Ligation-based detection of genetic variants

Abstract

The present invention provides assays systems and methods for detection of genetic variants in a sample, including copy number variation and single nucleotide polymorphisms. The invention preferably employs the technique of tandem ligation, i.e. the ligation of two or more fixed sequence oligonucleotides and one or more bridging oligonucleotides complementary to a region between the fixed sequence oligonucleotides.

We claim: 
     
         1 . A method for detecting nucleic acid regions of interest in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing at least two fixed sequence oligonucleotides to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in each nucleic acid region of interest, wherein both ends of each fixed sequence oligonucleotide are complementary to a single nucleic acid region of interest, and wherein upon hybridization each fixed sequence oligonucleotide forms a pre-circle oligonucleotide;   introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the one or more bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid region of interest complementary to the ends of the fixed sequence oligonucleotides, and wherein the one or more bridging oligonucleotides hybridize continguously between the ends of the fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create circular ligation products, a portion of which is complementary to the nucleic acid region of interest;   amplifying the circular ligation product to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and   detecting and quantifying the amplification products.   
     
     
         2 . The method of  claim 1 , further comprising after the ligating step and before the amplifying step, cleaving the circular ligation products. 
     
     
         3 . The method of  claim 2 , wherein the fixed sequence oligonucleotides comprise universal primer regions that are used in amplification of the cleaved ligation products. 
     
     
         4 . The method of  claim 1 , wherein uncircularized fixed sequence oligonucleotides are removed after the ligating step and before the amplifying step. 
     
     
         5 . The method of  claim 1 , wherein the fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides. 
     
     
         6 . The method of  claim 5 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid regions of interest to which they hybridize are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         7 . The method of  claim 1 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the fixed sequence oligonucleotides. 
     
     
         8 . The method of  claim 1 , wherein the amplification products are quantified by next generation sequencing. 
     
     
         9 . The method of  claim 1 , wherein the fixed sequence oligonucleotides comprise one or more indices. 
     
     
         10 . The method of  claim 9 , wherein the amplification products are detected and quantified by next generation sequencing of the one or more indices. 
     
     
         11 . The method of  claim 9 , wherein the fixed sequence oligonucleotides comprise a locus index. 
     
     
         12 . The method of  claim 11 , wherein the amplification products are detected and quantified by hybridization of the locus index to an array. 
     
     
         13 . The method of  claim 11 , wherein each locus index is associated with a different label, and the amplification products are detected and quantified by the labels. 
     
     
         14 . The method of  claim 13 , wherein the labels is fluorescent. 
     
     
         15 . The method of  claim 9 , wherein the one or more indices comprises an allele index and wherein a bridging oligonucleotide complementary for a specific polymorphism is used with a corresponding allele index. 
     
     
         16 . The method of  claim 1 , wherein at least one fixed sequence oligonucleotide is specific for a polymorphism in a nucleic acid region of interest. 
     
     
         17 . The method of  claim 16 , wherein the fixed sequence oligonucleotide specific for a polymorphism in a nucleic acid region of interest further comprises an allele index. 
     
     
         18 . A method for detecting nucleic acid regions of interest in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing at least two fixed sequence oligonucleotides to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in each nucleic acid region of interest, wherein both ends of the fixed sequence oligonucleotides are complementary to a single nucleic acid region of interest, wherein upon hybridization each fixed sequence oligonucleotide forms a pre-circle oligonucleotide and wherein each fixed sequence oligonucleotide comprises a locus index specific for a nucleic acid region of interest;   introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid region of interest complementary to the ends of the fixed sequence oligonucleotides, and wherein the one or more bridging oligonucleotides hybridize continguously between the ends of the fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create a circular ligation product, a portion of which is complementary to the nucleic acid region of interest;   amplifying the circular ligation product to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and detecting and quantifying the circular amplification products.   
     
     
         19 . The method of  claim 18 , wherein the fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         20 . The method of  claim 19 , further comprising after the ligating step and before the amplifying step, cleaving the circular ligation products 
     
     
         21 . The method of  claim 20 , wherein the universal primer regions are used to amplify the cleaved ligation products and resulting amplification products are quantified by next generation sequencing of the locus indices. 
     
     
         22 . The method of  claim 20 , wherein the universal primer regions are used to amplify the cleaved ligation products and resulting amplification products are quantified by hybridization of the locus indices to an array.