Multiplex Design Research Articles

Clinical Evaluation of BioPlex 2200 HIV Ag-Ab, an Automated Screening Method Providing Discrete Detection of HIV-1 p24 Antigen, HIV-1 Antibody, and HIV-2 Antibody

Early and accurate diagnosis is essential for optimal therapeutic outcomes in patients infected with HIV. Currently, none of the commercially available fourth-generation assays differentiate HIV-1 and HIV-2 antibodies (Ab) or the HIV-1 p24 antigen (Ag). The aim of this study was to evaluate the performance of a novel assay, the BioPlex 2200 HIV Ag-Ab. This assay uses a multiplex flow immunoassay design allowing the simultaneous detection and identification of antibodies to HIV-1 (groups M and O), HIV-2, and the HIV-1 p24 antigen, in addition to providing a traditional composite result. A total of 1,505 routine serum samples were prospectively tested. Results were compared with those from the Architect HIV Combo assay. The sensitivity of the BioPlex 2200 was 100%. The specificity assessed on repeated false-positive samples was 99.5%. In addition, 524 frozen specimens from patients known to be infected with HIV-1 or HIV-2 were tested. Of these specimens, 420 were infected with HIV-1, including 156 of known genotypes, 86 were infected with HIV-2, 7 were infected with HIV-1 and HIV-2, and 11 were from patients with acute HIV infection. Sensitivity was 100% for the HIV genotypes tested. The differentiation capabilities of the BioPlex 2200 HIV Ag-Ab assay for HIV-1, HIV-2, dual HIV-1/HIV-2, and early infections were 100%, 90.7%, 100%, and 90.9%, respectively. The BioPlex 2200 is a sensitive and specific assay that offers advantages over conventional HIV combo assays, also referred to as fourth-generation assays, to accurately differentiate and report HIV-1 p24 antigen and HIV-1 and HIV-2 antibodies.

Real-Image Generation in Normal Viewing Disk-Type Multiplex Holography and Design of Replication Method

In this study, the fabrication method and the parameter design for real-image display in normal viewing image-plane disk-type multiplex holography are described. Concept of two-step holographic copying for this kind of hologram is theoretically and experimentally demonstrated. The quantitative analysis for image quality of finished hologram is also presented.

Abstract 2020: Next-generation sequencing for mutation discovery in Ewing sarcoma.

Abstract Background: Ewing sarcoma (EWS) is the second most common type of primary bone tumor to affect children and adolescent and accounts for 2.9% of all childhood cancers. Despite advances in multidisciplinary treatment leading to improved outcomes over time, long term survival still remains less than 50% for patients older than 15 years of age and only 65% in younger patients. In an effort to identify novel prognostic and therapeutic targets, we utilized next generation sequencing techniques to evaluate EWS samples for somatic mutation. Methods: Whole genome paired-end sequencing using the Complete Genomics method was performed on the DNA from 6 EWS matched tumor-normal samples. Somatic mutations were identified and classified using standard bioinformatics techniques and mutations of interest were verified using capillary sequencing. To extend discoveries and evaluate for mutational recurrence, targeted sequencing was performed on a validation cohort of 61 EWS tumors and 22 EWS cell lines using a custom multiplex PCR design followed by high-coverage sequencing on the Ion Torrent system. Results: In the discovery cohort, we detected an average of 361 somatic mutations per tumor in non-repetitive regions and an average of 6 somatic mutations per tumor in protein coding regions (=0.15 mutations/Mb of coding sequence). The well-established FLI1-EWSR1 gene fusion was detected by the whole genome sequencing in all 6 samples. Additional previously reported genetic events were also seen including frequent chromosomal trisomy and deletion of the CDKN2A containing locus on chromosomal region 9p21 in 2 of 6 samples. In the validation cohort of tumors, known recurrent mutations such as in TP53 (3/61 = 4.9%) were seen at rates similar to that previously reported in EWS. Continued efforts are underway to verify novel recurrent mutations. Conclusion: Next-generation sequencing provides a powerful tool to characterize the genetic landscape and evaluate for recurrent mutations in cancer. We utilize this tool in Ewing sarcoma in a project that provides both a comprehensive genomic evaluation from whole genome sequencing data and as well evaluates for recurrent mutations using a targeted sequencing approach. Citation Format: Andrew S. Brohl, Young Song, Laura Hurd, Hongling Liao, Li Chen, Rajesh Patidar, John F. Shern, Jun S. Wei, Javed Khan. Next-generation sequencing for mutation discovery in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2013-2020

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

BackgroundQuantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design.MethodsPrimers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings.ResultsUpon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R2=0.985), MAL (R2=0.986) and hsa-miR-124-2 (R2=0.944).ConclusionMultiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions.

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

BackgroundRNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods.ResultsDifferential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices.ConclusionsThis work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates.

