Simple algorithm for quantifying polymorphisms in electropherograms

In a method to quantify DNA methylation at a particular site from bisulfite genomic sequencing using data from four-dye-trace trace value electropherograms from fluorescent dye terminator sequencing, an improvement comprising:

• selecting a target CpG site;
• determining the mean T area (T bar) from a plurality of Ts surrounding the target CpG site;
• subtracting the area of the T at the target CpG site from T bar,
• wherein the subtracting yields delta T;
• and
• calculating the level of methylation on the site as the proportion of (delta T)/(T bar).

An algorithm for quantifying DNA methylation at a particular site from bisulfite genomic sequencing using data from four-dye-trace trace value electropherograms from fluorescent dye terminator sequencing, comprising:

• choosing the target CpG site;
• determining the mean T area (T bar) from a plurality of Ts surrounding the target CpG site.
• subtracting the area of the T at the target CpG site from T bar to yield delta T (i.e. T bar−CpG T=delta T);
• and
• calculating the level of methylation on the site as the proportion, (delta T)/(T bar).

An algorithm for quantifying DNA methylation at a particular site from bisulfite genomic sequencing using data from four-dye-trace trace value electropherograms from fluorescent dye terminator sequencing, comprising:

• choosing the target CpG site;
• determining the mean T area (T bar) from 10 Ts surrounding the target CpG site
• wherein Ts used to calculate T bar are at least 10 times the area of their secondary base (C, G or A),
• wherein the Ts used to calculate T bar have a T signal-to-noise ratio of 10 or better with respect to their secondary peak, and
• wherein an equal number of T's from each side of the target CpG (i.e. 5 on each side) are used;
• subtracting the area of the T at the target CpG site from T bar to yield delta T (i.e. T bar−CpG T=delta T);
• and
• calculating the level of methylation on the site as the proportion, (delta T)/(T bar), or the percentage, 100×(delta T)/(T bar).

An algorithm for quantifying the level of a nucleotide signal at a polymorphic site in a sequencing electropherogram using trace value data, comprising:

• choosing the target polymorphic site and nucleotide (N1);
• choosing a second nucleotide (N2) at the polymorphic site (N2p);
• determining the mean area (N2 bar) from a plurality of examples of the second nucleotide surrounding the target polymorphic site;
• subtracting the area of the second nucleotide at the target polymorphic site from N2 bar to yield delta N2 (i.e. N2 bar−N2p=delta N2);
• and
• calculating the level of N1 at the polymorphic site as the proportion, (delta N2)/(N2 bar).