Systems and Methods for Detection of Aneuploidy

Abstract

Provided herein are improved methods for detecting aneuploidy in a sample. The methods in certain embodiments are used for the analysis of circulating DNA in serum samples, such as circulating fetal DNA or circulating tumor DNA. In certain embodiments, chromosome or chromosome segments of interest are used to set a bias model and/or a control value for a z-score determination, in illustrative examples without the use of a control chromosome.

What is claimed is: 
     
         1 . A method for preparing a deoxyribonucleic acid (DNA) fraction useful for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a target cell, comprising
 (a) extracting a mixture of cell-free DNA comprising (1) DNA derived from the target cell and (2) DNA derived from one or more non-target cells from a biological sample of each member of a set of patients;   (b) producing a fraction of DNA extracted in (a) by performing targeted multiplex amplification on the mixture of cell-free DNA to amplify at least 100 target loci on the chromosome or chromosome segment of interest together in a single reaction volume; and   (c) analyzing the fraction of DNA produced in (b) by performing high-throughput sequencing to generate genetic data representative of (1) DNA derived from the target cell and (2) DNA derived from one or more non-target cells, wherein the genetic data is obtained from each member of a set of patients, and wherein the genetic data comprises a representation of an amount of DNA corresponding to each locus in a set of loci wherein the loci are present on the chromosome or chromosome segment of interest, and wherein the target and the non-target cells in at least one patient have non-identical genomes, the method comprising:   for each patient in the set of patients,   creating a plurality of first hypotheses wherein each first hypothesis is associated with a specific copy number for the chromosome or chromosome segment in the genome of the target cell, and   calculating a first probability value for each first hypothesis, wherein the first probability value indicates the likelihood that the genome of the target cell has the number of copies of the chromosome or chromosome segment that is associated with the first hypothesis, wherein the first probability values are derived from the genetic data associated with that patient,   selecting a subset of patients consisting of those patients that are the source of genetic data that is found to match a specific copy number hypothesis with at least a specified level of confidence,   determining an amplification bias for each of the loci based on the genetic data from the subset of patients,   adjusting the genetic data, for one or more patients in the set of patients, at each locus, to correct for the determined amplification bias, to give normalized genetic data for the one or more patients,   creating a plurality of second hypotheses wherein each second hypothesis is associated with a specific copy number for the chromosome or chromosome segment in the genome of the target cell,   calculating a second probability value for each second hypothesis, wherein the second probability value indicates the likelihood that the genome of the target cell has the number of copies of the chromosome or chromosome segment that is associated with the second hypothesis, wherein the second probability values are derived from the normalized genetic data, and   determining which second hypothesis is more likely to be correct by selecting the second hypothesis with the maximum likelihood, thereby determining the copy number for the chromosome or chromosome segment in the genome of the target cell.   
     
     
         2 . The method of  claim 1  where the selection of the subset of patients is further refined by:
 calculating a depth of read for the sequencing genetic data for each polymorphic locus in the set of loci from each patient in the first subset of patients, whereby a depth of read value is created for each locus from each patient in the first subset, by: 
 calculating bias parameter that is the average depth of read for each polymorphic locus and the standard deviation between the depth of reads, 
 calculating a goodness of fit value based on the parameter for the depth of read compared to the average depth of read and the standard deviation for each for each patient in the first subset, and 
 creating a second subset of patients wherein those patients in the first subset of patients having a goodness of fit value below a specified threshold are removed, whereby a second subset of patients is created. 
 
     
     
         3 . The method of  claim 1  wherein the first plurality of hypotheses is the same as the second plurality of hypotheses. 
     
     
         4 . The method of  claim 1  wherein the genetic data comprises quantitative allelic data from a plurality of polymorphic loci in the set of loci, and wherein each of first hypotheses specifies an expected distribution of quantitative allelic data at the plurality of polymorphic loci, and wherein the first probability values are determined by calculating, for each of the first hypotheses, the fit between the expected genetic data and the obtained genetic data. 
     
     
         5 . The method of  claim 1  wherein the genetic data comprises quantitative data from a plurality of non-polymorphic loci in the set of loci, and wherein each of first hypotheses specifies an expected distribution of quantitative data at the plurality of non-polymorphic loci, and wherein the first probability values are determined by calculating, for each of the first hypotheses, the fit between the expected genetic data and the obtained genetic data. 
     
     
         6 . The method of  claim 1  wherein the genetic data comprises quantitative data from a plurality of non-polymorphic loci, and wherein each of second hypotheses specifies an expected distribution of quantitative data at the plurality of non-polymorphic loci, and wherein the second probability values are determined by calculating, for each of the second hypotheses, the fit between the expected genetic data and the normalized genetic data. 
     
     
         7 . The method of  claim 1 , wherein the genetic data from the set of patients is obtained by simultaneously sequencing a mixture comprising (1) DNA derived from one or more target cell and (2) DNA derived from one or more non-target cells to give genetic data at the set of loci from each member of the set of patients. 
     
     
         8 . The method of  claim 1 , wherein the mixture comprises cell free DNA, wherein the target cell is a fetal cell and the non-target cell is a cell from the mother of the fetus, wherein the DNA from the target cell is fetal cell free DNA and is obtained from the blood of the mother of the fetus, and wherein the DNA from the non-target cell is maternal cell free DNA obtained from the blood of the mother of the fetus. 
     
