Quantitative technologies for allele frequency estimation of SNPs in DNA pools
TL;DR: It is found that the influence of the error variance attributed to pool construction on quantification accuracy is insignificant and is SNP dependent, while the methods can all serve for quantification of allele frequency in DNA pools with reasonable accuracy.
About: This article is published in Molecular and Cellular Probes.The article was published on 2002-12-01. It has received 56 citations till now. The article focuses on the topics: Allele frequency & Restriction fragment length polymorphism.
Citations
More filters
TL;DR: A general description of SNP typing protocols and a summary of current methods for each step of the protocol are pointed out, pointing out the unique features and weaknesses of these techniques as well as comparing the cost and throughput structures of the technologies.
Abstract: The large number of single nucleotide polymorphism (SNP) markers available in the public databases makes studies of association and fine mapping of disease loci very practical. To provide information for researchers who do not follow SNP genotyping technologies but need to use them for their research, we review here recent developments in the fields. We start with a general description of SNP typing protocols and follow this with a summary of current methods for each step of the protocol and point out the unique features and weaknesses of these techniques as well as comparing the cost and throughput structures of the technologies. Finally, we describe some popular techniques and the applications that are suitable for these techniques.
304 citations
TL;DR: Cis-acting eQTL serve as an important new resource for the identification of positional candidates in QTL studies in mice and the analysis of the correlation structures between genotypes, gene expression traits, and phenotypic traits is used to further characterize genes expressed in liver that are under cis-acting control.
Abstract: We previously reported the analysis of genome-wide expression profiles and various diabetes-related traits in a segregating cross between inbred mouse strains C57BL/6J (B6) and DBA/2J (DBA). By considering transcript levels as quantitative traits, we identified several thousand expression quantitative trait loci (eQTL) with LOD score >4.3. We now experimentally address the problem of multiple comparisons by estimating the fraction of false-positive eQTL that are under cis-acting regulation. For this, we have utilized a classic cis–trans test with (B6 × DBA)F1 mice to determine the relative levels of transcripts from the B6 and DBA alleles. The results suggest that at least 64% of cis-acting eQTL with LOD >4.3 are true positives, while the remaining 36% could not be confirmed as truly cis-acting. Moreover, we find that >96% of apparent cis-acting eQTL occur in regions that do not share SNP haplotypes. Cis-acting eQTL serve as an important new resource for the identification of positional candidates in QTL studies in mice. Also, we use the analysis of the correlation structures between genotypes, gene expression traits, and phenotypic traits to further characterize genes expressed in liver that are under cis-acting control, and highlight the advantages and disadvantages of integrating genetics and gene expression data in segregating populations.
294 citations
Cites background or result from "Quantitative technologies for allel..."
...Previous studies have shown comparable results and accurate allelic quantification using primer extension by Pyrosequencing, single-base extention, and RFLP analysis (Shifman et al. 2002)....
[...]
...Correlation patterns for genes with strong cis-acting eQTL and their experimental value Strong cis-acting eQTL can obscure the true relationship between genes....
[...]
TL;DR: A variety of the molecular markers used to physically map genomes have now been successfully adapted for detection of food substitution, and successes include the speciation of meats, fish and fruit in processed food products, the identification of the geographical origin of olive oil and the detection of dilution of Basmati rice with non-Basmati varieties.
291 citations
TL;DR: A review of the applications of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry has been presented in this article.
Abstract: Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.
287 citations
TL;DR: Arrays of haplotype-tagged candidate genes, such as this addictions-focused array, represent a cost-effective approach to generate high-quality SNP genotyping data useful for the haplotypes-based analysis of panels of genes such as these 130 genes of interest to alcohol and addictions researchers.
