Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies

Abstract

The present invention provides a method capable of detecting single or multiple fetal chromosomal aneuploidies in a maternal sample comprising fetal and maternal nucleic acids, and verifying that the correct determination has been made. The method is applicable to determining copy number variations (CNV) of any sequence of interest in samples comprising mixtures of genomic nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest. The method is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of conditions associated with a difference in sequence representation in healthy versus diseased individuals.

1 - 35 . (canceled) 
     
     
         36 . A computer processing system comprising a computer readable medium having stored thereon computer-readable instructions for determining the presence or absence of a fetal chromosomal aneuploidy, the program instructions comprising:
 (a) instructions for using sequence information obtained from human fetal and maternal nucleic acids in a maternal test sample to identify a number of sequence tags for a chromosome of interest and a number of sequence tags for at least two normalizing chromosomes;   (b) instructions for using the numbers of sequence tags to calculate a first normalizing value and a second normalizing value for the chromosome of interest; and   (c) instructions for comparing the first normalizing value for the chromosome of interest to a first threshold value and comparing the second normalizing value for the chromosome of interest to a second threshold value to determine the presence or absence of a fetal aneuploidy in the sample.   
     
     
         37 . The computer processing system of  claim 36 , wherein the first normalizing value for the chromosome of interest is a first chromosome dose, the first chromosome dose being a ratio of the number of sequence tags for the chromosome of interest and a first normalizing chromosome, and wherein the second normalizing value for the chromosome of interest is a second chromosome dose, the second chromosome dose being a ratio of the number of sequence tags for the chromosome of interest and a second normalizing chromosome. 
     
     
         38 . A computer processing system comprising a computer readable medium having stored thereon computer-readable instructions for determining the presence or absence of a fetal chromosomal aneuploidy, the program instructions comprising:
 (a) instructions for using sequence information obtained from fetal and maternal nucleic acids in a sample to identify a number of sequence tags for a chromosome of interest and a number of sequence tags for at least two normalizing chromosomes;   (b) instructions for using the number of sequence tags for the chromosome of interest and the number of sequence tags for a first normalizing chromosome to determine a first normalizing value for the chromosome of interest, and using the number of sequence tags for the first normalizing chromosome and the number of sequence tags for a second normalizing chromosome to determine a second normalizing value for the first normalizing chromosome; and   (c) instructions for comparing the first normalizing value for the chromosome of interest to a first threshold value and comparing the second normalizing value for the first normalizing chromosome to a second threshold value to determine the presence or absence of a fetal aneuploidy in the sample.   
     
     
         39 . The computer processing system of  claim 38 , wherein the first normalizing value for the chromosome of interest is a first chromosome dose, the first chromosome dose being a ratio of the number of sequence tags for the chromosome of interest and a first normalizing chromosome, and wherein the second normalizing value for the chromosome of interest is a second chromosome dose, the second chromosome dose being a ratio of the number of sequence tags for the first normalizing chromosome and a second normalizing chromosome. 
     
     
         40 . The computer processing system of  claim 36  or  claim 38 , further comprising instructions for determining a first and a second normalized chromosome value (NCV), wherein said first NCV relates said first chromosome dose to the mean of the corresponding first chromosome dose in a set of qualified samples, and wherein said second NCV relates said second chromosome dose to the mean of the corresponding second chromosome dose in a set of qualified samples as: 
       
         
           
             
               
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         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. 
       
     
     
         41 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 21, and said normalizing chromosomes are selected from chromosomes 9, 11, 14, and 1. 
     
     
         42 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 18, and said normalizing chromosomes are selected from chromosomes 8, 3, 2, and 6. 
     
     
         43 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 13, and said normalizing chromosomes are selected from chromosome 4, the group of chromosomes 2-6, chromosome 5, and chromosome 6. 
     
     
         44 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome X, and said normalizing chromosomes are selected from chromosomes 6, 5, 13, and 3. 
     
     
         45 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome Y, and said normalizing chromosomes are selected from the group of chromosomes 2-6, chromosome 3, chromosome 4, and chromosome 5. 
     
