IP Library › Granted Patent US 12,606,819
Granted Patent B2
US 12,606,819 · App. 18/347,440 · Granted Apr 21, 2026

PCR-free library preparation using double-stranded splint adaptors and methods of use

Inventors: Junhua Zhao (San Diego, CA); Xiaodong Qi (San Diego, CA); Shawn Levy (San Diego, CA)
Assignee: Element Biosciences, Inc.
C12N15/1065
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Quick Facts
Patent No.
US 12,606,819
App. No.
18/347,440
Filed
Jul 5, 2023
Granted
Apr 21, 2026
Kind
B2
Art Unit
1684
USPC
506/26
Abstract

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors, e.g., PCR-free workflows. 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 and/or an index sequence.

Claims (56)

1 . A method for forming a plurality of library-splint complexes ( 500 ) comprising:

a) providing a plurality of double-stranded splint adaptors ( 200 ), wherein individual double-stranded splint adaptors ( 200 ) in the plurality comprise a first splint strand ( 300 ) hybridized to all or a portion of a second splint strand ( 400 ),

wherein the double-stranded splint adaptor includes a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region ( 320 ), an internal region ( 310 ) comprising a universal adaptor sequence for a surface capture primer binding site, and a second region ( 330 ), and wherein:

ii) the internal region of the first splint strand ( 310 ) is hybridized to all or a portion of the second splint strand ( 400 ), and

(ii) the internal region ( 310 ) of the first splint strand ( 300 ) comprises at least three sub-regions comprising sub-region ( 311 ), sub-region ( 312 ) and sub-region ( 313 ),

wherein the first splint strand, or both splint strands comprise sample index sequence comprising a 3-mer random sequence (NNN), and

wherein sub-region ( 311 ), subregion ( 312 ) or subregion ( 313 ) comprise the sample index sequence of the first splint strand, and the sample index sequence of the first splint strand comprises an 18-carbon spacer and/or an 18-carbon spacer and at least one deoxyinosine; and

b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual single-stranded nucleic acid library molecules comprise a sequence of interest ( 110 ) flanked on a first side by a universal adaptor sequence for a forward sequencing primer binding site ( 120 ) and flanked on a second side by a universal adaptor sequence for a reverse sequencing primer binding site ( 130 ), wherein the first region of the first splint strand ( 320 ) hybridizes to the universal adaptor sequence for the forward sequencing primer binding site ( 120 ) and the second region of the first splint strand ( 330 ) hybridizes to the universal adaptor sequence for the reverse sequencing primer binding site ( 130 ), wherein the first splint strand ( 300 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 7-10 and 12-14,

thereby circularizing the plurality of single-stranded nucleic acid library molecules to form a plurality of library-splint complexes ( 500 ), individual library-splint complexes having two nicks.

2 . The method of claim 1 , further comprising: (c) contacting the plurality of library-splint complexes ( 500 ) with a ligase to generate a plurality of covalently closed circular library molecules ( 600 ).

3 . The method of claim 1 , wherein the sub-region ( 311 ) comprises the universal adaptor sequence for a surface capture primer binding site, a universal adaptor sequence for a surface pinning primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

4 . The method of claim 1 , wherein the sub-region ( 312 ) comprises the universal adaptor sequence for a surface capture primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

5 . The method of claim 1 , wherein the sub-region ( 313 ) comprises the universal adaptor sequence for a surface capture primer binding site, a universal adaptor sequence for a surface pinning primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

6 . The method of claim 2 , further comprising:

i) distributing the plurality of covalently closed circular library molecules ( 600 ) onto a support having a plurality of the surface capture primers immobilized to the support, under a condition suitable for hybridizing individual covalently closed circular library molecules ( 600 ) to individual immobilized surface capture primers thereby immobilizing the plurality of covalently closed circular library molecules ( 600 ) to the support.

7 . The method of claim 6 , wherein the support further comprises a plurality of surface pinning primers immobilized to the support.

