Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.     

Kinetic FP-TDI assay for SNP allele frequency determination

Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.     

Validation of SNP Allele Frequencies Determined by Pooled Next-Generation Sequencing in Natural Populations of a Non-Model Plant Species

Sequencing of pooled samples (Pool-Seq) using next-generation sequencing technologies has become increasingly popular, because it represents a rapid and cost-effective method to determine allele frequencies for single nucleotide polymorphisms (SNPs) in population pools. Validation of allele frequencies determined by Pool-Seq has been attempted using an individual genotyping approach, but these studies tend to use samples from existing model organism databases or DNA stores, and do not validate a realistic setup for sampling natural populations. Here we used pyrosequencing to validate allele frequencies determined by Pool-Seq in three natural populations of Arabidopsis halleri (Brassicaceae). The allele frequency estimates of the pooled population samples (consisting of 20 individual plant DNA samples) were determined after mapping Illumina reads to (i) the publicly available, high-quality reference genome of a closely related species (Arabidopsis thaliana) and (ii) our own de novo draft genome assembly of A. halleri. We then pyrosequenced nine selected SNPs using the same individuals from each population, resulting in a total of 540 samples. Our results show a highly significant and accurate relationship between pooled and individually determined allele frequencies, irrespective of the reference genome used. Allele frequencies differed on average by less than 4%. There was no tendency that either the Pool-Seq or the individual-based approach resulted in higher or lower estimates of allele frequencies. Moreover, the rather high coverage in the mapping to the two reference genomes, ranging from 55 to 284x, had no significant effect on the accuracy of the Pool-Seq. A resampling analysis showed that only very low coverage values (below 10-20x) would substantially reduce the precision of the method. We therefore conclude that a pooled re-sequencing approach is well suited for analyses of genetic variation in natural populations.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential-for example, in linkage disequilibrium analysis.     

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.     

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.     

Estimation of allele frequency in pooled DNA by using PCR–RFLP combined with microchip electrophoresis

Biallelic marker, most commonly single nucleotide polymorphism (SNP), is widely utilized in genetic association analysis, which can be speeded up by estimating allele frequency in pooled DNA instead of individual genotyping. Several methods have shown high accuracy and precision for allele frequency estimation in pools. Here, we explored PCR restriction fragment length polymorphism (PCR–RFLP) combined with microchip electrophoresis as a possible strategy for allele frequency estimation in DNA pools. We have used the commercial available Agilent 2100 microchip electrophoresis analysis system for quantifying the enzymatically digested DNA fragments and the fluorescence intensities to estimate the allele frequencies in the DNA pools. In this study, we have estimated the allele frequencies of five SNPs in a DNA pool composed of 141 previously genotyped health controls and a DNA pool composed of 96 previously genotyped gastric cancer patients with a frequency representation of 10–90% for the variant allele. Our studies show that accurate, quantitative data on allele frequencies, suitable for investigating the association of SNPs with complex disorders, can be estimated from pooled DNA samples by using this assay. This approach, being independent of the number of samples, promises to drastically reduce the labor and cost of genotyping in the initial association analysis.     

Candidate genes for non-diabetic ESRD in African Americans: a genome-wide association study using pooled DNA

