Duplicating DNA with contiguity barcodes for genome and epigenome sequencing

Patent metadata

Field	Value
Publication number	US-10428373-B2
Application number	US-201415035957-A
Country	US
Kind code	B2
Filing date	Nov 13, 2014
Priority date	Nov 15, 2013
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

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

What is claimed is:

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, damaging the 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 , 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.

Assignees

• Univ California

Inventors

• Huang Xiaohua

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• B01L2200/0631

  Purification arrangements, e.g. solid phase extraction [SPE] · CPC title

• B01L2300/0645

  Electrodes · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• C12Q1/6846 Primary

  Common amplification features · CPC title