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.

Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification

“…However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations, which occur when two or more different allele combinations produce identical consensus sequences or if alleles are defined by a polymorphism outside the regions analyzed. Although handicapped by short sequence lengths, pyrosequencing has often been used for genotyping and allele frequency analysis [23,24], as well as in HLA typing [8].…”

THE INTENSITY OF SELECTION ACTING ON THECOUCH POTATOGENE-SPATIAL-TEMPORAL VARIATION IN A DIAPAUSE CLINE

“…We used pyrosequencing in the bulk DNA preps to estimate SNP frequency (Doostzadeh et al 2008;Lavebratt and Sengul 2006). Fifteen polymorphic sites, all in exons in cpo-RV transcript, were screened by bulk pyrosequencing (Fig.…”

A rapid and cost‐effective quantitative microsatellite genotyping protocol to estimate intraspecific competition in protist microcosm experiments

“…Gruber, Colligan & Wolford ; Lavebratt & Sengul ; Doostzadeh et al . ) because of convenient genotyping methodology and as SNPs are the most abundant genetic marker in the genome (Schlotterer ). For model protists where whole‐genome data are available for multiple strains (e.g.…”