Comparison of random and SSR‐enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization inAcacia harpophyllaF. Muell. Ex Benth

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.

Validation of multiplex microbead immunoassay for simultaneous serodetection of multiple infectious agents in laboratory mouse

Multiplex methodologies enable simultaneous detection of antibodies against several infectious agents allowing sample conservation, cost effectiveness, and amenability to high-throughput/automation. We have previously described a multiplex microbead immunoassay for serodetection of ten, high-priority mouse infectious pathogens. Here, we present a validation of this multiplex diagnostic system using approximately four hundred serum samples from different groups of mice. Computer assisted multivariate analysis of the resulting high volume data (8000 data points) was performed. This computational approach enabled presentation of data in a variety of easily interpretable formats ( e.g., correlation tables and heat maps). Importantly, this computer aided approach was instrumental for the evaluation of assay accuracy, sensitivity, specificity, and robustness during the study. Crucial pieces of information were obtained to make timely adjustments for assay refinement. This progressive approach to developing an implementation-ready clinical assay, facilitated by computational analysis, produced a highly efficient, accurate and dependable serodiagnostics system. This system has effectively replaced the current state-of-the-art methodology (ELISA) used in mouse colony health management at the University of California and the Jackson Laboratory. A pathway to develop multiplex serology tests for infectious disease diagnosis described here serves as a model for multiplex immunoassay design, clinical validation, refinement and implementation.

Improvement of Multiplex PCR Efficiency by Using The MPprimer to Design Primers and Optimizing The Amplification Conditions*

Multiplex PCR was used in many biological fields. Primer design and amplification conditions are critical for enhancing the efficiency of multiplex PCR. In order to improve the multiplex PCR assay, five house-keeping genes of mouse were selected to design primers for multiplex PCR analysis by using the MPprimer program, followed with optimizing the conditions for PCR reactions. Result showed that MPprimer is a valuable tool for multiplex PCR primer design. In addition, by optimizing the conditions for multiplex PCR such as the annealing temperature and extension time, the efficiency of multiplex PCR assay could be significantly improved. This work can be used to advance large-scale gene expression analysis in the post-genome era.

MPprimer: a program for reliable multiplex PCR primer design

BackgroundMultiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility.ResultsA program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions.ConclusionsMPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

The Organizational Design of Transnational Professional Service Firms

Large transnational professional service firms (PSFs) are highly influential in today's global economy because they underpin the integrity of financial markets, enable complex international transactions, and deliver ideas and advice to the world's largest corporations and governments. They sell expertise – that most intangible of outputs – and they seek to provide customized solutions to demanding clients on a global basis. Many other businesses will face similar challenges in the 21st century, as the world becomes more globally connected and customers become more demanding, seeking customized products or services that fit their particular needs. As such, professional service firms – of which accounting, law, and consulting are prime examples – are critical corporate players in the 21st century. PSFs also face significant managerial challenges. Over the past few decades, they have grown in complexity, both geographically and in size, to the point where traditional organizational arrangements have proven inadequate. How have PSFs responded, and what lessons can we draw? Our ongoing studies of PSFs show the emergence of a “multiplex” organizational form. Organizations that are successfully implementing the multiplex design are responding to their complex environments by developing highly differentiated structures in order to capture the benefits of deep specialization along several axes. The trick, however, is in pulling these differentiated parts together. The risk that high differentiation will overpower attempts at coordination was responsible for the earlier failure in various industries of the two-dimensional matrix organizational form. But transnational PSFs have gone further by using three axes of differentiation – and appear to have done so successfully. They have discovered how to gain the powerful benefits of multidimensional specialization without losing overall coordinated effort. First, transnational PSFs have developed multiple axes of deep expertise with respect to their professional services, clients, and markets. Second, professionals within these firms are members of multiple practice groups and teams that criss-cross the multiple axes of expertise, thereby creating a landscape of nascent communities in which personal networks can flourish and expert knowledge can be identified. Third, the deep specialization and experience embodied in a firm's resources are pulled together and harnessed via a client management system, an integrative mechanism which classifies and sorts clients according to various strategic and operational criteria, assigns responsibility for each to appropriate senior professionals (partners), and assists in creating semi-permanent or bespoke client teams to address the needs of a client or specific engagement. Fourth, and perhaps most important, cooperation and coordination are achieved through a culture of reciprocity that is developed and sustained by a number of strategic, organizational, and normative processes within the firm. In this article, we elaborate on this promising organizational design for the 21st century. We use accounting firms as our example, because they are exemplars of the knowledge-intensive, multiplex organizational form, but our research in other professional service organizations confirms that they, too, are using the same practices to develop the same design template. We begin by outlining the challenges for which this organizational form is the emerging solution.

