Methods and compositions for nucleic acid analysis
DCFirst Claim
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1. A method comprising:
- a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets in a fluid that is immiscible to the first droplets;
b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is greater than the first volume, wherein the second partitions are second droplets in a immiscible fluid that is immiscible to the second droplets;
c. applying a treatment to fuse the at least one second partition with at least one first droplet to form a fused partition; and
d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof.
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Abstract
Provided herein are methods, compositions, and kits for assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR. The assays may be used for such applications as sequencing, copy number variation analysis, and others. In some cases, the assays involve subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes.
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Citations
36 Claims
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1. A method comprising:
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a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets in a fluid that is immiscible to the first droplets; b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is greater than the first volume, wherein the second partitions are second droplets in a immiscible fluid that is immiscible to the second droplets; c. applying a treatment to fuse the at least one second partition with at least one first droplet to form a fused partition; and d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof. - View Dependent Claims (3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31)
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2. A method comprising:
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a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets in a fluid that is immiscible to the first droplets; b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is less than the first volume, wherein the second partitions are second droplets in a fluid that is immiscible to the second droplets; c. applying a treatment to fuse the at least one first partition with the at least one second droplet to form a fused partition; and d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof. - View Dependent Claims (5, 7, 28, 32, 33)
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34. A method comprising:
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a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets; b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is greater than the first volume or the second volume is less than the first volume, wherein the second partitions are second wells; c. applying a treatment to fuse the at least one second partition with at least one first droplet to form a fused partition; and d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof. - View Dependent Claims (35, 36)
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Specification