Dynamic model based algorithms for screening and genotyping over 100 K SNPs on oligonucleotide microarrays

MOTIVATION: A high density of single nucleotide polymorphism (SNP) coverage on the genome is desirable and often an essential requirement for population genetics studies. Region-specific or chromosome-specific linkage studies also benefit from the availability of as many high quality SNPs as possible. The availability of millions of SNPs from both Perlegen and the public domain and the development of an efficient microarray-based assay for genotyping SNPs has brought up some interesting analytical challenges. Effective methods for the selection of optimal subsets of SNPs spanning the genome and methods for accurately calling genotypes from probe hybridization patterns have enabled the development of a new microarray-based system for robustly genotyping over 100,000 SNPs per sample. RESULTS: We introduce a new dynamic model-based algorithm (DM) for screening over 3 million SNPs and genotyping over 100,000 SNPs. The model is based on four possible underlying states: Null, A, AB and B for each probe quartet. We calculate a probe-level log likelihood for each model and then select between the four competing models with an SNP-level statistical aggregation across multiple probe quartets to provide a high-quality genotype call along with a quality measure of the call. We assess performance with HapMap reference genotypes, informative Mendelian inheritance relationship in families, and consistency between DM and another genotype classification method. At a call rate of 95.91% the concordance with reference genotypes from the HapMap Project is 99.81% based on over 1.5 million genotypes, the Mendelian error rate is 0.018% based on 10 trios, and the consistency between DM and MPAM is 99.90% at a comparable rate of 97.18%. We also develop methods for SNP selection and optimal probe selection. AVAILABILITY: The DM algorithm is available in Affymetrix's Genotyping Tools software package and in Affymetrix's GDAS software package. See http://www.affymetrix.com for further information. 10 K and 100 K mapping array data are available on the Affymetrix website.     

Gene expression profiling in a mouse model identifies fetal liver- and placenta-derived potential biomarkers for Down Syndrome screening

Background

As a first step to identify novel potential biomarkers for prenatal Down Syndrome screening, we analyzed gene expression in embryos of wild type mice and the Down Syndrome model Ts1Cje. Since current Down Syndrome screening markers are derived from placenta and fetal liver, these tissues were chosen as target.

Methodology/Principal Findings

Placenta and fetal liver at 15.5 days gestation were analyzed by microarray profiling. We confirmed increased expression of genes located at the trisomic chromosomal region. Overall, between the two genotypes more differentially expressed genes were found in fetal liver than in placenta. Furthermore, the fetal liver data are in line with the hematological aberrations found in humans with Down Syndrome as well as Ts1Cje mice. Together, we found 25 targets that are predicted (by Gene Ontology, UniProt, or the Human Plasma Proteome project) to be detectable in human serum.

Conclusions/Significance

Fetal liver might harbor more promising targets for Down Syndrome screening studies. We expect these new targets will help focus further experimental studies on identifying and validating human maternal serum biomarkers for Down Syndrome screening.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

Redox regulation of neuronal migration in a Down Syndrome model

Down Syndrome (DS), one of the major genetic causes of mental retardation, is characterized by disrupted corticogenesis produced, in part, by an abnormal layering of neurons in cortical laminas II and III. Because defects in the normal migration of neurons during corticogenesis can result in delayed cortical radial expansion and abnormalities in cortical layering, we have examined neuronal migration in murine trisomy 16 (Ts16), a mouse model for DS. Using an in vitro assay for chemotaxis, our data demonstrate that the number of acutely dissociated Ts16 cortical neurons migrating in response to glutamate or N-methyl-D-aspartate (NMDA), known chemotactic factors, was decreased compared to normal littermates, suggesting a defect in NMDA receptor- (NMDAR-) mediated events. Ts16 neurons did not lack NMDAR since expression of mRNA and protein for NMDAR subunits was observed in Ts16 cells. However, the number of cells that generated an observable current in response to NMDA was decreased compared to normal littermates. Similar to DS, Ts16 CNS demonstrated an inherent oxidative stress likely caused by the triplication of genes such as SOD1. To determine if the abnormal redox state was a factor in the failure of NMDAR-mediated migration in Ts16, we treated Ts16 neurons with either n-acetyl cysteine (NAC) or dithiothrietol (DTT), known antioxidants. The reduction in NMDAR-mediated migration observed in Ts16 neurons was returned to normal littermate values by NAC or DTT. Our data indicate that oxidative stress may play a key role in the abnormal glutamate-mediated responses during cortical development in the Ts16 mouse and may have an impact on neuronal migration at critical stages.     