Development of a laboratory project to determine human ABO genotypes—Limitations lead to further student explorations

A multiplex allele-specific PCR analysis was developed to identify six "common" genotypes: AA, AO, BB, BO, OO, and AB. This project included a pre-laboratory exercise that provided active learning experiences and developed critical thinking skills. This laboratory resulted in many successful analyses, which were verified by student knowledge of their phenotypes. However, the design was found to be deficient for the analysis of variants. The limitations in the original allied-health multiplex design were verified through a student designed problem-based laboratory project in an advanced level biochemistry class. Variants were further analyzed in an undergraduate research project using SSCP analyses. The topic of ABO genotyping provides several opportunities for student-centered explorations.

A 26plex Autosomal STR Assay to Aid Human Identity Testing*†

A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex-typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.

STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design.

BackgroundMicrosatellites (MSs) are DNA markers with high analytical power, which are widely used in population genetics, genetic mapping, and forensic studies. Currently available software solutions for high-throughput MS design (i) have shortcomings in detecting and distinguishing imperfect and perfect MSs, (ii) lack often necessary interactive design steps, and (iii) do not allow for the development of primers for multiplex amplifications. We present a set of new tools implemented as extensions to the STADEN package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the STADEN package) is important to avoid developing redundant markers, a feature missing from most other similar tools.ResultsOur extensions to the STADEN package provide the following functionality to facilitate microsatellite (and also minisatellite) marker design: The new modules (i) integrate the state-of-the-art tandem repeat detection and analysis software PHOBOS into workflows, (ii) provide two separate repeat detection steps – with different search criteria – one for masking repetitive regions during assembly of sequencing reads and the other for designing repeat-flanking primers for MS candidate loci, (iii) incorporate the widely used primer design program PRIMER3 into STADEN workflows, enabling the interactive design and visualization of flanking primers for microsatellites, and (iv) provide the functionality to find optimal locus- and primer pair combinations for multiplex primer design. Furthermore, our extensions include a module for storing analysis results in an SQLite database, providing a transparent solution for data access from within as well as from outside of the STADEN Package.ConclusionThe STADEN package is enhanced by our modules into a highly flexible, high-throughput, interactive tool for conventional and multiplex microsatellite marker design. It gives the user detailed control over the workflow, enabling flexible combinations of manual and automated analysis steps. The software is available under the OpenBSD License [1,2]. The high efficiency of our automated marker design workflow has been confirmed in three microsatellite development projects.

Multiplex PCR electrospray-ionization mass spectrometry (ESI-MS): Application to forensic mitochondrial DNA examinations

Mitochondrial DNA (mtDNA) examinations play an important role in criminal investigations, identification of victims of mass disasters, and association of unidentified remains with family members. Typically, HV1 and HV2 are amplified via polymerase chain reaction (PCR) followed by fluorescent sequencing. While this method produces the highest level of resolution, it is labor intensive and unable to distinguish components of a mixture. Previously, an electrospray-ionization mass spectrometry (ESI-MS) method was described to determine the base composition profile of enzymatically digested PCR amplified fragments derived from the HV1 and HV2 regions. Advantages of ESI-MS compared to sequencing include speed of analysis, automation, and increased sensitivity, while retaining a high degree of resolution. Here, we report the next generation of this method in which a base composition profile is determined from 24 overlapping PCR reactions. Because ESI-MS provides the relative abundance of each component present, this method allows for the quantitative typing of mixtures. This ESI-MS method does not rely on a priori knowledge of variable sites, allowing the capture of private mutations and individual-specific variation. Due to the multiplex design, automation, speed of analysis, and ability to interrogate mixtures, this method provides a powerful and rapid tool for forensic mtDNA examinations.

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants

The sequential deletion method is generally used to locate the functional domain of a protein. With this method, in order to find the various N-terminal truncated mutants, researchers have to investigate the ATG-like codons, to design various multiplex polymerase chain reaction (PCR) forward primers and to do several PCR experiments. This web server (N-terminal Truncated Mutants Generator for cDNA) will automatically generate groups of forward PCR primers and the corresponding reverse PCR primers that can be used in a single batch of a multiplex PCR experiment to extract the various N-terminal truncated mutants. This saves much time and money for those who use the sequential deletion method in their research. This server is available at http://oblab.cs.nchu.edu.tw:8080/WebSDL/.

