Methods for linking polynucleotides

Patent metadata

Field	Value
Publication number	US-10982278-B2
Application number	US-201816608769-A
Country	US
Kind code	B2
Filing date	Apr 26, 2018
Priority date	Apr 26, 2017
Publication date	Apr 20, 2021
Grant date	Apr 20, 2021

Abstract

The present disclosure generally relates to methods and compositions of linking, amplifying, and sequencing nucleic acid molecules. Also disclosed is the use of 5′-5′linked oligonucleotides for linking nucleic acid molecules for sequencing of the ends of long nucleic acid template molecules, or for sequencing polymorphism or different target genes or different RNAs simultaneously.

First claim

What is claimed is:

1. A method for linking nucleic acid molecules or fragments thereof, comprising:
(a) segregating individual nucleic acid molecules labeled on both terminal ends with a first adapter pair comprising a forward sequence (F) and a reverse sequence (R), into individual discrete volumes;
(b) inserting, within the individual discrete volumes, at least a second adapter into two or more interior locations of the nucleic acid molecule;
(c) fragmenting the nucleic acid molecules to generate nucleic acid fragments of the nucleic acid molecule of which a least a portion are labeled with both the first adapter pair and the second adapter;
(d) contacting the nucleic acid fragments with at least a first and a second primer, wherein the first primer comprises at least two 5′-5′-linked arms, wherein a first arm of the at least two 5′-5′-linked arms comprises a sequence that hybridizes to the forward (F) sequence of the first adapter pair and a second arm of the at least two 5′-5′-linked arms comprises a sequence that hybridizes to the reverse (R) sequence of the first adapter pair, and wherein the second primer comprises a sequence that hybridizes to the second adapter; and
(e) amplifying the nucleic acid molecules using both the first and second primers by PCR amplification or isothermal amplification.

2. The method of claim 1 , further comprising:
(f) pooling the amplified nucleic acid fragments from each individual discrete volume; and
(g) circularizing the amplified nucleic acid fragments by joining the second adapters.

3. The method of claim 2 , further comprising isolating the amplified nucleic acid fragments labeled with the first primer prior to the circularization step.

4. The method of claim 1 , further comprising:
(h) PCR amplification to generate linearized nucleic acid molecules comprising the second adapter; and
(i) sequencing the linearized nucleic acid molecules to generate a set of nucleic acid reads.

5. The method of claim 4 , further comprising:
exonuclease digestion prior to the PCR amplification step;
removing the first adapter pair sequence from the circularized nucleic acid molecules to generate linearized nucleic acid molecules comprising the second adapter prior to the PCR amplification step; or
assembling a nucleic acid sequence of the nucleic acid molecules based, at least in part, on the set of nucleic acid sequencing reads.

6. The method of claim 3 , wherein the amplified nucleic acid fragments labeled with the first primer are isolated via the binding tag.

7. The method of claim 1 , wherein the forward (F) sequence and the reverse (R) sequence are:
between 6 and 5000 nucleotides in length;
the same; or
different.

8. The method of claim 1 , wherein the forward (F) sequence, the reverse (R) sequence, or the second adapter:
further comprises a restriction site; or
is removed or shortened from the circularized nucleic acid fragments by a restriction enzyme recognizing the restriction site.

9. The method of claim 8 , wherein the restriction site is a Type IIS restriction site.

10. The method of claim 9 , wherein the Type IIS restriction site is a SapI, AcuI, BpuEI, BsgI, BseRI, or Ecil restriction site.

11. The method of any of claim 1 , wherein the first arm of the first primer comprises a forward sequencing adapter sequence or a fragment thereof, and the second arm of the first primer comprises a reverse sequencing adapter sequence or a fragment thereof.

12. The method of claim 1 , wherein the end-labeled nucleic acid molecules are fragmented by a transposase.

13. The method of claim 1 , wherein the individual discrete volume is:
a droplet generated by emulsification;
a droplet generated by vortexing or shaking;
a droplet generated on a microfluidic device;
a droplet that comprises the transposase, the second adapter, and the first and the second primers;
a hollow particle of sufficient size to hold reaction mixture; or
a particle that is a section of a thin capillary tube and has a sufficient size to hold reaction mixture.

14. The method of claim 1 , wherein the nucleic acid molecules:
are DNA molecules;
are RNA molecules;
are 5 kb or longer;
are 40-100 kb or longer; or
encode T-cell receptor, B-cell receptor, or immunoglobulin heavy or light chain.

Assignees

• Broad Inst Inc

Inventors

• Xin Xiaofeng
• Nicol Robert

Classifications

• C12N5/0636

  T lymphocytes · CPC title

• C12N5/0635

  B lymphocytes · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/68 Primary

  involving nucleic acids · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title