Double-stranded splint adaptors with universal long splint strands and methods of use

Patent metadata

Field	Value
Publication number	US-12365892-B2
Application number	US-202318465687-A
Country	US
Kind code	B2
Filing date	Sep 12, 2023
Priority date	Sep 12, 2022
Publication date	Jul 22, 2025
Grant date	Jul 22, 2025

Abstract

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors ( 200 ) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor ( 200 ) comprises a first splint strand (long splint strand ( 300 )) and a second splint strand (short splint strand ( 400 )), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor ( 200 ) having a double-stranded region and two flanking single-stranded regions. The second splint strand ( 400 ) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence, an index sequence and/or a random sequence.

First claim

What is claimed:

1. A library-splint complex ( 500 ) comprising:
(i) a single-stranded nucleic acid library molecule ( 100 ) comprising a sequence of interest ( 110 ) flanked on one side by at least a first left universal adaptor sequence ( 120 ), and flanked on the other side by at least a first right universal adaptor sequence ( 130 ); and
(ii) a double-stranded splint adaptor ( 200 ) comprising a first splint strand ( 300 ) and a second splint strand ( 400 ), wherein the double-stranded splint adaptor ( 200 ) comprises a double-stranded region and two single-stranded regions, one on either side of the double-stranded region, wherein the first splint strand ( 300 ) comprises a first region ( 320 ), an internal region ( 310 ), and a second region ( 330 ), and wherein the first splint strand comprises at least one abasic site or uracil;
wherein the internal region ( 310 ) of the first splint strand ( 300 ) is hybridized to the second splint strand ( 400 ), wherein the first region ( 320 ) of the first splint strand ( 300 ) is hybridized to the at least first left universal adaptor sequence ( 120 ) of the single-stranded nucleic acid library molecule, and wherein the second region ( 330 ) of the first splint strand ( 300 ) is hybridized to the at least first right universal sequence ( 130 ) of the single-stranded nucleic acid library molecule, thereby circularizing the single-stranded nucleic acid library molecule to generate a library-splint complex ( 500 ).

2. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a second left universal adaptor sequence ( 140 ).

3. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a second right universal adaptor sequence ( 150 ).

4. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a first left index sequence ( 160 ).

5. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a first right index sequence ( 170 ).

6. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a first left unique identification sequence ( 180 ).

7. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises: a first right unique identification sequence ( 190 ).

8. The library-splint complex ( 500 ) of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises any combination of two or more of:
(i) a second left universal adaptor sequence ( 140 );
(ii) a second right universal adaptor sequence ( 150 );
(iii) a first left index sequence ( 160 );
(iv) a first right index sequence ( 170 );
(v) a first left unique identification sequence ( 180 ); and/or
(vi) a first right unique identification sequence ( 190 ).

9. The library-splint complex ( 500 ) of claim 2 , wherein the at least first left universal adaptor sequence ( 120 ) and/or the second left universal adaptor sequence ( 140 ) comprises:
(i) a universal binding sequence for a forward sequencing primer;
(ii) a universal binding sequence for a reverse sequencing primer;
(iii) a universal binding sequence for a first surface primer;
(iv) a universal binding sequence for a second surface primer;
(v) a universal binding sequence for a forward amplification primer;
(vi) a universal binding sequence for a reverse amplification primer; and/or
(vii) a universal binding sequence for a compaction oligonucleotide.

10. The library-splint complex ( 500 ) of claim 9 , wherein the second splint strand ( 400 ) includes at least a first sub-region and a second sub-region, wherein the first sub-region comprises a universal binding sequence for a third surface primer and the second sub-region comprises a universal binding sequence for a fourth surface primer, and wherein the first and second sub-regions do not hybridize or exhibit very little hybridization to the first surface primer and second surface primer.

11. The library-splint complex ( 500 ) of claim 10 , wherein the second splint strand ( 400 ) comprises third sub-region, wherein the third sub-region comprises a sample index sequence having 5-20 bases and/or a unique identification sequence having 2-10 or more bases.

12. The library-sprint complex ( 500 ) of claim 11 , wherein the unique identification sequence comprises a random sequence.

13. The library-splint complex ( 500 ) of claim 10 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises at least a fourth sub-region and a fifth sub-region, wherein the fourth sub-region hybridizes to the first sub-region of the second splint strand ( 400 ), wherein the fifth sub-region hybridizes to the second sub-region of the second splint strand ( 400 ), and wherein the fourth and fifth sub-regions do not hybridize or exhibit very little hybridization to the first and second surface primers.

14. The library-splint complex ( 500 ) of claim 11 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises a sixth sub-region comprising a sample index sequence having 5-20 bases and/or a unique identification sequence having 2-10 or more bases, and wherein the sixth sub-region hybridizes to the third sub-region of the second splint strand ( 400 ).

15. The library-splint complex ( 500 ) of claim 14 , wherein the unique identification sequence comprises a random sequence.

16. The library-splint complex ( 500 ) of claim 1 , wherein the first right universal adaptor sequence ( 130 ) and/or the second right universal adaptor sequence ( 150 ) comprises:
(i) a universal binding sequence for a forward sequencing primer;
(ii) a universal binding sequence for a reverse sequencing primer;
(iii) a universal binding sequence for a first surface primer;
(iv) a universal binding sequence for a second surface primer;
(v) a universal binding sequence for a forward amplification primer;
(vi) a universal binding sequence for a reverse amplification primer; and/or
(vii) a universal binding sequence for a compaction oligonucleotide.

17. The library-splint complex ( 500 ) of claim 16 , wherein the second splint strand ( 400 ) includes at least a first sub-region and a second sub-region, wherein the first sub-region comprises a universal binding sequence for a third surface primer and the second sub-region comprises a universal binding sequence for a fourth surface primer, and wherein the first and second sub-regions do not hybridize or exhibit very little hybridization to the first and second surface primers.

18. The library-splint complex ( 500 ) of claim 17 , wherein the second splint strand ( 400 ) comprises a third sub-region, wherein the third sub-region comprises a sample index sequence having 5-20 bases and/or a unique identification sequence having 2-10 or more bases.

19. The library-sprint complex ( 500 ) of claim 18 , wherein the unique identification sequence comprises a random sequence.

20. The library-splint complex ( 500 ) of claim 17 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises at least a fourth sub-region and a fifth sub-region, wherein the fourth sub-region hybridizes to the first sub-region of the second splint strand ( 400 ), and the fifth sub-region hybridizes to the second sub-region of the second splint strand ( 400 ), and where

Assignees

• Element Biosciences Inc

Inventors

• Zhao Junhua
• Qi Xiaodong
• Wiseman Kelly
• Yu Hua
• Kellinger Matthew

Classifications

• C12Q2535/125

  Allele specific primer extension · CPC title

• C12Q2565/50

  Detection characterised by immobilisation to a surface · CPC title

• C12Q2525/307

  Circular oligonucleotides · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12N15/09 Primary

  Recombinant DNA-technology · CPC title