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 four or more different complete fetal chromosomal aneuploidies in a maternal test blood sample comprising fetal and maternal nucleic acids, 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 each of said any four or more chromosomes of interest and to identify a number of sequence tags for a normalizing sequence for each of said any four or more chromosomes of interest;   (c) using said number of sequence tags identified for each of said any four or more chromosomes of interest and said number of sequence tags identified for each said normalizing sequence to calculate a single chromosome dose for each of said any four or more chromosomes of interest; and   (d) comparing each of said single chromosome dose for each of said any four or more chromosomes of interest to a threshold value for each of said four or more chromosomes of interest, and thereby determining the presence or absence of four or more complete different fetal chromosomal aneuploidies in said sample.   
     
     
         2 . The method of  claim 1 , wherein said any four or more chromosomes of interest are selected from chromosomes 1-22. 
     
     
         3 . The method of  claim 2 , wherein said four or more chromosomes of interest further comprise chromosomes X and Y. 
     
     
         4 . The method of  claim 1 , wherein said any four or more chromosomes of interest comprise at least twenty chromosomes, and wherein the presence or absence of at least twenty complete fetal chromosomal aneuploidies is determined. 
     
     
         5 . The method of  claim 1 , wherein said any four or more chromosomes of interest is all of chromosomes 1-22, X, and Y, and wherein the presence or absence of complete fetal chromosomal aneuploidies of all of chromosomes 1-22, X, and Y is determined. 
     
     
         6 . The method of  claim 1 , wherein said different complete fetal chromosomal aneuploidies are selected from complete chromosomal monosomies and complete chromosomal polysomies. 
     
     
         7 . The method of  claim 1 , wherein said different complete fetal chromosomal aneuploidies are selected from trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, trisomy 18, trisomy 22, 47,XXY, 47,XXX, 47,XYY, and monosomy X. 
     
     
         8 . The method of  claim 1 , said normalizing sequence is a single chromosome. 
     
     
         9 . The method of  claim 1 , wherein said normalizing sequence is a group of chromosomes. 
     
     
         10 . The method of  claim 1 , wherein step (c) comprises calculating a single chromosome dose for each of said chromosomes of interest as the ratio of the number of sequence tags identified for each of said chromosomes of interest and the number of sequence tags identified for said normalizing sequence for each of said chromosomes of interest. 
     
     
         11 . The method of  claim 1 , wherein step (c) comprises:
 (i) calculating a sequence tag density ratio for each of said chromosomes of interest, by relating the number of sequence tags identified for each of said chromosomes of interest in step (b) to the length of each of said chromosomes of interest;   (ii) calculating a sequence tag density ratio for each said normalizing sequence by relating the number of sequence tags identified for said normalizing sequence in step (b) to the length of each said normalizing sequence; and   (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of said chromosomes of interest, wherein said chromosome dose is calculated as a ratio of the sequence tag density ratio for each of said chromosomes of interest and the sequence tag density ratio for said normalizing sequence for each of said chromosomes of interest.   
     
     
         12 . The method of  claim 1 , wherein each said normalizing sequence for each of said chromosome of interest is determined by a method comprising systematically calculating multiple chromosome doses for each of said chromosome of interest in a set of qualified samples. 
     
     
         13 . 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 four or more different complete fetal chromosomal aneuploidies in each of said samples. 
     
     
         14 . The method of  claim 1 , further comprising calculating a normalized chromosome value (NCV), wherein said NCV relates said chromosome dose to the mean of the corresponding chromosome dose in a set of qualified samples 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 dose in a set of qualified samples, and x ij  is the observed j-th chromosome dose for test sample i. 
       
     
     
         15 . The method of  claim 1 , wherein said test blood sample is the plasma fraction thereof. 
     
     
         16 . The method of  claim 1 , wherein said fetal and maternal nucleic acids are a mixture of fetal and maternal cell-free DNA. 
     
     
         17 . The method of  claim 1 , wherein said sequencing is next generation sequencing (NGS). 
     
     
         18 . The method of  claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators. 
     
     
         19 . The method of  claim 1 , wherein said sequencing is sequencing-by-ligation. 
     
     
         20 . The method of  claim 1 , wherein said sequencing comprises an amplification. 
     
     
         21 . The method of  claim 1 , wherein said sequencing is single molecule sequencing.