8 . The method of claim 6 , further comprising:

ii) contacting the plurality of covalently closed circular library molecules ( 600 ) immobilized to the support with a plurality of strand-displacing polymerases and a plurality of nucleotides, under a condition suitable to conduct a rolling circle amplification reaction on the support using the plurality of surface capture primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 600 ) as template molecules,

thereby generating a plurality of nucleic acid concatemer molecules immobilized to the surface capture primers.

9 . The method of claim 8 , further comprising:

iii) sequencing the plurality of nucleic acid concatemer molecules immobilized to the surface capture primers, wherein the sequencing comprises (i) sequencing the sample index sequence and (ii) sequencing the sequence of interest ( 110 ).

10 . The method of claim 8 , further comprising:

iii) sequencing the plurality of nucleic acid concatemer molecules immobilized to the surface capture primers, wherein the sequencing comprises (A) sequencing one or more short random sequences (NNN), (B) sequencing one or more sample index sequences, and (C) sequencing the sequence of interest ( 110 ).

11 . The method of claim 3 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises one sample index sequence.

12 . The method of claim 1 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 1-6 and 43-44.

13 . The method of claim 1 , wherein the second splint strand ( 400 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 18-26, 45-47, 49, 5′-CATGTAAT-3′, 5′-GTAGGAGCCNNN-3′, 5′-CCGCTGCTANNN-3′, 5′-AACAACAAGNNN-3′, 5′-GGTGGTCTANNN-3′, 5′-TTGGCCAACNNN-3′, 5′-CAGGAGTGCNNN-3′, and 5′-ATCACACTANNN-3′.

14 . The method of claim 1 , wherein the first splint strand ( 300 ) is a universal long splint strand comprising:

i) at least one sub-region only partially hybridized to the second splint strand ( 400 ), and/or

iii) a sub-region comprising a spacer sequence.

15 . The method of claim 1 , wherein the first splint strand ( 300 ) and the second splint strand ( 400 ) each comprise one or more sub-regions, and wherein the second splint strand comprises a sub-region that does not hybridize to the first splint strand, thereby providing a duplex formed by hybridization of the first splint strand to the second splint strand comprising a loop of the sub-region of the second splint strand.

16 . A method for forming a plurality of library-splint complexes ( 500 ) comprising:

a) providing a plurality of double-stranded splint adaptors ( 200 ), wherein individual double-stranded splint adaptors ( 200 ) in the plurality comprise a first splint strand ( 300 ) hybridized to all or a portion of a second splint strand ( 400 ),

wherein the double-stranded splint adaptor includes a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region ( 320 ), an internal region ( 310 ) comprising a universal adaptor sequence for a surface capture primer binding site, and a second region ( 330 ),

wherein the first splint strand, the second splint strand, or both splint strands comprise a sample index sequence comprising a 3-mer random sequence (NNN), and

wherein the first splint strand ( 300 ) and the second splint strand ( 400 ) each comprise one or more sub-regions, and wherein the second splint strand comprises a sub-region that does not hybridize to the first splint strand, thereby providing a duplex formed by hybridization of the first splint strand to the second splint strand comprising a loop of the sub-region of the second splint strand; and

b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual single-stranded nucleic acid library molecules comprise a sequence of interest ( 110 ) flanked on a first side by a universal adaptor sequence for a forward sequencing primer binding site ( 120 ) and flanked on a second side by a universal adaptor sequence for a reverse sequencing primer binding site ( 130 ), wherein the first region of the first splint strand ( 320 ) hybridizes to the universal adaptor sequence for the forward sequencing primer binding site ( 120 ) and the second region of the first splint strand ( 330 ) hybridizes to the universal adaptor sequence for the reverse sequencing primer binding site ( 130 ), wherein the first splint strand ( 300 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 7-10 and 12-14,

thereby circularizing the plurality of single-stranded nucleic acid library molecules to form a plurality of library-splint complexes ( 500 ), individual library-splint complexes having two nicks.

17 . The method of claim 16 , further comprising: (c) contacting the plurality of library-splint complexes ( 500 ) with a ligase to generate a plurality of covalently closed circular library molecules ( 600 ).

