Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis

Sequencing targeted DNA regions in large samples is necessary to discover the full spectrum of rare variants. We report an effective Illumina sequencing strategy utilizing pooled samples with novel quality (Srfim) and filtering (SERVIC ⁴ E) algorithms. We sequenced 24 exons in two cohorts of 480 samples each, identifying 47 coding variants, including 30 present once per cohort. Validation by Sanger sequencing revealed an excellent combination of sensitivity and specificity for variant detection in pooled samples of both cohorts as compared to publicly available algorithms.     

Spatial normalization improves the quality of genotype calling for Affymetrix SNP 6.0 arrays

Background  

Microarray measurements are susceptible to a variety of experimental artifacts, some of which give rise to systematic biases that are spatially dependent in a unique way on each chip. It is likely that such artifacts affect many SNP arrays, but the normalization methods used in currently available genotyping algorithms make no attempt at spatial bias correction. Here, we propose an effective single-chip spatial bias removal procedure for Affymetrix 6.0 SNP arrays or platforms with similar design features. This procedure deals with both extreme and subtle biases and is intended to be applied before standard genotype calling algorithms.     

Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500 K GeneChip

Background

Cystic fibrosis (CF) mice, created with a genetically engineered mutation in the Cystic fibrosis transmembrane conductance regulator (Cftr) gene, may develop intestinal plugs which limit their survival past weaning. In a studied population of genetically mixed CF mice differences in allelic ratios at particular loci, between surviving CF mice and mice with the lethal intestinal defect, were used to map cystic fibrosis modifier gene one, Cfm1. Using this approach, we previously identified an X chromosome locus which may influence the survival to weaning of C57BL/6J × BALB/cJ F2 CF mice. We also detected two regions of transmission ratio distortion, independent of Cftr genotype, in a limited dataset. To investigate these findings, in this study we have genotyped 1208 three-week old F2 mice, and 186 day E15.5 embryos, derived from a congenic (C57BL/6J × BALB/cJ) F1 Cftr +/- intercross, for the putative distortion regions.

Results

An excess of homozygous BALB genotypes, compared to Mendelian expectations, was detected on chromosomes 5 (p = 5.7 × 10^-15) and X (p = 3.0 × 10^-35) in three-week old female mice but transmission ratio distortion was not evident in the tested region of chromosome 3 (p = 0.39). Significant pre-weaning lethality of CF mice occurred as 11.3% (137/1208) of the three-week old offspring were identified as CF mice. X chromosome genotypes were not, however, distorted in the female CF mice (p = 0.62), thus the significant non-Mendelian inheritance of this locus was dependent on CF status. The survival of CF embryos to day E15.5 was consistent with Mendelian expectations (42/186 = 23%), demonstrating the loss of CF mice to have occurred between E15.5 and three weeks of age. The excess of X chromosome homozygous BALB genotypes was recorded in female embryos (p = 0.0048), including CF embryos, indicating the distortion to be evident at this age.

Conclusion

Two of three previously suggested loci of transmission ratio distortion were replicated as distorted in this mouse cross. The non-Mendelian inheritance of X chromosome genotypes implicates this region in the survival to weaning of non-CF mice.     

Gene expression profiling of chromophobe renal cell carcinomas and renal oncocytomas by Affymetrix GeneChip using pooled and individual tumours

Due to overlapping morphology, malignant chromophobe renal cell carcinomas (RCC) and benign renal oncocytomas (RO) may pose a diagnostic problem. In the present study, we have applied different algorithms to evaluate the data sets obtained by hybridisation of pooled and also individual samples of renal cell tumours (RCT) onto two different gene expression platforms. The two approaches revealed high similarities in the gene expression profiles of chromophobe RCCs and ROs but also some differences. After identifying the differentially expressed genes by statistic analyses, the candidate genes were further selected by a real time and normal RT-PCR and their products were analysed by immunohistochemistry. We have identified CD82 and S100A1 as valuable markers for chromophobe RCC as well as AQP6 for ROs. However, these genes are expressed at the protein level in other types of RCTs as well albeit at a low frequency and low intensity. As none of the selected genes marks exclusively one type of RCTs, for the differential diagnosis of chromophobe RCCs and ROs, a set of markers such as CD82, S100A1 and AQP6 as well as some others would be an option in routine histological laboratories.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

The opposing effects of lipopolysaccharide on the antitumor therapeutic efficacy of DNA vaccine

DNA vaccine represents a novel method to elicit immunity against infectious disease. Lipopolysaccharide (LPS) copurified with plasmid DNA may affect therapeutic efficacy and immunological response. We aimed to study the effect of LPS on the therapeutic efficacy of HER-2/neu DNA vaccine in a mouse tumor animal model. Plasmid DNA purified from commercial EndoFree plasmid purification kits functioned as a better therapeutic DNA vaccine than that purified from Non-EndoFree purification kit, which contains >or=0.5 microg LPS per 100 mg DNA plasmid. To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine, increasing amount of LPS was added to endotoxin-free plasmid DNA, and inoculated on mice with established tumors. One mug of LPS significantly attenuated the therapeutic effect of neu DNA vaccine and increased Th2 immune responses bias with interleukin-4 cytokine production. In contrast, high amount (100 microg) of LPS enhanced the therapeutic efficacy of neu DNA vaccine with an increase of cytotoxic T lymphocyte response and Th1 immune response. The effect of LPS on DNA vaccine was diminished when the tumor was grown in toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice. Our results indicate that variation in the LPS doses exerts opposing effects on the therapeutic efficacy of DNA vaccine, and the observed effect is TLR4 dependent.     

