Detecting mutations and ploidy in chromosomal segments

Abstract

The invention provides methods, systems, and computer readable medium for detecting ploidy of chromosome segments or entire chromosomes, for detecting single nucleotide variants and for detecting both ploidy of chromosome segments and single nucleotide variants. In some aspects, the invention provides methods, systems, and computer readable medium for detecting cancer or a chromosomal abnormality in a gestating fetus.

1 . (canceled) 
     
     
         2 . A method for monitoring the progression of cancer comprising:
 (a) performing a multiplex amplification reaction to amplify a plurality of target loci from cell-free DNA isolated from a first biological sample of a subject, wherein the target loci each spans a tumor-specific mutation identified in a tumor biopsy sample of the subject, and wherein the multiplex amplification comprises nested PCR;   (b) determining the sequences of the amplified target loci; and   (c) determining the presence or absence of the tumor-specific mutations based on the sequencing results, thereby monitoring the progression of cancer.   
     
     
         3 . The method of  claim 2 , wherein the nested PCR comprises a first PCR reaction with a first universal primer and a first set of target-specific primers and a second PCR reaction with a second universal primer and a second set of target-specific primers. 
     
     
         4 . The method of  claim 2 , wherein the method further comprises ligating tagged adaptors to the isolated cell-free DNA prior to the multiplex amplification, wherein individual DNA molecules are tagged with different tags. 
     
     
         5 . The method of  claim 2 , further comprising:
 (d) repeating steps (a)-(c) on cell-free DNA isolated from a second biological sample obtained from the subject at a subsequent time point.   
     
     
         6 . The method of  claim 2 , wherein the cell-free DNA comprises circulating tumor DNA. 
     
     
         7 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more single nucleotide variant (SNV) mutations. 
     
     
         8 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more copy number variation (CNV) mutations. 
     
     
         9 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more SNV mutations in a gene selected from the group consisting of TP53, PTEN, PIK3CA, APC, EGFR, NRAS, NF2, FBXW7, ERBBs, ATAD5, KRAS, BRAF, VEGF, EGFR, HER2, ALK, p53, BRCA, BRCA1, BRCA2, SETD2, LRP1B, PBRM, SPTA1, DNMT3A, ARID1A, GRIN2A, TRRAP, STAG2, EPHA3/5/7, POLE, SYNE1, C20orf80, CSMD1, CTNNB1, ERBB2. FBXW7, KIT, MUC4, ATM, CDH1, DDX11, DDX12, DSPP, EPPK1, FAM186A, GNAS, HRNR, KRTAP4-11, MAP2K4, MLL3, NRAS, RB1, SMAD4, TTN, ABCC9, ACVR1B, ADAM29, ADAMTS19, AGAP10, AKT1, AMBN, AMPD2, ANKRD30A, ANKRD40, APOBR, AR, BIRC6, BMP2, BRAT1, BTNL8, C12orf4, C1QTNF7, C20orf186, CAPRIN2, CBWD1, CCDC30, CCDC93, CD5L, CDC27, CDC42BPA, CDH9, CDKN2A, CHD8, CHEK2, CHRNA9, CIZ1, CLSPN, CNTN6, COL14A1, CREBBP, CROCC, CTSF, CYP1A2, DCLK1, DHDDS, DHX32, DKK2, DLEC1, DNAH14, DNAH5, DNAH9, DNASE1L3, DUSP16, DYNC2H1, ECT2, EFHB, RRN3P2, TRIM49B, TUBB8P5, EPHA7, ERBB3, ERCC6, FAM21A, FAM21C, FCGBP, FGFR2, FLG2, FLT1, FOLR2, FRYL, FSCB, GAB1, GABRA4, GABRP, GH2, GOLGA6L1, GPHB5, GPR32, GPX5, GTF3C3, HECW1, HIST1H3B, HLA-A, HRAS, HS3ST1, HS6ST1, HSPD1, IDH1, JAK2, KDM5B, KIAA0528, KRT15, KRT38, KRTAP21-1, KRTAP4-5, KRTAP4-7, KRTAP5-4, KRTAP5-5, LAMA4, LATS1, LMF1, LPAR4, LPPR4, LRRFIP1, LUM, LYST, MAP2K1, MARCH1, MARCO, MB21D2, MEGF10, MMP16, MORC1, MRE11A, MTMR3, MUC12, MUC17, MUC2, MUC20, NBPF10, NBPF20, NEK1, NFE2L2, NLRP4, NOTCH2, NRK, NUP93, OBSCN, OR11H1, OR2B11, OR2M4, OR4Q3, OR5D13, OR812, OXSM, PIK3R1, PPP2R5C, PRAME, PRF1, PRG4, PRPF19, PTH2, PTPRC, PTPRJ, RAC1, RAD50, RBM12, RGPD3, RGS22, ROR1, RP11-671M22.1, RP13-996F3.4, RP1L1, RSBN1L, RYR3, SAMD3, SCN3A, SEC31A, SF1, SF3B1, SLC25A2, SLC44A1, SLC4A1, SMAD2, SPTA1, ST6GAL2, STK11, SZT2, TAF1L, TAX1BP1, TBP, TGFBI, TIF1, TMEM14B, TMEM74, TPTE, TRAPPC8, TRPS1, TXNDC6, USP32, UTP20, VASN, VPS72, WASH3P, WWTR1, XPO1, ZFHX4, ZMIZ1, ZNF167, ZNF436, ZNF492, ZNF598, ZRSR2, ABL1, AKT2, AKT3, ARAF, ARFRP1, ARID2, ASXL1, ATR, ATRX, AURKA, AURKB, AXL, BAP1, BARD1, BCL2, BCL2L2, BCL6, BCOR, BCORL1, BLM, BRIP1, BTK, CARD11, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD79A, CD79B, CDC73, CDK12, CDK4, CDK6, CDK8, CDKN1B, CDKN2B, CDKN2C, CEBPA, CHEK1, CIC, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, DAXX, DDR2, DOT1L, EMSY (C11orf30), EP300, EPHA3, EPHA5, EPHB1, ERBB4, ERG, ESR1, EZH2, FAM123B (WTX), FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FLT4, FOXL2, GATA1, GATA2, GATA3, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GPR124, GSK3B, HGF, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL7R, INHBA, IRF4, IRS2, JAK1, JAK3, JUN, KAT6A (MYST3), KDM5A, KDM5C, KDM6A, KDR, KEAP1, KLHL6, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MET, MITF, MLH1, MLL, MLL2, MPL, MSH2, MSH6, MTOR, MUTYH, MYC, MYCL1, MYCN, MYD88, NF1, NFKBIA, NKX2-1, NOTCH1, NPM1, NRAS, NTRK1, NTRK2, NTRK3, PAK3, PALB2, PAX5, PBRM1, PDGFRA, PDGFRB, PDK1, PIK3CG, PIK3R2, PPP2R1A, PRDM1, PRKAR1A, PRKDC, PTCH1, PTPN11, RAD51, RAF1, RARA, RET, RICTOR, RNF43, RPTOR, RUNX1, SMARCA4, SMARCB1, SMO, SOCS1, SOX10, SOX2, SPEN, SPOP, SRC, STAT4, SUFU, TET2, TGFBR2, TNFAIP3, TNFRSF14, TOP1, TP53, TSC1, TSC2, TSHR, VHL, WISP3, WT1, ZNF217, and ZNF703. 
     
