Array-based methods for analysing mixed samples using different allele-specific labels, in particular for detection of fetal aneuploidies

Abstract

Provided includes methods and systems useful in array-based analysis of mixed nucleic acid populations, including for genotyping and copy number analysis of the various subpopulations of the mixed nucleic acid population. Also provided includes methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken from an organism.

1 . A system for analyzing a nucleic acid sample obtained from a pregnant female, the system comprising 
 (a) a probe microarray comprising 
 a plurality of probes configured to hybridize to a target nucleic acid from a target chromosome, the target nucleic acid comprising a polymorphic site, the plurality of probes comprising first probes configured to hybridize to a first allelic variant (“A allele”) of the target nucleic acid and comprising second probes configured to hybridize to a second allelic variant (“B allele”) of the target nucleic acid, wherein the polymorphic site can be homozygous for the A allele (“AA”), homozygous for the B allele (“BB”), or heterozygous (“AB”), 
 the probe microarray being configured to generate first signals indicative of hybridization of one or more first probes to one or more target nucleic acids comprising the first allelic variant, and to generate second signals indicative of hybridization of one or more second probes to one or more target nucleic acids comprising the second allelic variant; 
   (b) a scanner configured to detect the signals generated by the probe microarray according to a first channel for the first signals and a second channel for the second signals; and   (c) a computer communicatively coupled to the scanner, the computer comprising a processor and memory, the memory encoded with instructions which are executable by the processor to cause the computer to at least: 
 receive first signal and second signal data from the scanner, and 
 determine, using the first signal and second signal data, one or more of: 
 (i) a copy number of the target chromosome in a fetus, 
 (ii) a fetal genotype for the SNP, 
 (iii) a maternal genotype for the SNP, or 
 (iv) a fetal fraction of the sample. 
 
   
     
     
         2 . The system of  claim 1 , wherein the executable instructions further cause the computer to calculate the observed B- allele frequency (BAF) for the allelic variants present in the sample. 
     
     
         3 . The system of  claim 2 , wherein the fetal fraction of the sample is determined using the BAF. 
     
     
         4 . The system of  claim 1 , wherein the first signals in the first channel indicate an amount of A allele present in the nucleic acid sample and the second signals in the second channel indicate an amount of B allele present in the nucleic acid sample. 
     
     
         5 . The system of  claim 1 , wherein copy number of the target chromosome is determined by determining a ratio of a first value to a second value. 
     
     
         6 . The system of  claim 5 , further comprising calculating the first value by normalizing and summarizing the first signals to obtain a first normalized and summarized signal value, normalizing the second signals to obtain a normalized and summarized second signal value, and adding the normalized and summarized first signal value to normalized and summarized second signal value to obtain the first value. 
     
     
         7 . The system of  claim 6 , wherein the second value is obtained by:
 conducting the first assay on additional biological samples serving as reference samples and identifying reference samples having an SNP genotype corresponding to the first maternal SNP genotype;   from conducting the first assay on the reference samples, obtaining first signals reference signals detected in the first channel indicating an amount of A allele present in the polymorphic site of the SNP with respect to the additional biological samples and obtaining second reference signals indicating the amount of B allele present at the polymorphic site.   
     
     
         8 . The system of  claim 7 , wherein the additional biological samples are from non-pregnant individuals. 
     
     
         9 . The system of  claim 7 , wherein the additional biological samples include some samples from pregnant females and some samples from non-pregnant individuals. 
     
     
         10 . The system of  claim 7 , wherein the additional biological samples include samples from pregnant females assayed on the same probe microarray as the biological sample from the subject pregnant female. 
     
     
         11 . The system of  claim 7 , wherein the nucleic acid sample includes maternal blood, plasma or serum and the nucleic acid of both maternal and fetal origin includes cell-free DNA (ctDNA). 
     
     
         12 . The system of  claim 1 , wherein the fetal DNA is no greater than 30% of total DNA in the nucleic acid sample. 
     
     
         13 . A system for analyzing a mixed nucleic acid sample comprising a major subpopulation and a minor subpopulation, the system comprising:
 (a) a probe microarray comprising: 
 a plurality of probes configured to hybridize to a target nucleic acid from a target chromosome, the target nucleic acid comprising a polymorphic site, the plurality of probes comprising first probes configured to hybridize to a first allelic variant (“A allele”) of the target nucleic acid and comprising second probes configured to hybridize to a second allelic variant (“B allele”) of the target nucleic acid, wherein the polymorphic site can be homozygous for the A allele (“AA”), homozygous for the B allele (“BB”), or heterozygous (“AB”), 
 the probe microarray being configured to generate first signals indicative of hybridization of one or more first probes to one or more target nucleic acids comprising the first allelic variant, and to generate second signals indicative of hybridization of one or more second probes to one or more target nucleic acids comprising the second allelic variant; 
   (b) a scanner configured to detect the signals generated by the probe microarray according to a first channel for the first signals and a second channel for the second signals; and   (c) a computer communicatively coupled to the scanner, the computer comprising a processor and memory, the memory encoded with instructions which are executable by the processor to cause the computer to at least:
 receive first signal and second signal data from the scanner, and 
 determine the copy number of the target chromosome in the minor subpopulation using the first signal and the second signal. 
   
     
     
         14 . The system of  claim 13 , wherein the executable instructions further cause the computer to determine the cop number of the target chromosome in the major subpopulation using the first signal and the second signal. 
     
     
         15 . The system of  claim 13 , wherein the executable instructions further cause the computer to determine the genotype for the minor subpopulation using the first signal and the second signal. 
     
     
         16 . The system of  claim 13 , wherein the executable instructions further cause the computer to determine the genotype for the major subpopulation using the first signal and the second signal. 
     
     
         17 . The system of  claim 13 , wherein the executable instructions further cause the computer to determine the relative amounts of the major subpopulation and the minor subpopulation using the first signal and the second signal. 
     
     
         18 . The system of  claim 13 , wherein the major subpopulation includes or is derived from normal tissue and the minor subpopulation includes or is derived from the tumor. 
     
     
         19 . The system of  claim 13 , wherein the mixed nucleic acid population is cell-free nucleic acid obtained from a pregnant female’s blood, wherein the major subpopulation is maternal nucleic acid and the minor subpopulation includes or is derived from fetal nucleic acid. 
     
     
         20 . The system of  claim 19 , wherein fetal DNA is no greater than 30% of total DNA in the nucleic acid sample.