Methods and compositions for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-11319534-B2
Application number	US-202016735348-A
Country	US
Kind code	B2
Filing date	Jan 6, 2020
Priority date	Mar 13, 2013
Publication date	May 3, 2022
Grant date	May 3, 2022

Abstract

Embodiments of the present invention relate to sequencing nucleic acids. In particular, embodiments of the methods and compositions provided herein relate to preparing nucleic acid templates and obtaining sequence data therefrom.

First claim

What is claimed is:

1. A device for preparing a nucleic acid library, comprising:
a substrate comprising a plurality of vessels among which a target nucleic acid is distributed, wherein each vessel of the plurality of vessels comprises:
a portion of the target nucleic acid in an amount less than about one haploid equivalent; and
a plurality of transposomes comprising an integration enzyme and a nucleic acid comprising a transposase recognition site integrated into the target nucleic acid, each transposome of the plurality of transposomes comprising a first transposon sequence comprising a first index unique to each vessel of the plurality of vessels, and a second transposon sequence, wherein the first transposon sequence and the second transposon sequence are contiguous, wherein the first transposon sequence and the second transposon sequence are in a 5′ to 5′ orientation and coupled by a single-stranded linker, and wherein the integration enzyme is associated with the first transposon sequence and the second transposon sequence; and
an integration enzyme removing agent in an amount sufficient to dislodge the integration enzyme from the target nucleic acid.

2. The device of claim 1 , wherein the first transposon sequence and the second transposon sequence of at least some of the plurality of transposomes are integrated into the target nucleic acid.

3. The device of claim 1 , wherein each transposome of the plurality of transposomes comprises a fragmentation site disposed between the first transposon sequence and the second transposon sequence.

4. The device of claim 1 , wherein the first transposon sequence comprises a first primer site and the second transposon sequence comprises a second primer site.

5. The device of claim 4 , wherein the first primer site further comprises a first barcode and the second primer site further comprises a second barcode.

6. The device of claim 5 , wherein the first barcode and the second barcode are different.

7. The device of claim 1 , wherein each vessel of the plurality of vessels further comprises a plurality of tagmented nucleic acids in each vessel, wherein the first transposon sequence and the second transposon sequence of some of the plurality of transposomes are integrated into the plurality of tagmented nucleic acids.

8. The device of claim 7 , wherein the substrate comprises a second plurality of vessels into which pooled tagmented nucleic acids from the plurality of vessels are configured to be re-distributed.

9. The device of claim 8 , wherein the second plurality of vessels and the plurality of vessels are about equal in number.

10. The device of claim 1 , wherein the integration enzyme is a transposase having a first subunit associated with the first transposon sequence and a second subunit associated with the second transposon sequence.

11. The device of claim 1 , wherein the integration enzyme removing agent comprises detergent or protease configured to remove the integration enzyme.

12. The device of claim 1 , wherein the substrate comprises spheres, microparticles, beads, membranes, slides, plates, micromachined chips, capillary tubes, microwells, microfluidic devices, channels, or filters.

13. A device for preparing a nucleic acid library, comprising:
pooled tagmented nucleic acids wherein each individual tagmented nucleic acid of the pooled tagmented nucleic acids comprises an integrated transposon sequence comprising a first index, and wherein a first individual tagmented nucleic acid and a second individual tagmented nucleic acid of the pooled tagmented nucleic acids comprise different first indexes that are different from one another such that a plurality of different first indexes are represented in the pooled tagmented nucleic acids;
a substrate comprising a plurality of vessels among which the pooled tagmented nucleic acids are distributed, wherein each vessel of the plurality of vessels comprises:
a portion of the pooled tagmented nucleic acids in an amount greater than about a haploid equivalent, wherein each different first index is present in the portion in an amount less than about a haploid equivalent; and
a plurality of nucleic acids comprising a second index sequence unique to each vessel of the plurality of vessels.

14. The device of claim 13 , wherein the plurality of nucleic acids are ligated to primers configured to bind to the pooled tagmented nucleic acids.

15. The device of claim 13 , wherein each vessel of the plurality of vessels further comprises dual-indexed nucleic acids comprising the first index and the second index sequence.

16. The device of claim 13 , wherein the pooled tagmented nucleic acids comprise a second integrated transposon sequence.

17. The device of claim 13 , wherein the plurality of vessels are configured to receive the pooled tagmented nucleic acids from a different plurality of vessels of the substrate.

18. The device of claim 13 , wherein the substrate comprises spheres, microparticles, beads, membranes, slides, plates, micromachined chips, capillary tubes, microwells, microfluidic devices, channels, or filters.

19. The device of claim 17 , wherein the plurality of vessels are reduced in number relative to the different plurality of vessels.

20. The device of claim 17 , wherein the plurality of vessels are equivalent in number relative to the different plurality of vessels.

21. The device of claim 17 , wherein the nucleic acid library is prepared from a diploid nucleic acid sample and wherein the pooled tagmented nucleic acids in at least one vessel of the plurality of vessels comprise a nucleic acid region from a maternal chromosome and the same nucleic acid region from a paternal chromosome having different first indexes relative to one another.

22. The device of claim 1 , wherein the integration enzyme removing agent comprises one or both of a chaperone, or a polymerase.

23. The device of claim 11 , wherein the integration enzyme removing agent comprises SDS detergent.

24. A device for preparing a nucleic acid library, comprising:
a substrate comprising a plurality of vessels among which a target nucleic acid is distributed, wherein each vessel of the plurality of vessels comprises:
a portion of the target nucleic acid in an amount less than about one haploid equivalent; and
a plurality of transposomes comprising an integration enzyme and a nucleic acid comprising a transposase recognition site integrated into the target nucleic acid, each transposome of the plurality of transposomes comprising a first transposon sequence comprising a first index unique to each vessel of the plurality of vessels, a second transposon sequence, wherein the first transposon sequence and the second transposon sequence are contiguous, wherein the first transposon sequence and the second transposon sequence are in a 5′ to 5′ orientation and coupled by a single-stranded linker, and wherein the integration enzyme is associated with the first transposon sequence and the second transposon sequence; and
an integration enzyme removing system coupled to the substrate, wherein the integration enzyme removing system is configured to change a temperature of the substrate an amount sufficient to dislodge the integration enzyme from the target nucleic acid.

Assignees

• Illumina Inc

Inventors

• Steemers Frank J
• Amini Sasan
• Gunderson Kevin L
• Pignatelli Natasha
• Goryshin Igor

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6806

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

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title