     
         10 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more SNV mutations in a gene selected from the group consisting of CYFIP1, FAT1, MLLT4, RASA1, HERC4, JAK2, MSH2, MTOR, PLCG2, GABRG1, and TRIM67. 
     
     
         11 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more clonal SNV mutations. 
     
     
         12 . The method of  claim 11 , wherein at least one clonal SNV mutation is in a gene selected from the group consisting of BRIP1, CARS, FAT1, MLLT4, NFE2L2, TP53, TP53, EGFR, EGFR, TP53, KDM6A, and ROS1. 
     
     
         13 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more subclonal SNV mutations. 
     
     
         14 . The method of  claim 13 , wherein at least one subclonal SNV mutation is in a gene selected from the group consisting of CIC, KDM6A, NF1, and TRIM67. 
     
     
         15 . The method of  claim 2 , wherein the tumor-specific mutations comprise one or more clonal SNV mutations and one or more subclonal SNV mutations. 
     
     
         16 . The method of  claim 2 , wherein the method further comprises whole exome sequencing of the tumor biopsy sample to identify the tumor-specific mutations. 
     
     
         17 . The method of  claim 16 , wherein the method further comprises determining clonal heterogeneity of the tumor biopsy sample. 
     
     
         18 . The method of  claim 16 , wherein the method further comprises designing target-specific PCR primers for the tumor-specific mutations. 
     
     
         19 . The method of  claim 2 , wherein step (a) comprises targeted multiplex PCR amplification of 10 to 500 target loci from the cell-free DNA. 
     
     
         20 . The method of  claim 2 , wherein the method further comprises detecting recurrence and/or metastases of the cancer from the tumor-specific mutations detected in the cell-free DNA. 
     
     
         21 . The method of  claim 2 , wherein the cancer is colorectal cancer, lung cancer, bladder cancer, or breast cancer. 
     
     
         22 . The method of  claim 2 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         23 . The method of  claim 2 , wherein the sequences of the amplified target loci are determined by high-throughput sequencing. 
     
     
         24 . The method of  claim 2 , wherein the sequences of the amplified target loci are determined by microarray.