DNA Pooling Strategy Research Articles

A whole genome scan for QTL affecting milk protein percentage in Italian Holstein cattle, applying selective milk DNA pooling and multiple marker mapping in a daughter design

We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP.

Identification of SPOCK2 As a Susceptibility Gene for Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia is the most common chronic respiratory disease in premature infants. Genetic factors might contribute to bronchopulmonary dysplasia susceptibility. To identify genetic variants involved in bronchopulmonary dysplasia through a genome-wide association study. We prospectively evaluated 418 premature neonates (gestational age <28 wk), of whom 22% developed bronchopulmonary dysplasia. Two discovery series were created, using a DNA pooling strategy in neonates from white and African ancestry. Polymorphisms associated with the disease were confirmed in an independent replication population. Genes were then explored by fine mapping and associations were replicated in an external Finnish population of 213 neonates. Validated genes expression patterns were studied in rat lung, after air or hyperoxia exposure. SPOCK2 gene was identified by both discovery series. The most significant polymorphism (rs1245560; P = 1.66 × 10(-7)) was confirmed by individual genotyping, and in the replication population (P = 0.002). Fine mapping confirmed the association of rs1245560 with bronchopulmonary dysplasia in both white and African populations with adjusted odds ratios of 2.96 (95% confidence interval [CI], 1.37-6.40) and 4.87 (95% CI, 1.88-12.63), respectively. In white neonates, rs1049269 was also associated with the disease (odds ratio, 3.21; 95% CI, 1.51-6.82). These associations were replicated in the Finnish population. In newborn rat lungs, SPOCK2 mRNA levels markedly increased during the alveolar stage of lung development. After rat exposure to hyperoxia, SPOCK2 expression increased relative to air-exposed controls. We identified SPOCK2 as a new possible candidate susceptibility gene for bronchopulmonary dysplasia. Its lung expression pattern points toward a potential role in alveolarization.

The influence of sampling strategies and spatial variation on the detected soil bacterial communities under three different land-use types

To determine the influence of pooling strategies on detected soil bacterial communities, we sampled 45 soil cores each from a eucalypt woodland, a sown pasture and a revegetated site in an Australian landscape. We assessed the spatial variation within each land-use plot, including the influence of sampling distance, soil chemical characteristics and, where appropriate, proximity to trees on the soil bacterial community, by generating terminal restriction fragment length polymorphism profiles of the bacterial 16S rRNA genes. The soil bacterial community under the revegetated site was more similar to the original woodland than the pasture, and this result was found regardless of the soil- or the DNA-pooling strategy used. Analyzing as few as eight cores per plot was sufficient to detect significant differences between the bacterial communities under the different plots to be distinguished. Soil pH was found to be most strongly associated with soil bacterial community composition within the plots and there was no association found with proximity to trees. This study has investigated sampling strategies for further research into the transitions of soil microbial communities with land-use change across broader temporal and spatial scales.

Estimation of haplotypes based on DNA pooling

DNA pooling, a fast and economic study strategy, is widely used in areas of scientific research. In spite of various limits, researchers are making their efforts to improve DNA pooling toward a more perfect direction, including allele frequency detection and estimation of haplotypes. In haplotype estimation, more and more analyzing Methods originated from the expectation-maximization algorithm have appeared, with improved accuracy and practicality, such as HaploPool algorithm and PoooL algorithm.

Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy.

Scanning for Genomic Regions Subject to Selective Sweeps Using SNP-MaP Strategy

Population genomic approaches, which take advantages of high-throughput genotyping, are powerful yet costly methods to scan for selective sweeps. DNA-pooling strategies have been widely used for association studies because it is a cost-effective alternative to large-scale individual genotyping. Here, we performed an SNP-MaP (single nucleotide polymorphism microarrays and pooling) analysis using samples from Eurasia to evaluate the efficiency of pooling strategy in genome-wide scans for selection. By conducting simulations of allelotype data, we first demonstrated that the boxplot with average heterozygosity (HET) is a promising method to detect strong selective sweeps with a moderate level of pooling error. Based on this, we used a sliding window analysis of HET to detect the large contiguous regions (LCRs) putatively under selective sweeps from Eurasia datasets. This survey identified 63 LCRs in a European population. These signals were further supported by the integrated haplotype score (iHS) test using HapMap II data. We also confirmed the European-specific signatures of positive selection from several previously identified genes (KEL, TRPV5, TRPV6, EPHB6). In summary, our results not only revealed the high credibility of SNP-MaP strategy in scanning for selective sweeps, but also provided an insight into the population differentiation.