     
         9 . The method of  claim 1 , wherein the selected hypothesis is that a specific chromosome is disomic. 
     
     
         10 . The method of  claim 1  wherein the chromosome is selected from the group consisting of chromosomes 13, 18, 21, X, and Y. 
     
     
         11 . The method of  claim 1  wherein the chromosome segment is selected from the group consisting of chromosome 22q11.2, chromosome 1p36, chromosome 15q 11-q13, chromosome 4p16.3, chromosome Sp15.2, chromosome 17p13.3, chromosome 22q13.3, chromosome 2q37, chromosome 3q29, chromosome 9q34, chromosome 17q21.31, and the terminus of a chromosome. 
     
     
         12 . The method of  claim 1 , wherein the set of loci are present on a selected region of a chromosome. 
     
     
         13 . The method of  claim 1 , wherein the method is performed independently for different chromosomes or chromosome segments. 
     
     
         14 . The method of  claim 1 , wherein the set of patients comprises at least 24 patients. 
     
     
         15 . The method of  claim 4 , wherein the first probability value is derived from the genetic data obtained from polymorphic loci that comprise alleles present in the target cells that are not present in the non-target cells. 
     
     
         16 . The method of  claim 1 , wherein the target cell is a tumor and the non-target cell is a non-tumor cell, wherein the tumor cell is a cancerous tumor cell and optionally a malignant tumor cell. 
     
     
         17 . The method of  claim 1 , wherein the mixture comprises cell free DNA, and wherein the cell free DNA comprises DNA that that has been released by apoptosis. 
     
     
         18 . The method of  claim 1 , wherein the amplification bias is determined by a method comprising presuming that the selected first hypothesis is correct for the subset of patients, wherein the amplification bias, for each locus, is the difference between the expected genetic data for the selected first hypothesis and the obtained genetic data, for the subset of patients. 
     
     
         19 . A method for preparing a deoxyribonucleic acid (DNA) fraction useful for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a target cell, the method comprising:
 (a) extracting a mixture of cell-free DNA comprising (1) DNA derived from the target cell and (2) DNA derived from non-target cells from a biological sample of each member of a set of patients;   (b) producing a fraction of DNA extracted in (a) by performing targeted multiplex amplification on the mixture of cell-free DNA to amplify at least 1,000 target loci on the chromosome or chromosome segment of interest together in a single reaction volume; and   (c) analyzing the fraction of DNA produced in (b) by performing high-throughput sequencing to generate genetic data representative of (1) DNA derived from the target cell and (2) DNA derived from non-target cells, wherein the genetic data is obtained from a set of patients and wherein the target cell and the non-target cells in at least one patient have non-identical genomes,   deriving a chromosome or chromosome segment reference value for depth of sequencing reads indicative of a specific copy number for a specific chromosome or chromosome segment of interest, wherein the reference value is derived from the chromosome or chromosome segment of interest in a subset of the patients known to be disomic at the chromosome or chromosome segment of interest or determined to be disomic at the chromosome or chromosome segment of interest by selecting between 5 and 75% of patients where the depth of sequencing reads of DNA in the obtained genetic data is closest to the median of the relative fractions for the set of patients, and   comparing the genetic data derived from the specific chromosome or chromosome segment of interest from a selected patient in the set of patients to the reference value, wherein the comparison indicates the copy number of the specific chromosome or chromosome segment of interest from the selected patient.   
     
     
         20 . A method for preparing a deoxyribonucleic acid (DNA) fraction useful for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a target cell, the method comprising:
 (a) extracting a mixture of cell-free DNA comprising (1) DNA derived from the target cell and (2) DNA derived from non-target cells from a biological sample of each member of a set of patients;   (b) producing a fraction of DNA extracted in (a) by performing targeted multiplex amplification on the mixture of cell-free DNA to amplify at least 1,000 target loci on the chromosome or chromosome segment of interest together in a single reaction volume; and   (c) analyzing the fraction of DNA produced in (b) by performing high-throughput sequencing to generate genetic data representative of (1) DNA derived from the target cell and (2) DNA derived from non-target cells, wherein the genetic data is obtained from a set of patients, and wherein the genetic data comprises the relative fraction of DNA in the obtained genetic data that corresponds to the chromosome or chromosome segment of interest and wherein the target and the non-target cells in at least one patient have non-identical genomes,   selecting a subset of patients, by choosing those patients where the relative fraction of DNA in the obtained genetic data for that patient is closest to the median of the relative fractions for the set of patients,   determining a reference value for the relative fraction of DNA in the obtained genetic data that corresponds to the chromosome or chromosome segment of interest from the subset of the patients, wherein the reference value is determined from genetic data obtained from the chromosome or chromosome segment of interest, and   comparing the reference value to the relative fraction of DNA that corresponds to the chromosome or chromosome segment of interest in the obtained genetic data from a selected patient in the set of patients, wherein the comparison produces an experimental value indicative of the presence or absence of genetic abnormality in chromosome copy number or chromosome segment copy number in the target cell of the selected patient.