Granted Patent B2

US 12,686,863 · App. 17/536,839 · Granted Jul 21, 2026

Inventor: Keith Brown (Carlsbad, CA)

Assignee: Twist Bioscience Corporation

C12N15/1093C12Q1/6806C12Q1/6874G16B30/00C12Q1/6844C12Q1/686C40B40/06G16B30/20

Abstract

Claims (26)

1 . A kit for generating a library from a target nucleic acid sample, the kit comprising:

an extension mixture comprising dNTPs and ddNTPs;

a first primer library comprising a first plurality of random primers configured to generate a population of non-identical sequences each comprising a subset of a target nucleic acid sample and at least one mismatch between a random primer of the first plurality of random primers and the target nucleic acid sequence; and

a second primer library comprising a second plurality of random primers configured to extend the population of non-identical sequences to obtain double-stranded sequences.

2 . The kit of claim 1 , wherein a molar ratio of dNTPs:ddNTPs is about 999 dNTPs to 1 ddNTP, about 199 dNTPs to 1 ddNTP, about 99 dNTPs to 1 ddNTPs, or about 49 dNTPs to 1 ddNTP.

3 . The kit of claim 1 , wherein the ddNTPs comprise biotin-labeled ddNTPs.

4 . The kit of claim 1 , wherein the first primer library is selected based at least in part on a GC content of the target nucleic acid sample.

5 . The kit of claim 4 , wherein a GC content of the first primer library is biased to match a relative abundance of the GC content in the target nucleic acid sample.

6 . The kit of claim 1 , further comprising DNA polymerase.

7 . The kit of claim 6 , wherein the DNA polymerase comprises phage29 polymerase, sequenase, thermosequenase, bst DNA polymerase, or Taq DNA polymerase.

8 . The kit of claim 1 , further comprising a polymerase extension buffer comprising reagents consistent with polymerase activity.

9 . The kit of claim 8 , wherein the polymerase activity comprises displacement activity, ddNTP incorporation activity, biotin-labeled nucleotide incorporation activity, or any combination thereof.

10 . The kit of claim 8 , wherein a volume ratio of the polymerase extension buffer to the extension mixture is about 1:2.

11 . The kit of claim 8 , wherein a molar ratio of the extension mixture to the first primer library is about 1:1.

12 . The kit of claim 1 , wherein the first primer library further comprises a plurality of adapter sequences and labels.

13 . The kit of claim 12 , wherein the labels comprise molecular tag sequences.

14 . The kit of claim 1 , further comprising magnetic beads to capture one or more of the population of non-identical sequences.

15 . The kit of claim 14 , wherein the one or more of the population of non-identical sequences comprises a molecular tag at its 3′ end.

16 . The kit of claim 15 , wherein the molecular tag is biotin.

17 . The kit of claim 14 , wherein the magnetic beads are streptavidin-coated magnetic beads.

18 . A kit for generating a library from a target nucleic acid sample, the kit comprising:

an extension mixture comprising dNTPs and ddNTPs, wherein a molar ratio of dNTPs:ddNTPs is at least about 49 dNTPs to 1 ddNTP; and

a primer library comprising a plurality of random primers, wherein the primer library is configured to generate a population of non-identical sequences each comprising a subset of a target nucleic acid sample and at least one mismatch between a random primer of the plurality of random primers and the target nucleic acid sample.

19 . A kit for generating a library from a target nucleic acid sample, the kit comprising:

an extension mixture comprising dNTPs and ddNTPs; and

a primer library comprising a plurality of random primers, wherein a GC content of the primer library is biased to match a relative abundance of GC content in a target nucleic acid sequence, and wherein the primer library is configured to generate a population of non-identical sequences each comprising a subset of the target nucleic acid sample and at least one mismatch between a random primer of the plurality of random primers and the target nucleic acid sequence.

Assignments (2)

Continuity (7)

References Cited (124)