after hybridization by using specific probes. The Tc I genotype was not detected. The prevalent sublineage was Tc IId in triatomines (27 of 33) and infant blood (36 of 58) as assessed by amplification of the 24Salpha ribosomal RNA and the mini-exon region genes. The Tc IIc genotype was detected in 20 infant blood samples and in 1 triatomine. This study shows T. cruzi II is the predominant lineage circulating in triatomines and humans in endemic areas of eastern region of Paraguay.
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Peters, M; Kilwinski, J; Beringhoff, A; Reckling, D; Genersch, E
2006-03-01
Between March 2003 and October 2004, Paenibacillus larvae, the aetiological agent of American foulbrood disease of the honey bee, was isolated from broodcombs and honey samples of 54 apiaries in the administrative district of Arnsberg (North Rhine-Westphalia, Germany). Genotyping of 176 P. larvae isolates with repetitive element polymerase chain reaction fingerprinting (rep-PCR) using BOX A1R and MBO REP1 primers revealed five different genotypes (AB, Ab, ab, ass, Acapital BE, Cyrillic). In samples of three apiaries, more than one genotype was detected. A combination of two genotypes was isolated from honey samples of the same hive two times (ab/ass and Ab/ab). The five genotypes were not randomly distributed in the district, but revealed a certain geographical clustering. Possible factors with impact on the genotype diversity and the distribution pattern are discussed.
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Bocanegra-Ibarias, Paola; Flores-Treviño, Samantha; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Villarreal-Treviño, Licet; Llaca-Díaz, Jorge; Martínez-Landeros, Erik Alan; Rodríguez-Noriega, Eduardo; Calzada-Güereca, Andrés; Maldonado-Garza, Héctor Jesús; Garza-González, Elvira
2016-01-01
Enterococcus faecium has emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. Our aim was to determine the antimicrobial susceptibility, biofilm production, and clonal relatedness of vancomycin-resistant E. faecium (VREF) clinical isolates from two hospitals in Mexico. Consecutive clinical isolates (n=56) were collected in two tertiary care hospitals in Mexico from 2011 to 2014. VREF isolates were characterized by phenotypic and molecular methods including pulsed-field gel electrophoresis (PFGE). VREF isolates were highly resistant to vancomycin, erythromycin, norfloxacin, high-level streptomycin, and teicoplanin, and showed lower resistance to tetracycline, nitrofurantoin and quinupristin-dalfopristin. None of the isolates were resistant to linezolid. The vanA gene was detected in all isolates. Two VanB phenotype-vanA genotype isolates, highly resistant to vancomycin and susceptible to teicoplanin, were detected. Furthermore, 17.9% of the isolates were classified as biofilm producers, and the espfm gene was found in 98.2% of the isolates. A total of 37 distinct PFGE patterns and 6 clones (25% of the isolates as clone A, 5.4% as clone B, and 3.6% each as clone C, D, E, and F) were detected. Clone A was detected in 5 different wards of the same hospital during 14 months of surveillance. The high resistance to most antimicrobial agents and the moderate cross-transmission of VREF detected accentuates the need for continuous surveillance of E. faecium in the hospital setting. This is also the first reported incidence of the E. faecium VanB phenotype-vanA genotype in the Americas. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.
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Thibault, Vincent; Gaudy-Graffin, Catherine; Colson, Philippe; Gozlan, Joël; Schnepf, Nathalie; Trimoulet, Pascale; Pallier, Coralie; Saune, Karine; Branger, Michel; Coste, Marianne; Thoraval, Francoise Roudot
2013-03-15
Chronic hepatitis B (CHB) is a clinical concern in human immunodeficiency virus (HIV)-infected individuals due to substantial prevalence, difficulties to treat, and severe liver disease outcome. A large nationwide cross-sectional multicentre analysis of HIV-HBV co-infected patients was designed to describe and identify parameters associated with virological and clinical outcome of CHB in HIV-infected individuals with detectable HBV viremia. A multicenter collaborative cross-sectional study was launched in 19 French University hospitals distributed through the country. From January to December 2007, HBV load, genotype, clinical and epidemiological characteristics of 223 HBV-HIV co-infected patients with an HBV replication over 1000 IU/mL were investigated. Patients were mostly male (82%, mean age 42 years). Genotype distribution (A 52%; E 23.3%; D 16.1%) was linked to risk factors, geographic origin, and co-infection with other hepatitis viruses. This genotypic pattern highlights divergent contamination event timelines by HIV and HBV viruses. Most patients (74.7%) under antiretroviral treatment were receiving a drug with anti-HBV activity, including 47% receiving TDF. Genotypic lamivudine-resistance detected in 26% of the patients was linked to duration of lamivudine exposure, age, CD4 count and HIV load. Resistance to adefovir (rtA181T/V) was detected in 2.7% of patients. Advanced liver lesions were observed in 54% of cases and were associated with an older age and lower CD4 counts but not with viral load or genotype. Immune escape HBsAg variants were seldom detected. Despite the detection of advanced liver lesions in most patients, few were not receiving anti-HBV drugs and for those treated with the most potent anti-HBV drugs, persistent replication suggested non-optimal adherence. Heterogeneity in HBV strains reflects epidemiological differences that may impact liver disease progression. These findings are strong arguments to further optimize clinical management and to promote vaccination in HIV-infected patients.
