Digital analyte analysis
The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.
1. A method for analyzing a nucleic acid template in a biological sample, the method comprising the steps of:
forming a plurality of microdroplets, each comprising a nucleic acid template and a plurality of sets of primer pairs, each set of primer pairs being specific for a different target site on a different nucleic acid template, binding only a single primer pair to the target site on the nucleic acid template in each microdroplet;
amplifying a single target site from the nucleic acid template in each of the microdroplets using one of the sets of primer pairs specific for the single target site to generate amplification products;
attaching a sequencing adaptor and a barcode sequence to the amplification products; and
sequencing the amplification products, thereby to analyze the nucleic acid template.
2. The method according to claim 1 , wherein the amplification products are attached to a bead.
3. The method according to claim 2 , wherein the amplification products are attached to a surface.
4. The method according to claim 3 , wherein the surface is a surface of a flow cell.
5. The method according to claim 3 , wherein the amplification products are covalently attached to the surface.
6. The method according to claim 1 , wherein sequencing is sequencing-by-synthesis.
7. The method according to claim 6 , wherein sequencing-by-synthesis is single-molecule sequencing-by-synthesis.
8. The method according to claim 1 , wherein amplifying is by the polymerase chain reaction.
9. The method according to claim 1 , wherein the single nucleic acid template is a DNA or an RNA.
10. The method according to claim 1 , wherein the nucleic acid template is associated with a cancer.