Methods and compositions for rapid nucleic acid library preparation
View Patent ↗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 composition comprising a population of nucleic acid molecules, wherein each molecule of said population independently comprises:
a first strand comprising
a first sequencer specific adapter sequence,
a molecular tag sequence comprising a barcode, and
an independent target sequence,
wherein each independent target sequence comprises a random primer sequence and a subset of a sample nucleic acid sequence, wherein at least one of said population comprises at least one mismatch between said random primer sequence and said sample nucleic acid sequence, wherein at least a first molecule of said population comprises a first independent target sequence comprising a first subset of said sample nucleic acid sequence, and wherein at least a second molecule of said population comprises a second independent target sequence that comprises a second subset of said sample nucleic acid sequence.
2. The composition of claim 1 , wherein said first sequencer specific adapter of each first strand of said population is identical.
3. The composition of claim 1 , wherein said molecular tag sequence of each molecule of said population comprises at least 6 nucleotide bases.
4. The composition of claim 1 , wherein a first member of said population and a second member of said population comprise non-identical molecular tag sequences.
5. The composition of claim 1 , wherein each first strand comprises a 3′-deoxynucleotide base at its 3′ end.
6. The composition of claim 1 , wherein each first strand comprises a molecular ligand at its 5′ end.
7. The composition of claim 1 , wherein each first strand comprises a molecular ligand attached at a non-terminal position.
8. The composition of claim 1 , wherein each first strand comprises a molecular ligand at its 3′ end.
9. The composition of claim 8 , wherein said molecular ligand is biotin.
10. The composition of claim 1 , wherein each molecule of said population comprises a second strand comprising:
a second sequencer specific adapter sequence, and
a second molecular tag sequence comprising a barcode.
11. The composition of claim 10 , wherein said second strand of at least one molecule of said population is annealed to a first strand via at least partial base pairing of a second molecular tag sequence of said second strand to the independent target sequence of said first strand.
12. The composition of claim 1 , wherein at least one molecule of said population is bound to a molecular ligand binder.
13. The composition of claim 1 , wherein said composition comprises unincorporated nucleic acid triphosphates.
14. The composition of claim 1 , wherein said composition comprises molecular ligand binder wash buffer.
15. The composition of claim 1 , wherein said composition comprises polymerase extension buffer.
16. The composition of claim 1 , wherein said composition comprises nucleic acid polymerase.
17. The composition of claim 16 , wherein said composition comprises nucleic acid polymerase possessing nucleic acid helicase activity.
18. The composition of claim 16 , wherein said composition comprises nucleic acid polymerase possessing nucleic acid strand displacement activity.
19. The composition of claim 16 , wherein said composition comprises nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 2.
20. The composition of claim 1 , wherein the composition is contained in a single tube.