Pyrosequencing

Ulrich Lehmann

Pyrosequencing(®) offers the opportunity to quantify the amount of nucleotides incorporated during the sequencing reaction. This enables the precise and sensitive quantification of DNA methylation, allele-specificexpression, or mutation load. However, only a few studies addressed the question how precise and sensitive Pyrosequencing really is. Many publications claiming precise measurement (with two decimal places) fail to demonstrate performance figures like precision or reproducibility. And most publications reporting the detection (and quantification) of very weak signals by Pyrosequencing do omit to report the technical sensitivity of the assay used (i.e., limit of blank, limit of detection, limit of quantification).Therefore, this chapter aims at providing (1) an introduction to important basic concepts defining quantitative analytical assays, (2) an outline of a minimal set of validation measurements every study employing Pyrosequencing should include, especially if values close to the analytical threshold are reported.