A triple combination strategy corrects population stratification bias and saves genotyping cost

Objective The case-control association study has gained popularity for mapping disease-susceptibility gene(s) of complex human diseases. However, the study is prone to population stratification bias. Study Design and Setting The authors proposed a triple combination strategy of stratum matching, genomic control, and multiple DNA pooling for population-based case-control association studies. Monte Carlo simulation showed that the type I error rates are well controlled using this strategy. Results As for power, it increases under the following situations: (1) more accurate stratum matching, (2) more null markers for genomic control, (3) more DNA pooling sets, and (4) more accurate DNA pooling measurement. Conclusion The triple combination strategy corrects population stratification bias and saves genotyping cost.

Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

BackgroundNephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative...

Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals.

Bardet-Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1-BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1-BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.

Extremes of Unexplained Variation as a Phenotype

Received December 23, 2009; accepted February 22, 2010. Because cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the Western world,1 reduction of CVD is the focus of a large body of research. Ever since the initial Framingham study, researchers have sought to identify and validate CVD risk factors, now often referred to as “traditional” risk factors, including age, sex, blood pressure, lipid parameters, diabetes, and smoking.2 More recently, extraordinary efforts have been undertaken to construct large study samples to carry out population-based genome-wide association studies (GWAS) to further deconstruct the heritable component of CVD risk, with the successful identification of molecular determinants, such as the robustly associated chromosome 9p21 locus.3–6 However, a substantial component of heritable CVD risk still exists,7 and new study designs and statistical methods will be necessary for uncovering additional associated variants. Mounting evidence suggests that common complex disorders represent the extremes of quantitative traits.8 The evaluation of individuals with extreme values of a continuously distributed trait in studies seeking to identify quantitative trait loci has a long history in genetics. “Selective genotyping” was first described by Lander and Botstein9 in the context of animal models, and later Carey and Williamson10 and then Risch and Zhang11 described the approach in family-based linkage studies. Schork et al12 demonstrated formally that selective genotyping, or genotyping of the extremes, showed substantial efficiency gains in human genetic association studies. Since the publication by Schork et al, a number of authors have extended the extreme sampling strategy to the use of more general selection strategy designs,13 considerations when loci are multiallelic and exhibit varying strengths of linkage disequilibrium,14 and for use in DNA pooling strategies.15 The various ultrasound techniques used to monitor carotid atherosclerosis, including intima-media …

Combining DNA pooling with selective recombinant genotyping for increased efficiency in fine mapping

One of the key steps in positional cloning and marker-aided selection is to identify marker(s) tightly linked to the target gene (i.e., fine mapping). Selective genotyping such as selective recombinant genotyping (SRG) is commonly used in fine mapping for cost-saving. To further decrease genotyping effort and rapidly screen for tightly linked markers, we propose here a combined DNA pooling and SRG strategy. A two-stage pooled genotyping can be used for identifying recombinants between a pair of flanking markers more efficiently, and a joint use of bulked DNA analysis and two-stage pooling can also save cost for genotyping recombinants. The combined DNA pooling and SRG strategy can further be extended to fine mapping for polygenic traits. The numerical results based on hypothetical scenarios and an illustrative application to fine mapping of a mutant gene, called xl(t), in rice suggest that the proposed strategy can remarkably reduce genotyping amount compared with the conventional SRG.

SSR analysis for genetic structure and diversity determination of maize local populations from former Yugoslavia territories

A collection of 2178 local populations from ex-Yugoslavia territories is maintained in Maize Research Institute (MRI) gene bank. These populations were characterized mainly by morphological markers. In this work 21 local populations belonging to seven different agro-ecological groups have been subjected to SSR analysis using a DNA-pooling strategy. The objective of this work was to develop genetic fingerprints for characterization, identification and classification of the populations, as well as for estimation of their genetic diversity. Also, a DNA-pooling strategy was employed with the aim to certify if it could be applied for population analysis with SSR markers. Statistical analysis of 25 informative SSR primers revealing 224 alleles (bands) showed that the average within-population mean number of alleles was 2.55, the average values for total and within-population diversity were 0.784 and 0.502, respectively and G(ST) value was 0.360. Genetic distance values calculated using Modified Rogers' Distance were in the range from 0.35 to 0.60. The silver staining method of DNA used for bulked samples showed some weakness that could be overcome with a more sensitive staining method. Nevertheless, the results in this work indicate that the SSR analysis of bulks could be used for characterizing a large number of populations in gene banks.

