Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided.
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1. A method of multiplex PCR-based enrichment of a target substrate comprising the steps of: (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction com
1. A method of multiplex PCR-based enrichment of a target substrate comprising the steps of: (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a plurality of different target-specific primer pairs for amplifying a plurality of different loci of the nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein each of the plurality of different target-specific primer pairs comprise a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3′ complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, wherein the first sequence and second sequence is different for each of the plurality of different target-specific primer pairs, and wherein the forward primer and the reverse primer of each of the plurality of different target-specific primer pairs further comprise a 5′ terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′ terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence, and wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs;(ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a plurality of different target specific amplicons, wherein each of the plurality of different target specific amplicons comprise the 5′ terminal sequence at their 5′ terminal regions;(iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles to further amplify the plurality of different target specific amplicons, wherein the additional PCR cycles comprise the first annealing temperature for a second annealing duration, wherein the first annealing duration is greater than the second annealing duration. 2. The method of claim 1 wherein either or both of the forward primer and the reverse primer of each of the plurality of different target specific primer pair further comprise a degenerate sequence in their 5′ terminal sequence, wherein the degenerate sequence is positioned between the 3′ complementary sequence and the universal adaptor sequence. 3. The method of claim 1 wherein the modified base is deoxyuridine, RNA, deoxyinosine, or inosine. 4. The method of claim 3 wherein the DNA polymerase is a high-fidelity DNA polymerase that is tolerant of the modified base. 5. The method of claim 1 wherein the universal primer further comprises a nuclease resistant modification at its 3′ termini. 6. The method of claim 5 wherein the nuclease resistant modification is a phosphorothioate linkage. 7. The method of claim 1 wherein the forward primer and the reverse primer of each of the plurality of different target-specific primer pairs further comprise a nuclease resistant modification at their 3′ termini. 8. The method of claim 1 wherein the molar ratio of each of the plurality of different target-specific primer pairs to universal primer is at least 1:100. 9. The method of claim 1 wherein the plurality of different target-specific amplicons comprise a first target-specific amplicon and a second target-specific amplicon, wherein the first target-specific amplicon comprises a region that overlaps with the second target-specific amplicon and a region that does not overlap with the second target-specific amplicon. 10. The method of claim 1 wherein at least one of the plurality of different target-specific amplicons comprises two regions that overlap with two other of the plurality of different target-specific amplicons and a region that does not overlap with any other of the plurality of different target-specific amplicons. 11. The method of claim 1 wherein the first annealing duration is 5 minutes or more. 12. The method of claim 1 wherein the second annealing duration is 1 minute or less. 13. The method of claim 1 wherein step (ii) is two PCR cycles. 14. The method of claim 1 wherein step (ii) is at least four PCR cycles. 15. The method of claim 1 wherein step (iii) is from one PCR cycle to forty PCR cycles. 16. The method of claim 1 further comprising (iv) purifying the plurality of different target specific amplicons from the PCR mixture to yield a purified target specific amplicon sample. 17. The method of claim 16 further comprising the following steps: (v) applying a ligase, a 5′ adaptor, a 3′ adaptor, an enzyme having 5′ flap endonuclease activity, and an endonuclease to the purified target specific amplicon sample, wherein the 5′ adaptor comprises a 3′ sequence that is identical to a 3′ portion of the universal adaptor sequence, wherein the 5′ adaptor further comprises a 5′ portion that is not present in the universal adaptor sequence, wherein the 3′ adaptor comprises a sequence that is identical to the 5′ portion of the universal adaptor sequence, and wherein the 3′ adaptor further comprises a 3′ portion that is not present in the universal adaptor sequence; and(vi) incubating the purified target specific amplicon sample of step (iv) under conditions sufficient to permit the following: (a) cleavage of the universal primers comprising the modified base present in the plurality of different target specific amplicons;(b) annealing of the 5′ adaptor and 3′ adaptor to each of the plurality of different target specific amplicons; and(c) ligation of the 5′ adaptor and 3′ adaptor to each of the plurality of different target specific amplicons, preceded by a flap endonuclease cleavage if the endonuclease digestion results in any remaining bases of the universal primer. 18. The method of claim 17 wherein the conditions sufficient comprise 37° C. for at least 10 minutes. 