Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.
1. A system, comprising: a reaction mixture comprising a sample nucleic acid molecule, a plurality of nucleotides, a polymerizing enzyme, an excising enzyme, a bead comprising a plurality of nucleic acid barcode molecules, and a primer separate from said bead, wherein said plurality of nucleotides comprises a uridine-containing nucleotide,
wherein said primer comprises a random sequence,
wherein said polymerizing enzyme is configured to use said sample nucleic acid molecule and said plurality of nucleotides, including said uridine-containing nucleotide, to generate a double-stranded nucleic acid molecule comprising a uracil,
wherein said excising enzyme is configured to excise said uracil from said double-stranded nucleic acid molecule, and
wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is configured to couple to said double-stranded nucleic acid molecule or derivative thereof.
2. The system of claim 1 , wherein said primer is configured to anneal to said sample nucleic acid molecule.
3. The system of claim 1 , wherein said random sequence of said primer is 5 to 25 nucleotides in length.
4. The system of claim 1 , further comprising a ligating enzyme, wherein said ligating enzyme is configured to ligate said nucleic acid barcode molecule to said double-stranded nucleic acid molecule or derivative thereof.
5. The system of claim 4 , wherein said ligating enzyme is a deoxyribonucleic acid (DNA) ligase.
6. The system of claim 5 , wherein said ligating enzyme is a T4 DNA ligase.
7. The system of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is double stranded.
8. The system of claim 1 , wherein said polymerizing enzyme has strand displacement activity.
9. The system of claim 8 , wherein said polymerizing enzyme is a phi 29 polymerase.
10. The system of claim 1 , wherein said excising enzyme is a uracil DNA glycosylase.
11. The system of claim 1 , wherein said bead is a gel bead.
12. The system of claim 11 , wherein said gel bead is a degradable gel bead.
13. The system of claim 1 , wherein said plurality of nucleic acid barcode molecules is releasably coupled to said bead.
14. The system of claim 1 , wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are covalently-linked to said bead.
15. The system of claim 1 , wherein said uracil is present in only one strand of said double-stranded nucleic acid molecule.
16. The system of claim 1 , wherein said nucleic acid barcode molecule further comprises one or more functional sequences selected from the group consisting of an adapter sequence, a primer sequence, a primer annealing sequence, an attachment sequence, a sequencing primer sequence, and a partial sequencing primer sequence.
17. The system of claim 1 , further comprising a partition comprising said reaction mixture.
18. The system of claim 17 , wherein said partition is a droplet.
19. The system of claim 17 , wherein said partition is a well.
20. A system, comprising: a reaction mixture comprising a double-stranded deoxyribonucleic acid (DNA) molecule comprising a uracil, an excising enzyme configured to excise said uracil from said double-stranded DNA molecule, a DNA polymerase having strand displacement activity, and a bead comprising a plurality of nucleic acid barcode molecules.
21. The system of claim 20 , further comprising a ligating enzyme, wherein said ligating enzyme is configured to ligate a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to said double-stranded DNA molecule or derivative thereof.
22. The system of claim 20 , wherein said DNA polymerase having strand displacement activity is configured to generate single-stranded nucleic acid molecules from said double-stranded DNA molecule or derivative thereof.
23. The system of claim 20 , wherein said excising enzyme is configured to excise said uracil from said double-stranded DNA molecule to generate a nicked, double-stranded DNA molecule.
24. The system of claim 20 , wherein said uracil is present in only one strand of said double-stranded DNA molecule.
25. The system of claim 20 , wherein said bead is a gel bead.
26. The system of claim 25 , wherein said gel bead is a degradable gel bead.
27. The system of claim 20 , wherein said plurality of nucleic acid barcode molecules is releasably coupled to said bead.
28. The system of claim 20 , wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are covalently-linked to said bead.
29. The system of claim 20 , wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is configured to couple to a strand of said double-stranded DNA molecule or derivative thereof.
30. A system, comprising: a reaction mixture comprising a sample nucleic acid molecule, a plurality of nucleotides, a DNA polymerase, an excising enzyme, and a bead comprising a plurality of nucleic acid barcode molecules, wherein said plurality of nucleotides comprise a uridine-containing nucleotide,
wherein said DNA polymerase has strand-displacement activity and is configured to use said sample nucleic acid molecule and said plurality of nucleotides, including said uridine-containing nucleotide, to generate a double-stranded nucleic acid molecule comprising a uracil,
wherein said excising enzyme is configured to excise said uracil from said double-stranded nucleic acid molecule, and
wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is configured to couple to said double-stranded nucleic acid molecule or derivative thereof.
31. The system of claim 30 , further comprising a ligating enzyme, wherein said ligating enzyme is configured to ligate said nucleic acid barcode molecule to said double-stranded nucleic acid molecule or derivative thereof.
32. The system of claim 31 , wherein said ligating enzyme is a deoxyribonucleic acid (DNA) ligase.
33. The system of claim 32 , wherein said ligating enzyme is a T4 DNA ligase.
34. The system of claim 30 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is double stranded.
35. The system of claim 30 , wherein said DNA polymerase is a phi 29 polymerase.
36. The system of claim 30 , wherein said excising enzyme is a uracil DNA glycosylase.
37. The system of claim 30 , wherein said bead is a gel bead.
38. The system of claim 37 , wherein said gel bead is a degradable gel bead.
39. The system of claim 30 , wherein said plurality of nucleic acid barcode molecules is releasably coupled to said bead.