18 . The method of claim 16 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises at least three sub-regions.

19 . The method of claim 18 , wherein the at least three sub-regions comprise sub-region ( 311 ), sub-region ( 312 ) and sub-region ( 313 ).

20 . The method of claim 19 , wherein the sub-region ( 311 ) comprises the universal adaptor sequence for a surface capture primer binding site, a universal adaptor sequence for a surface pinning primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

21 . The method of claim 19 , wherein the sub-region ( 312 ) comprises the universal adaptor sequence for a surface capture primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

22 . The method of claim 19 , wherein the sub-region ( 313 ) comprises the universal adaptor sequence for a surface capture primer binding site, a universal adaptor sequence for a surface pinning primer binding site, the sample index sequence comprising the short random sequence (NNN) and/or a unique molecule index (UMI).

23 . The method of claim 17 , further comprising:

i) distributing the plurality of covalently closed circular library molecules ( 600 ) onto a support having a plurality of the surface capture primers immobilized to the support, under a condition suitable for hybridizing individual covalently closed circular library molecules ( 600 ) to individual immobilized surface capture primers thereby immobilizing the plurality of covalently closed circular library molecules ( 600 ) to the support.

24 . The method of claim 23 , wherein the support further comprises a plurality of surface pinning primers immobilized to the support.

25 . The method of claim 23 , further comprising:

ii) contacting the plurality of covalently closed circular library molecules ( 600 ) immobilized to the support with a plurality of strand-displacing polymerases and a plurality of nucleotides, under a condition suitable to conduct a rolling circle amplification reaction on the support using the plurality of surface capture primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 600 ) as template molecules,

thereby generating a plurality of nucleic acid concatemer molecules immobilized to the surface capture primers.

26 . The method of claim 25 , further comprising:

iii) sequencing the plurality of nucleic acid concatemer molecules immobilized to the surface capture primers, wherein the sequencing comprises (i) sequencing the sample index sequence and (ii) sequencing the sequence of interest ( 110 ).

27 . The method of claim 25 , further comprising:

iii) sequencing the plurality of nucleic acid concatemer molecules immobilized to the surface capture primers, wherein the sequencing comprises (A) sequencing one or more short random sequences (NNN), (B) sequencing one or more sample index sequences, and (C) sequencing the sequence of interest ( 110 ).

28 . The method of claim 16 , wherein the internal region ( 310 ) of the first splint strand ( 300 ) comprises one sample index sequence.

29 . The method of claim 16 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 1-6 and 43-44.