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.     

Micro-colony array based high throughput platform for enzyme library screening

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.     

Gut Microbiome in Down Syndrome

Background

Premature aging seriously compromises the health status of Down Syndrome (DS) persons. Since human aging has been associated with a deterioration of the gut microbiota (GM)-host mutualism, here we investigated the composition of GM in DS.

Methods

The observational study presented involved 17 adult DS persons. We characterized the GM structure by 454 pyrosequencing of the V4 region of the 16S rRNA gene. DS microbiome was compared with that of age-matched healthy non-trisomic adults enrolled in the same geographic area.

Results and Conclusions

The dominant GM fraction of DS persons showed an overall mutualistic immune-modulatory layout, comparable to that of healthy controls. This makes GM a possible factor counteracting the genetic determined acceleration of immune senescence in DS persons. However, we also found detectable signatures specific for DS among subdominant GM components, such as the increase of Parasporobacterium and Sutterella. In particular, the abundance of this last microorganism significantly correlated with the Aberrant Behavior Checklist (ABC) total score, allowing us to hypothesize a possible role for this microbial genus in behavioral features in DS.     

Onychomycosis in Children with Down Syndrome

Purpose of Review

Fungal infection of the nail, known as onychomycosis, occurs more frequently in older age, showing a higher prevalence in pediatric age in recent years. A high rate of dermatological infections befalls in patients with Down syndrome, including onychomycosis, due to a decrease in T and B lymphocytes in number and function, resulting in a disarrangement of cellular and humoral immunity. This has led to several investigations on onychomycosis in children with Down syndrome, so the purpose of this review is to show the available evidence.

Recent Findings

The etiological agents of onychomycosis can be dermatophytes, non-dermatophyte molds, and yeasts. Most cases are related with dermatophytes; Trichophyton rubrum being the most common cause. In children with Down syndrome, T. rubrum has been reported as the main cause, followed by T. mentagrophytes. Distal lateral subungual onychomycosis is the most common variety of onychomycosis in children. The importance of identifying the fungus lies in selecting the appropriate treatment, since not all antifungals have the same spectrum of action against molds and Candida. Terbinafine has showed to be safe and effective for the treatment of onychomycosis in patients from special populations, including children with Down syndrome. In patients with Down syndrome, treatment for onychomycosis has not been completely studied; so far, terbinafine has shown the best results.

Summary

The clinical presentations of children with Down syndrome and the rest of the general pediatric population are similar. However, there are few studies about onychomycosis in children with Down syndrome. It is necessary to perform new onychomycosis research in this study population, in order to establish recommendations.

     

Postnatal lethality and cardiac anomalies in the Ts65Dn Down Syndrome mouse model

The Ts65Dn mouse is a well-studied model for Down syndrome (DS). The presence of the translocation chromosome T17¹⁶ (referred to as T65Dn) produces a trisomic dosage imbalance for over 100 genes on the distal region of mouse Chromosome 16. This dosage imbalance, with more than half of the orthologs of human Chromosome 21 (Hsa21), causes several phenotypes in the trisomic mice that are reminiscent of DS. Careful examination of neonates in a newly established Ts65Dn colony indicated high rates of postnatal lethality. Although the transmission rate for the T65Dn chromosome has been previously reported as 20%–40%, genotyping of all progeny indicates transmission at birth is near the 50% expected with Mendelian transmission and survival. Remarkably, in litters with maternal care that allowed survival of some pups, postnatal lethality occurred primarily in pups that inherited the T65Dn marker chromosome. This selective loss within 48 h of birth reduced the transmission of the marker chromosome from 49% at birth to 34% at weaning. Gross morphologic examination revealed cardiovascular anomalies, i.e., right aortic arch accompanied by septal defects, in 8.3% of the trisomic newborn cadavers examined. This is an intriguing finding because the orthologs of the DiGeorge region of HSA22, which are posited to contribute to the aortic arch abnormalities seen in trisomy 16 mice, are not triplicated in Ts65Dn mice. These new observations suggest that the Ts65Dn mouse models DS not only in its previously described phenotypes but also with elevated postnatal lethality and congenital heart malformations that may contribute to mortality.     

