Assay systems for genetic analysis
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.
1. An assay method for calculation of source contribution and detection of the presence or absence of copy number variations (CNVs) in one or more genomic regions within a mixed sample, the assay comprising the steps of:
interrogating selected loci in the mixed sample by:
introducing a first set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region;
introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on at least one polymorphic locus;
introducing a pool of one or more bridging oligonucleotides under conditions that allow one or more of the bridging oligonucleotides within the pool to specifically hybridize to complementary regions in the two or more selected loci, wherein the one or more bridging oligonucleotides are complementary to a region of the loci between and adjacent to the regions complementary to the fixed sequence oligonucleotides of each set and the one or more bridging oligonucleotides are designed to have a melting temperature in a range of +/− 5° C.;
ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the loci;
amplifying the contiguous ligation products to create amplification products;
analysing the individual amplification products through sequence determination or hybridization techniques, wherein one or more selected loci are interrogated for copy number and one or more selected loci are interrogated for polymorphisms of interest in nucleic acids corresponding to a major source or a minor source in the mixed sample;
calculating source contribution by determining loci content from amplification products derived from fixed sequence oligonucleotides that identify one or more selected loci interrogated for polymorphisms of interest in nucleic acids corresponding to a major source or a minor source in the mixed sample; and
detecting CNVs by determining loci frequency from amplification products derived from fixed sequence oligonucleotides that identify one or more selected loci interrogated for copy number.
2. The assay method of claim 1 , wherein the fixed sequence oligonucleotides comprise universal primer regions.
3. The assay method of claim 2 , wherein the first and second sets of fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides.
4. The assay method of claim 1 , wherein the unhybridized fixed sequence oligonucleotides are removed prior to introduction of the bridging oligonucleotides.
5. The assay method of claim 1 , wherein the pool of bridging oligonucleotides are introduced simultaneously with the first and second sets of fixed sequence oligonucleotides.
6. The assay method of claim 1 , wherein the hybridized fixed sequence oligonucleotides and the loci are isolated prior to introduction of the pool of bridging oligonucleotides.
7. The assay method of claim 1 , wherein the amplification products are isolated after the amplifying step.
8. The assay method of claim 7 , wherein the amplification products are isolated as individual molecules after the amplifying step.
9. The assay method of claim 8 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products after the amplifying step.
10. The assay method of claim 8 , wherein the individual isolated amplification products are further amplified to create identical copies of molecules complementary to all or a portion of the individual amplification products after the amplifying step.
11. The assay method of claim 1 , wherein at least one fixed sequence oligonucleotide from each of the first and second sets of fixed sequence oligonucleotides comprises a sample index.
12. The assay method of claim 1 , wherein the mixed sample is a maternal sample comprising maternal and fetal cfDNA.
13. The assay method of claim 1 , wherein (a) the genomic region is located on a chromosome selected from the group consisting of chromosomes 13, 18, and 21; or (b) the genomic region is located on a Y or X chromosome.
14. The assay method of claim 1 , wherein (a) the mixed sample is a maternal sample comprising maternal and fetal cfDNA or the cfDNA from the maternal sample and the assay system identifies the presence or absence of chromosomal abnormalities in the sample, wherein the chromosomal abnormalities are selected from Down Syndrome (trisomy 21), Edwards Syndrome (trisomy 18), Patau syndrome (trisomy 13), Klinefelter's Syndrome (XXY), Triple X syndrome, XYY syndrome, Trisomy 8, Trisomy 16, Turner Syndrome, Robertsonian syndrome, DiGeorge Syndrome and Wolf-Hirschhorn Syndrome; and/or
(b) wherein the mixed sample is a maternal sample comprising maternal and fetal cfDNA of the cfDNA from the maternal sample and the assay system identifies percent fetal contribution in the sample, and wherein the fetus is male, and where percent fetal DNA in the sample is determined through detection of Y-specific loci; and/or (c) wherein optionally quantities of an amplified Y-specific locus are determined from the sample and compared to one or more amplified selected loci which are present in both maternal DNA and fetal DNA and which are from an autosomal region that is not on chromosome 13, 18, or 21.
15. The assay method of claim 1 , wherein the at least one polymorphic locus is located on a chromosome selected from the group consisting of chromosomes 1-16.