LABELING AND SAMPLE PREPARATION FOR SEQUENCING

• US 20130225418A1
• Filed: 02/22/2013
• Published: 08/29/2013
• Est. Priority Date: 02/24/2012
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Watson, Andrew
• Granted Patent: US 9,176,031 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Abstract

The invention provides methods for sequencing and sample preparation for sequencing and amplification.

Claims

1. A method for preparing nucleic acid for analysis, the method comprising the steps of:

fragmenting nucleic acid into long fragment and short fragments, wherein two or more of said short fragments are capable of hybridizing to a single long fragment;

attaching an adaptor to each of 3′ and

5′

ends of said short fragments;

hybridizing said short fragments to said long fragments in order to form hybridized fragments;

removing unhybridized short fragments;

exposing said hybridized fragments to barcoded primers capable of hybridizing to a portion of one or more of said short fragments; and

analyzing said hybridized fragments.

2. The method according to claim 1, wherein removing comprises conducting a gel purification assay that separates the hybridized fragments from the unhybridized short fragments.

3. The method according to claim 1, wherein prior to the hybridizing step, the method further comprises attaching a first member of a binding pair to the long fragments.

4. The method according to claim 3, wherein removing comprises exposing the hybridized fragments and the unhybridized short fragments to a solid support comprising a second member of the binding pair such that the hybridized fragments bind to the solid support via the binding pair and are separated from the unhybridized short fragments that do not bind the support.

5. The method according to claim 4, wherein the binding pair is selected from the group consisting of:

biotin/streptavidin;

carbohydrate/lectin; and

antibody/antigen.

6. The method according to claim 1, wherein exposing comprises:

compartmentalizing the hybridized fragments into compartmentalized portions; and

introducing the barcoded primers to the compartmentalized portions.

7. The method according to claim 6, wherein each compartmentalized portion comprises only one hybridized fragment.

8. The method according to claim 6, wherein the compartmentalized portions are aqueous droplets surrounded by an immiscible fluid.

9. The method according to claim 8, wherein compartmentalizing comprises partitioning the aqueous fluid as it is flowing through a channel with the immiscible fluid.

10. The method according to claim 9, wherein the immiscible fluid is oil.

11. The method according to claim 10, wherein the oil is a fluorinated oil.

12. The method according to claim 10, wherein the oil comprises a surfactant.

13. The method according to claim 12, wherein the surfactant is a fluorosurfactant.

14. The method according to claim 9, wherein introducing comprises merging the droplet comprises the hybridized fragment with a droplet comprising the barcoded primers.

15. The method according to claim 14, wherein merging occurs in the presence of an electric field.

16. The method according to claim 6, wherein introducing comprises merging a portion of a fluid stream comprising the hybridized fragment with a droplet comprising the barcoded primers.

17. The method according to claim 16, wherein merging occurs in the presence of an electric field.

18. The method according to claim 1, wherein analyzing comprises sequencing the short fragments and aligning obtained sequence reads.

19. The method according to claim 1, wherein prior to analyzing, the method further comprises amplifying the short fragments hybridized to the long fragment.

20. The method according to claim 16, wherein amplifying is by the polymerase chain reaction.

21. The method according to claim 19, wherein analyzing comprises sequencing the short fragments and aligning obtained sequence reads.

22. The method according to claim 1, wherein the barcode primer further comprises a universal priming site.

23. The method according to claim 22, wherein the universal priming site is attached to the barcode primer via polymerase chain reaction.

24. The method according to claim 22, wherein the universal priming site is attached to the barcode primer via ligation.