Tagmentation using immobilized transposomes with linkers

Patent metadata

Field	Value
Publication number	US-12234451-B2
Application number	US-202318327187-A
Country	US
Kind code	B2
Filing date	Jun 1, 2023
Priority date	Feb 21, 2017
Publication date	Feb 25, 2025
Grant date	Feb 25, 2025

Abstract

The present disclosure relates to methods, compositions, and kits for treating target nucleic acids, including methods and compositions for fragmenting and tagging nucleic acid (e.g., DNA) using transposome complexes bound to a solid support.

First claim

What is claimed is:

1. A method for generating a library of tagged nucleic acid fragments from a double-stranded, target nucleic acid, comprising incubating the target nucleic acid with a transposome complex, wherein the transposome complex comprises:
a. a transposase,
b. a first transposon comprising:
i. a 3′ portion comprising a first transposon end sequence; and
ii. a first adaptor sequence at the 5′ end of the first transposon end sequence;

c. a second transposon comprising a second transposon end sequence complementary to at least a portion of the first transposon end sequence; and
d. a non-nucleic acid linker having a first end attached to the 3′ end of the second transposon and a second end attached to an affinity element,
under conditions sufficient to fragment the target nucleic acid into a plurality of target fragments, and to join the 3′ end of the first transposon to the 5′ ends of the target fragments to produce a plurality of 5′ tagged target fragments.

2. The method of claim 1 , wherein the affinity element is bound to an affinity binding partner on a solid support, whereby the complex is bound to the solid support.

3. The method of claim 2 , wherein the affinity element is biotin and the affinity binding partner is streptavidin.

4. The method of claim 2 , wherein the solid support is a tube, a well of a plate, a slide, a bead, or a flowcell, optionally wherein the solid support is a paramagnetic bead.

5. The method of claim 1 , wherein:
a. the first adaptor sequence comprises a universal sequence, primer sequence, or sequencing related sequence;
b. the first transposon end sequence and/or second transposon end sequence further comprises a universal sequence, primer sequence, or sequencing related sequence;
c. the first transposon end sequence and the second transposon end sequence are a mosaic end sequence (ME) and a complementary mosaic end sequence (ME′);
d. the transposase is a Tn5 transposase, optionally wherein the Tn5 transposase is a wild-type Tn5 transposase or a hyperactive Tn5 transposase, or a mutant thereof; and/or
e. the transposase is conjugated to a purification tag.

6. The method of claim 1 , wherein the non-nucleic acid linker and affinity element have the structure of Formula (I):

wherein:
AE is the affinity element;
Y is C 2-6 alkylene;
X 1 is O, NR 1 , or S; wherein R 1 is H or C 1-10 alkyl;
n is an integer from 1 to 6;
X 2 is O, CH 2 , or S;
R a is H or —OH; and
Z is absent when R a is H, or is CH 2 when R a is H or OH;
wherein the marks the connection point to the second transposon.

7. The method of claim 6 , wherein the phosphate group in Formula (I) is connected to a 3′ hydroxyl of the terminal nucleotide of the second transposon and/or AE comprises or is an optionally substituted biotin or an amino group.

8. The method of claim 6 , wherein the non-nucleic acid linker and affinity element have the structure of Formula (I′):

wherein Z is absent or is CH 2 .

9. The method of claim 6 , wherein the non-nucleic acid linker and affinity element have the structure of Formula (Ia):

10. The method of claim 6 , wherein the non-nucleic acid linker and affinity element have the structure of Formula (Ib) or (Ic):

where n is 1 or 2;
X 2 is O or CH 2 ; and
Z is absent or is CH 2 .

11. The method of claim 6 , wherein the non-nucleic acid linker and affinity element have a structure selected from the group consisting of:

12. The method of claim 1 , wherein the nucleic acid is chosen from at least one of:
a. DNA, optionally where the DNA is double-stranded, optionally where the double-stranded DNA is genomic DNA, optionally where the genomic DNA is from a single cell, tissue, tumor, blood, plasma, urine, or cell-free nucleic acid; or
b. RNA or a derivative thereof; or
c. CDNA.

13. The method of claim 2 , further comprising amplifying one or more of the 5′ tagged target fragments.

14. The method of claim 13 , wherein the amplifying comprises generating and/or amplifying fully duplexed 5′ tagged target fragments, wherein generating the one or more fully duplexed 5′ tagged target fragments comprises:
removing the second transposon from the plurality of 5′ tagged target fragments; and
extending the plurality of 5′ tagged target fragments after removing the second transposon from the plurality of 5′ tagged target fragments.

15. The method of claim 14 , wherein the amplifying comprises incubating at least one fully duplexed 5′ tagged target fragment of the one or more 5′ tagged target fragments comprising a primer sequence at each end with a secondary adaptor carrier, single nucleotides, and a polymerase, under conditions sufficient to amplify the target fragments and incorporate the secondary adaptor carrier, wherein the secondary adaptor carrier comprises the complement to the primer sequence and a secondary adaptor sequence, thereby producing a library of sequencing fragments.

16. The method of claim 15 , wherein the secondary adaptor carrier comprises:
a. a primer sequence, an index sequence, a barcode sequence, a purification tag, or a combination thereof, or
b. an index sequence and a primer sequence.

17. The method of claim 14 , wherein the one or more fully duplexed 5′ tagged target fragments comprise a different primer sequence at each end, optionally wherein the different primer sequences are A14 (SEQ ID NO: 6) and B15 (SEQ ID NO: 7).

18. The method of claim 15 , wherein the secondary adaptor carriers each comprise one of two primer sequences, optionally where the two primer sequences are a P5 primer sequence (SEQ ID NO: 1) and a P7 primer sequence (SEQ ID NO: 2), and one of a plurality of index sequences.

19. The method of claim 18 , wherein the fragments are hybridized to complementary primers grafted to a flow cell or solid support.

20. The method of claim 13 , further comprising sequencing one or more of the 5′ tagged target fragments or amplification products thereof.

21. The method of claim 5 , wherein the ME sequence is SEQ ID NO: 8 and the ME′ sequence is SEQ ID NO: 5.

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Desantis Grace
• Gross Stephen M
• Li jian-sen
• Morrell Natalie
• Slatter Andrew
• Shen Kevin
• Snow Samantha

Classifications

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N11/06 Primary

  attached to the carrier via a bridging agent · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title