Modified transposases for improved insertion sequence bias and increased DNA input tolerance

• US 9,790,476 B2
• Filed: 04/15/2015
• Issued: 10/17/2017
• Est. Priority Date: 04/15/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Gloeckner, Christian, Kia, Amirali, Bomati, Erin, He, Molly, Grunenwald, Haiying Li, Kuersten, Scott, Osothprarop, Trina Faye, Haskins, Darin, Burgess, Joshua, Khanna, Anupama, Schlingman, Daniel, Vaidyanathan, Ramesh
• Primary Examiner(s): Ramirez, Delia
• Application Number:
  US14/686,961
• Publication Number:
  US 20150291942A1
• Time in Patent Office: 916 Days
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

Presented herein are transposase enzymes and reaction conditions for improved fragmentation and tagging of nucleic acid samples, in particular altered transposases and reaction conditions which exhibit improved insertion sequence bias, as well as methods and kits using the same.

Claims

1. A mutant Tn5 transposase having transposase activity, wherein said mutant Tn5 transposase comprises all of SEQ ID NO:

1 or SEQ ID NO;

11 except for;

(a) mutations at one or more positions corresponding to positions 214 and 338 of SEQ ID NO;

1 or SEQ ID NO;

11, and optionally

(b) mutations at one or more positions corresponding to positions 54, 56, 212, 251 and 372 of SEQ ID NO;

1 or SEQ ID NO;

11.

2. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises all of SEQ ID NO:

1 or SEQ ID NO;

11 except for;

(a) mutations at one or more positions corresponding to positions 214 and 338 of SEQ ID NO;

1 or SEQ ID NO;

11,

(b) mutations at one or more positions corresponding to positions 212 and 251 of SEQ ID NO;

1 or SEQ ID NO;

11, and optionally

(c) mutations at one or more positions corresponding to positions 54, 56, and 372 of SEQ ID NO;

1 or SEQ ID NO;

11.

3. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises all of SEQ ID NO:

1 or SEQ ID NO;

11 except for;

(a) mutations at one or more positions corresponding to positions 212, 214 and 338 of SEQ ID NO;

1 or SEQ ID NO;

11, and optionally

(b) mutations at one or more positions corresponding to positions 54, 56, 251, and 372 of SEQ ID NO;

1 or SEQ ID NO;

11.

4. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises arginine at the position corresponding to position 214 of SEQ ID NO:

1 or SEQ ID NO;

11.

5. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises valine at the position corresponding to position 338 of SEQ ID NO:

1 or SEQ ID NO;

11.

6. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises arginine at the position corresponding to position 251 of SEQ ID NO:

1 or SEQ ID NO;

11.

7. A fusion protein comprising the mutant Tn5 transposase of claim 1 and a polypeptide fusion domain.

8. The fusion protein of claim 7, wherein the polypeptide fusion domain is fused to the N-terminus of the mutant Tn5 transposase.

9. The fusion protein of claim 7, wherein the polypeptide fusion domain is fused to the C-terminus of the mutant Tn5 transposase.

10. The fusion protein of claim 7, wherein the polypeptide fusion domain comprises a tag to increase solubility.

11. The fusion protein of claim 7, wherein the polypeptide fusion domain comprises a domain selected from the group consisting of maltose binding protein (MBP), elongation factor Ts (Tsf), 5-methyl cytosine binding domain, and protein A.

12. A kit for performing an in vitro transposition reaction, wherein the kit comprises a transposome complex, wherein the transposome complex comprises the mutant Tn5 transposase of claim 1 and

a polynucleotide, wherein the 3′

portion of the polynucleotide comprises a transposon end sequence.

13. The mutant Tn5 transposase of claim 3, wherein the mutant Tn5 transposase comprises a substitution at the position corresponding to position 212 of SEQ ID NO:

1 or SEQ ID NO;

11.

14. The mutant Tn5 transposase of claim 13, wherein the mutant Tn5 transposase comprises arginine at the position corresponding to position 212 of SEQ ID NO:

1 or SEQ ID NO;

11.

15. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises all of SEQ ID NO:

1 or SEQ ID NO;

11 except for;

(a) mutations at one or more positions corresponding to positions 212, 214, 251 and 338 of SEQ ID NO;

1 or SEQ ID NO;

11, and

(b) mutations at one or more positions corresponding to positions 54, 56, and 372 of SEQ ID NO;

1 or SEQ ID NO;

11.

16. The mutant Tn5 transposase of claim 15, wherein the mutant Tn5 transposase comprises arginine, arginine, arginine, and valine, respectively, at the positions corresponding to positions 212, 214, 251, and 338 of SEQ ID NO:

1 or SEQ ID NO;

11.

17. The fusion protein of claim 11, wherein the polypeptide fusion domain comprises elongation factor Ts (Tsf) and wherein the mutant Tn5 transposase comprises arginine, arginine, arginine, and valine, respectively, at the positions corresponding to positions 212, 214, 251, and 338 of SEQ ID NO:

1 or SEQ ID NO;

11.

18. The fusion protein of claim 7, wherein said fusion protein comprises SEQ ID NO:

26.

19. The mutant Tn5 transposase of claim 1, wherein said mutant Tn5 transposase comprises all of SEQ ID NO:

1 or SEQ ID NO;

11 except for mutations at positions corresponding to positions 54, 56, 212, 214, 251, 338, and 372 of SEQ ID NO;

1 or SEQ ID NO;

11.

20. The mutant Tn5 transposase of claim 1, wherein the mutant Tn5 transposase comprises SEQ ID NO:

26.