Method for determining copy number variations

Abstract

The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the present method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.

1 . A method for determining the presence or absence of a fetal chromosomal aneuploidy of a chromosome of interest in a maternal test blood sample comprising fetal and maternal nucleic acid molecules, said method comprising:
 (a) sequencing at least a portion of said fetal and maternal nucleic acids in said sample to obtain sequence information;   (b) using said sequence information to identify a number of sequence tags for a chromosome of interest and a number of sequence tags for a normalizing sequence, wherein said normalizing sequence is a group of segments of one or more chromosomes;   (c) calculating a chromosome ratio of said number of sequence tags for said chromosome of interest to said number of sequence tags for said normalizing sequence; and   (d) comparing said ratio obtained in step (c) to a threshold, thereby determining the presence or absence of said fetal chromosomal aneuploidy in said maternal blood sample.   
     
     
         2 . The method of  claim 1 , where said chromosome of interest is selected from chromosomes 1-22, X, and Y. 
     
     
         3 . The method of  claim 1 , wherein step (c) further comprises relating said number of sequence tags for said chromosome of interest to the length of said chromosome of interest, and relating said number of sequence tags for said normalizing sequence to the length of said normalizing sequence. 
     
     
         4 . The method of  claim 1 , wherein step (d) comprises calculating a Z statistic. 
     
     
         5 . The method of  claim 4 , wherein calculating said Z-statistic comprises determining a normalized chromosome value (NCV), wherein said NCV relates said chromosome ratio obtained in step (c) for said maternal test sample to the mean of the corresponding chromosome ratio in a set of samples known to be diploid for said chromosome of interest as: 
       
         
           
             
               
                 NCV 
                 ij 
               
               = 
               
                 
                   
                     x 
                     ij 
                   
                   - 
                   
                     
                       μ 
                       ^ 
                     
                     j 
                   
                 
                 
                   
                     σ 
                     ^ 
                   
                   j 
                 
               
             
           
         
       
       where {circumflex over (μ)} j  and {circumflex over (σ)} j  are the estimated mean and standard deviation, respectively, for the j-th chromosome ratio in a set of qualified samples, and x ij  is the observed j-th chromosome ratio for test sample i. 
     
     
         6 . The method of  claim 1 , wherein the presence or absence of a fetal aneuploidy is determined for more than one chromosome of interest in said maternal sample. 
     
     
         7 . The method of  claim 6 , wherein said more than one chromosome of interest comprises chromosome 21 and chromosome 18. 
     
     
         8 . The method of  claim 1 , wherein the presence or absence of a fetal aneuploidy is determined for at least four chromosomes of interest. 
     
     
         9 . The method of  claim 1 , wherein the presence or absence of a fetal aneuploidy is determined for at least twenty chromosomes of interest. 
     
     
         10 . The method of  claim 1 , wherein the presence or absence of a fetal aneuploidy is determined for all chromosomes. 
     
     
         11 . The method of  claim 1 , wherein said fetal chromosomal aneuploidy is a polysomy. 
     
     
         12 . The method of  claim 8 , wherein said polysomy is a trisomy. 
     
     
         13 . The method of  claim 9 , wherein said trisomy is selected from trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, trisomy 18, trisomy 22, 47,XXY, 47,XXX, and 47,XYY. 
     
     
         14 . The method of  claim 1 , wherein said fetal chromosomal aneuploidy is a monosomy. 
     
     
         15 . The method of  claim 11 , wherein said monosomy is monosomy X. 
     
     
         16 . The method of  claim 1 , wherein steps (a)-(d) are repeated for test samples from different maternal subjects, and wherein the method comprises determining the presence or absence of any one or more fetal chromosomal aneuploidies in each of said samples. 
     
     
         17 . The method of  claim 1 , wherein said fetal and maternal nucleic acid molecules are plasma cell-free DNA (cfDNA) molecules. 
     
     
         18 . The method of  claim 1 , wherein said sequencing is next generation sequencing (NGS). 
     
     
         19 . The method of  claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators. 
     
     
         20 . The method of  claim 1 , wherein said sequencing is sequencing-by-ligation. 
     
     
         21 . The method of  claim 1 , wherein said sequencing comprises an amplification. 
     
     
         22 . The method of  claim 1 , wherein said sequencing is single molecule sequencing.