METHODS FOR DETECTING MUTATIONS
The invention generally relates to methods for detecting mutations in a nucleic acid. In certain embodiments, the invention provides methods that involve forming a plurality of droplets, such that on average, each droplet includes a ratio of one nucleic acid template per bead, amplifying the template in the droplet to produce bead-bound amplicons, and sequencing at least one amplicon detect a mutation.
1 . A method for detecting a mutation in a nucleic acid, the method comprising:
forming a plurality of droplets, wherein on average, each droplet comprises a ratio of one nucleic acid template per bead;
amplifying the template in the droplet to produce bead-bound amplicons; and
sequencing at least one amplicon detect a mutation.
2 . The method according to claim 1 , wherein the droplet is an aqueous droplet surrounded by an immiscible fluid.
3 . The method according to claim 2 , wherein the immiscible fluid is oil.
4 . The method of claim 1 , wherein prior to amplifying, the templates are denatured.
5 . The method according to claim 1 , wherein the amplifying step is conducted with a limiting amount of amplification primers, thereby decreasing an overall amount of amplicon that binds to an individual bead during the amplifying step.
6 . The method according to claim 1 , wherein the method is performed in the presence of a control genomic DNA.
7 . The method according to claim 1 , wherein the method is performed in the presence of an artificially introduced amount of nucleic acid comprising a known mutation.
8 . The method according to claim 1 , wherein prior to sequencing, the droplets are ruptured.
9 . The method according to claim 8 , wherein prior to sequencing, bead-bound amplicons are separated from remaining components of the droplets.
10 . The method according to claim 1 , wherein the nucleic acid template is obtained from a sample.
11 . The method of claim 10 , wherein the sample is blood, sputum, saliva, urine, sweat, tissue, biopsy tissue, or stool.
12 . The method of claim 1 , wherein prior to the forming step, the method further comprises amplifying the nucleic acid template.
13 . The method of claim 1 , wherein prior to the forming step, the method further comprises attaching a unique barcode sequence to the template.
14 . The method according to claim 1 , wherein the nucleic acid template represents the FGFR3 gene.
15 . The method according to claim 13 , wherein the mutation in the template is indicative of bladder cancer.
16 . A method for detecting a mutation in a nucleic acid, the method comprising:
performing an amplification reaction in a plurality of droplets, each droplet comprising nucleic acid templates and beads, wherein the template to bead ratio is such that less than 2% of the beads comprise more than one template after completion of the amplification reaction; and
sequencing at least one amplification product to detect a mutation.
17 . The method according to claim 16 , wherein the droplet is an aqueous droplet surrounded by an immiscible fluid.
18 . The method according to claim 17 , wherein the immiscible fluid is oil.
19 . The method of claim 16 , wherein prior to amplifying, the templates are denatured.
20 . The method according to claim 16 , wherein the amplification reaction is conducted with a limiting amount of amplification primers, thereby decreasing an overall amount of amplicon that binds to an individual bead during the amplification reaction.
21 . The method according to claim 16 , wherein the method is performed in the presence of a control genomic DNA.
22 . The method according to claim 16 , wherein the method is performed in the presence of an artificially introduced amount of nucleic acid comprising a known mutation.
23 . The method according to claim 16 , wherein prior to sequencing, the droplets are ruptured.
24 . The method according to claim 23 , wherein prior to sequencing, bead-bound amplification products are separated from remaining components of the droplets.
25 . The method according to claim 16 , wherein the nucleic acid template is obtained from a sample.
26 . The method of claim 25 , wherein the sample is blood, sputum, saliva, urine, sweat, tissue, biopsy tissue, or stool.
27 . The method of claim 16 , wherein prior to the performing step, the method further comprises attaching a unique barcode sequence to the template.
28 . The method according to claim 16 , wherein the nucleic acid template represents the FGFR3 gene.
29 . The method according to claim 28 , wherein the mutation in the template is indicative of bladder cancer.