African Americans have increased susceptibility to non-diabetic (non-DM) forms of end-stage renal disease (ESRD) and extensive evidence supports a genetic contribution. A genome-wide association study (GWAS) using pooled DNA was performed in 1,000 African Americans to detect associated genes. DNA from 500 non-DM ESRD cases and 500 non-nephropathy controls was quantified using gel electrophoresis and spectrophotometric analysis and pools of 50 case and 50 control DNA samples were created. DNA pools were genotyped in duplicate on the Illumina HumanHap550-Duo BeadChip. Normalization methods were developed and applied to array intensity values to reduce inter-array variance. Allele frequencies were calculated from normalized channel intensities and compared between case and control pools. Three SNPs had p values of <1.0E−6: rs4462445 (ch 13), rs4821469 (ch 22) and rs8077346 (ch 17). After normalization, top scoring SNPs (n = 65) were genotyped individually in 464 of the original cases and 478 of the controls, with replication in 336 non-DM ESRD cases and 363 non-nephropathy controls. Sixteen SNPs were associated with non-DM ESRD (p < 7.7E−4, Bonferroni corrected). Twelve of these SNPs are in or near the MYH9 gene. The four non-MYH9 SNPs that were associated with non-DM ESRD in the pooled samples were not associated in the replication set. Five SNPs that were modestly associated in the pooled samples were more strongly associated in the replication and/or combined samples. This is the first GWAS for non-DM ESRD in African Americans using pooled DNA. We demonstrate strong association between non-DM ESRD in African Americans with MYH9, and have identified additional candidate loci.     

A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples

A practical way to reduce the cost of surveying single-nucleotide polymorphism (SNP) in a large number of individuals is to measure the allele frequencies in pooled DNA samples. Pyrosequencing(TM) has been frequently used for this application because signals generated by this approach are proportional to the amount of DNA templates. The Pyrosequencing(TM) pyrogram is determined by the dispensing order of dNTPs, which is usually designed based on the known SNPs to avoid asynchronistic extensions of heterozygous sequences. Therefore, utilizing the pyrogram signals to identify de novo SNPs in DNA pools has never been undertook. Here, in this study we developed an algorithm to address this issue. With the sequence and pyrogram of the wild-type allele known in advance, we could use the pyrogram obtained from the pooled DNA sample to predict the sequence of the unknown mutant allele (de novo SNP) and estimate its allele frequency. Both computational simulation and experimental Pyrosequencing(TM) test results suggested that our method performs well. The web interface of our method is available at http://life.nctu.edu.tw/～yslin/PSM/.     

Rapid Assessment of Genetic Ancestry in Populations of Unknown Origin by Genome-Wide Genotyping of Pooled Samples

As we move forward from the current generation of genome-wide association (GWA) studies, additional cohorts of different ancestries will be studied to increase power, fine map association signals, and generalize association results to additional populations. Knowledge of genetic ancestry as well as population substructure will become increasingly important for GWA studies in populations of unknown ancestry. Here we propose genotyping pooled DNA samples using genome-wide SNP arrays as a viable option to efficiently and inexpensively estimate admixture proportion and identify ancestry informative markers (AIMs) in populations of unknown origin. We constructed DNA pools from African American, Native Hawaiian, Latina, and Jamaican samples and genotyped them using the Affymetrix 6.0 array. Aided by individual genotype data from the African American cohort, we established quality control filters to remove poorly performing SNPs and estimated allele frequencies for the remaining SNPs in each panel. We then applied a regression-based method to estimate the proportion of admixture in each cohort using the allele frequencies estimated from pooling and populations from the International HapMap Consortium as reference panels, and identified AIMs unique to each population. In this study, we demonstrated that genotyping pooled DNA samples yields estimates of admixture proportion that are both consistent with our knowledge of population history and similar to those obtained by genotyping known AIMs. Furthermore, through validation by individual genotyping, we demonstrated that pooling is quite effective for identifying SNPs with large allele frequency differences (i.e., AIMs) and that these AIMs are able to differentiate two closely related populations (HapMap JPT and CHB).     

DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia

Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.     

Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER)

A new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which is capable of detecting small allele frequency differences between two DNA pools for genetic association studies other than SNP typing. The method is based on specific primer extension reactions coupled with PPi detection. As the specificity of the primer-directed extension is not enough for quantitative SNP analysis, artificial mismatched bases are introduced into the 3′-terminal regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the primer 3′-terminus. Contamination with endogenous PPi, which produces a large background signal level in SNP analysis, was removed using PPase to degrade the PPi during the sample preparation process. It is possible to accurately and quantitatively analyze SNPs using a set of primers that correspond to the wild-type and mutant DNA segments. The termini of these primers are at the mutation positions. Various types of SNPs were successfully analyzed. It was possible to very accurately determine SNPs with frequencies as low 0.02. It is very reproducible and the allele frequency difference can be determined. It is accurate enough to detect meaningful genetic differences among pooled DNA samples. The method is sensitive enough to detect 14 amol ssM13 DNA. The proposed method seems very promising in terms of realizing a cost-effective, large-scale human genetic testing system.     