基于受体的药物高通量筛选研究进展

受体是药物筛选的重要靶标,基于受体的药物高通量筛选是药物筛选的主要类型之一。本文根据受体作用原理,按照检测对象的不同,从直接与间接检测的角度,将基于受体的药物高通量筛选进行了分类,总结了基于受体的几种不同的药物筛选模型,并简要介绍了高通量筛选技术在中药研究中的应用,对药物筛选的发展进行了展望。     

Adaptive learning style prediction in e-learning environment using levy flight distribution based CNN model

A learning style that focuses on individual learning is one of the most important aspects of any learning environment. Each learner has a unique manner of understanding, retaining, processing, and interpreting new information based on their learning styles. The ability of an e-learning system to automatically determine a student's learning style has become more essential. For learning events, the evolution of e-learning platforms provides students with higher opportunities online. In this paper, we proposed a Convolutional Neural Network-based Levy Flight Distribution (CNN-LFD) algorithm for learning style prediction. An adaptive e-learning system is divided into two sections: automatic learning style prediction and classification based on the number of learning styles included. Initially, the student logs in with their user ID, and the data is saved in the database. The features such as questionnaire score, login credentials (session ID, learner ID, and course ID), and login time (location, session ID) are extracted along with the sequence of the user's learning behavior. After that, the CNN-LFD algorithm predicts the learning styles of the learners namely Active/reflective, Sensing/intuitive, visual/verbal, sequential/global based on the extracted features. The dataset information are gathered from a Massive Open Online Course (MOOC), and the proposed model is built in JAVA software. The experimental results demonstrate higher classification accuracy during learning style prediction. The proposed CNN-LFD algorithm accomplishes 97.09% accuracy, 94.76% specificity, 92.12% sensitivity, and 97.56%, precision values than other methods.

     

基于CNN与LSTM模型的蛋白质二级结构预测

蛋白质结构的预测在理解蛋白质结构组成和蛋白质的生物学功能有重要意义,而蛋白质二级结构预测是蛋白质结构预测的重要环节。当PSSM位置特异性进化矩阵被广泛应用于将蛋白质初级结构序列编码作为输入样本后,每个残基可以被表示成二维空间的数据平面,由此文中尝试利用卷积神经网络对其进行训练。文中还设计了另一种卷积神经网络,利用长短记忆网络感知了CNN最后卷积特征面的横向特征和纵向特征后连同卷积神经网络的全连接共同完成分类,最后用ensemble方法对两类卷积神经网络模型进行了整合,最终ensemble方法中包含两类卷积神经网络的六个模型,在CB513蛋白质数据集测得的Q3结果为77.2。     

Centromeric genotyping and direct analysis of nondisjunction in humans: Down syndrome

In species with chiasmate meioses, alterations in genetic recombination are an important correlate of nondisjunction. In general, these alterations fall into one of two categories: either homologous chromosomes fail to pair and/or recombine at meiosis I, or they are united by chiasmata that are suboptimally positioned. Recent studies of human nondisjunction suggest that these relationships apply to our species as well. However, methodological limitations in human genetic mapping have made it difficult to determine whether the important determinant(s) in human nondisjunction is absent recombination, altered recombination, or both. In the present report, we describe somatic cell hybrid studies of chromosome 21 nondisjunction aimed at overcoming this limitation. By using hybrids to “capture” individual chromosomes 21 of the proband and parent of origin of trisomy, it is possible to identify complementary recombinant meiotic products, and thereby to uncover crossovers that cannot be detected by conventional mapping methods. In the present report, we summarize studies of 23 cases. Our results indicate that recombination in proximal 21q is infrequent in trisomy-generating meioses and that, in a proportion of the meioses, recombination does not occur anywhere on 21q. Thus, our observations indicate that failure to recombine is responsible for a proportion of trisomy 21 cases. Received: 12 January 1997; in revised form: 16 February 1998 / Accepted: 19 February 1998     

以酵母嗜杀系统为基础的抗病毒药物筛选模型的建立

根据嗜杀酵母T158c/S14a中L-A病毒-1移码效率改变影响M1病毒的存活,导致K1毒素减少,在低pH的美蓝平板上用杯碟法通过抑菌圈的大小检测酵母K1毒素的嗜杀活性,建立了一个以酵母嗜杀系统为基础的抗病毒药物筛选模型。研究了杯碟法检测酵母毒素嗜杀活性的各种条件。对不同pH和温度下酵母的嗜杀活性进行了研究,确定了模型用于筛选的最适pH范围为4.3~4.7,最适温度范围为20~22℃。运用该模型研究了几种中药对嗜杀活性的抑制作用,发现了金银花和升麻具有一定的抗病毒作用。该模型为抗病毒药物的高通量初筛奠定了基础。     

Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array

We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.