Compositions and methods for molecular labeling
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 obtaining genetic sequence data for assembling a haplotype, the method comprising:
providing a plurality of aqueous fluid-in-oil droplets, each aqueous fluid-in-oil droplet comprising:
a single copy of a polynucleotide template; and
a plurality of primers not bound to microbeads each comprising
a functional N-mer portion configured to bind to the polynucleotide template and
a unique N-mer portion, wherein the unique N-mer portions within an aqueous fluid-in-oil droplet are identical to each other, and wherein the unique N-mer portions differ between aqueous fluid-in-oil droplets;
hybridizing the primers to the templates;
extending the hybridized primers to produce a plurality of extension products comprising the unique N-mers, wherein the extension products are not bound to microbeads;
pooling the plurality of aqueous fluid-in-oil droplets;
breaking the plurality of aqueous fluid-in-oil droplets to combine the extension products in a liquid aqueous phase;
performing a second amplification on the extension products to produce amplified extension products not bound to microbeads;
sequencing the amplified extension products not bound to microbeads; and
combining sequences with matching unique N-mer portions to generate a haplotype.
2. The method of claim 1 , wherein the plurality of aqueous fluid-in-oil droplets are water-in-oil droplets.
3. The method of claim 2 , wherein the primers are members of PCR primer pairs, each primer pair having a first primer and a second primer.
4. The method of claim 3 , wherein each member of the PCR primer pair comprises the unique N-mer portion.
5. The method of claim 2 , wherein the aqueous fluid-in-oil droplets are formed by merging a droplet comprising the single copy of the polynucleotide template with a droplet comprising the plurality of primers.
6. The method of claim 1 , wherein the aqueous fluid-in-oil droplets comprise at least two polynucleotide templates that differ in nucleotide sequence from each other, wherein a single molecule of each template is present in each aqueous fluid-in-oil droplet.
7. The method of claim 1 , wherein the functional N-mer portions hybridize to different regions of the polynucleotide template.
8. The method of claim 1 , wherein the functional N-mer portions hybridize at random locations on the polynucleotide template.
9. A method of producing barcoded polynucleotides derived from a polynucleotide template, the method comprising:
providing a library of polynucleotide barcode primers not bound to microbeads, each barcode primer comprising a functional N-mer portion and a barcode comprising a unique N-mer portion, in a microfluidic channel forming a plurality of aqueous fluid-in-oil droplets, each aqueous fluid-in-oil droplet comprising a single copy of at least one polynucleotide template and a barcode primer from the library;
hybridizing the barcode primers to the polynucleotide templates in the aqueous fluid-in-oil droplets;
extending the hybridized barcode primers to produce a plurality of extension products comprising the unique N-mers, wherein the extension products are not bound to microbeads;
breaking the plurality of aqueous fluid-in-oil droplets to combine the extension products comprising the unique N-mers in a liquid aqueous phase;
performing an amplification on the extension products comprising the unique N-mers to produce amplified extension products not bound to microbeads; and
sequencing the amplified extension products not bound to microbeads.
10. The method of claim 9 , wherein the aqueous fluid-in-oil droplets are water-in-oil droplets.
11. The method of claim 10 , wherein the barcode primers are members of PCR primer pairs, each PCR primer pair having a first barcode primer and a second barcode primer.
12. The method of claim 10 , wherein the aqueous fluid-in-oil droplets are formed by merging a droplet comprising a single copy of a polynucleotide template with a droplet comprising a barcode primer from the library.
13. The method of claim 10 , wherein each aqueous fluid-in-oil droplet comprises a plurality of polynucleotide templates that differ in sequence from each other.
14. The method of claim 10 , wherein the barcode primers in each aqueous fluid-in-oil droplet comprise the same unique N-mer.
15. The method of claim 10 , wherein all the barcode primers in a aqueous fluid-in-oil droplet are identical.
16. The method of claim 9 , wherein the aqueous fluid-in-oil droplets comprise at least two polynucleotide templates that differ in nucleotide sequence from each other, wherein a single molecule of each template is present in each compartment.
17. The method of claim 9 , wherein the aqueous fluid-in-oil droplets comprise a plurality of barcode primers that hybridize to different regions of the polynucleotide template and comprise the same barcode sequence.
18. The method of claim 9 , wherein the barcode primers hybridize at random locations on the polynucleotide template.
19. The method of claim 9 , wherein the aqueous fluid-in-oil droplets comprise a plurality of barcode primers, and wherein the functional N-mer portions comprise random sequences, and wherein the unique N-mer portions are identical.