Optimal DNA Pooling-Based Two-Stage Designs in Case-Control Association Studies

Study cost remains the major limiting factor for genome-wide association studies due to the necessity of genotyping a large number of SNPs for a large number of subjects. Both DNA pooling strategies and two-stage designs have been proposed to reduce genotyping costs. In this study, we propose a cost-effective, two-stage approach with a DNA pooling strategy. During stage I, all markers are evaluated on a subset of individuals using DNA pooling. The most promising set of markers is then evaluated with individual genotyping for all individuals during stage II. The goal is to determine the optimal parameters (π^p_sample, the proportion of samples used during stage I with DNA pooling; and π^p_marker, the proportion of markers evaluated during stage II with individual genotyping) that minimize the cost of a two-stage DNA pooling design while maintaining a desired overall significance level and achieving a level of power similar to that of a one-stage individual genotyping design. We considered the effects of three factors on optimal two-stage DNA pooling designs. Our results suggest that, under most scenarios considered, the optimal two-stage DNA pooling design may be much more cost-effective than the optimal two-stage individual genotyping design, which use individual genotyping during both stages.

Applying polynomial standard curve method to correct bias encountered in estimating allele frequencies using DNA pooling strategy

DNA pooling approach is a cost-saving strategy which is crucial for multiple-SNP association study and particularly for laboratories with limited budget. However, the biased allele frequency estimates cannot be completely abolished by κ correction. Using the SNaPshot™, we systematically examined the relations between actual minor allele frequencies (AMiAFs) levels and estimates obtained from the pooling process for all six types of SNPs. We applied principle of polynomial standard curves method (PSCM) to produce allele frequency estimates in pooled DNA samples and compared it with the κ method. The results showed that estimates derived from the PSCM were in general closer to AMiAFs than those from the κ method, particularly for C/G and G/T polymorphisms at the range of AMiAF between 20–40%. We demonstrated that applying PSCM in the SNaPshot™ platform is suitable for multiple-SNP association study using pooling strategy, due to its cost effectiveness and estimation accuracy.

A novel mutation in Prph2, a gene regulated by Nr2e3, causes retinal degeneration and outer-segment defects similar to Nr2e3 ( rd7/rd7 ) retinas.

The nmf193 mutant was generated by a large-scale ENU mutagenesis screen and originally described as having a dominantly inherited phenotype characterized by fundus abnormalities. We determined that nmf193 mice exhibit outer-segment defects and progressive retinal degeneration. Clinical examination revealed retinal spotting apparent at 6 weeks of age. Histologic analysis of homozygous mutant mice at 6 weeks indicated an absence of outer segments (OS) and a 50% reduction of photoreceptor cells which progressed to complete loss of photoreceptors by 10 months. Mice heterozygous for the nmf193 mutation had a less severe phenotype of shortened outer segments at 2 months with progressive loss of photoreceptor cells to 50% by 10 months. A positional cloning approach using a DNA pooling strategy was performed to identify the causative mutation in nmf193 mice. The nmf193 mutation was linked to chromosome 17 and fine mapped to an interval containing the peripherin/rds (Prph2) gene. Mutation analysis identified a single base change in Prph2 that causes aberrant splicing between exons 1 and 2. Interestingly, a comparative histologic analysis demonstrated that Prph2 ( nmf193/+ ) mutants have similar photoreceptor degeneration to that of Nr2e3 ( rd7/rd7 ). We show that Prph2 mRNA and protein levels are reduced in the Nr2e3 ( rd7/rd7 ) mutant compared to control littermates. Chromatin immunoprecipitation analysis shows that Prph2 is a direct target of NR2E3. In addition, the downregulation of Prph2 gene expression is similar in both the Nr2e3 ( rd7/rd7 ) and Prph2 ( nmf193/+ ) mutants, suggesting that the reduction of Prph2 may contribute to the degenerative pathology seen in Nr2e3 ( rd7/rd7 ).

An optimal DNA pooling strategy for progressive fine mapping

We present a cost-effective DNA pooling strategy for fine mapping of a single Mendelian gene in controlled crosses. The theoretical argument suggests that it is potentially possible for a single-stage pooling approach to reduce the overall experimental expense considerably by balancing costs for genotyping and sample collection. Further, the genotyping burden can be reduced through multi-stage pooling. Numerical results are provided for practical guidelines. For example, the genotyping effort can be reduced to only a small fraction of that needed for individual genotyping at a small loss of estimation accuracy or at a cost of increasing sample sizes slightly when recombination rates are 0.5% or less. An optimal two-stage pooling scheme can reduce the amount of genotyping to 19.5%, 14.5% and 6.4% of individual genotyping efforts for identifying a gene within 1, 0.5, and 0.1 cM, respectively. Finally, we use a genetic data set for mapping the rice xl(t) gene to demonstrate the feasibility and efficiency of the DNA pooling strategy. Taken together, the results demonstrate that this DNA pooling strategy can greatly reduce the genotyping burden and the overall cost in fine mapping experiments.

