SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis

Nucleic Acids Research, Aug 2002

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.

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SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis

Ste phanie Le Hellard 2 Ste phane J. Ballereau 2 Peter M. Visscher 1 Helen S. Torrance 2 Jeni Pinson 2 Stewart W. Morris 2 Marian L. Thomson 2 Colin A. M. Semple 0 2 Walter J. Muir 3 Douglas H. R. Blackwood 3 David J. Porteous 2 Kathryn L. Evans 2 0 MRC Human Genetics Unit, Western General Hospital , Crewe Road, Edinburgh EH4 2XU, UK 1 Institute of Cell, Animal and Population Biology, University of Edinburgh , West Mains Road, Edinburgh EH9 3JT, UK 2 Medical Genetics Section, Molecular Medicine Centre, University of Edinburgh, Western General Hospital , Crewe Road, Edinburgh EH4 2XU, UK 3 Department of Psychiatry, University of Edinburgh, Royal Edinburgh Hospital , Edinburgh EH10 5HF, UK We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies. - Single nucleotide polymorphisms (SNPs) are the most common type of polymorphism in the human genome, with an approximate frequency of one every kilobase (1). These biallelic variants are relatively easy to genotype compared with VNTRs and microsatellites. For these reasons SNPs are thought to have a promising future in a wide range of human genetics applications including pharmacogenomics, the study of population evolution, analysis of forensic samples and the identification of susceptibility genes involved in complex diseases. Hence, a large proportion of the effort of genome centres is now focused on the identification and the mapping of a large collection of SNPs: to date about 1 260 000 have been mapped onto the human draft sequence (http://snp. cshl.org/). The study of complex common diseases and quantitative traits is confounded by the effects of disease heterogeneity, genegene and geneenvironment interactions. This means that large numbers of SNPs must be surveyed in large numbers of individuals in order to detect single gene variants with a small to moderate effect size (2,3). The use of pooled samples, comprised of equal amounts of genomic DNA from up to 1000 individuals, has been proposed as a means of reducing the number of genotyping reactions required. The method used to genotype SNPs in pooled DNAs must provide accurate estimates of allele frequencies, and must be time and cost effective. The spectra of methods currently available for genotyping SNPs in individual samples [for an extensive review of SNP genotyping methods see Syvanen (4)] can be divided into three classes. First, methods such as SSCP or dHPLC that are based on the physicalchemical properties of the alleles. Secondly, methods such as TAQMAN (Applied Biosystems); oligo-ligation assay; Invader assay (Third Wave Technologies Inc.); and allele-specific amplification and padlock probes that are based on hybridisation, amplification or ligation of an allele-specific probe. Thirdly, methods based on allele-specific extension or minisequencing from a primer adjacent to the site of the SNP such as SNaPshot (Applied Biosystems); primer extension read by dHPLC or by mass spectrometry; primer extension performed on microarrays; fluorescence polarisation; bioluminometric assay coupled with modified primer extension reactions (BAMPER) and Pyrosequencing (Pyrosequencing). Previous studies have shown that allelic frequencies can be accurately estimated from pools using primer extension followed by dHPLC (5); TAQMAN and RFLP analysis (6); allele-specific amplification with real-time PCR (7); SSCP (8); BAMPER (9) and MassARRAY (10). In common with many other groups, we wish to screen a large candidate region for evidence of genetic association. The preferred strategy is to assay small numbers of pooled DNA samples with large numbers of SNPs. Consequently, methods such as Pyrosequencing , TAQMAN or BAMPER that use modified primers are too expensive. Methods based on hybridisation or on physicalchemical properties are ruled out as each assay must be optimised. We therefore chose to compare the robustness, accuracy and cost of three methods based on minisequencing: SNaPshot (Applied Biosystems) and primer extension followed either by dHPLC, or mass spectrometry (MassARRAY system by Sequenom). We have a (...truncated)


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Stéphanie Le Hellard, Stéphane J. Ballereau, Peter M. Visscher, Helen S. Torrance, Jeni Pinson, Stewart W. Morris, Marian L. Thomson, Colin A. M. Semple, Walter J. Muir, Douglas H. R. Blackwood, David J. Porteous, Kathryn L. Evans. SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis, Nucleic Acids Research, 2002, pp. e74-e74, 30/15, DOI: 10.1093/nar/gnf070