Methods for multiplex pcr

Abstract

Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided.

What is claimed is: 
     
         1 . A method of multiplex PCR amplification of a specific target locus on a nucleic acid substrate for preparing a targeted next generation sequencing library comprising the steps of:
 (i) combining a plurality of target-specific primers with the nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′ complementary sequence that is fully complementary to a sequence of the specific target locus and a 5′ noncomplementary sequence that is not complementary to a sequence of the nucleic acid substrate, wherein the 3′ complementary sequence for each of the first and second forward and reverse primers is different, wherein the 3′ complementary sequence is between 10 and 40 bases in length, wherein the nucleic acid substrate is human genomic DNA, and wherein the specific target locus is a gene known to have clinical relevance in oncology   (ii) subjecting the PCR reaction mixture to a multiplex polymerase chain reaction thereby generating at least three amplicons within the specific target locus, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and the first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein the third amplicon is shorter in length than the first and second amplicons, wherein at least a portion of the 5′ noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third amplicon comprises a 3′ sequence and a 5′ sequence that are complementary to each other, wherein the third amplicon possesses overlapping sequence with the first and second amplicons, wherein the first amplicon possesses overlapping sequence with the second amplicon, wherein when the third amplicon is denatured, each strand of the third amplicon forms a secondary structure as a result of the 3′ sequence being complementary to the 5′ sequence, wherein the secondary structure is stable during a primer annealing step of the multiplex polymerase chain reaction.   
     
     
         2 . The method of  claim 1 , wherein the multiplex polymerase chain reaction comprises a first series of cycle conditions and a second series of cycle conditions, wherein each of the first and second series of cycle conditions include two or more cycles, wherein the first series of cycle conditions generates the first, second, and third amplicons, and wherein the first series of cycle conditions is different than the second series of cycle conditions. 
     
     
         3 . The method of  claim 2 , wherein the second series of cycle conditions yield a PCR reaction product, and wherein the second series of cycle conditions are sufficient to amplify the first and second amplicons more efficiently than the third amplicon. 
     
     
         4 . The method of  claim 3 , wherein the first and second amplicons are each present in the PCR reaction product at a greater amount than the third amplicon. 
     
     
         5 . The method of  claim 2 , wherein the first series of cycle conditions comprises 2 cycles, and wherein the second series of cycle conditions comprises 14 to more than 20 cycles. 
     
     
         6 . The method of  claim 2 , wherein the first series of cycle conditions comprises 4 cycles, and wherein the second series of cycle conditions comprises 14 to more than 20 cycles. 
     
     
         7 . The method of  claim 1 , wherein the 3′ complementary sequence is between 16 and 30 bases in length. 
     
     
         8 . The method of  claim 1 , wherein the first and second amplicon is between 90 bases and 700 bases in length. 
     
     
         9 . The method of  claim 1 , wherein the gene known to have clinical relevance in oncology is selected from the group consisting of ABU, ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, AKT1, AKT2, ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BUB1B, Cl2orf9, Cl5orf21, Cl5orf55, Cl6orf75, C2orf44, CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNBHP1, CCND1, CCND2, CCND3, CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C, CDKN2a(p14), CDX2, CEBPA, CEP1, CEP89, CHCHD7, CHEK2, CHIC2, CHN1, CIC, CIITA, CLIP1, CLTC, CLTCL1, CMKOR1, CNOT3, COL1A1, COL2A1, COPEB, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CSF3R, CTNNB1, CUX1, CYLD, D1OS170, DAXX, DCTN1, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, DUX4, EBF1, ECT2L, EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4, EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETV1, ETV4, ETV5, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7, FCGR2B, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FH, FHIT, FIP1L1, FLIT, F1127352, FLT3, FNBP1, FOXA1, FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1, FSTL3, FUBP1, FUS, FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA1 1, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB, HERPUD1, HEY1, HIP1, HIS T1H3B , HIS T1H4I, HLA-A, HLF, HLXB 9, HMGA1, HMGA2, HNRNPA2B1, HOOKS, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSPCA, HSPCB, IDHL IDH2, IGH\, IGK, IGL, IKZFL IL2, IL21R, IL6ST, IL7R, IRF4, IRTAL ITK, JAKL JAK2, JAK3, JAZFL JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA1549, KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D, KRAS, KTN1, LAF4, LASP1, LCK, LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2, LPP, LRIG3, LSM14A, LYL1, MAF, MAFB, MALAT1, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDS1, MDS2, MECT1, MED12, MEN1, MET, MITF, MKL1, MLF1, MLH1, MLL, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYO5A, MYST4, NAB2, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2, NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG1, PLCG1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPFIBP1, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1A, PSIP1, PTCH1, PTEN, PTPN11, PTPRB, PTPRC, PTPRK, PWWP2A, RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS, RANBP17, RAP1GD51, RARA, RB1, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC, SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STATS, STAT5B, STAT6, STK11, STL, SUFU, SUZ12, SYK, TAF15, TALL TAL2, TBL1XR1, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBRS, USP6, VHL, VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9 and ZRSR2. 
     
     
         10 . The method of  claim 1 , wherein the gene known to have clinical relevance in oncology is BRCA1 or BRCA2. 
     
     
         11 . The method of  claim 1 , wherein the gene known to have clinical relevance in oncology is TP53. 
     
     
         12 . The method of  claim 1 , further comprising (iii) incubating a sample comprising the first and second amplicons with a 3′ adaptor, a 5′ adaptor and a ligase under conditions sufficient to permit ligation of the 3′ adaptor to a 3′ end of the first and second amplicons and 5′ adaptor to a 5′ end of the first and second amplicons thereby yielding; the targeted next generation sequencing library. 
     
