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 simultaneous detection of the presence or absence of copy number variation (CNV) of a genomic region and presence or absence of one or more fetal polymorphisms in a maternal sample using a single assay, comprising the steps of:
 providing a maternal sample comprising fetal and maternal cell-free DNA;   introducing a first set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a first genomic region, wherein at least one of the fixed sequence oligonucleotides comprise a universal primer region, and wherein the fixed sequence oligonucleotides of the first set hybridize to non-adjacent regions in the locus;   introducing a second set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a second genomic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the second set hybridize to non-adjacent regions in the locus;   introducing a third set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to complementary regions on a locus in a polymorphic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the third set hybridize to non-adjacent regions in the locus;   extending the region between the hybridized first and second fixed sequence oligonucleotides of each set with a polymerase and dNTPs resulting in two adjacently hybridized oligonucleotides from each set;   ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the loci in the first genomic region, the second genomic region, and the polymorphic region;   amplifying the contiguous ligation products using primers complementary to the universal primer regions to create amplification products that are representative of the DNA content of the genomic regions in the maternal sample;   performing high throughput sequence determination on the amplification products;   quantifying sequence reads from the amplification products from the loci of the first and second genomic regions; and   comparing the quantified sequence reads of the amplification products of the first and second genomic regions to identify a copy number variation in the maternal sample;   wherein the presence or absence of a fetal polymorphism in the polymorphic region is detected from the sequence reads of the amplification products from the locus of the polymorphic region.   
     
     
         2 . The method of  claim 1 , wherein the universal primer regions are used for sequence determination of the amplification products. 
     
     
         3 . The method of  claim 1 , wherein the two fixed sequence oligonucleotides of one or more sets are linked to form a precircle probe. 
     
     
         4 . The method of  claim 1 , wherein the amplification products are isolated prior to performing high throughput sequence determination. 
     
     
         5 . The method of  claim 4 , wherein the amplification products are isolated as individual molecules prior to performing high throughput sequence determination. 
     
     
         6 . The method of  claim 5 , wherein the isolated amplification products are further amplified to create identical copies of all or a portion of the isolated amplification products prior to performing high throughput sequence determination. 
     
     
         7 . The method of  claim 6 , wherein the isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the isolated amplification products prior to performing high throughput sequence determination. 
     
     
         8 . The method of  claim 1 , wherein at least one locus interrogated for the presence or absence of copy number variation is different from all loci interrogated for the presence or absence of polymorphisms. 
     
     
         9 . The method of  claim 8 , wherein both loci interrogated for copy number variation are different from the locus interrogated for polymorphisms. 
     
     
         10 . The method of  claim 1 , wherein the genomic region is a chromosome. 
     
     
         11 . The method of  claim 1 , wherein the genomic region is a sub-chromosomal region. 
     
     
         12 . The method of  claim 1 , wherein the genomic region is a single locus. 
     
     
         13 . The method of  claim 1 , wherein at least one of the first or second fixed sequence oligonucleotides in each set comprises one or more indices. 
     
     
         14 . The method of  claim 13 , wherein the amplification products are sequenced by sequencing the one or more indices. 
     
     
         15 . The method of  claim 13 , wherein the amplification products are quantified by counting the one or more indices. 
     
     
         16 . The method of  claim 13 , wherein the indices comprise a locus index. 
     
     
         17 . The method of  claim 13 , wherein the indices comprise a sample index. 
     
     
         18 . The method of  claim 13 , wherein the indices comprise an allele index. 
     
     
         19 . The method of  claim 13 , wherein the indices comprise both a sample index and an allele index, and wherein the sample index and the allele index are present on the same fixed oligonucleotide in a set. 
     
     
         20 . The method of  claim 13 , wherein the indices comprise both a sample index and an allele index, and wherein the sample index and the allele index are on different fixed oligonucleotides in a set. 
     
     
         21 . The method of  claim 1 , wherein the CNV is of fetal origin. 
     
     
         22 . A method for statistically determining the likelihood of a copy number variation (CNV) of a genomic region and presence or absence of one or more fetal polymorphisms in a maternal sample using a single assay, comprising the steps of:
 providing a maternal sample comprising fetal and maternal cell-free DNA;   introducing a first set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a first genomic region, wherein at least one of the fixed sequence oligonucleotides comprise a universal primer region, and wherein the fixed sequence oligonucleotides of the first set hybridize to non-adjacent regions in the locus;   introducing a second set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a second genomic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the second set hybridize to non-adjacent regions in the locus;   introducing a third set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to complementary regions on a locus in a polymorphic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the third set hybridize to non-adjacent regions in the locus;   extending the region between the hybridized first and second fixed sequence oligonucleotides of each set with a polymerase and dNTPs resulting in two adjacently hybridized oligonucleotides from each set;   ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the loci in the first genomic region, the second genomic region, and the polymorphic region;   amplifying the contiguous ligation products using primers complementary to the universal primer regions to create amplification products are representative of the DNA content of the genomic regions in the maternal sample;   performing high throughput sequence determination on the amplification products;   quantifying sequence reads from the amplification products from the loci of the first and second genomic regions;   comparing the quantified sequence reads from the amplification products from the loci of the first and second genomic regions; and   calculating a statistical likelihood of a CNV using a quantity of sequence reads corresponding to the first genomic region and the quantity of sequence reads corresponding to the second genomic region;   wherein the presence or absence of a fetal polymorphism in the polymorphic region is detected from the sequence reads of the amplification products from the locus of the polymorphic region.   
     
     
         23 . The method of  claim 22 , wherein the CNV is of fetal origin. 
     
     
         24 . The method of  claim 22 , wherein the universal primer regions are used for sequence determination of the amplification products. 
     