Estimation of single nucleotide polymorphism allele frequency in DNA pools by using Pyrosequencing

Positional cloning of genes underlying complex diseases, such as type 2 diabetes mellitus (T2DM), typically follows a two-tiered process in which a chromosomal region is first identified by genome-wide linkage scanning, followed by association analyses using densely spaced single nucleotide polymorphic markers to identify the causal variant(s). The success of genome-wide single nucleotide polymorphism (SNP) detection has resulted in a vast number of potential markers available for use in the construction of such dense SNP maps. However, the cost of genotyping large numbers of SNPs in appropriately sized samples is nearly prohibitive. We have explored pooled DNA genotyping as a means of identifying differences in allele frequency between pools of individuals with T2DM and unaffected controls by using Pyrosequencing technology. We found that allele frequencies in pooled DNA were strongly correlated with those in individuals (r=0.99, P<0.0001) across a wide range of allele frequencies (0.02-0.50). We further investigated the sensitivity of this method to detect allele frequency differences between contrived pools, also over a wide range of allele frequencies. We found that Pyrosequencing was able to detect an allele frequency difference of less than 2% between pools, indicating that this method may be sensitive enough for use in association studies involving complex diseases where a small difference in allele frequency between cases and controls is expected.     

Software for machine-independent quantitative interpretation of SSCP in capillary array electrophoresis (QUISCA)

PCR single-stranded conformational polymorphism (SSCP) analysis is a simple and rapid electrophoretic technique for the sensitive detection of sequence variants of PCR products. Here we describe a cross-platform program package, quantitative interpretation of SSCP in capillary array (QUISCA), which allows semi-automated quantitative detection of sequence variants separated by multicolor fluorescence-based SSCP electrophoresis using various capillary array apparatus. The program, together with the QUISCAview as a graphical user interface, takes trace data in ASCII format and processes them with three modules: signal denoising/baseline subtraction, color-matrix construction/application, and calibration of peak positions between multiple capillary runs using internal standard peaks. QUISCA is compatible with data from various widely used capillary array sequencers and is suitable not only for finding or typing SNPs in individual DNAs but also for the accurate estimation of the allele frequencies of many SNPs using a pooled DNA strategy. QUISCA can also serve as a versatile core program for various fragment analyses.     

Estimation of haplotype frequencies,linkage-disequilibrium measures,and combination of haplotype copies in each pool by use of pooled DNA data

Inference of haplotypes is important for many genetic approaches, including the process of assigning a phenotype to a genetic region. Usually, the population frequencies of haplotypes, as well as the diplotype configuration of each subject, are estimated from a set of genotypes of the subjects in a sample from the population. We have developed an algorithm to infer haplotype frequencies and the combination of haplotype copies in each pool by using pooled DNA data. The input data are the genotypes in pooled DNA samples, each of which contains the quantitative genotype data from one to six subjects. The algorithm infers by the maximum-likelihood method both frequencies of the haplotypes in the population and the combination of haplotype copies in each pool by an expectation-maximization algorithm. The algorithm was implemented in the computer program LDPooled. We also used the bootstrap method to calculate the standard errors of the estimated haplotype frequencies. Using this program, we analyzed the published genotype data for the SAA (n=156), MTHFR (n=80), and NAT2 (n=116) genes, as well as the smoothelin gene (n=102). Our study has shown that the frequencies of major (frequency >0.1 in a population) haplotypes can be inferred rather accurately from the pooled DNA data by the maximum-likelihood method, although with some limitations. The estimated D and D' values had large variations except when the /D/ values were >0.1. The estimated linkage-disequilibrium measure rho2 for 36 linked loci of the smoothelin gene when one- and two-subject pool protocols were used suggested that the gross pattern of the distribution of the measure can be reproduced using the two-subject pool data.     

