Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.
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 allelefrequencies determined in DNA pools. We have developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals). The measured correlation (R2) in large DNA pools was 0.980. In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (e.g., 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 1.5 +/- 0.9%. It may be concluded that the allele frequency determination protocol could reliably detect over 4% differences between populations. The method is economical in regard to amounts of DNA, PCR, and primer extension reagents required. Furthermore, it allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.
Neve
B
B
Genetics of Multifactorial Diseases, CNRS UPRES A 8090, Institut Biologie de Lille, Institut Pasteur de Lille, France. bernadette,neve@mail-good
Froguel
P
P
Corset
L
L
Vaillant
E
E
Vatin
V
V
Boutin
P
P
D003920:Diabetes Mellitus / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics; D005787:Gene Frequency / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009765:Obesity; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards