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Analysis of Trace Data from Fluorescence Based Sanger Sequencing

By David John Thornley and Thomas Edison

Abstract

The processes underlying trace production in fluorescence based Sanger DNA sequencing methods are examined in detail, and modelled mathematically. It is proposed that polymerase specificity for incorporating deoxynucleotides over dideoxynucleotides is affected by a dependency on sequence local to the incorporation point. This dependency is summarised in a model which calculates numbers of sequencing fragments, and has the capability to take into account a dependency of fluorescent dye spectra on local sequence when generating fluorescence measurements from these fragment bodies. A new method for interpreting trace data is constructed which allows direct sequencing of general heterogeneous samples of DNA which may contain more than one unknown sequence. Possible modes of dependency of polymerase activity and fluorescent dye spectral emissions on local sequence are described, and practical results based on these assumptions verify their applicability. The new sequencing method hypothesises options for the sequence composition of the sample, and tests these hypotheses against the traces by generating synthetic fluorescence measurements to compare with the target trace data. Potential applications are mutation characterisation, multiple allele sequencing, and prenatal foetal examination using genetic material in the maternal bloodstream

Publisher: G
Year: 1997
OAI identifier: oai:CiteSeerX.psu:10.1.1.352.5199
Provided by: CiteSeerX
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