     
         13 . The method of  claim 12 , further comprising (iv) sequencing the targeted next generation sequencing library. 
     
     
         14 . The method of  claim 1 , wherein the melting temperature of the target-specific primers is from about 60.5° C. to about 65.5° C. 
     
     
         15 . A method of multiplex PCR amplification of a specific target locus on a nucleic acid substrate for preparing a targeted next generation sequencing library comprising the steps of:
 (i) combining a plurality of target-specific primers with the nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′ complementary sequence that is fully complementary to a sequence of the specific target locus and a 5′ noncomplementary sequence that is not complementary to a sequence of the nucleic acid substrate, wherein the 3′ complementary sequence for each of the first and second forward and reverse primers is different, wherein the 3′ complementary sequence is between 16 and 30 bases in length, and wherein the specific target locus is a gene known to have clinical relevance in oncology;   (ii) subjecting the PCR reaction mixture to a multiplex polymerase chain reaction thereby generating at least three amplicons within the specific target locus, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and the first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein the third amplicon is shorter in length than the first and second amplicons, wherein at least a portion of the 5′ noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third amplicon comprises a 3′ sequence and a 5′ sequence that are complementary to each other, wherein the third amplicon possesses overlapping sequence with the first and second amplicons, wherein the first amplicon possesses overlapping sequence with the second amplicon, wherein when the third amplicon is denatured, each strand of the third amplicon forms a secondary structure as a result of the 3′ sequence being complementary to the 5′ sequence, wherein at the end of the multiplex polymerase chain reaction, the first and second amplicons are each present at a greater amount than the third amplicon, and wherein the nucleic acid substrate is human genomic DNA.   
     
     
         16 . The method of  claim 15 , wherein the gene known to have clinical relevance in oncology is selected from the group consisting of ABU, ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, AKT1, AKT2, ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BUB1B, Cl2orf9, Cl5orf21, Cl5orf55, Cl6orf75, C2orf44, CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNBHP1, CCND1, CCND2, CCND3, CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C, CDKN2a(p14), CDX2, CEBPA, CEP1, CEP89, CHCHD7, CHEK2, CHIC2, CHN1, CIC, CIITA, CLIP1, CLTC, CLTCL1, CMKOR1, CNOT3, COL1A1, COL2A1, COPEB, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CSF3R, CTNNB1, CUX1, CYLD, D105170, DAXX, DCTN1, DDB2, DDIT3, DDX10, DDXS, DDX6, DEK, DICER1, DNM2, DNMT3A, DUX4, EBF1, ECT2L, EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4, EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCCS, ERG, ETV1, ETV4, ETVS, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7, FCGR2B, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FH, FHIT, FIP1L1, FLIT, F1127352, FLT3, FNBP1, FOXA1, FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1, FSTL3, FUBP1, FUS, FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA1 1, GNAQ, GNAS, GOLGAS, GOPC, GPC3, GPHN, GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB, HERPUD1, HEY1, HIP1, HIS T1H3B , HIS T1H4I, HLA-A, HLF, HLXB 9, HMGA1, HMGA2, HNRNPA2B1, HOOKS, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSPCA, HSPCB, IDHL IDH2, IGH\, IGK, IGL, IKZFL IL2, IL21R, IL6ST, IL7R, IRF4, IRTAL ITK, JAKL JAK2, JAK3, JAZFL JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA1549, KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D, KRAS, KTN1, LAF4, LASP1, LCK, LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2, LPP, LRIG3, LSM14A, LYL1, MAF, MAFB, MALAT1, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDS1, MDS2, MECT1, MED12, MEN1, MET, MITF, MKL1, MLF1, MLH1, MLL, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYO5A, MYST4, NAB2, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2, NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG1, PLCG1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPFIBP1, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1A, PSIP1, PTCH1, PTEN, PTPN11, PTPRB, PTPRC, PTPRK, PWWP2A, RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS, RANBP17, RAP1GD51, RARA, RB1, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC, SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STATS, STAT5B, STAT6, STK11, STL, SUFU, SUZ12, SYK, TAF15, TALL TAL2, TBL1XR1, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBRS, USP6, VHL, VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9 and ZRSR2. 
     
     
         17 . The method of  claim 15 , wherein the melting temperature of the target-specific primers is from about 60.5° C. to about 65.5° C. 
     
     
         18 . The method of  claim 15 , wherein the gene known to have clinical relevance in oncology is BRCA1 or BRCA2. 
     
     
         19 . The method of  claim 15 , wherein the gene known to have clinical relevance in oncology is TP53. 
     
     
         20 . A method of multiplex PCR amplification of a specific target locus on a nucleic acid substrate for preparing a targeted next generation sequencing library comprising the steps of:
 (i) combining a plurality of target-specific primers with the nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′ complementary sequence that is fully complementary to a sequence of the specific target locus and a 5′ noncomplementary sequence that is not complementary to a sequence of the nucleic acid substrate, wherein the 3′ complementary sequence for each of the first and second forward and reverse primers is different, wherein the 3′ complementary sequence is between 16 and 30 bases in length;   (ii) subjecting the PCR reaction mixture to a multiplex polymerase chain reaction thereby generating at least three amplicons within the specific target locus, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and the first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein the third amplicon is shorter in length than the first and second amplicons, wherein at least a portion of the 5′ noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third amplicon comprises a 3′ sequence and a 5′ sequence that are complementary to each other, wherein the third amplicon possesses overlapping sequence with the first and second amplicons, wherein the first amplicon possesses overlapping sequence with the second amplicon, wherein when the third amplicon is denatured, each strand of the third amplicon forms a secondary structure as a result of the 3′ sequence being complementary to the 5′ sequence, wherein the secondary structure is stable during a primer annealing step of the multiplex polymerase chain reaction.