Reagents and methods for normalization

Abstract

Provided herein are compositions and methods for normalizing sequencing libraries. Further provided herein are adapter conjugates and hybrid circular adapters, composition comprising the same, and methods of generating the same. Further provided herein are methods of using the adapter conjugates, hybrid circular adapters, and compositions comprising the same to normalize genomic DNA libraries for next-generation sequencing.

Description

This article provides compositions and methods for normalizing sequencing libraries. It also provides adaptor conjugates and hybrid circular adaptors, compositions containing them, and methods for generating them. Furthermore, this article provides methods for normalizing genomic DNA libraries for next-generation sequencing using adaptor conjugates, hybrid circular adaptors, and compositions containing them. Abstract

Claims

WHAT IS CLAIMED IS:

1. A composition comprising:(a) a first polynucleotide adapter comprising: a first strand, wherein the first strand comprises a first terminal adapter region, a first non-complementary region, and a first yoke region; a second strand, wherein the second strand comprises a second terminal adapter region, a second non-complementary region, and a second yoke region; wherein the first yoke region and the second yoke region are complementary, wherein the first non-complementary region and the second non-complementary region are not complementary.(b) a second polynucleotide adapter comprising: a third strand, wherein the third strand comprises a third terminal adapter region, a third non-complementary region, and a third yoke region; and a fourth strand, wherein the fourth strand comprises a fourth terminal adapter region, a fourth non-complementary region, and a fourth yoke region, wherein the third yoke region and the fourth yoke region arc complementary, and wherein the third non-complementary region and the fourth non-complementary region are not complementary, and wherein the first polynucleotide adapter and the second polynucleotide adapter are comrected via linker.

2. The composition of claim 1, wherein the linker is attached to the first terminal adapter region and the third terminal adapter region.

3. The composition of claim 1, wherein the linker is attached to the second terminal adapter region and the fourth terminal adapter region.

4. The composition of claim 1, wherein the linker comprises: an alkene, alkyne, triazine, triazole, oxime, sulfide, amide, and / or cyclooctane; and / or at least one PEG unit; and / or at least one Spacer 18 (Spl8).

5. The composition of claim 1, wherein the linker comprises a polynucleotide.

6. The composition of claim 5, wherein the polynucleotide comprises: at least one cleavable base; and / or a duplex region; and / or at least one splint polynucleotide.

7. The composition of claim 6, wherein the polynucleotide comprises at least one splint polynucleotide, wherein the at least one splint polynucleotide is at least partially complementary to a portion of the first polynucleotide adapter or the second polynucleotide adapter.

8. The composition of claim 7. wherein the polynucleotide comprises at least two splint polynucleotides, wherein the at least two splint polynucleotides are at least partially overlapped with each other.

9. The composition of claim 7, wherein the polynucleotide comprises at least two splint polynucleotides, wherein the at least two splint polynucleotides are at least partially overlapped with a portion of the first polynucleotide adapter or the second polynucleotide adapter.

10. The composition of claim 1, wherein one or more of the first polynucleotide adapter and the second polynucleotide adapter comprises at least one barcode.

11. The composition of claim 10, wherein the at least one barcode comprises one or more of a cell index, sample index, or unique molecular identifier (UMI).

12. Air adapter-ligated sample polynucleotide comprising: the composition of any one of claims 1-11; and at least one sample polynucleotide.

13. The adapter-ligated sample polynucleotide of claim 12, wherein the at least one sample polynucleotide is attached to the 3’ end of the first strand and the 3’ end or the 5’ end of the second strand.

14. The adapter-ligated sample polynucleotide of claim 12, wherein the at least one sample polynucleotide is attached to the 3’ end or the 5’ end of the third strand and the 5’ end of the fourth strand.

15. A method of generating a composition of any one of claims 1-11 , the method comprising: contacting the first strand with the second strand, wherein the first strand comprises a first reactive group and the second strand comprises a second reactive group, wherein reaction of the first reactive group and the second reactive group generates the conjugate.

16. A method of generating a composition of any one of claims 1-11, the method comprising: (a) coupling a nucleotide monomer to a growing chain on a surface;(b) repeating step (a) to generate the first strand of any one of claims 1-11;(c) coupling a linker comprising a reactive group to a terminus of the first strand; and(d) repeating step (a) to generate the third strand of any one of claims 1-11.

17. The method of claim 16, wherein the method further comprises hybridizing the second strand to the first strand.

18. The method of claim 1 , wherein the method further comprises hybridizing the fourth strand to the third strand.

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