Duplicating dna with contiguity barcodes for genome and epigenome sequencing

Patent metadata

Field	Value
Publication number	US-2016265039-A1
Application number	US-201415035957-A
Country	US
Kind code	A1
Filing date	Nov 13, 2014
Priority date	Nov 15, 2013
Publication date	Sep 15, 2016
Grant date	—

Abstract

Provided herein are methods and devices for accurate sequencing and detection of epigenetic information from template polynucleotides. Also provided are methods for long-range strand displacement amplification of polynucleotides, microfluidic devices with selectively permeable barriers for multistep processing, and methods for polynucleotide amplification using the microfluidic devices.

First claim

1 . A method for sequencing a template polynucleotide comprising:
(a) contacting the template polynucleotide with a plurality of oligonucleotide pairs, wherein each member of each oligonucleotide pair comprises (i) an adaptor sequence, (ii) a unique barcode sequence that hybridizes to its complement on the other member of the oligonucleotide pair, and wherein one or both of the oligonucleotide pairs comprises (iii) a primer sequence that hybridizes to the template polynucleotide; (b) contacting the template polynucleotide and plurality of oligonucleotide pairs with a polymerase lacking strand displacement activity and reagents necessary for polymerization, and (c) allowing extension of a polynucleotide strand from the 3′ end of the primer sequence to produce an extended polynucleotide comprising components (i)-(iii) and a sequence complementary to the template polynucleotide; (d) collecting the extended polynucleotides; (e) sequencing the extended polynucleotides; and (f) assembling the sequences of the extended polynucleotides based on the unique barcodes, thereby sequencing the template polynucleotide, wherein the method does not include fragmenting the template polynucleotide or removing epigenetic markers on the template polynucleotide.

2 . The method of claim 1 , further comprising denaturing the template polynucleotide before step (a).

3 . The method of claim 1 or 2 , further comprising allowing the plurality of oligonucleotide pairs to hybridize to the template polynucleotide, and washing away unhybridized oligonucleotide pairs between steps (a) and (b).

4 . The method of claim 1 , wherein each member of the oligonucleotide pair further comprises (iv) at least one linker sequence, and the extended polynucleotide comprises components (i)-(iv) and a sequence complementary to the template polynucleotide.

5 . The method of claim 1 , wherein the template polynucleotide is genomic DNA.

6 . The method of claim 5 , further comprising detecting methylated bases on the genomic DNA.

7 . The method of claim 1 , wherein both members of the oligonucleotide pair comprise (iii) a primer sequence that hybridizes to the template polynucleotide.

8 . The method of claim 1 , wherein the primer sequence (iii) is a random primer sequence.

9 . The method of claim 1 , wherein the adaptor sequence is complementary to a predetermined primer sequence.

10 . The method of claim 1 , wherein the adaptor sequence is attached to an affinity reagent.

11 . A method for amplifying a template polynucleotide comprising:
(a) contacting the template polynucleotide with a plurality of primers; (b) contacting the template polynucleotide and plurality of primers with a polymerase having strand displacement activity and reagents necessary for polymerization; (c) allowing extension of a polynucleotide strand from the 3′ end of at least one of the plurality of primers hybridized to the template polynucleotide to produce elongated amplification product, thereby amplifying the template polynucleotide.

12 . The method of claim 11 , further comprising denaturing the template polynucleotide before step (a).

13 . The method of claim 11 , further comprising allowing the plurality of primers to hybridize to the template polynucleotide, and washing away unhybridized primers between steps (a) and (b).

14 . The method of claim 11 , wherein step (c) comprises allowing extension for a predetermined time to produce partially elongated amplification product, washing away unhybridized primers, adding polymerase having strand displacement activity and reagents necessary for polymerization, and allowing extension to continue.

15 . The method of claim 11 , wherein the primers are random primers.

16 . The method of claim 11 , wherein the primers are attached to an affinity reagent.

17 . The method of claim 16 , further comprising affinity purifying the elongated amplification product.

18 . The method of claim 11 , further comprising separating the elongated amplification product based on size.

19 . The method of claim 17 , further comprising repeating steps (a)-(c) after removal of the elongated amplification product by affinity purification or separation.

20 . (canceled)

21 . A microfluidic device comprising:
(a) at least one chamber; (b) a plurality of fluid channels operably connected to the at least one chamber; (c) at least one polymer barrier separating the at least one chamber from (i) one of the plurality of fluid channels and (ii) an electrode configured to produce an electric field in the fluid channel and chamber; wherein the at least one chamber includes at least one valve, each valve separating the at least one chamber from one of the plurality of fluid channels that is not separated from the at least one chamber by the polymer barrier, and wherein the polymer barrier is semipermeable and has a size cutoff so that nucleic acids larger than the size cutoff are retained in the at least one chamber when the electrode produces an electric field in the fluid channel and chamber.

22 - 28 . (canceled)

Assignees

• Univ California

Inventors

• Huang Xiaohua

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• B01L2300/0681

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• B01L2200/0668

  Trapping microscopic beads · CPC title

• B01L3/502738

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title