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Minagawa, Hiroko; Yasui, Yoshihiro; Adachi, Hirokazu; Ito, Miyabi; Hirose, Emi; Nakamura, Noriko; Hata, Mami; Kobayashi, Shinichi; Yamashita, Teruo
2015-11-09
Japan was verified as having achieved measles elimination by the Measles Regional Verification Commission in the Western Pacific Region in March 2015. Verification of measles elimination implies the absence of continuous endemic transmission. After the last epidemic in 2007 with an estimated 18,000 cases, Japan introduced nationwide case-based measles surveillance in January 2008. Laboratory diagnosis for all suspected measles cases is essentially required by law, and virus detection tests are mostly performed by municipal public health institutes. Despite relatively high vaccination coverage and vigorous response to every case by the local health center staff, outbreak of measles is repeatedly observed in Aichi Prefecture, Japan. Measles virus N and H gene detection by nested double RT-PCR was performed with all specimens collected from suspected cases and transferred to our institute. Genotyping and further molecular epidemiological analyses were performed with the direct nucleotide sequence data of appropriate PCR products. Between 2010 and 2014, specimens from 389 patients suspected for measles were tested in our institute. Genotypes D9, D8, H1 and B3 were detected. Further molecular epidemiological analyses were helpful to establish links between patients, and sometimes useful to discriminate one outbreak from another. All virus-positive cases, including 49 cases involved in three outbreaks without any obvious epidemiological link with importation, were considered as import-related based on the nucleotide sequence information. Chain of transmission in the latest outbreak in 2014 terminated after the third generations, much earlier than the 2010-11 outbreak (6th generations). Since 2010, almost all measles cases reported in Aichi Prefecture are either import or import-related, based primarily on genotypes and nucleotide sequences of measles virus detected. In addition, genotyping and molecular epidemiological analyses are indispensable to prove the interruption of endemic transmission when the importations of measles are repeatedly observed. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Hassine-Zaafrane, M; Kaplon, J; Ben Salem, I; Sdiri-Loulizi, K; Sakly, N; Pothier, P; Aouni, M; Ambert-Balay, K
2015-11-01
To ascertain the viral load, the distribution of G and P types of group A rotaviruses (RV-A) in sewage samples and to compare strains in clinical, animal and environmental samples. During our study from April 2007 to April 2010, 518 samples of raw and treated sewage were collected from two biological sewage treatment plants (STPs) located in the Monastir region, Tunisia. RV-A was detected by real-time RT-PCR in 375 (72·4%) sewage samples. According to the quantification results of RV-A, it appears that the viral load in raw and treated sewage of the two STPs was quite similar (P = 0·735). The genotyping of RV-A strains detected in sewage samples showed a great diversity with 10 G types and 8 P types. Most of them were described as common in humans, but we also detected genotypes commonly found in animals. All the genotypes detected in two previous studies performed in our laboratory on clinical and bovine samples were also found in environmental samples. However, some genotypes commonly found in animal were only found in sewage samples. The comparison of environmental, clinical and animal data suggests that STPs may convey not only human sewage but also animal wastes, both of them contaminated with numerous RV-A strains which are not efficiently eliminated by the sewage treatment process and may spread to surface waters. This work demonstrates the potential release of human and animal RV-A into water sources, representing a public health risk, by inducing gastroenteritis in population, but also by increasing the risk of zoonotic transmission and formation of reassortant viruses which could get a higher infectious potential. Our findings also suggest that monitoring of sewage may provide an additional tool to determine the epidemiology of RV-A circulating in a given community. © 2015 The Society for Applied Microbiology.
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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.
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Al-Jarallah, Khaled F.; Shehab, Diaa K.; Haider, Mohammad Z.