Highly cost-efficient genome-wide association studies using DNA pools and dense SNP arrays

Genome-wide association (GWA) studies to map genes for complex traits are powerful yet costly. DNA-pooling strategies have the potential to dramatically reduce the cost of GWA studies. Pooling using Affymetrix arrays has been proposed and used but the efficiency of these arrays has not been quantified. We compared and contrasted Affymetrix Genechip HindIII and Illumina HumanHap300 arrays on the same DNA pools and showed that the HumanHap300 arrays are substantially more efficient. In terms of effective sample size, HumanHap300-based pooling extracts >80% of the information available with individual genotyping (IG). In contrast, Genechip HindIII-based pooling only extracts ∼30% of the available information. With HumanHap300 arrays concordance with IG data is excellent. Guidance is given on best study design and it is shown that even after taking into account pooling error, one stage scans can be performed for >100-fold reduced cost compared with IG. With appropriately designed two stage studies, IG can provide confirmation of pooling results whilst still providing ∼20-fold reduction in total cost compared with IG-based alternatives. The large cost savings with Illumina HumanHap300-based pooling imply that future studies need only be limited by the availability of samples and not cost.

Fine mapping of pss1, a pollen semi-sterile gene in rice (Oryza sativa L.)

During routine seed increase procedures in rice, semi-sterile plants are common; however, such semi-sterility mutants in rice varieties have been only rarely analyzed genetically. W207-2 is a semi-sterile selection from the japonica rice variety Nipponbare. In this report, we found the female gamete of W207-2 was normal, and its semi-sterility was unaffected by growth duration but was conditioned by a recessive nuclear gene whose action leads to pollen semi-sterility and anther indehiscence, and the gene was named as pss1 (pollen semi-sterile). Using an F(2) population derived from the two parents W207-2 and Dular and a pooled DNA strategy, pss1 was mapped to an interval on chromosome 8 defined by the two SSR loci RM6356 and RS41. The position of pss1 was confirmed in another F(2) population derived from the cross W207-2 x Nipponbare. Over 2,000 homozygous pss1 segregants from the large W207-2 x Dular F(2) population were used to fine map pss1 to a 0.04 cM segment flanked by a CAPs marker L2 and a dCAPs L3 marker. Sequences for both markers are present on a single PAC clone, and the physical distance between them is about 28 kb. Analysis of the PAC sequence predicts the presence of five open reading frames, they are as follows: putative ribonuclease PH, putative avr9 elicitor response protein, kinesin1-like protein, putative protein RNP-D precursor and putative 40S ribosomal protein S13. This result would be helpful in cloning the pss1 gene.

The slick hair coat locus maps to chromosome 20 in Senepol‐derived cattle

The ability to maintain normal temperatures during heat stress is an important attribute for cattle in the subtropics and tropics. Previous studies have shown that Senepol cattle and their crosses with Holstein, Charolais and Angus animals are as heat tolerant as Brahman cattle. This has been attributed to the slick hair coat of Senepol cattle, which is thought to be controlled by a single dominant gene. In this study, a genome scan using a DNA-pooling strategy indicated that the slick locus is most likely on bovine chromosome 20 (BTA20). Interval mapping confirmed the BTA20 assignment and refined the location of the locus. In total, 14 microsatellite markers were individually genotyped in two pedigrees consisting of slick and normal-haired cattle (n = 36), representing both dairy and beef breeds. The maximum LOD score was 9.4 for a 4.4-cM support interval between markers DIK2416 and BM4107. By using additional microsatellite markers in this region, and genotyping in six more pedigrees (n = 86), the slick locus was further localized to the DIK4835 - DIK2930 interval.

Bovine chromosome 20: milk production QTL and candidate gene analysis in the Italian Holstein-Friesian breed

Bovine chromosome 20 (BTA20) was studied to identify QTL for milk yield and protein percentage in the Italian Holstein-Friesian breed using a selective milk DNA pooling strategy in a daughter design with sire haplotype analysis. Several QTL were identified. The effect of the known GHR F279Y and PRLR S18N mutations were in for the most part confirmed. However, it was also shown that these markers cannot explain all significant effects observed on BTA20 for the investigated traits.