     
         46 . The method of any one of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 1, and said normalizing chromosomes are selected from chromosomes 10, 11, 9 and 15. 
     
     
         47 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 2, and said normalizing chromosomes are selected from chromosomes 8, 7, 12, and 14. 
     
     
         48 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 3, and said normalizing chromosomes are selected from chromosomes 6, 5, 8, and 18. 
     
     
         49 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 4, and said normalizing chromosomes are selected from chromosomes 3, 5, 6, and 13. 
     
     
         50 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 5, and said normalizing chromosomes are selected from chromosomes 6, 3, 8, and 18. 
     
     
         51 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 6, and said normalizing chromosomes are selected from chromosomes 5, 3, 8, and 18. 
     
     
         52 . The method of any one of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 7, and said normalizing chromosomes are selected from chromosomes 12, 2, 14 and 8. 
     
     
         53 . The computer processing system of  claim 36  or  claim 38 , wherein; said normalizing chromosomes are used to normalize chromosome 8, and said normalizing chromosomes are selected from chromosomes 2, 7, 12, and 3. 
     
     
         54 . The computer processing system of  claim 36  or  claim 38 , wherein; said normalizing chromosomes are used to normalize chromosome 9, and said normalizing chromosomes for chromosome 9 are selected from chromosomes 11, 10, 1, and 14. 
     
     
         55 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 10, and said normalizing chromosomes are selected from chromosomes 1, 11, 9, and 15. 
     
     
         56 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 11, and said normalizing chromosomes are selected from chromosomes 1, 10, 9, and 15. 
     
     
         57 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 12, and said normalizing chromosomes are selected from chromosomes 7, 14, 2, and 8. 
     
     
         58 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 14, and said normalizing chromosomes are selected from chromosomes 12, 7, 2, and 9. 
     
     
         59 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 15, and said normalizing chromosomes are selected from chromosomes 1, 10, 11, and 9. 
     
     
         60 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 16, and said normalizing chromosomes are selected from chromosomes 20, 17, 15, and 1. 
     
     
         61 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 17, and said normalizing chromosomes are selected from chromosomes 16, 20, 19 and 22. 
     
     
         62 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 19, and said normalizing chromosomes are selected from 22, 17, 16, and 20. 
     
     
         63 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 20, and said normalizing chromosomes are selected from chromosomes 16, 17, 15, and 1. 
     
     
         64 . The computer processing system of  claim 36  or  claim 38 , wherein said normalizing chromosomes are used to normalize chromosome 22, and said normalizing chromosomes are selected from chromosomes 19, 17, 16, and 20. 
     
     
         65 . The computer processing system of  claim 36  or  claim 38 , further comprising instructions for repeating the steps of  claim 36  or  claim 38  for at least two chromosomes of interest to determine the presence or absence of said different fetal chromosomal aneuploidies. 
     
     
         66 . The computer processing system of  claim 65 , further comprising instructions for repeating the steps of  claim 36  or  claim 38  for all chromosomes to determine the presence or absence of different fetal chromosomal aneuploidies. 
     
     
         67 . The computer processing system of  claim 36  or  claim 38 , wherein said fetal chromosomal aneuploidy is selected from T21, T13, T18, and monosomy X. 
     
     
         68 . The computer processing system of claim  1  or claim  3 , wherein said chromosomal aneuploidy is a partial or complete chromosomal aneuploidy. 
     
     
         69 . The computer processing system of  claim 36  or  claim 38 , wherein said maternal test sample is a plasma sample obtained from a pregnant woman and said nucleic acid molecules are cell-free DNA (cfDNA) molecules. 
     
     
         70 . An apparatus configured to perform a method for determining the presence or absence of a fetal chromosomal aneuploidy, said apparatus comprising:
 (a) a sequencing device configured for sequencing at least a portion of fetal and maternal nucleic acid molecules in a maternal blood sample, to generate sequence information; and   (b) the computer processing system of  claim 36  or  claim 38 .   
     
     
         71 . The apparatus of claim  35 , wherein said sequencing device generates:
 sequence information by sequencing-by-synthesis using reversible dye terminators;   sequence information by sequencing-by-ligation; or   sequence information by single molecule sequencing.