2011-01-01
BACKGROUND AND OBJECTIVES: Peroxisome proliferator–activated receptors (PPARs) play an important role in a number of cellular and metabolic functions. This study was carried out to determine the prevalence of a missense mutation (Pro12Ala) in the PPARG2 gene in Kuwaiti Arab patients with primary knee osteoarthritis (OA) and healthy controls with the aim of identifying a possible association. DESIGN AND SETTING: A prospective cross-sectional study carried out at three major teaching hospitals (referral centers) in the country over a one-year period. PATIENTS AND METHODS: The prevalence of PPARG2 gene Pro12Ala missense mutation was determined in 104 Kuwaiti Arab patients with primary knee OA and 111 ethnically matched healthy controls. The prevalence of this Pro12Ala missense mutation was also determined in clinical subgroups of OA patients divided on the basis of age at onset, function and radiologic grading. RESULTS The Pro-Pro genotype of the PPARG2 gene Pro12Ala missense mutation was detected in 95/104 (91.3%) cases compared to 111/111 (100%) in the control subjects. The heterozygous Pro-Ala genotype was detected in 9/104 (8.7%) of the OA patients, while it was not detected in any of the controls. The Ala-Ala genotype was not detected in any of the OA patients or the controls. No significant differences were detected in the PPARG2 gene Pro12Ala genotypes in the subgroups of patients classified on the basis of age at onset, functional assessment using Lequesne’s functional index, and radiological grading using Kellgren-Lawrence (K-L) grading. CONCLUSIONS This study found no significant association between the PPARG2 gene Pro12Ala missense mutation and knee OA. However, the presence of the Pro-Pro genotype of the PPARG2 gene mutation has a protective effect against development of OA. PMID:21245597
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Poveda, Eva; Seclén, Eduardo; González, María del Mar; García, Federico; Chueca, Natalia; Aguilera, Antonio; Rodríguez, Jose Javier; González-Lahoz, Juan; Soriano, Vincent
2009-05-01
Genotypic tools may allow easier and less expensive estimation of HIV tropism before prescription of CCR5 antagonists compared with the Trofile assay (Monogram Biosciences, South San Francisco, CA, USA). Paired genotypic and Trofile results were compared in plasma samples derived from the maraviroc expanded access programme (EAP) in Europe. A new genotypic approach was built to improve the sensitivity to detect X4 variants based on an optimization of the webPSSM algorithm. Then, the new tool was validated in specimens from patients included in the ALLEGRO trial, a multicentre study conducted in Spain to assess the prevalence of R5 variants in treatment-experienced HIV patients. A total of 266 specimens from the maraviroc EAP were tested. Overall geno/pheno concordance was above 72%. A high specificity was generally seen for the detection of X4 variants using genotypic tools (ranging from 58% to 95%), while sensitivity was low (ranging from 31% to 76%). The PSSM score was then optimized to enhance the sensitivity to detect X4 variants changing the original threshold for R5 categorization. The new PSSM algorithms, PSSM(X4R5-8) and PSSM(SINSI-6.4), considered as X4 all V3 scoring values above -8 or -6.4, respectively, increasing the sensitivity to detect X4 variants up to 80%. The new algorithms were then validated in 148 specimens derived from patients included in the ALLEGRO trial. The sensitivity/specificity to detect X4 variants was 93%/69% for PSSM(X4R5-8) and 93%/70% for PSSM(SINSI-6.4). PSSM(X4R5-8) and PSSM(SINSI-6.4) may confidently assist therapeutic decisions for using CCR5 antagonists in HIV patients, providing an easier and rapid estimation of tropism in clinical samples.
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Error-Analysis for Correctness, Effectiveness, and Composing Procedure.
ERIC Educational Resources Information Center
Ewald, Helen Rothschild
The assumptions underpinning grammatical mistakes can often be detected by looking for patterns of errors in a student's work. Assumptions that negatively influence rhetorical effectiveness can similarly be detected through error analysis. On a smaller scale, error analysis can also reveal assumptions affecting rhetorical choice. Snags in the…
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[Evaluation of hepatitis B virus genotyping EIA kit].
Tanaka, Yasuhito; Sugauchi, Fuminaka; Matsuuraa, Kentaro; Naganuma, Hatsue; Tatematsu, Kanako; Takagi, Kazumi; Hiramatsu, Kumiko; Kani, Satomi; Gotoh, Takaaki; Wakimoto, Yukio; Mizokami, Masashi
2009-01-01
Clinical significance of Hepatitis B virus(HBV) genotyping is increasingly recognized. The aim of this study was to evaluate reproducibility, accuracy, and sensitivity of an enzyme immunoassay (EIA) based HBV genotyping kit, which designed to discriminate between genotypes to A, B, C, or D by detecting genotype-specific epitopes in PreS2 region. Using the four genotypes panels, the EIA demonstrated complete inter and intra-assay genotyping reproducibility. Serum specimens had stable results after 8 days at 4 degrees C, or 10 cycles of freezing-thawing. In 91 samples that have been genotyped by DNA sequencing, 87(95.6%) were in complete accordance with EIA genotyping. Of examined 344 HBsAg-positive serum specimens, genotypes A, B, C and D were determined in 26 (7.6%), 62 (18.0%), 228 (66.3%), and 9 (2.6%) cases, respectively. Of 19 (5.5%) specimens unclassified by the EIA, 13 were found to have low titer of HBsAg concentration (< 3 IU/ml), and the other 5 had amino acid mutations or deletions within targeted PreS2 epitopes. The EIA allowed genotyping even in HBV DNA negative samples (96.2%). In conclusion, HBV genotype EIA is reliable, sensitive and easy assay for HBV genotyping. The assay would be useful for clinical use.
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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.