Procédé pour la différentiation de variante utilisant une commande d'exception de pyro-séquençage

Method for variant differentiation utilizing a pyrosequencing dispensation order

Verfahren zur Variantendifferenzierung unter Benutzung einer pyrosequenzierenden Abgabereihenfolge

Abstract

The present invention relates to a method for differentiating between at least two sequence variants of a polynucleotide by pyrosequencing, wherein at least one sequence variant comprises at least one mutation, said method comprising a) generating a dispensation order based on a nucleotide sequence encompassing the nucleotides affected by said variants, said dispensation order comprising dispensations corresponding to each nucleotide occurring in at least one of the variants, said nucleotide dispensations ordered in a sequence i) generating on average a pyrogram peak for at least every second nucleotide dispensation for each variant and ii) generating the maximal possible number of sequence pyrogram peaks for each variant; b) generating according to the dispensation order of a) a first pyrogram comprising expected intensity values for a first variant and at least a second pyrogram comprising expected intensity values for at least one further variant; c) comparing a sample pyrogram comprising measured intensity values, said sample pyrogram being derived from pyrosequencing analysis of a sample polynucleotide according to the nucleotide dispensation order of a), with the said first and at least the said second pyrogram generated in step b); and d) differentiating between said at least two sequence variants, wherein the sample polynucleotide corresponds to the first variant if the sample pyrogram is essentially identical with the first pyrogram and wherein the sample polynucleotide corresponds to a further variant if the sample pyrogram is essentially identical with a further pyrogram.

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