Neurocognitive Endophenotypes in a Multiplex Multigenerational Family Study of Schizophrenia

Genetic factors contribute to the development of schizophrenia where cognitive dysfunction is a hallmark. The purpose of this article was to examine computerized neurocognitive measures as candidate endophenotypic markers of liability for schizophrenia in a genetically informative cohort. European Americans from 35 multiplex multigenerational families (N=349) and healthy participants (N=154) underwent clinical assessments and neurocognitive measurements and provided blood samples. The neurocognitive measures included performance (accuracy and speed) from a computerized battery that assessed abstraction/mental flexibility; attention; verbal, face, and spatial memory; spatial processing; sensorimotor processing; and emotion intensity discrimination. Probands, relatives, and comparison subjects differed from each other in performance. Probands demonstrated greatest impairment relative to comparison subjects, followed by family members. Liability for schizophrenia affected the speed-accuracy tradeoff differently for specific neurocognitive domains. Significant heritability estimates were obtained for accuracy of verbal, facial, and spatial memory and spatial and emotion processing. For speed, estimates of heritability were significant for abstraction/mental flexibility, attention, face memory, and spatial and sensorimotor processing. In a multigenerational multiplex design, the authors demonstrated that neurocognitive measures are associated with schizophrenia, differentiate unaffected relatives from comparison subjects, and may have significant presumed heritability. Therefore, they are endophenotypes suitable for genetic studies. Accuracy and speed can be differentially sensitive to presumed genetic liability.

Fetal Hemoglobin in Sickle Cell Anemia: Associations with Single Nucleotide Polymorphisms in Quantitative Trait Loci on Chromsomes 8q12 and Xp22.

Fetal hemoglobin (HbF) inhibits the polymerization of sickle hemoglobin modulating some of the subphenotypes of sickle cell anemia. HbF concentrations vary considerably among patients and this variation is regulated as a multigenic trait; some regulatory regions previously identified are linked to the β-globin gene-like cluster and are also within quantitative trait loci (QTL) on chromosomes 6q, 8q and Xp. We genotyped panels of haplotype tagging (ht)SNPs in the β-globin gene-like cluster and covering the 8q and Xp QTL in two independent patient samples and applied two independent analytical methods to study their association with HbF levels. The first sample included 327 patients (135 htSNPs) and the second sample had 987 patients (102 htSNPs). Genotyping was done using a custom 384 multiplex design and the Illumina platform. Single SNP association was investigated by multiple linear regression analysis with simultaneous adjustment for age, sex and the co-existence of α thalassemia; we performed a permutation procedure to correct for multiple testing. The nonlinear regression Random Forest method was used for a joint analysis of covariates and all SNPs. In the smaller patient sample, 7 SNPs in TOX (thymus high mobility protein; 8q12.1) gene showed significant association with HbF (p-value <0.05), but none passed the multiple test correction. Another 11 SNPs in TOX also showed significant association in the larger patient sample and SNP rs7817609 passed the multiple test correction. This SNP in TOX was significantly associated with HbF under codominant and dominant models (empirical p-values 0.0154 and 0.0067) with the C allele associated with a high level of HbF expression; the level of HbF in subjects with CC and CG genotypes was on average 25 percent higher than that in subjects with GG genotype. Three SNPs, including the Xmn I, −158 C T SNP 5′ to HBG2 within the β-globin gene-like cluster, also showed association with HbF. This SNP was the 2nd most important SNP in predicting HbF expression using Random Forest analysis indicating that it might interact non-linearly with other regulatory factors. Joint analysis of all SNPs and covariates revealed that the most important variables for predicting HbF matched most of the SNPs identified by single SNP association studies in both data sets. Random Forest analysis also identified SNPs in EGFL6, GPM6B and FIGF that have a strong effect on predicting HbF level and that lie within the Xp22.2–p22.3 QTL. This suggests that genes within the Xp QTL might be involved in interaction with other genes to regulate the expression of HbF. TOX belongs to one of the high mobility group (HMG) box protein families, which contains proteins with a single HMG box that bind with high sequence specificity to variants of the DNA sequence (A/T)(A/T)CAAAG, inducing a sharp bend in DNA, altering local chromatin structure and modulating the formation of multi-protein regulatory complexes. Within 50 kb 5′ and 40 kb 3′ to the HBG2 there were 50 matches to this binding sequence. TOX might bind to the β-globin gene cluster forming multi-protein regulatory complexes with other proteins to regulate the expression of HbF. We have identified new candidate genes, or linkage disequilibrium with candidate genes, that might play a role in HbF regulation, but further genetic and biological studies are required for validation.

A multiplex PCR design for simultaneous genotyping of X chromosome short tandem repeat markers

A tetraplex system for the X chromosome genetic markers DXS7423, DXS101, DXS8377 and HPRTB (human phosphoribosyl transferase) was optimized in a single PCR reaction. These short tandem repeat (STR) markers were typed for 65 individuals (29 female and 36 male samples) from a Galician population sample (Northern Spain). Allele frequencies were estimated for all loci. Optimization of STR multiplexes is a practical and simple method to obtain large amounts of information important in forensic and population genetic studies, therefore, in this context, they should be the tool of approach for X-STR studies.

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.