Compositions and methods for improving library enrichment

Patent metadata

Field	Value
Publication number	US-12435362-B2
Application number	US-201917267107-A
Country	US
Kind code	B2
Filing date	Aug 13, 2019
Priority date	Aug 15, 2018
Publication date	Oct 7, 2025
Grant date	Oct 7, 2025

Abstract

This disclosure describes a hybridization buffer including a crowding agent, a method that includes using the hybridization buffer, and a kit including the hybridization buffer. This disclosure also describes blockers for use in hybrid capture methods, methods of using those blockers, and a kit including those blockers. Additionally, this disclosure describes a method of hybrid capture that does not include amplifying the library members using PCR prior to sequencing the library members.

First claim

What is claimed is:

1. A hybridization buffer comprising: a crowding agent, and human Cot-1 DNA, a destabilizing agent, salt, and a blocker, wherein the blocker comprises at least one of:
(a) an oligonucleotide and is capable of binding to an adaptor, wherein the adaptor comprises a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), wherein a first region of the blocker capable of binding to the index region and/or UMI of the adaptor comprises: at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases, and wherein a second region of the blocker capable of binding to a non-index region of the adaptor comprises modified nucleotides; or
(b) two non-connected oligonucleotides; wherein the blocker is capable of binding to at least a portion of an adaptor, wherein the adaptor comprises a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); and wherein the non-connected oligonucleotides comprise bases that do not correspond to the index region and/or the UMI of the adaptor.

2. The hybridization buffer of claim 1 , wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine.

3. The hybridization buffer of claim 1 , comprising a crowding agent, human Cot-1 DNA, a blocker, a destabilizing agent, a buffer, salt, and a detergent.

4. The hybridization buffer of claim 1 , wherein the hybridization buffer comprises a buffer and the buffer is a phosphate buffer.

5. The hybridization buffer of claim 1 , wherein the hybridization buffer comprises salt and the salt is NaCl or sodium citrate.

6. The hybridization buffer of claim 1 , wherein the hybridization buffer comprises a destabilizing agent and the destabilizing agent is formamide or urea or a mixture thereof.

7. The hybridization buffer of claim 1 , wherein the hybridization buffer comprises a detergent and the detergent is TWEEN 20, TWEEN 80, or sodium dodecyl sulfate (SDS).

8. The hybridization buffer of claim 1 , wherein the hybridization buffer comprises:
0.5% to 10% dextran sulfate,
0.05 mg/mL to 0.5 mg/mL human Cot-1 DNA,
1% to 15% (v/v) formamide,
40 mM to 80 mM KH 2 PO 4 —K 2 HPO 4 ,
0.1 M to 4 M NaCl, and
0.001% to 10% (v/v) TWEEN 20.

9. A method of preparing an enriched library comprising forming a hybridization mixture comprising library members, a hybridization buffer of claim 1 , and a probe to form a hybridization mixture, under conditions sufficient for the probe to hybridize to a region of interest within a library member, wherein the probe comprises a ligand.

10. The method of claim 9 , comprising:
capturing the probe after it has hybridized using a capture means, and
eluting the captured library members from the capture means into an eluant comprising enriched library members, wherein the DNA concentration of the eluant is in the range of 1.3 pM to 250 pM.

11. The method of claim 9 , wherein the hybridization mixture comprises samples from at least two library preparations, wherein at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, or at least 500 ng of DNA from each library preparation are combined.

12. The method of claim 9 , wherein the DNA concentration of the hybridization mixture is in a range of 0.1 ng/uL to 120 ng/uL.

13. The method of claim 10 , comprising sequencing the enriched library members and wherein the method does not comprise amplifying the library members prior to capturing the library members.

14. The method of claim 13 , wherein the sequencing occurs on a flow cell and the method comprises loading the enriched library members onto the flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM and at a concentration of up to 100 pM, up to 200 pM, up to 250 pM, or up to 300 pM.

15. The method of claim 14 , comprising loading the enriched library members onto a flow cell using a direct flow cell loading jig.

16. A kit comprising the hybridization buffer of claim 1 and instructions for use.

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Slatter Andrew F
• Zhao Junhua
• Desantis Grace
• Gross Stephen M
• Hogan Hayley Marianne
• Li jian-sen
• Miller Oliver Jon

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2527/101

  Temperature · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title