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.

What is claimed is: 
     
         1 . A method for detecting tumor-specific mutations, comprising:
 (a) performing whole exome sequencing to identify a plurality of tumor-specific mutations from a tumor biopsy cancer sample of a subject;   (b) designing PCR primers for amplifying at least 10 target loci encompassing the tumor-specific mutations identified in (a);   (c) performing a multiplex amplification reaction to amplify the at least 10 target loci encompassing the tumor-specific mutations using the PCR primers designed in (b), from cell-free DNA isolated from a biological sample of the subject, wherein the biological sample is a blood, serum, plasma, or urine sample, wherein the target loci are amplified together in the same reaction volume; and   (d) performing high-throughput sequencing to determine the sequences of the amplified target loci, thereby detecting the tumor-specific mutations in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the depth of read for each of the target loci is at least 50,000. 
     
     
         3 . The method of  claim 1 , wherein the method further comprises isolating cell-free DNA from the biological sample of the subject. 
     
     
         4 . The method of  claim 1 , wherein the tumor-specific mutations comprise one or more single nucleotide variant (SNV) mutations. 
     
     
         5 . The method of  claim 1 , wherein the tumor-specific mutations comprise one or more copy number variation (CNV) mutations. 
     
     
         6 . The method of  claim 1 , wherein the tumor-specific mutations comprise one or more clonal SNV mutations and/or one or more subclonal SNV mutations. 
     
     
         7 . The method of  claim 1 , wherein step (a) further comprises determining clonal heterogeneity of the tumor biopsy sample. 
     
     
         8 . The method of  claim 1 , wherein step (c) comprises targeted multiplex PCR amplification of at least 50 target loci from the cell-free DNA. 
     
     
         9 . The method of  claim 1 , wherein the cancer is colorectal cancer, lung cancer, bladder cancer, or breast cancer. 
     
     
         10 . The method of  claim 1 , wherein the method further comprises repeated testing of biological samples obtained from the subject at multiple time points to monitor the progression of cancer or the remission or reoccurrence of cancer. 
     
     
         11 . A method for determining the presence or absence of one or more tumor-specific mutations, comprising:
 (a) performing a multiplex amplification reaction to amplify at least 10 target loci from cell-free DNA isolated from a first biological sample of a subject, wherein the first biological sample is a blood, serum, plasma, or urine sample, wherein the target loci are amplified together in the same reaction volume, wherein the target loci each spans a tumor-specific mutation previously identified in a tumor biopsy sample of the subject by whole exome sequencing of the tumor biopsy sample;   (b) determining the sequences of the amplified target loci by high-throughput sequencing; and   (c) determining the presence or absence of one or more tumor-specific mutations based on the sequencing results, thereby monitoring the progression of cancer.   
     
     
         12 . The method of  claim 11 , further comprising:
 (d) repeating steps (a) to (c) on cell-free DNA isolated from a second biological sample obtained from the subject at a subsequent time point, wherein second biological sample is a blood, serum, plasma, or urine sample.   
     
     
         13 . The method of  claim 11 , wherein the sequences of the amplified target loci are determined by high-throughput sequencing, and wherein the depth of read for each of the target loci is at least 50,000. 
     
     
         14 . The method of  claim 11 , wherein the cell-free DNA comprises circulating tumor DNA. 
     
     
         15 . The method of  claim 11 , wherein the tumor-specific mutations comprise one or more single nucleotide variant (SNV) mutations. 
     
     
         16 . The method of  claim 11 , wherein the tumor-specific mutations comprise one or more copy number variation (CNV) mutations. 
     
     
         17 . The method of  claim 11 , wherein the tumor-specific mutations comprise one or more clonal SNV mutations and/or one or more subclonal SNV mutations. 
     
     
         18 . The method of  claim 11 , wherein the method further comprises determining clonal heterogeneity of the tumor biopsy sample. 
     
     
         19 . The method of  claim 11 , wherein step (a) comprises targeted multiplex PCR amplification of at least 50 target loci from the cell-free DNA. 
     
     
         20 . The method of  claim 11 , wherein the cancer is colorectal cancer, lung cancer, bladder cancer, or breast cancer. 
     
     
         21 . A method for detecting tumor-specific mutations, comprising:
 (a) performing whole exome sequencing on a tumor biopsy sample of a subject to identify a plurality of tumor-specific SNV mutations;   (b) designing PCR primers for amplifying at least 50 target loci encompassing the tumor-specific SNV mutations identified in (a);   (c) isolating cell-free DNA from a biological sample of the subject;   (d) performing a multiplex amplification reaction to amplify the at least 50 target loci encompassing the tumor-specific SNV mutations using the PCR primers designed in (b), from cell-free DNA isolated from a biological sample of the subject, wherein the biological sample is a blood, serum, plasma, or urine sample, wherein the target loci are amplified together in the same reaction volume; and   (e) determining the sequences of the amplified target loci for the presence or absence of the tumor-specific SNV mutations, wherein the sequences of the amplified target loci are determined by high-throughput sequencing, and wherein the depth of read for each of the target loci is at least 50,000.