30 . The method of claim 16 , wherein the second splint strand ( 400 ) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 18-26, 45-47, 49, 5′-CATGTAAT-3′, 5′-GTAGGAGCCNNN-3′, 5′-CCGCTGCTANNN-3′, 5′-AACAACAAGNNN-3′, 5′-GGTGGTCTANNN-3′, 5′-TTGGCCAACNNN-3′, 5′-CAGGAGTGCNNN-3′, and 5′-ATCACACTANNN-3′.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 26, 2023
From: ZHAO, JUNHUA; QI, XIAODONG; LEVY, SHAWN
To: ELEMENT BIOSCIENCES, INC.
Reel/Frame 064385/0169 →
Continuity (3)
Provisional Application 63508833 · Jun 16, 2023
Provisional Application 63358491 · Jul 5, 2022
Related Publication 20240011022A1 · Jan 11, 2024
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GenBank Accession AAB52611.1; “DNA polymerase I [Geobacillus stearothermophilus],” Apr. 21, 1997; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/AAB52611.1, 2 pages. [cited by applicant]
GenBank Accession KUO42443.1; “MAG: hypothetical protein APZ16_03045 [Candidatus Hadarchaeum yellowstonense],” Jan. 14, 2016; [retrieved online Sep. 23, 2024], URL: www.ncbi.nlm.nih.gov/protein/KUO42443.1, 2 pages. [cited by applicant]
GenBank Accession MBC7218772.1; “MAG: DNA polymerase [Hadesarchaea archaeon],” Sep. 1, 2020; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/mbc7218772.1, 2 pages. [cited by applicant]
GenBank Accession NOZ58130.1; “MAG: DNA polymerase [Euryarchaeota archaeon],” Mar. 17, 2023 [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/NOZ58130, 2 pages. [cited by applicant]
GenBank Accession NOZ77387.1; “MAG: DNA polymerase, partial [Euryarchaeota archaeon],” Mar. 17, 2023 [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/NOZ77387.1, 2 pages. [cited by applicant]
GenBank Accession RLF78286.1; “MAG: DNA polymerase [Thermococci archaeon],” Oct. 15, 2018; URL: www.ncbi.nlm.nih.gov/protein/RLF78286.1, 2 pages. [cited by applicant]
GenBank Accession RLF89458.1; “MAG: DNA polymerase [Thermococci archaeon],” Oct. 15, 2018; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/RLF89458.1, 2 pages. [cited by applicant]
GenBank Accession RLI89578.1; “MAG: DNA polymerase [Candidatus Altiarchaeales archaeon],” Oct. 15, 2018; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/RLI89578.1, 2 pages. [cited by applicant]
GenBank Accession RMF90817.1; “MAG: DNA polymerase [Euryarchaeota archaeon],” Oct. 29, 2018; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/RMF90817.1, 2 pages. [cited by applicant]
Greenough, L., et al.; “Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes,” Nucleic Acids Research, 44(2):e15, pp. 1-11 (2016). [cited by applicant]
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Illumina “Overview of Illumina Sequencing by Synthesis Workflow,” Oct. 5, 2016 (Oct. 5, 2016) [retrieved online Oct. 9, 2024] https://www.youtube.com/watch?v=fCd6B5HRaZ8, 2 pages. [cited by applicant]
Kao, H-I, et al.; “Cleavage specificity of [cited by applicant]
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Lee, B-I., et al.; “The RAD2 domain of human exonuclease 1 exhibits 5′ to 3′ exonuclease and flap structure-specific endonuclease activities,” Journal of Biological Chemistry, 274(53):37763-37769 (1999). [cited by applicant]
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NCBI Reference Sequence: NP_041963.1; Accession NC_001604.1; “DNA ligase [ [cited by applicant]
NCBI Reference Sequence: NP_049813.1; Accession NC_000866.4; “DNA ligase [ [cited by applicant]
NCBI Reference Sequence: NP_523305.1; Accession NC_003298.1; “DNA ligase [Enterobacteria phage T3],” Jan. 7, 2023; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/17570796, 2 pages. [cited by applicant]
NCBI Reference Sequence: WP_042693257.1; Accession WP_042693257; “ATP-dependent DNA ligase [Thermococcus nautili],” Jun. 2, 2024; [retrieved online Sep. 23, 2024] URL: www.ncbi.nlm.nih.gov/protein/757139009, 1 page. [cited by applicant]
NCBI Reference Sequence: WP_175059460.1; Accession WP_175059460; “DNA-directed DNA polymerase [ [cited by applicant]
Pettersson, E., et al.; “Generations of sequencing technologies,” Genomics 93(2):105-111 (2009). [cited by applicant]
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Tsutakawa, S.E., et al.; “Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability,” Nature Communications, 8:15855, pp. 1-15 (2017). [cited by applicant]
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UniProtKB: P0CL77—DPOL_PYRAB; “DNA polymerase 1,” Last Updated: Apr. 5, 2011; [retrieved online Sep. 23, 2024] URL: www.uniprot.org/uniprotkb/P0CL77/entry, 5 pages. [cited by applicant]
UniProtKB: P30317—DPOL_THELI; “DNA polymerase,” Last Updated: Apr. 1, 1993 [retrieved online Sep. 23, 2024] URL: www.uniprot.org/uniprotkb/P30317/entry, 6 pages. [cited by applicant]
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UniProtKB: Q56366—DPOL_THES9; “DNA polymerase,” Last Updated: Nov. 1, 1996 [retrieved online Sep. 23, 2024] URL: www.uniprot.org/uniprotkb/Q56366/entry, 7 pages. [cited by applicant]
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