Compositions and methods for molecular labeling
View Patent ↗The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.
1. A method of making a barcode library comprising:
obtaining a first nucleic acid construct wherein the first construct comprises a forward sequence specific primer, a first barcode sequence, and a first universal primer recognition site;
compartmentalizing the first construct into a fluid compartment;
introducing a second nucleic acid construct into the fluid compartment, wherein the second construct comprises a reverse sequence specific primer, a second barcode sequence, and a second universal primer recognition site;
introducing a third construct into the fluid compartment and hybridizing the third construct to the first construct to form a hybrid construct; and
ligating the hybrid construct to form a new construct.
2. The method of claim 1 , wherein the constructs are within the fluid compartment in the absence of any target to make a barcode library.
3. The method of claim 1 , wherein the first construct and the second construct each comprises a sticky end.
4. The method of claim 1 , wherein the fluid compartment is a water-in-oil droplet.
5. The method of claim 1 , further comprising:
(a) preparing N of the fluid compartments containing the constructs; and
(b) repeating step (a) M times, thereby producing MxN fluid compartments.
6. The method of claim 1 , wherein introducing comprises merging droplets in a microfluidic device.
7. The method of claim 1 , wherein either the first or second construct further comprises a sequencing platform-specific adaptor.
8. The method of claim 1 , further comprising using either the hybrid or second construct in digital PCR.
9. The method of claim 1 , wherein each of the barcode sequences is between 3 and 30 nucleotides in length.
10. The method of claim 1 , wherein the hybrid or second construct is linked to an antibody.
11. The method of claim 1 , wherein the first and second barcode sequences are the same.
12. The method of claim 1 , wherein the first and second barcode sequences are different.
13. The method of claim 1 , wherein the first and second universal primer recognition sites are the same.
14. The method of claim 1 , wherein the first and second universal primer recognition sites are different.