Assay systems for genetic analysis

Abstract

The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.

What is claimed is: 
     
         1 . A method for detecting a presence or absence of copy number variations of one or more nucleic acid regions of interest and the presence of absence of polymorphisms in one or more nucleic acid regions of interest in a single assay, comprising the steps of:
 providing a genetic sample;   introducing at least two first sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the first sets of fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acid regions of interest;   introducing at least two second sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the second sets of fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acid regions of interest, wherein the first fixed sequence oligonucleotides of the second sets of fixed sequence oligonucleotides are complementary to alternative sequences of a polymorphism of interest in the nucleic acid regions of interest;   extending the region between the first fixed sequence oligonucleotides of the first and second sets and the second fixed sequence oligonucleotides of the first and second sets with a polymerase and dNTPs to create adjacently-hybridized oligonucleotides in each set in the nucleic acid regions of interest;   ligating the adjacently-hybridized oligonucleotides to create ligation products complementary to the nucleic acid regions of interest;   amplifying the ligation products to create amplification products that are representative of the original content of the nucleic acid regions of interest in the genetic sample; and   detecting and quantifying the amplification products from the first sets of fixed sequence oligonucleotides to detect the presence or absence of copy number variations in the nucleic acid regions of interest and detecting and quantifying the amplification products from the second sets of fixed sequence oligonucleotides to detect the presence of absence of polymorphisms in the nucleic acid regions of interest.   
     
     
         2 . The method of  claim 1 , wherein one or both of the first or second fixed sequence oligonucleotides of the first and second sets of fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         3 . The method of  claim 1 , wherein unhybridized first and second sets of fixed sequence oligonucleotides are removed prior to amplification of the ligation products. 
     
     
         4 . The method of  claim 1 , wherein the first and second sets of fixed sequence oligonucleotides are introduced simultaneously. 
     
     
         5 . The method of  claim 1 , wherein the hybridization products of the first sets of fixed sequence oligonucleotides and the nucleic acid regions of interest to which they hybridize are isolated prior to introduction of the second sets of fixed sequence oligonucleotides. 
     
     
         6 . The method of  claim 1 , wherein the amplification products are detected and quantified by next generation sequencing. 
     
     
         7 . The method of  claim 1 , wherein the first or second fixed sequence oligonucleotide of each set of fixed sequence oligonucleotides comprises one or more indices. 
     
     
         8 . The method of  claim 7 , wherein the amplification products are detected and quantified by next generation sequencing of the one or more indices. 
     
     
         9 . The method of  claim 7 , wherein the first or second fixed sequence oligonucleotide of the first sets of fixed sequence oligonucleotides comprises a locus index and the first or second fixed sequence oligonucleotide of the second sets of fixed sequence oligonucleotides comprises an allele index. 
     
     
         10 . The method of  claim 9 , wherein the amplification products are detected and quantified by hybridization of the locus index or allele index to an array. 
     
     
         11 . The method of  claim 9 , wherein the amplification products are detected and quantified by next generation sequencing of the locus index or allele index. 
     
     
         12 . The method of  claim 1 , wherein the amplification products are isolated prior to the detecting and quantifying step. 
     
     
         13 . The method of  claim 1 , wherein the amplification products from the second sets of fixed sequence oligonucleotides are used with the first sets of fixed sequence oligonucleotides to detect the presence or absence of copy number variations of a genomic region. 
     
     
         14 . The method of  claim 1 , wherein the copy number variations are chromosomal aneuploidies. 
     
     
         15 . The method of  claim 14 , wherein the chromosomal aneupolidies are trisomy 13, trisomy 18, trisomy 21, or monosomy X. 
     
     
         16 . The method of  claim 1 , wherein the first sets of first and second fixed sequence oligonucleotides are complementary to loci on different chromosomes than the chromosomes complementary to the second sets of first and second fixed sequence oligonucleotides. 
     
     
         17 . A method for detecting a presence or absence of copy number variations of one or more nucleic acid regions of interest and the presence of absence of polymorphisms in one or more nucleic acid regions of interest in a single assay, comprising the steps of:
 providing a genetic sample;   introducing at least two first sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the first sets of fixed sequence oligonucleotides to specifically hybridize to adjacent complementary regions in nucleic acid regions of interest;   introducing at least two second sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the second sets of fixed sequence oligonucleotides to specifically hybridize to adjacent complementary regions in nucleic acid regions of interest to create adjacently-hybridized oligonucleotides, wherein the first fixed sequence oligonucleotides of the second sets of fixed sequence oligonucleotides are complementary to alternative sequences of a polymorphism of interest in the nucleic acid regions of interest;   ligating the adjacently-hybridized oligonucleotides to create ligation products complementary to the nucleic acid regions of interest;   amplifying the ligation products to create amplification products that are representative of the original content of the nucleic acid regions of interest in the genetic sample; and   detecting and quantifying the amplification products from the first sets of fixed sequence oligonucleotides to detect the presence or absence of copy number variations in the nucleic acid regions of interest and detecting and quantifying the amplification products from the second sets of fixed sequence oligonucleotides to detect the presence of absence of polymorphisms in the nucleic acid regions of interest.   
     
     
         18 . The method of  claim 17 , wherein the first and second sets of fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         19 . The method of  claim 17 , wherein unhybridized first and second sets of fixed sequence oligonucleotides are removed prior to amplification of the ligation products. 
     
     
         20 . The method of  claim 17 , wherein the first or second fixed sequence oligonucleotide of each set of fixed sequence oligonucleotides comprises one or more indices. 
     
     
         21 . The method of  claim 20 , wherein the first or second fixed sequence oligonucleotide of the first sets of fixed sequence oligonucleotides comprises a locus index and the first or second fixed sequence oligonucleotide of the second sets of fixed sequence oligonucleotides comprises an allele index. 
     
     
         22 . The method of  claim 21 , wherein the amplification products are detected and quantified by hybridization of the locus index or allele index to an array. 
     
     
         23 . The method of  claim 21 , wherein the amplification products are detected and quantified by next generation sequencing of the locus index or allele index. 
     
     
         24 . The method of  claim 17 , wherein the amplification products are isolated prior to the detecting and quantifying step. 
     
     
         25 . The method of  claim 17 , wherein the amplification products from the second sets of fixed sequence oligonucleotides are used with the first sets of fixed sequence oligonucleotides to detect the presence or absence of copy number variations of a genomic region. 
     
     
         26 . The method of  claim 17 , wherein the copy number variations are chromosomal aneuploidies. 
     
     
         27 . The method of  claim 26 , wherein the chromosomal aneupolidies are trisomy 13, trisomy 18 or trisomy 21. 
     
     
         28 . The method of  claim 17 , wherein the first sets of first and second fixed sequence oligonucleotides are complementary to loci on different chromosomes than the chromosomes complementary to the second sets of first and second fixed sequence oligonucleotides.