A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power.

BACKGROUND: Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. "Next generation" high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose. RESULTS: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807-819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123-139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic. CONCLUSIONS: The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

Single primer enrichment technology as a tool for massive genotyping: a benchmark on black poplar and maize.

BACKGROUND AND AIMS: The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. METHODS: Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. KEY RESULTS: Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. CONCLUSION: The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.

Cyber and Physical Security Vulnerability Assessment for IoT-Based Smart Homes.

The Internet of Things (IoT) is an emerging paradigm focusing on the connection of devices, objects, or "things" to each other, to the Internet, and to users. IoT technology is anticipated to become an essential requirement in the development of smart homes, as it offers convenience and efficiency to home residents so that they can achieve better quality of life. Application of the IoT model to smart homes, by connecting objects to the Internet, poses new security and privacy challenges in terms of the confidentiality, authenticity, and integrity of the data sensed, collected, and exchanged by the IoT objects. These challenges make smart homes extremely vulnerable to different types of security attacks, resulting in IoT-based smart homes being insecure. Therefore, it is necessary to identify the possible security risks to develop a complete picture of the security status of smart homes. This article applies the operationally critical threat, asset, and vulnerability evaluation (OCTAVE) methodology, known as OCTAVE Allegro, to assess the security risks of smart homes. The OCTAVE Allegro method focuses on information assets and considers different information containers such as databases, physical papers, and humans. The key goals of this study are to highlight the various security vulnerabilities of IoT-based smart homes, to present the risks on home inhabitants, and to propose approaches to mitigating the identified risks. The research findings can be used as a foundation for improving the security requirements of IoT-based smart homes.

Development of a high-density sub-species-specific targeted SNP assay for Rocky Mountain bighorn sheep (Ovis canadensis canadensis).

Due to their abundance and relative ease of genotyping, single nucleotide polymorphisms (SNPs) are a commonly used molecular marker for contemporary population genetic and genomic studies. A high-density and cost-effective way to type SNP loci is Allegro targeted genotyping (ATG), which is a form of targeted genotyping by sequencing developed and offered by Tecan genomics. One major drawback of this technology is the need for a reference genome and information on SNP loci when designing a SNP assay. However, for some non-model species genomic information from other closely related species can be used. Here we describe our process of developing an ATG assay to target 50,000 SNPs in Rocky Mountain bighorn sheep, using a reference genome from domestic sheep and SNP resources from prior bighorn sheep studies. We successfully developed a high accuracy, high-density, and relatively low-cost SNP assay for genotyping Rocky Mountain bighorn sheep that genotyped ~45,000 SNP loci. These loci were relatively evenly distributed throughout the genome. Furthermore, the assay produced genotypes at tens of thousands of SNP loci when tested on other mountain sheep species and subspecies.

EAST in Children with Allergic rhinitis: Experience from Indian Tertiary Centre.

Allergic rhinitis (AR) is recognized as a growing global health disease with considerable importance among children and adolescents. This study aims to study the clinical and sensitization profile of children with allergic rhinitis using EAST. All children presenting to pediatric ENT outpatient with a clinical diagnosis of AR were prospectively recruited. Detailed demographic and clinical history including self-reported allergens, predominant symptoms and associated comorbid conditions were obtained. Severity of symptoms was graded on a visual analogue scale. Specific Ig E antibodies to 20 inhalant allergens was measured using EAST (EUROIMMUN, Germany).The pattern of sensitization was analyzed with respect to age, symptoms, associated comorbid conditions and urbanization. We recruited 328 children with a clinical diagnosis of AR (Mean age 10.3 year, IQR 8-13 years) Nasal block was the predominant symptom across all age groups, sneezing became more troublesome during adolescence. In 191 children sera were tested for allergen specific IgE, 119 (62.3%) showed positive sensitization. The most common sensitization noted was for cockroach, followed by dust mite and pollens. Majority had polysensitization (73%). Those who were predominantly sneezers were more likely to be sensitized with indoor allergens (p < 0.05). Among the comorbid conditions, asthma and atopic dermatitis accounted for maximum non ENT physician visits. The pattern of sensitization did not vary with age, urbanization or comorbid condition. This study highlights the clinical and sensitization profile of children with AR in South East India. Various peculiarities of this community has been presented which needs further attention.

Cylinder Axis Agreement: Unexpected Scenarios.

BACKGROUND: The purpose of this retrospective study was to evaluate cylinder axis agreement between manifest refraction (MR), cycloplegic refraction (CR), Allegro Oculyzer ІІ and Allegro Topolyzer-Vario. METHODS: We included 82 patients (32 males and 50 females, 28.1 ± 8.7 years old), with 156 eyes scheduled for wavefront optimized laser refractive surgery, photorefractive keratectomy (PRK) in 50 eyes and laser-assisted in situ keratomileusis (LASIK) in 106 eyes, for correction of simple, myopic, hyperopic or mixed astigmatism. Cylinder axis was determined under manifest and cycloplegic refractions and using Allegro Occulyzer ІІ and Allegro Topolyzer-Vario platforms. Cylinder axis agreement was assessed by intraclass correlation coefficient, Pearson correlation coefficient and by the method described by Bland and Altman. RESULTS: Intraclass correlation coefficient and Pearson correlation coefficient showed statistically significant cylinder axis agreement between manifest refraction, cycloplegic refraction, Allegro Oculyzer ІІ and Allegro Topolyzer-Vario (p <0.001). Despite statistically significant cylinder axis agreement between the four measuring tools, 4 of 156 eyes (2.5%) showed unexpected discrepancy between Allegro Oculyzer ІІ and Allegro Topolyzer-Vario cylinder axis. CONCLUSION: Although cylinder axis shows statistically significant agreement between manifest refraction, cycloplegic refraction, Allegro Oculyzer ІІ and Allegro Topolyzer-Vario, unexpected discrepancies occur.