Compositions and methods for molecular labeling

• US 10,584,332 B2
• Filed: 01/14/2016
• Issued: 03/10/2020
• Est. Priority Date: 02/18/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Samuels, Michael L., Olson, Jeffrey Charles, Watson, Andrew, Brown, Keith, Link, Darren R.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/995,744
• Publication Number:
  US 20160201125A1
• Time in Patent Office: 1,517 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/10 Nucleotidyl transfering
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

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.

Claims

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.