Non-invasive fetal sex determination

1. A method for measuring a risk for Y chromosomal frequency abnormalities in a fetus comprising the steps of:

(a) annealing sets of two fixed sequence oligonucleotides to maternal and fetal nucleic acids from at least one maternal sample,

wherein oligonucleotides in the sets of two fixed sequence oligonucleotides are specific to selected non-polymorphic nucleic acid regions on the Y chromosome and to selected polymorphic and non-polymorphic nucleic acid regions on at least one non-Y chromosome,

wherein a portion of each of the two fixed sequence oligonucleotides is complementary to one of the selected non-polymorphic or polymorphic nucleic acid regions,

at least one of the two fixed sequence oligonucleotides in each set specific to a selected non-polymorphic nucleic acid region comprises a locus index,

at least one of the two fixed sequence oligonucleotides in each set specific to a selected at least one of the two fixed sequence oligonucleotides in each set specific to a selected polymorphic nucleic acid region comprises an allele index,

at least one of the two fixed sequence oligonucleotides in each set comprises an amplification universal primer sequence, and

wherein the oligonucleotides specific to the selected polymorphic nucleic acid regions on the at least one non-Y chromosome in the maternal and fetal nucleic acids are specific for different alleles in the selected polymorphic nucleic acid region;

(b) annealing bridging oligonucleotides to the selected non-polymorphic and polymorphic nucleic acid regions, wherein the bridging oligonucleotides hybridize between the two fixed sequence oligonucleotides of each set;

(c) ligating the fixed sequence oligonucleotides and bridging oligonucleotides to generate a contiguous oligonucleotide sequence spanning the two fixed sequence oligonucleotides of each set and the contiguous oligonucleotide sequence is complementary to the selected nucleic acids regions from the Y chromosome and the at least one non-Y chromosome;

(d) amplifying the selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome to generate amplified selected nucleic acid regions using the universal primer sequence, wherein at least eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified;

(e) sequencing the amplified selected nucleic acid regions formed in step (d);

(f) measuring a percent of fetal nucleic acids from the at least one maternal sample by quantifying the amplified selected polymorphic nucleic acid regions from the at least one non-Y chromosome, wherein the maternal nucleic acids are homozygous for one allele and the fetal nucleic acids are heterozygous; and

(g) measuring a probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities by (i) quantifying the amplified selected non-polymorphic nucleic acid regions from the Y chromosome and (ii) quantifying the amplified selected non-polymorphic nucleic acid regions from the at least one non-Y chromosome, wherein the fetus is a normal male fetus if the non-polymorphic nucleic acid regions on the Y chromosome contribute no more than 1/46 th of the percent of fetal nucleic acids from the at least one maternal sample, and the fetus is at risk for Y chromosomal frequency abnormalities if the non-polymorphic nucleic acid regions on the Y chromosome contribute 1/23 rd or more of the percent of fetal nucleic acids from the at least one maternal sample.

2. The method of claim 1 , wherein at least forty-eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified.

3. The method of claim 2 , wherein at least ninety-six selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified.

4. The method of claim 1 , wherein the Y chromosomal frequency abnormality arises from a Y chromosome aneuploidy, a Y chromosome mosaicism of the fetus or sample contamination.

5. The method of claim 1 , wherein selected polymorphic and non-polymorphic nucleic acid regions from at least two non-Y chromosomes are amplified, sequenced and quantified.

6. The method of claim 5 , wherein selected polymorphic and non-polymorphic nucleic acid regions from at least four non-Y chromosomes are amplified, sequenced and quantified.

7. The method of claim 6 wherein selected polymorphic and non-polymorphic nucleic acid regions from at least six non-Y chromosomes are amplified, sequenced and quantified.

8. The method of claim 1 , wherein the at least one non-Y chromosome is selected from chromosome 13, 18 or 21.

9. The method of claim 1 , wherein at least one of the fixed sequence oligonucleotides in each set comprises at least one sample index.

10. The method of claim 9 , wherein the at least one maternal sample comprises a plurality of maternal samples.

11. The method of claim 10 , wherein before the amplification step the maternal and fetal nucleic acids from each of the plurality of maternal samples are in different vessels for the reactions, and after the amplification step the reactions are pooled.

12. The method of claim 1 , wherein at least one of the fixed sequence oligonucleotides in each set further comprises a locus index and a sample index, and wherein the locus index and sample index are located on a same fixed sequence oligonucleotide in a set.

13. The method of claim 1 , wherein at least one of the fixed sequence oligonucleotides in each set further comprises a locus index and a sample index, and wherein the locus index and sample index are located on different fixed sequence oligonucleotides in a set.

14. The method of claim 1 , wherein at least one of the fixed sequence oligonucleotides in each set used to interrogate a selected polymorphic nucleic acid region in the at least one non-Y chromosome comprises an allele index.

15. The method of claim 1 , where the selected nucleic acid regions are amplified in a single vessel.

16. The method of claim 1 , where the amplified selected nucleic acid regions are each counted an average of at least five times.

17. The method of claim 16 , where the amplified selected nucleic acid regions are each counted an average of at least 250 times.

18. The method of claim 1 , wherein the at least one non-Y chromosome is selected from chromosome 1, 2, 3, 4, 5, 7, 10, 11, 12, 13, 17, 18, 20, 21 or 23.

19. The method of claim 1 , where the amplified selected nucleic acid regions are each counted an average of at least 50 times.

20. The method of claim 1 , where the amplified selected nucleic acid regions are each counted an average of at least 100 times.