Methods and compositions for rapid nucleic library preparation
Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.
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.