Abstract: Aims: To develop a panel of markers able to extract full haplotype information for candidate genes in alcoholism, other addictions and disorders of mood and anxiety. Methods: A total of 130 genes were haplotype tagged and genotyped in 7 case/control populations and 51 reference populations using Illumina GoldenGate SNP genotyping technology, determining haplotype coverage. We also constructed and determined the efficacy of a panel of 186 ancestry informative markers. Results: An average of 1465 loci were genotyped at an average completion rate of 91.3%, with an average call rate of 98.3% and replication rate of 99.7%. Completion and call rates were lowered by the performance of two datasets, highlighting the importance of the DNA quality in high throughput assays. A comparison of haplotypes captured by the Addictions Array tagging SNPs and commercially available whole-genome arrays from Illumina and Affymetrix shows comparable performance of the tag SNPs to the best whole-genome array in all populations for which data are available. Conclusions: Arrays of haplotype-tagged candidate genes, such as this addictions-focused array, represent a cost-effective approach to generate high-quality SNP genotyping data useful for the haplotype-based analysis of panels of genes such as these 130 genes of interest to alcohol and addictions researchers. The inclusion of the 186 ancestry informative markers allows for the detection and correction for admixture and further enhances the utility of the array.
230 citations
Cites background from "Quantitative technologies for allel..."
...The relatively high per sample cost and the requirement for large numbers of cases and control subjects to identify alleles of modest effect size with associations that are able to withstand correction for multiple testing, make the widespread use of this approach impractical and financially burdensome for many research groups unless pooling approaches are adopted (Shifman et al., 2002; Liu et al., 2006; Johnson et al., 2006)....
[...]
...…effect size with associations that are able to withstand correction for multiple testing, make the widespread use of this approach impractical and financially burdensome for many research groups unless pooling approaches are adopted (Shifman et al., 2002; Liu et al., 2006; Johnson et al., 2006)....
[...]
References
More filters
TL;DR: These issues are discussed, together with suggestions for optimal post-genome strategies, which are being presented for unravelling the complex genetic basis of non-mendelian disorders based on large-scale genome-wide studies.
Abstract: Human genetics is now at a critical juncture. The molecular methods used successfully to identify the genes underlying rare mendelian syndromes are failing to find the numerous genes causing more common, familial, non-mendelian diseases. With the human genome sequence nearing completion, new opportunities are being presented for unravelling the complex genetic basis of non-mendelian disorders based on large-scale genome-wide studies. Considerable debate has arisen regarding the best approach to take. In this review I discuss these issues, together with suggestions for optimal post-genome strategies.
1,960 citations
TL;DR: By providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
Abstract: We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
439 citations
TL;DR: PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically are described.
Abstract: To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5′ nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11–β hydroxylase gene. The genotyping method is accurate—we estimate an error rate of fewer than 1 in 2000 genotypes, rapid—with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible—a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three “pseudo-SNPs” (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.
381 citations
TL;DR: This work addresses the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System using pyrosequencing, a real-time pyrophosphate detection technology and provides extensive flexibility in regard to the positioning of sequencing primers.
Abstract: The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.
288 citations
TL;DR: Use of pooled DNA amplifications is reported for the accurate determination of marker-disease associations for both case-control and nuclear family-based data, including application of correction methods for stutter artifact and preferential amplification.
Abstract: Genomic screening to map disease loci by association requires automation, pooling of DNA samples, and 3,000-6,000 highly polymorphic, evenly spaced microsatellite markers. Case-control samples can be used in an initial screen, followed by family-based data to confirm marker associations. Association mapping is relevant to genetic studies of complex diseases in which linkage analysis may be less effective and to cases in which multigenerational data are difficult to obtain, including rare or late-onset conditions and infectious diseases. The method can also be used effectively to follow up and confirm regions identified in linkage studies or to investigate candidate disease loci. Study designs can incorporate disease heterogeneity and interaction effects by appropriate subdivision of samples before screening. Here we report use of pooled DNA amplifications-the accurate determination of marker-disease associations for both case-control and nuclear family-based data-including application of correction methods for stutter artifact and preferential amplification. These issues, combined with a discussion of both statistical power and experimental design to define the necessary requirements for detecting of disease loci while virtually eliminating false positives, suggest the feasibility and efficiency of association mapping using pooled DNA screening.
220 citations