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 stud...

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Bibliographic Details
Published in:PloS one 2008-07, Vol.3 (7), p.e2693-e2693
Main Authors: Doostzadeh, Julie, Shokralla, Shadi, Absalan, Farnaz, Jalili, Roxana, Mohandessi, Sharareh, Langston, James W, Davis, Ronald W, Ronaghi, Mostafa, Gharizadeh, Baback
Format: Article
Language:English
Subjects:
Accuracy
Alleles
Automation
Biotechnology
Biotin
Biotin - chemistry
Case-Control Studies
Chemical synthesis
Deoxyribonucleic acid
Diphosphates - chemistry
DNA
DNA Mutational Analysis - methods
DNA Primers - chemistry
DNA sequencing
Frequency analysis
Frequency estimation
Gene Frequency
Gene sequencing
Genetic Techniques
Genetic testing
Genetics and Genomics/Population Genetics
Genomes
Genotype
Genotyping
Humans
Molecular Biology - methods
Parkinson Disease - genetics
Population genetics
Population studies
Public Health and Epidemiology/Screening
Reproducibility of Results
Sampling error
Sequence Analysis, DNA - methods
Studies
Technology application
Work stations
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Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0002693