Pyrosequencing technology for Allele Quantification

Pyrosequencing technology uniquely presents qualitative sequence data with quantitative allele representation

Pyrosequencing technology was used to measure the relative abundance of D. melanogaster and D. simulans alleles in cDNA and DNA samples.

Because both alleles are extracted and measured in a single sample, this method is insensitive to differences in extraction efficiency and eliminates the need for 'control' genes or quantification of total RNA recovery.

(Extracted from Wittkopp PJ, Haerum BK, Clark AG. Evolutionary changes in cis and trans gene regulation. Nature, Jul 2004; 430: 85 - 88.)

Quantitative Genetic Data

100 % quantitative data

Pyrosequencing technology not only gives real sequence information, but the data is also a quantitative measure of each detected nucleotide. The data is ideal for measuring the relative amounts of alleles. This property allows the quantification of DNA methylation, heterozygosity, ploidy levels, multi-copy genes, pooled DNA samples, hematopoeitic chimerism, and mixed genotypes in heterogeneous samples (e.g. tumor and normal cells).

CpG Methylation
Based on Pyrosequencing technology's quantitative properties, assessment of DNA (CpG) methylation is more accurate, sensitive and reproducible than by any other technique. It is also simple to perform, with results available 30 minutes after PCR.

Among the numerous technologies for methylation analysis, Pyrosequencing technology represents a breakthrough by combining the processivity of PCR-based technologies with the ability to analyze all the individual CpGs of a given region.

(Extracted from Jean-Michel Dupont, Jörg Tost, Hélène Jammes, and Ivo Glynn Gut. De novo quantitative bisulfite sequencing using the Pyrosequencing technology. Analytical Biochemistry 333 (2004) 119-127.)

Automatic allele quantification

A simple click displays the raw data. Each of the two alleles is displayed as a percentage of its presence in the reaction.

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Each sample is assigned a frequency with simple color codes to indicate the confidence levels based on the data quality. Allele frequencies are calculated from the peak heights of the polymorphic position. Sample details (genotype, allele frequency and peak heights) can be displayed at any time.

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A complete statistical report is available specifically designed for pooled DNA studies. Data are grouped by specific polymorphism, showing the mean allele frequencies and standard deviations of replicate reactions.

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Under the tab Peak Heights, the numerical heights of all peaks are displayed, enabling calculation of allele statistics in a spreadsheet program for customised applications.

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