Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using Pyrosequencing™

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Abstract
Identifying the genetic variation underlying complex disease requires analysis of many single nucleotide polymorphisms (SNPs) in a large number of samples. Several high-throughput SNP genotyping techniques are available; however, their cost promotes the use of association screening with pooled DNA. This protocol describes the estimation of SNP allele frequencies in pools of DNA using the quantitative sequencing method Pyrosequencing™ (PSQ™). PSQ is a relatively recently described high-throughput method for genotyping, allele frequency estimation and DNA methylation analysis based on the detection of real-time pyrophosphate release during synthesis of the complementary strand to a PCR product. The protocol involves the following steps: (i) quantity and quality assessment of individual DNA samples; (ii) DNA pooling, which may be undertaken at the pre- or post-PCR stage; (iii) PCR amplification of PSQ template containing the variable sequence region of interest; and (iv) PSQ to determine the frequency of alleles at a particular SNP site. Once the quantity and quality of individual DNA samples has been assessed, the protocol usually requires a few days for setting up pre-PCR pools, depending on sample number. After PCR amplification, preparation and analysis of PCR amplicon by PSQ takes 1 h per plate.