Nucleic acid sequencing

A method for determining a plurality of target nucleic acid sequences, which method comprises the steps of:

• (a) contacting a preparation wherein the plurality of target nucleic acid sequences is comprised in the preparation which further comprises a plurality of corresponding non-target nucleic acid sequences, each target nucleic acid sequence in the preparation corresponds to one or more corresponding non-target nucleic acid sequences in the preparation, each target nucleic acid sequence and each corresponding non-target nucleic acid sequence has a first region of common sequence upstream of a first region of dissimilar sequence upstream of a second region of dissimilar sequence, the first region of common sequence of each target nucleic acid sequence is the same as the first region of common sequence of its corresponding non-target nucleic acid sequences, the first region of dissimilar sequence of each target nucleic acid sequence is different to the first region of dissimilar sequence of its corresponding non-target nucleic acid sequences, the second region of dissimilar sequence of each target nucleic acid sequence is different to the second region of dissimilar sequence of its corresponding-non-target nucleic acid sequences, —with a plurality of oligonucleotide primers, wherein each primer is complementary to at least a portion of the first region of common sequence of a target nucleic acid sequence and its corresponding non-target nucleic acid sequence, under conditions to hybridize the primer thereto;
• and
• (b) contacting the resulting preparation with a plurality of first labeled nucleotides wherein each first labeled nucleotide bears a different first label, wherein each first labeled nucleotide is complementary to a first template nucleotide comprised in the first region of dissimilar sequence of a target nucleic acid under conditions to incorporate the first labeled nucleotide into the primer hybridized to the target nucleic acid sequence;
• (c) subjecting the so treated preparation to a sequencing reaction, thereby extending each primer to form one or more first-labeled sequencing products comprising a first labeled nucleotide and one or more non-first-labeled sequencing products comprising no first labeled nucleotide;
• and
• (d) determining at least a portion of the sequence of each different first-labeled sequencing product, thereby determining at least the second region of dissimilar sequence of each target nucleic acid sequences.

A method of determining the haplotype of a subject comprising the steps of:

• providing a sample comprising DNA from the subject to sequence a target locus on a first chromosome of a chromosome pair and a non-target locus on the second chromosome of the chromosome pair, wherein the target locus and the non-target locus each comprise two or more single nucleotide polymorphisms, and wherein the subject is heterozygous at each single nucleotide polymorphism;
• providing a primer that hybridizes to the target locus and the non-target locus upstream of the first single nucleotide polymorphism;
• providing a first labeled nucleotide that is complementary to the nucleotide of the first single nucleotide polymorphism in either the target locus or the non-target locus wherein the label is a member of a ligand affinant pair;
• contacting the sample, the primer and the first labeled nucleotide under conditions to hybridize the primer to the target locus and the non-target locus and to incorporate the first labeled nucleotide into the primer hybridized to either the target locus or the non-target locus, but not both;
• separating the target locus/primer complex from the non-target locus/primer complex using the incorporated first labeled nucleotide;
• extending the primer of the target/locus primer complex and/or the non-target locus primer complex in a pyrophosphate sequencing reaction to thereby determining a haplotype of the subject.