19. The method of claim 17 wherein the endonuclease is selected from the group consisting of UDG+Endonuclease VIII, RNase HI, RNase H2, and Endonuclease V. 20. A method for generating a sequencing library comprising the steps of: (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a target-specific primer pair, a universal primer, a DNA polymerase, and dNTPs, wherein a forward primer and a reverse primer of the target-specific primer pair comprise a 3′ complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, and wherein each primer of the target-specific primer pair comprises a 5′ terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′ terminal sequence comprises an universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence, and wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of the target-specific primer pair;(ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a set of target specific amplicons that comprise the 5′ terminal sequence at their 5′ terminal regions;(iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles sufficient to further amplify the set of target specific amplicons, wherein the additional PCR cycles comprise the first annealing temperature for a second annealing duration, wherein the first annealing duration is greater than the second annealing duration;(iv) applying a ligase, a 5′ adaptor, a 3′ adaptor, an enzyme having 5′ flap endonuclease activity, and an endonuclease to a second sample comprising the set of target specific amplicons purified from the PCR reaction mixture following step (iii), wherein the 5′ adaptor comprises a 3′ sequence that is identical to a 3′ portion of the universal adaptor sequence, wherein the 5′ adaptor further comprises a 5′ portion that is not present in the universal adaptor sequence, wherein the 3′ adaptor comprises a sequence that is identical to the 5′ portion of the universal adaptor sequence, and wherein the 3′ adaptor further comprises a 3′ portion that is not present in the universal adaptor sequence; and(v) incubating the second sample of step (iv) under conditions sufficient to permit the following:(a) cleavage of the universal primers comprising the modified base present in the set of target specific amplicons;(b) annealing of the 5′ adaptor and 3′ adaptor to each of the set of target specific amplicons; and(c) ligation of the 5′ adaptor and 3′ adaptor to each of the set of target specific amplicons, preceded by a flap endonuclease cleavage if the endonuclease digestion results in any remaining bases of the universal primer. 21. A polymerase chain reaction system for generating a sequencing library comprising: a target-specific primer pair comprising a forward primer and a reverse primer, wherein the forward primer and reverse primer each comprise a 3′ complementary sequence that is complementary to a first sequence and a second sequence, respectively, of a nucleic acid substrate and a 5′ terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′ terminal sequence comprises a universal adaptor sequence;a universal primer, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, and wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence;dNTPs;a DNA polymerase for amplifying the nucleic acid substrate;an endonuclease for cleaving the universal primer comprising the modified base;a 3′ adaptor polynucleotide comprising a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises the second oligonucleotide hybridized to the first oligonucleotide, and wherein the 3′ adaptor polynucleotide comprises a sequence that is identical to the 5′ portion of the universal adaptor sequence, and wherein the 3′ adaptor polynucleotide further comprises a 3′ portion that is not present in the universal adaptor sequence;a 5′ adaptor polynucleotide comprising a 3′ sequence that is identical to a 3′ portion of the universal adaptor sequence, wherein the 5′ adaptor polynucleotide further comprises a 5′ portion that is not present in the universal adaptor sequence;an enzyme having 5′ flap endonuclease activity for cleaving any bases remaining of the universal primer; anda ligase for ligating the 3′ adaptor polynucleotide to a 3′ terminus of a target specific amplicon and for ligating the 5′ adaptor polynucleotide to a 5′ terminus of the target specific amplicon. 22. A method of PCR-based enrichment of a target substrate comprising the steps of: (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a target-specific primer pair for amplifying a nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3′ complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, and wherein the forward primer and the reverse primer each further comprise a 5′ terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′ terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, and wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence;(ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a target specific amplicon, wherein the target specific amplicon comprises the 5′ terminal sequence at its 5′ terminal region;(iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles to further amplify the target specific amplicon. 23. The method of claim 22, wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs. 24. The method of claim 22, wherein the additional PCR cycles comprise the first annealing temperature for a second annealing duration, wherein the first annealing duration is greater than the second annealing duration. 25. The method of claim 22, wherein the modified base is deoxyuridine, RNA, deoxyinosine, or inosine. 26. The method of claim 22 further comprising (iv) purifying the target specific amplicon from the PCR mixture to yield a purified target specific amplicon sample. 27. The method of claim 26 further comprising the following steps: (v) applying a ligase, a 5′ adaptor, a 3′ adaptor, an enzyme having 5′ flap endonuclease activity, and an endonuclease to the purified target specific amplicon sample, wherein the 5′ adaptor comprises a 3′ sequence that is identical to a 3′ portion of the universal adaptor sequence, wherein the 5′ adaptor further comprises a 5′ portion that is not present in the universal adaptor sequence, wherein the 3′ adaptor comprises a sequence that is identical to the 5′ portion of the universal adaptor sequence, and wherein the 3′ adaptor further comprises a 3′ portion that is not present in the universal adaptor sequence; and(vi) incubating the purified target specific amplicon sample of step (iv) under conditions sufficient to permit the following: (a) cleavage of the universal primers comprising the modified base present in the plurality of different target specific amplicons;(b) annealing of the 5′ adaptor and 3′ adaptor to each of the plurality of different target specific amplicons; and(c) ligation of the 5′ adaptor and 3′ adaptor to each of the plurality of different target specific amplicons, preceded by a flap endonuclease cleavage if the endonuclease digestion results in any remaining bases of the universal primer. 28. The method of claim 9, wherein the second target-specific amplicon forms a stable secondary structure.
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