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. Any aneuploidy can be determined from sequencing information that is obtained by sequencing only once the nucleic acids of a test sample.

1 - 6 . (canceled) 
     
     
         7 . In a method of performing prenatal diagnosis using massively parallel sequencing of a mixture of fetal and maternal cell-free DNA extracted from the plasma portion of a human maternal test blood sample, the method including steps of extraction, purification, library preparation and cluster amplification, each of which can cause variation of the number of sequence tags that map to the reference genome within and between sequencing runs,
 the improvement comprising:   (a) performing massively parallel sequencing of at least a portion of said fetal and maternal nucleic acids in said test sample to obtain sequence information, comprising sequence information for any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and for a normalizing sequence for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y, said normalizing sequence having previously been systematically determined to minimize the variation of the chromosome doses within and between sequencing runs;   (b) receiving said sequence information in a computer readable medium;   (c) using computer readable logic, utilizing said sequence information to identify a number of sequence tags for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for said normalizing sequence;   (d) using computer readable logic, utilizing said number of sequence tags identified for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and said number of sequence tags identified for said normalizing sequence obtained in step (d) to calculate a chromosome dose for said one or more chromosome of interest selected from chromosomes 1-22, X, and Y; and   (e) calculating a differential between the chromosome dose for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and a threshold value, wherein a statistically significant differential indicates the presence or absence of complete fetal chromosomal aneuploidy for said one or more chromosome of interest selected from chromosomes 1-22, X, and Y.   
     
     
         8 . The method of  claim 7 , wherein step (e) 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. 
     
     
         9 . The method of  claim 7 , wherein step (e) 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 of said normalizing sequence by relating the number of sequence tags identified for said normalizing sequence in step (d) 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 chromosome 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.   
     
     
         10 . The method of  claim 7 , wherein each said normalizing sequence for each of said chromosomes of interest is determined by a method comprising systematically calculating multiple chromosome doses for each of said chromosomes of interest in a set of qualified samples. 
     
     
         11 . The method of  claim 7 , wherein steps (a)-(e) are repeated for test samples from different maternal subjects, and wherein the method comprises determining the presence or absence of complete fetal chromosomal aneuploidy for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y in each of said samples. 
     
     
         12 . The method of  claim 7 , 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: 
       
         
           
             
               
                 N 
                 ⁢ 
                 C 
                 ⁢ 
                 
                   V 
                   ij 
                 
               
               = 
               
                 
                   
                     x 
                     
                       i 
                       ⁢ 
                       j 
                     
                   
                   - 
                   
                     
                       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. 
     
     
         13 . The method of  claim 7 , said one or more chromosome of interest comprises chromosome 13, 18, 21, X, and/or Y. 
     
     
         14 . The method of  claim 7 , said normalizing sequence is a single chromosome. 
     
     
         15 . The method of  claim 7 , wherein said normalizing sequence is a group of chromosomes. 
     
     
         16 . The method of  claim 7 , wherein said sequencing comprises:
 sequencing-by-hybridization;   sequencing-by-synthesis with reversible dye terminators;   sequencing-by-ligation; or   single molecule sequencing.   
     
     
         17 . The method of  claim 7 , wherein said cell free DNA is amplified using PCR before it is subject to cluster amplification. 
     
     
         18 . A method comprising:
 (a) preparing a sequencing library from a mixture of fetal and maternal nucleic acid molecules in a maternal test sample, wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids, wherein said consecutive steps exclude purifying the end-repaired products prior to said dA-tailing and exclude purifying the dA-tailing products prior to said adaptor-ligating;   (b) performing massively parallel sequencing of at least a portion of said fetal and maternal nucleic acids in said test sample to obtain sequence information, comprising sequence information for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and for a normalizing sequence for each said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y, said normalizing sequence having previously been systematically determined to minimize the variation of the chromosome doses within and between sequencing runs;   (c) receiving said sequence information in a computer readable medium;   (d) using computer readable logic, utilizing said sequence information to identify a number of sequence tags for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for said normalizing sequence;   (e) using computer readable logic, utilizing said number of sequence tags identified for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and said number of sequence tags identified for said normalizing sequence obtained in step (c) to calculate a chromosome dose for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y; and   (f) calculating a differential between the chromosome dose for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and a threshold value, wherein a statistically significant differential indicates the presence or absence of complete fetal chromosomal aneuploidy for said one or more chromosomes of interest selected from chromosomes 1-22, X, and Y.   
     
     
         19 . The method of  claim 18 , wherein step (f) 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. 
     
     
         20 . The method of  claim 18 , wherein step (f) 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 (e) 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.   
     
     
         21 . The method of  claim 18 , wherein each said normalizing sequence for each of said chromosomes of interest is determined by a method comprising systematically calculating multiple chromosome doses for each of said chromosomes of interest in a set of qualified samples. 
     
     
         22 . The method of  claim 18 , 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: 
       
         
           
             
               
                 N 
                 ⁢ 
                 C 
                 ⁢ 
                 
                   V 
                   ij 
                 
               
               = 
               
                 
                   
                     x 
                     
                       i 
                       ⁢ 
                       j 
                     
                   
                   - 
                   
                     
                       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. 
       
     
     
         23 . The method of  claim 18 , said one or more chromosomes of interest comprises chromosome 13, 18, 21, X, and/or Y. 
     
     
         24 . The method of  claim 18 , wherein said normalizing sequence is a single chromosome. 
     
     
         25 . The method of  claim 18 , wherein said normalizing sequence is a group of chromosomes.