Abstract
Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.
Highlights
Population-based studies are commonly used to locate genes that underlie complex diseases in genetic association studies, which have shown to be a crucial tool for mapping complex diseases and traits
The cost of primer biotinylation per amplification is relatively high for large-scale studies. Another issue that affects the accuracy of quantitative genotyping is the intensity of sequence signal peaks generated during the incorporation of nucleotide dATP-alpha-thio that are generally 10–15% higher
We have addressed these issues by developing a robust, cost-effective, accurate and reproducible assay for largescale genotyping of DNA pools based on a combination of robotic DNA pooling, universal biotin amplification, touchdown PCR, using lower DNA concentrations, and replacing dATPalpha-thio wtih dTTP readouts by redesigning the genotyping for accurate peak uniformity
Summary
Population-based studies are commonly used to locate genes that underlie complex diseases in genetic association studies, which have shown to be a crucial tool for mapping complex diseases and traits. The cost of individual SNP genotyping has been reduced significantly, the use of DNA pooling decreases the cost even further, especially for large-scale genetic studies. Pyrosequencing [1] is a DNA sequencing method, which allows quantitative measurement of population allelic frequencies [2,3,4,5,6].
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