Compositions and methods for molecular labeling
AssigneeBio-Rad Laboratories, Inc.
InventorsMichael L. Samuels, Jeffrey Charles Olson, Andrew Watson, Keith Brown, Darren R. Link
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Abstract
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.
Claims19
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.
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.
Dependencies are inferred from the claim text.