Determining a nucleic acid sequence imbalance using multiple markers

Abstract

Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The percentage of fetal DNA can be calculated from the same or different data used to determine the cutoff value, and can use a locus where the mother is homozygous and the fetus is heterozygous. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing (SPRT).

What is claimed is: 
     
         1 . A method for determining a presence or absence of a fetal aneuploidy in a biological sample of a female subject pregnant with a fetus, wherein the biological sample includes cell-free genomic DNA from the female subject and from the fetus, the method comprising:
 enriching the biological sample for DNA from first target loci on a first chromosome and second target loci on at least one second chromosome to obtain an enriched sample;   sequencing DNA molecules in the enriched sample to obtain sequence tags;   determining a first amount of sequence tags from the first target loci on the first chromosome;   determining a second amount of sequence tags from the second target loci on the at least one second chromosome; and   determining whether the fetal aneuploidy exists for the first chromosome based on a comparison of the first amount and the second amount.   
     
     
         2 . The method of  claim 1 , wherein at least one pair of a first target locus and a second target locus is paralogous. 
     
     
         3 . The method of  claim 1 , wherein enriching the biological sample includes:
 using a hybridization technique.   
     
     
         4 . The method of  claim 3 , wherein using the hybridization technique includes:
 using an oligonucleotide array to select DNA from the first target loci on the first chromosome and the second target loci on the at least one second chromosome.   
     
     
         5 . The method of  claim 1 , wherein determining the first amount includes:
 aligning the sequence tags to a human genome; and   counting a number of sequence tags that align to one of the first target loci.   
     
     
         6 . The method of  claim 1 , wherein the sequencing is random sequencing. 
     
     
         7 . The method of  claim 1 , wherein determining the first amount includes:
 determining a respective amount of sequence tags from each of the first target loci; and   performing a sum of the respective amounts.   
     
     
         8 . The method of  claim 7 , further comprising:
 correcting the respective amounts before calculating the sum by applying a probability distribution to the respective amounts to calculate corrected values for the respective amounts.   
     
     
         9 . The method of  claim 1 , wherein the sequencing involves ligation. 
     
     
         10 . The method of  claim 1 , wherein determining whether the fetal aneuploidy exists for the first chromosome based on the comparison of the first amount and the second amount includes:
 determining a parameter from the first amount and the second amount, wherein the parameter provides a relative amount between the first amount and the second amount; and   comparing the parameter to one or more cutoff values to determine a classification of whether the fetal aneuploidy exists for the first chromosome.   
     
     
         11 . The method of  claim 1 , wherein comparing the first amount and the second amount to determine whether the fetal aneuploidy exists for the first chromosome includes:
 determining a proportion of the first amount relative to the second amount; and   comparing the proportion to a reference value to determine whether the fetal aneuploidy exists for the first chromosome.   
     
     
         12 . The method of  claim 1 , wherein determining whether the fetal aneuploidy exists for the first chromosome based on the comparison of the first amount and the second amount includes:
 determining a fractional concentration of fetal DNA in the biological sample; and   using the fractional concentration to determine a cutoff value for a differential between the first amount and the second amount to indicate that the fetal aneuploidy exists for the first chromosome.   
     
     
         13 . The method of  claim 12 , wherein determining the fractional concentration of fetal DNA in the biological sample includes a quantification of a polymorphic difference between the female subject and the fetus. 
     
     
         14 . The method of  claim 13 , wherein the quantification includes:
 identifying a target polymorphic site at which the female subject is homozygous and the fetus is heterozygous; and   comparing an amount of a fetal-specific allele at the target polymorphic site and an amount of a common allele at the target polymorphic site to determine the fractional concentration of fetal DNA, the common allele not being fetal-specific.   
     
     
         15 . The method of  claim 12 , wherein the fetus is male, and wherein determining the fractional concentration of fetal DNA in the biological sample includes:
 determining a Y chromosome DNA concentration.   
     
     
         16 . The method of  claim 12 , wherein determining the fractional concentration of fetal DNA in the biological sample includes:
 comparing an amount of DNA molecules exhibiting a fetal-specific methylation pattern at a first locus to a total amount of DNA molecules at the first locus.   
     
     
         17 . The method of  claim 16 , wherein fetal-derived DNA molecules are hypermethylated at the first locus and maternally derived DNA molecules are hypomethylated at the first locus. 
     
     
         18 . The method of  claim 1 , wherein the first target loci comprise non-consecutive sequences on the first chromosome whose data is used additively.