Library preparation methods and compositions and uses therefor
Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.
1. A method for preparing a library of target nucleic acid sequences comprising
(a) contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences in the sample under conditions wherein the target nucleic acid(s) undergo a first amplification;
(b) digesting resulting first amplification products to reduce or eliminate resulting primer dimers and prepare partially digested target amplicons, producing gapped, double stranded amplicons, then repairing the partially digested target amplicons; and
(c) amplifying the repaired target amplicons in a second amplification using universal primers,
thereby producing a library of target nucleic acid sequences;
wherein each of the plurality of adaptors comprises a universal handle sequence and a target nucleic acid sequence and a cleavable moiety and optionally one or more tag sequences; wherein at least two and up to one hundred thousand target specific adaptor pairs are included; and
wherein the target nucleic acid sequence of the adaptor includes at least one cleavable moiety and the universal handle sequence does not include the cleavable moiety.
2. The method of claim 1 wherein an optional tag sequence is included in at least one adaptor, and the cleavable moieties are included in the adaptor sequence flanking either end of the tag sequence.
3. The method of claim 2 wherein all of the adaptors comprise tag sequences having cleavable groups flanking either end of the tag sequences.
4. The method of claim 2 wherein each target specific pair of the plurality of adaptors includes up to 16,777,216 different adaptor combinations comprising different tag sequences.
5. The method of claim 1 carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries.
6. The method of claim 1 wherein the digestion and repair of step (b) is carried out in a single step or is carried out in a temporally separate manner at different temperatures.
7. The method of claim 6 , wherein the plurality of gapped polynucleotide products in step (b) are contacted simultaneously with a digestion reagent and a repair reagent.
8. The method of claim 6 , wherein the plurality of gapped polynucleotide products in step (b) are contacted sequentially with a digestion reagent and a repair reagent.
9. The method of claim 6 , wherein a digestion and repair reagent comprises any one or a combination of one or a combination of uracil DNA glycosylase (UDG), apurinic endonuclease (APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and/or human DNA polymerase beta; and any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, Human DNA polymerase beta, T4 DNA polymerase and/or T7 DNA polymerase, SuperFiU DNA polymerase, E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, and/or 9° N DNA ligase.
10. The method of claim 9 wherein a digestion and repair reagent comprises any one or a combination of uracil DNA glycosylase (UDG), apurinic endonuclease (APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, and T7 DNA ligase.
11. The method claim 1 wherein one or more of the method steps is conducted in an automated mode.
12. The method of claim 1 further comprising at least one purification step.
13. The method of claim 12 wherein a purification step is carried out only after step (c) or wherein a first purification step is carried out after step (b) and a second purification step is carried out after step (c).
14. The method of claim 1 , further comprising analyzing the sequence of the resulting library of target nucleic acid sequences.
15. The method of claim 14 wherein analyzing comprises sequencing by traditional sequencing reactions, high throughput next generation sequencing, targeted multiplex array sequence detection, or any combination of two or more of the foregoing.
16. The method of claim 15 wherein sequencing is carried out in a bidirectional manner, thereby generating sequence reads in both forward and reverse strands for any given amplicon.
17. The method of claim 14 further comprising determining the abundance of at least one of the target nucleic acid sequences in the sample.
18. The method of claim 1 , wherein a digestion reagent comprises any one or a combination of uracil DNA glycosylase (UDG), apurinic endonuclease (APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and/or human DNA polymerase beta.
19. The method of claim 1 wherein a repair reagent comprises any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, Human DNA polymerase beta, T4 DNA polymerase and/or T7 DNA polymerase, SuperFiU DNA polymerase, E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, and/or 9° N DNA ligase.
20. A method for preparing a library of target nucleic acid sequences comprising
(d) contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences in the sample under conditions wherein the target nucleic acid(s) undergo a first amplification;
(e) digesting resulting first amplification products to reduce or eliminate resulting primer dimers and prepare partially digested target amplicons, producing gapped, double stranded amplicons, then repairing the partially digested target amplicons; and
(f) amplifying the repaired target amplicons in a second amplification using universal primers,
thereby producing a library of target nucleic acid sequences;
wherein each of the plurality of adaptors comprise a universal handle sequence, one or more tag sequences, a target nucleic acid sequence and a cleavable moiety; and wherein at least two and up to one hundred thousand target specific adaptor pairs are included;
wherein the target nucleic acid sequence of the adaptor includes at least one cleavable moiety, cleavable moieties are included in the flanking either end of the tag sequence and the universal handle sequence does not include the cleavable moiety.