Array-based methods for analysing mixed samples using differently labelled allele-specific probes

Abstract

This disclosure provides methods and systems useful in array-based analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics.

1 . A method for analyzing a mixed nucleic acid sample obtained from an organism, comprising:
 obtaining or deriving from an organism a nucleic acid sample containing a mixed nucleic acid population that includes a major subpopulation and a minor subpopulation, wherein the major and minor subpopulations each include a target sequence located in a first nucleic acid locus and containing a polymorphic site, wherein the polymorphic site can include combinations of a first nucleotide variant and a second nucleotide variant; genotyping the polymorphic site, wherein the genotyping includes:
 (a) hybridizing at least one nucleic acid fragment containing or derived from the nucleic acid population and containing the polymorphic site to an oligonucleotide probe of an oligonucleotide array; and 
 (b) detecting from the oligonucleotide array, using a detector, a first signal indicating the presence or absence of a first nucleotide variant (“A signal”) corresponding to the first allelic variant and a second signal indicating the presence or absence of a second nucleotide variant (“B signal”) corresponding to the second allelic variant; and 
 (c) contacting the nucleic acid sample with a pool of linear molecular inversion probes to provide an annealing mixture; 
 wherein the pool of linear molecular inversion probes binds DNA fragments from chromosomes 1, 5, 13, 18, 21, X, and Y. 
   
     
     
         2 . The method of  claim 1 , further including determining the copy number of the first nucleic acid locus in the minor subpopulation, the major subpopulation, or both the major and minor subpopulations, using the first signal and the second signal. 
     
     
         3 . The method of  claim 1 , further including determining the copy number of a chromosome containing the first nucleic acid locus in the major subpopulation, the minor subpopulation or both the major and minor subpopulations, using the first signal and the second signal. 
     
     
         4 . The method of  claim 1 , further including determining the genotype of the polymorphic site for the minor subpopulation using the first signal and the second signal. 
     
     
         5 . The method of  claim 1 , further including determining the genotype of the polymorphic site for the major subpopulation using the first signal and the second signal. 
     
     
         6 . The method of  claim 1 , further including determining the relative amounts of the major subpopulation and the minor subpopulation in the mixed nucleic acid population using the first signal and the second signal. 
     
     
         7 . The method of  claim 1 , wherein the major subpopulation and the minor subpopulation originate from different sources in the organism. 
     
     
         8 . The method of  claim 1 , wherein the mixed nucleic acid population includes cell-free DNA. 
     
     
         9 . The method of  claim 8 , wherein the cell-free DNA is obtained or derived from the organism's blood, plasma, serum, urine, stool or saliva. 
     
     
         10 . The method of  claim 1 , wherein the organism includes a tumor, the major subpopulation includes or is derived from normal tissue and the minor subpopulation includes or is derived from the tumor. 
     
     
         11 . The method of  claim 1 , wherein the organism is a pregnant female, the mixed nucleic acid population is cell-free DNA obtained from the pregnant female's blood, the major subpopulation includes or is derived maternal nucleic acid and the minor subpopulation includes or is derived from fetal nucleic acid. 
     
     
         12 - 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the polymorphic site includes a bi-allelic SNP, the first nucleotide variant is a first allelic variant of the SNP (“A allele”) and the second nucleotide variant is a second allelic variant of the SNP (“B allele”). 
     
     
         23 . The method of  claim 1 , wherein the detector includes a first detection channel and a second detection channel, and further including the steps of detecting the first signal in the first detection channel and the second signal in the second detection channel. 
     
     
         24 . The method of  claim 22 , wherein the SNP can include the A allele or the B allele, and wherein the SNP genotype can be homozygous for the A allele (“AA”), homozygous for the B allele (“BB”) or heterozygous (“AB”). 
     
     
         25 - 82 . (canceled) 
     
     
         83 . A method for detecting a copy number in a fetus, comprising:
 obtaining a biological sample from a subject who is a pregnant female, the biological sample including nucleic acid of both maternal and fetal origin containing a target nucleic acid sequence located on a first chromosome, the target nucleic acid sequence containing a polymorphic site for a single nucleotide polymorphism (SNP);   generating a population of nucleic acid fragments containing or derived from the target nucleic acid sequence;   conducting a first assay comprising (a) contacting the population of nucleic acid fragments with an oligonucleotide array containing a first oligonucleotide probe configured to hybridize to the target nucleic acid sequence containing the polymorphic site of the SNP; and (b) detecting, using a detector, first signals indicating hybridization of the oligonucleotide probe to one or more nucleic acid fragments of the population containing a first allelic variant (“A allele”) of the SNP and second signals indicating hybridization of the oligonucleotide probe to one or more nucleic acid fragments of the population containing a second allelic variant (“B allele”) of the SNP; and   determining, using the first signals and the second signals, any one or more of the following:   (i) the copy number of the first chromosome in the fetus;   (ii) a fetal genotype for the SNP;   (iii) a maternal genotype for the SNP; and   (iv) a fetal fraction of the sample.   
     
     
         84 . The method of  claim 83 , further comprising calculating the observed B-allele frequency (BAF) for the allelic variants of the SNP present in the sample. 
     
     
         85 . The method of  claim 84 , further including calculating the fetal fraction of the sample using the BAF. 
     
     
         86 . The method of  claim 84 , wherein the polymorphic site of the SNP can be homozygous for the A allele (“AA”), homozygous for the B allele (“BB”) or heterozygous (“AB”). 
     
     
         87 . The method of  claim 83 , wherein the detector has a first and a second detection channel, and the genotyping further includes detecting the first signals in the first channel and the second signals in the second channel. 
     
     
         88 . (canceled) 
     
     
         89 . The method of  claim 88 , wherein determining the copy number of the first chromosome in the fetus comprises determining a ratio of a first value to a second value. 
     
     
         90 - 138 . (canceled)