Evaluating the effects of genetic variants of DNA repair genes using cytogenetic mutagen sensitivity approaches

Mutagen sensitivity, measured in short-term cultures of peripheral blood lymphocytes by cytogenetic endpoints, is an indirect measure for DNA repair capacity and has been used for many years as a biomarker for intrinsic susceptibility for cancer. In this article, we briefly give an overview of the different cytogenetic mutagen sensitivity approaches that have been used successfully to evaluate the biological effects of polymorphisms in DNA repair genes based on a current review of the literature and based on the need for biomarkers that would allow the characterization of the biological and functional significance of such polymorphisms. We also address some of the future challenges facing this emerging area of research.     

A simple method for detecting drug effects on the DNA of mammalian cells

A simple method for detecting drug or X-ray effects on the DNA of intact cells is described. This method uses an easily constructed viscometer to measure changes in the viscosity of alkaline cell lysates. The method has been tested by comparing the effects of 4′-[(9-acridinyl)amino]methanesulfon-o-anisidide (oAMSA), 4′-[(9-acridinyl)amino]methanesulfon-m-anisidide (mAMSA), daunorubicin, and adriamycin on cellular DNA.     

Quantitative determination of allele frequency in pooled DNA by using sequencing method

Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r2 = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Deep sequencing of the Nicastrin gene in pooled DNA, the identification of genetic variants that affect risk of Alzheimer's disease

Nicastrin is an obligatory component of the γ-secretase; the enzyme complex that leads to the production of Aβ fragments critically central to the pathogenesis of Alzheimer's disease (AD). Analyses of the effects of common variation in this gene on risk for late onset AD have been inconclusive. We investigated the effect of rare variation in the coding regions of the Nicastrin gene in a cohort of AD patients and matched controls using an innovative pooling approach and next generation sequencing. Five SNPs were identified and validated by individual genotyping from 311 cases and 360 controls. Association analysis identified a non-synonymous rare SNP (N417Y) with a statistically higher frequency in cases compared to controls in the Greek population (OR 3.994, CI 1.105-14.439, p = 0.035). This finding warrants further investigation in a larger cohort and adds weight to the hypothesis that rare variation explains some of genetic heritability still to be identified in Alzheimer's disease.     

The possibilities of using variants of immunoenzyme analysis for detecting L-form Salmonella typhi bacteria in biological fluids

The possibility of using, on principle, the enzyme immunoassay (EIA) in different modifications for the detection of S. typhi L-forms in biological fluids (blood, urine) was established. The inhibiting variant of EIA showed the highest sensitivity: 1 ng/ml. The direct sandwich variant permitted the quantitative determination of the antigen of S. typhi L-form in the widest range of 20-500 ng/ml. The indirect enzyme immunometric variant permitted the detection of S. typhi L-forms with a sensitivity of 10(5) colony-forming units per ml only in urine.     

Model-based prediction of human hair color using DNA variants

Predicting complex human phenotypes from genotypes is the central concept of widely advocated personalized medicine, but so far has rarely led to high accuracies limiting practical applications. One notable exception, although less relevant for medical but important for forensic purposes, is human eye color, for which it has been recently demonstrated that highly accurate prediction is feasible from a small number of DNA variants. Here, we demonstrate that human hair color is predictable from DNA variants with similarly high accuracies. We analyzed in Polish Europeans with single-observer hair color grading 45 single nucleotide polymorphisms (SNPs) from 12 genes previously associated with human hair color variation. We found that a model based on a subset of 13 single or compound genetic markers from 11 genes predicted red hair color with over 0.9, black hair color with almost 0.9, as well as blond, and brown hair color with over 0.8 prevalence-adjusted accuracy expressed by the area under the receiver characteristic operating curves (AUC). The identified genetic predictors also differentiate reasonably well between similar hair colors, such as between red and blond-red, as well as between blond and dark-blond, highlighting the value of the identified DNA variants for accurate hair color prediction.     

The effects of corticosteroids on COPD lung macrophages: a pooled analysis

Background

There is large variation in the therapeutic response to inhaled corticosteroids (ICS) in COPD patients. We present a pooled analysis of our previous studies investigating the effects of corticosteroids on lung macrophages, in order to robustly determine whether corticosteroid sensitivity in COPD cells is reduced compared to controls, and also to evaluate the degree of between individual variation in drug response.

Methods

Data from 20 never smokers (NS), 27 smokers (S) and 45 COPD patients was used. Lung macropahges had been stimulated with lipopolysaccharide (LPS), with or without the corticosteroid dexamethasone, and tumour necrosis factor (TNF)-α, interleukin (IL)-6 and chemokine C-X-C motif ligand (CXCL) 8 production was measured.

Results

There was no difference in the anti-inflammatory effects of corticosteroids when comparing group mean data of COPD patients versus controls. The inhibition of TNF-α and IL-6 was greater than CXCL8. The effects of corticosteroids varied considerably between subjects, particularly at lower corticosteroid concentrations.

Conclusions

We confirm that overall corticosteroid sensitivity in COPD lung macrophages is not reduced compared to controls. The varied effect of corticosteroids between subjects suggests that some individuals have an inherently poor corticosteroid response. The limited suppression of lung macrophage derived CXCL8 may promote neutrophilic inflammation in COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0260-0) contains supplementary material, which is available to authorized users.