High throughput automated allele frequency estimation by pyrosequencing.
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.
Doostzadeh
Julie
J
The Parkinson's Institute, Sunnyvale, California, United States of America.
Shokralla
Shadi
S
Absalan
Farnaz
F
Jalili
Roxana
R
Mohandessi
Sharareh
S
Langston
James W
JW
Davis
Ronald W
RW
Ronaghi
Mostafa
M
Gharizadeh
Baback
B
D000483:Alleles; D001331:Automation; D001710:Biotin / Q000737:chemistry; D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry*; D005787:Gene Frequency; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D010300:Parkinson Disease / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D011756:Diphosphates; D001710:Biotin