Sample preparation method

A method of characterising a homopolynucleotide consisting of contiguous nucleotides of a single nucleotide species, comprising:

• a) contacting the homopolynucleotide with a polymerase and a population of free nucleotides under conditions in which the polymerase forms a modified polynucleotide using the homopolynucleotide as a template for synthesizing the modified polynucleotide,
• wherein the population of free nucleotides comprises a first modified nucleotide that is not the same as the nucleotide species that is complementary to the single nucleotide species of the homopolynucleotide, and a second modified nucleotide that is not the same as the nucleotide species that is complementary to the single nucleotide species of the homopolynucleotide or the first modified nucleotide,
• wherein forming the modified polynucleotide comprises using the polymerase to replace nucleotides of the nucleotide species that is complementary to the single nucleotide species of the homopolynucleotide with (i) the first modified nucleotide, or (ii) the second modified nucleotide, such that the modified polynucleotide comprises at least one of (i) and at least one of (ii);
• b) contacting the modified polynucleotide with a transmembrane pore in the presence of an electrical potential across the transmembrane pore, such that the modified polynucleotide moves through the pore;
• and
• c) taking one or more electrical measurements as the modified polynucleotide moves through the pore wherein the measurements are indicative of one or more characteristics of the modified polynucleotide and thereby characterising the homopolynucleotide.