     
         25 . The method of  claim 22 , wherein the two fixed sequence oligonucleotides of one or more sets are linked to form a precircle probe. 
     
     
         26 . The method of  claim 22 , wherein the amplification products are isolated prior to performing high throughput sequence determination. 
     
     
         27 . The method of  claim 26 , wherein the amplification products are isolated as individual molecules prior to performing high throughput sequence determination. 
     
     
         28 . The method of  claim 27 , wherein the isolated amplification products are further amplified to create identical copies of all or a portion of the isolated amplification products prior to performing high throughput sequence determination. 
     
     
         29 . The method of  claim 28 , wherein the isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the isolated amplification products prior to performing high throughput sequence determination. 
     
     
         30 . The method of  claim 22 , wherein at least one locus interrogated for the presence or absence of copy number variation is different from all loci interrogated for the presence or absence of polymorphisms. 
     
     
         31 . The method of  claim 30 , wherein both loci interrogated for copy number variation are different from the locus interrogated for polymorphisms. 
     
     
         32 . The method of  claim 22 , wherein the genomic region is a chromosome. 
     
     
         33 . The method of  claim 22 , wherein the genomic region is a sub-chromosomal region. 
     
     
         34 . The method of  claim 22 , wherein the genomic region is a single locus. 
     
     
         35 . The method of  claim 22 , wherein at least one of the first or second fixed sequence oligonucleotides in each set comprises one or more indices. 
     
     
         36 . The method of  claim 35 , wherein the amplification products are sequenced by sequencing the one or more indices. 
     
     
         37 . The method of  claim 35 , wherein the amplification products are quantified by counting the one or more indices. 
     
     
         38 . The method of  claim 35 , wherein the indices comprise a locus index. 
     
     
         39 . The method of  claim 35 , wherein the indices comprise a sample index. 
     
     
         40 . The method of  claim 35 , wherein the indices comprise an allele index. 
     
     
         41 . The method of  claim 35 , wherein the indices comprise both a sample index and an allele index, and wherein the sample index and the allele index are present on the same fixed oligonucleotide in a set. 
     
     
         42 . The method of  claim 35 , wherein the indices comprise both a sample index and an allele index, and wherein the sample index and the allele index are on different fixed oligonucleotides in a set. 
     
     
         43 . A method for simultaneous detection of the presence or absence of a fetal chromosomal aneuploidy and presence or absence of one or more fetal polymorphisms in a maternal sample using a single assay, comprising the steps of:
 providing a maternal sample comprising fetal and maternal cell-free DNA;   introducing a first set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a first genomic region, wherein at least one of the fixed sequence oligonucleotides comprise a universal primer region, and wherein the fixed sequence oligonucleotides of the first set hybridize to non-adjacent regions in the locus;   introducing a second set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a second genomic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the second set hybridize to non-adjacent regions in the locus;   introducing a third set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to complementary regions on a locus in a polymorphic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the third set hybridize to non-adjacent regions in the locus;   extending the region between the hybridized first and second fixed sequence oligonucleotides of each set with a polymerase and dNTPs resulting in two adjacently hybridized oligonucleotides from each set;   ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the loci in the first genomic region, the second genomic region, and the polymorphic region;   amplifying the contiguous ligation products using primers complementary to the universal primer regions to create amplification products that reflect are representative of the DNA content of the genomic regions in the maternal sample;   performing high throughput sequence determination on the amplification products;   quantifying sequence reads from the amplification products from the loci of the first and second genomic regions; and   comparing the quantified sequence reads of the amplification products of the first and second genomic regions to identify a fetal chromosomal aneuploidy in the maternal sample;   wherein the presence or absence of a fetal polymorphism in the polymorphic region is detected from the sequence reads of the amplification products from the locus of the polymorphic region.   
     
     
         44 . A method for statistically determining the likelihood of a fetal chromosomal aneuploidy and presence or absence of one or more fetal polymorphisms in a maternal sample using a single assay, comprising the steps of:
 providing a maternal sample comprising fetal and maternal cell-free DNA;   introducing a first set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a first genomic region, wherein at least one of the fixed sequence oligonucleotides comprise a universal primer region, and wherein the fixed sequence oligonucleotides of the first set hybridize to non-adjacent regions in the locus;   introducing a second set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to a complementary region on a locus in a second genomic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the second set hybridize to non-adjacent regions in the locus;   introducing a third set of first and second fixed sequence oligonucleotides to the maternal sample under conditions that allow a region of the fixed sequence oligonucleotides to specifically hybridize to complementary regions on a locus in a polymorphic region, wherein at least one of the fixed sequence oligonucleotides comprises a universal primer region, and wherein the fixed sequence oligonucleotides of the third set hybridize to non-adjacent regions in the locus;   extending the region between the hybridized first and second fixed sequence oligonucleotides of each set with a polymerase and dNTPs resulting in two adjacently hybridized oligonucleotides from each set;   ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the loci in the first genomic region, the second genomic region, and the polymorphic region;   amplifying the contiguous ligation products using primers complementary to the universal primer regions to create amplification products that are representative of the DNA content of the genomic regions in the maternal sample;   performing high throughput sequence determination on the amplification products;   quantifying sequence reads from the amplification products from the loci of the first and second genomic regions;   comparing the quantified sequence reads from the amplification products from the loci of the first and second genomic regions; and   calculating a statistical likelihood of a fetal chromosomal aneuploidy using a quantity of sequence reads corresponding to the first genomic region and the quantity of sequence reads corresponding to the second genomic region;   wherein the presence or absence of a fetal polymorphism in the polymorphic region is detected from the sequence reads of the amplification products from the locus of the polymorphic region.