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Journal ArticleDOI

Evaluation of a New Pooling Strategy Based on Leukocyte Count for Rapid Quantification of Allele Frequencies

01 May 2007-Clinical Chemistry (American Association for Clinical Chemistry)-Vol. 53, Iss: 5, pp 980-982

TL;DR: DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples and the leukocytes approach is much less tedious, quicker, and less expensive.

AbstractBackground: Allele frequencies of single-nucleotide polymorphisms (SNPs) can be quantified from DNA pools. The conventional preparation of DNA pools requires DNA isolation and quantification for each blood sample. We hypothesized that pooling of whole blood samples according to their leukocyte count, which determines DNA content, would be as reliable as the conventional pooling method but much less tedious to perform. Methods: We collected 100 whole blood samples and measured the leukocyte count. Samples were frozen until further use. After thawing, pools were generated by combining aliquots containing an equal number of leukocytes. In parallel, DNA was extracted from another aliquot, DNA concentration was measured, and DNA concentration-based pools were assembled. All original samples were genotyped directly using 4 different SNP assays to obtain the exact allele frequencies in the pool. In addition, samples of known genotypes were mixed according to the DNA concentration or the leukocyte count to generate artificial samples of known allele frequencies. We analyzed pools and mixes in triplicate by pyrosequencing and calculated allelic frequencies. Results: Leukocyte and DNA pooling provided equally accurate and precise SNP frequencies comparable to published data. Conclusion: DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples. The leukocyte pooling approach is much less tedious, quicker, and less expensive. It should be always considered if leukocyte counts are available.

Topics: DNA extraction (53%)

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Citations
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Journal ArticleDOI
TL;DR: The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.
Abstract: Context: Mutations in the four subunits of succinate dehydrogenase (SDH) are the cause for the hereditary paraganglioma (PGL) syndrome types 1–4 and are associated with multiple and recurrent pheochromocytomas and PGLs. SDHC mutations most frequently result in benign, nonfunctional head-and neck PGLs (HNPGLs). The malignant potential of SDHC mutations remains unclear to date. Objectives: We report a patient with malignant PGL carrying a SDHC mutation and compare her case with two others of the same genotype but presenting with classic benign HNPGLs. Loss of heterozygosity (LOH) was demonstrated in the malignant PGL tissue. Design: In three unrelated patients referred for routine genetic testing, SDHB, SDHC, and SDHD genes were sequenced, and gross deletions were excluded by multiplex ligation-dependent probe amplification (MLPA). LOH was determined by pyrosequencing-based allele quantification and SDHB immunohistochemistry. Results: In a patient with a nonfunctioning thoracic PGL metastatic to the bone, t...

20 citations


Cites methods from "Evaluation of a New Pooling Strateg..."

  • ...Arg133*) in the SDHC gene by pyrosequencing-based allele quantification as described previously (12)....

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Journal ArticleDOI
TL;DR: It is shown that G- quadruplex- or i-motif-like sequences can reproducibly cause ADO, and PCR products should be checked for G-quadruplex and i-Motifs to avoid the formation of ADO-causing secondary structures.
Abstract: Background: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex– or i-motif–like sequences on the reliability of PCR-based genetic analyses. Methods: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures. Results: We showed that G-quadruplex– or i-motif–like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected. Conclusions: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem. .

19 citations


References
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Journal ArticleDOI
TL;DR: Recent developments in quantitative genotyping assays and in the design and analysis of pooling studies are discussed.
Abstract: DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.

552 citations


Journal ArticleDOI
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.

285 citations


Journal ArticleDOI
TL;DR: This work has developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals), which allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.
Abstract: Individual genotyping of single nucleotide polymorphisms (SNPs) remains expensive, especially for linkage disequilibrium mapping strategies involving high-throughput SNP genotyping On one hand, current methods may suit scientific and laboratory needs in regard to accuracy, reproducibility/robustness, and large-scale application On the other hand, a cheaper and less time-consuming alternative to individual genotyping is the use of SNP allele frequencies determined in DNA pools We have developed an accurate and reproducible protocol for allele frequency determination using PyrosequencingTM technology in large genomic DNA pools (374 individuals) The measured correlation (R2) in large DNA pools was 0980 In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (eg, type 2 diabetes and obesity) and control groups detected by either individual genotyping or Pyrosequencing of DNA pools In large pools, the variation between the two methods was 15 ± 09% I

76 citations


Journal ArticleDOI
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.
Abstract: We have compared several genotyping methods to assess their applicability to single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools. The accuracy of these methods (restriction fragment length polymorphism, real-time pyrophosphate DNA sequencing, single base extension with fluorescently labeled ddNTPs, homogeneous 5'-nuclease assay, and MALDI-TOF mass spectrometry) was tested by calculating the standard deviation among heterozygous individuals (which are natural DNA pools with 50% representation of each allele) and by estimating allele frequency in artificial pools. We show that although the methods differ in their accuracy, they can all serve for quantification of allele frequency in DNA pools with reasonable accuracy. We found that the influence of the error variance attributed to pool construction on quantification accuracy is insignificant and is SNP dependent.

56 citations


Journal ArticleDOI
TL;DR: Pyrosequencing™ can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios with accuracy and precision values in ranges comparable with those of other SNP typing techniques.
Abstract: Association screening involving numerous genetic markers is facilitated by the analysis of pooled DNA samples rather than individual samples. Several genotyping methods have shown high accuracy and precision of allele frequency estimation in pools. Here, we expand the validation of SNP allele frequency estimation in DNA pools using Pyrosequencing by analyzing 186 pools for three SNPs representing complex sequencing cases. The correlation coefficient between estimated and true allele frequencies ranged between 0.979 and 0.996 and tended to increase with pool size, whereas the difference between estimated and true allele frequencies was 2.37+/-0.11%, in post-PCR pools. The precision was 1.73%. Pool size had no significant effect on accuracy and precision. A comparison between post-PCR and pre-PCR pools showed that for pre-PCR pooling efforts to accurately quantify the genomic DNA samples to be pooled and subsequently amplified are critical. To conclude, Pyrosequencing can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios with accuracy and precision values in ranges comparable with those of other SNP typing techniques. Considering the ease of use, short run and analysis times, and little instrument maintenance requirements, Pyrosequencing may even be a preferred option.

50 citations