title: High throughput automated allele frequency estimation by pyrosequencing.
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.
authors.affiliation: The Parkinson's Institute, Sunnyvale, California, United States of America.
authors.forename: Julie
authors.initials: J
authors.lastname: Doostzadeh
authors.forename: Shadi
authors.initials: S
authors.lastname: Shokralla
authors.forename: Farnaz
authors.initials: F
authors.lastname: Absalan
authors.forename: Roxana
authors.initials: R
authors.lastname: Jalili
authors.forename: Sharareh
authors.initials: S
authors.lastname: Mohandessi
authors.forename: James W
authors.initials: JW
authors.lastname: Langston
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ronaghi
authors.forename: Baback
authors.initials: B
authors.lastname: Gharizadeh
mesh_terms: 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*
chemical_list: D017931:DNA Primers; D011756:Diphosphates; D001710:Biotin