Empirical Validation of Pooled Whole Genome Population Re-Sequencing in Drosophila melanogaster

The sequencing of pooled non-barcoded individuals is an inexpensive and efficient means of assessing genome-wide population allele frequencies, yet its accuracy has not been thoroughly tested. We assessed the accuracy of this approach on whole, complex eukaryotic genomes by resequencing pools of largely isogenic, individually sequenced Drosophila melanogaster strains. We called SNPs in the pooled data and estimated false positive and false negative rates using the SNPs called in individual strain as a reference. We also estimated allele frequency of the SNPs using "pooled" data and compared them with "true" frequencies taken from the estimates in the individual strains. We demonstrate that pooled sequencing provides a faithful estimate of population allele frequency with the error well approximated by binomial sampling, and is a reliable means of novel SNP discovery with low false positive rates. However, a sufficient number of strains should be used in the pooling because variation in the amount of DNA derived from individual strains is a substantial source of noise when the number of pooled strains is low. Our results and analysis confirm that pooled sequencing is a very powerful and cost-effective technique for assessing of patterns of sequence variation in populations on genome-wide scales, and is applicable to any dataset where sequencing individuals or individual cells is impossible, difficult, time consuming, or expensive.     

Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds

We propose the use of single nucleotide polymorphisms (SNPs) instead of polymorphic microsatellite markers for individual identification and parentage control in cattle. To this end, we present an initial set of 37 SNP markers together with a gender-specific SNP for identity control and parentage testing in the Holstein, Fleckvieh and Braunvieh breeds. To obtain suitable SNPs, a total of 91.13 kb of random genomic DNA was screened yielding 531 SNPs. These, and 43 previously identified SNPs, were subjected to the following selection criteria: (1) the frequency of the minor allele must be larger than 0.1 in at least two of the three examined breeds, and (2) markers should not be linked closely. Allele frequencies were estimated by analysing sequencing traces of pooled DNA or by genotyping individual DNA samples. The selected SNP loci were physically mapped by radiation hybrid mapping or by fluorescence in situ hybridization, and tested against the neutral mutation hypothesis. The presented marker set theoretically allows probabilities of identity less than 10(-13) for individual verification and exclusion powers exceeding 99.99% for parentage testing.     

DNA Pooling Base Genome-Wide Association Study Identifies Variants at NRXN3 Associated with Delayed Encephalopathy after Acute Carbon Monoxide Poisoning

Delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) is more characteristic of anoxic encephalopathy than of other types of anoxia. Those who have the same poisoning degree and are of similar age and gender have a greater risk of getting DEACMP. This has made it clear that there are obvious personal differences. Genetic factors may play a very important role. The authors performed a genome-wide association study involving pooling of DNA obtained from 175 patients and 244 matched acute carbon monoxide poisoning without delayed encephalopathy controls. The Illumina HumanHap 660 Chip array was used for DNA pools. Allele frequencies of all SNPs were compared between delayed encephalopathy after acute carbon monoxide poisoning and control groups and ranked. A total of 123 SNPs gave an OR >1.4. Of these, 46 mapped in or close to known genes. Forty-eight SNPs located in 19 genes were associated with DEACMP after correction for 5% FDR in the genome-wide association of pooled DNA. Two SNPs (rs11845632 and rs2196447) locate in the Neurexin 3 gene were selected for individual genotyping in all samples and another cohort consisted of 234 and 271 controls. There were significant differences in the genotype and allele frequencies of rs11845632 and rs2196447 between the DEACMP group and controls group (all P-values <0.05). This study describes a positive association between Neurexin 3 and controls in the Han Chinese population, and provides genetic evidence to support the susceptibility of DEACMP, which may be the resulting interaction of environmental and genetic factors.