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. A method of detecting a presence or absence of a copy number variation (CNV) in a mixed sample from a single subject, said mixed sample comprising cell-free nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:
a) hybridizing at least 24 first sets of two fixed sequence oligonucleotides to the cell-free nucleic acids in the mixed sample, wherein each first set of two fixed sequence oligonucleotides is complementary to a locus in a first region of interest, each first set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein melting temperatures (T m s) of first fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;
b) hybridizing at least 24 second sets of two fixed sequence oligonucleotides to the cell-free nucleic acids in the mixed sample, wherein each second set of two fixed sequence oligonucleotides is complementary to a locus in a second region of interest, each second set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein T m s of first fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;
c) introducing to the cell-free nucleic acids in the mixed sample one or more bridging oligonucleotides for each set of two fixed sequence oligonucleotides under conditions that allow the bridging oligonucleotides to hybridize to complementary regions between and immediately adjacent to the regions complementary to the first and second fixed sequence oligonucleotides to create adjacently hybridized fixed sequence oligonucleotides and bridging oligonucleotides;
d) ligating the adjacently hybridized bridging oligonucleotides and fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides to create contiguous ligation products and ligating the adjacently hybridized bridging oligonucleotides and fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides to create contiguous ligation products;
e) amplifying the contiguous ligation products using the universal primer regions on each first and second sets of two fixed sequence oligonucleotides to produce amplification products;
f) detecting the amplification products; and
g) detecting a CNV of the first region of interest relative to the second region of interest.
2. The method of claim 1 , wherein the subject comprises a pregnant female such that the cell-free nucleic acids of the major source comprise maternal nucleic acids and the cell-free nucleic acids of the minor source comprise fetal nucleic acids.
3. The method of claim 1 , wherein the subject comprises an individual having normal cells and cancerous cells such that the cell-free nucleic acids of the major source comprise cell-free nucleic acids of the normal cells and the cell-free nucleic acids of the minor source comprise nucleic acids of the cancerous cells.
4. The method of claim 1 , wherein the subject comprises a transplant patient such that the cell-free nucleic acids of the major source comprise nucleic acids of the patient and the cell-free nucleic acids of the minor source comprise nucleic acids of a donor organ, and wherein said CNV is detected in said major source or said minor source.
5. The method of claim 1 , wherein the subject comprises an individual infected with an organism such that the cell-free nucleic acids of the major source comprise nucleic acids of the individual and the cell-free nucleic acids of the minor source comprise nucleic acids of the infectious organism, and wherein said CNV is detected in said major source or said minor source.
6. The method of claim 1 , wherein each locus in the second region of interest comprises one or more polymorphisms.
7. The method of claim 1 , wherein the first region of interest and the second region of interest are on different chromosomes.
8. The method of claim 1 , wherein at least one locus does not comprise a polymorphism.
9. The method of claim 1 , wherein one or both sets of the fixed sequence oligonucleotides comprises at least one index.
10. The method of claim 9 , wherein the amplification products are detected by sequencing the at least one index.
11. The method of claim 9 , wherein the at least one index comprises a locus index.
12. The method of claim 9 , wherein the at least one index comprises a sample index.
13. The method of claim 1 , wherein the amplification products are detected by high throughput sequencing.
14. The method of claim 1 , wherein the amplification products are detected on an array.
15. The method of claim 1 , wherein one or both sets of fixed sequence oligonucleotides comprises an oligonucleotide 5′ of each locus, an oligonucleotide 3′ of each locus, and a bridging oligonucleotide.
16. The method of claim 1 , wherein the amplification products are isolated as individual molecules prior to detection.
17. The method of claim 16 , wherein the individual isolated amplification products are further amplified to create identical copies of all or a portion of the individual amplification products prior to detection.
18. A method of detecting a presence or absence of a copy number variation in a mixed sample from a single subject, said mixed sample comprising cell-free nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:
a) hybridizing at least 24 sets of two fixed sequence oligonucleotides to the cell-free nucleic acids in the mixed sample, wherein each set of two fixed sequence oligonucleotides is complementary to a polymorphic locus in a region of interest, each set of two fixed sequence oligonucleotides comprising universal primer regions, and each locus in the region of interest having different genotypes in the major source and the minor source, and wherein T m s of first fixed sequence oligonucleotides of each set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;
b) introducing one or more bridging oligonucleotides for each set of two fixed sequence oligonucleotides under conditions that allow the bridging oligonucleotides to hybridize to complementary regions between and immediately adjacent to the regions complementary to the first and second fixed sequence oligonucleotides to create adjacently hybridized fixed sequence oligonucleotides and bridging oligonucleotides;
c) ligating the adjacently hybridized oligonucleotides to create contiguous ligation products complementary to the locus;
d) amplifying the contiguous ligation products using the universal primer regions on the two fixed sequence oligonucleotides of each set of fixed sequence oligonucleotides to produce amplification products;
e) detecting the amplification products; and
f) detecting a presence or absence of a copy number variation in the locus of interest between the major and minor sources from the detected amplified products.