Methods for non-invasive prenatal ploidy calling

• US 10,216,896 B2
• Filed: 09/25/2015
• Issued: 02/26/2019
• Est. Priority Date: 09/30/2009
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Rabinowitz, Matthew, Ryan, Allison, Gemelos, George, Banjevic, Milena, Demko, Zachary
• Primary Examiner(s): Riggs, II, Larry D
• Application Number:
  US14/866,223
• Publication Number:
  US 20160024564A1
• Time in Patent Office: 1,250 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G06N 7/01 Probabilistic graphical models, e.g. probabilistic networks
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Disclosed herein are methods for determining the copy number of a chromosome in a fetus in the context of non-invasive prenatal diagnosis. In an embodiment, the measured genetic data from a sample of genetic material that contains both fetal DNA and maternal DNA is analyzed, along with the genetic data from the biological parents of the fetus, and the copy number of the chromosome of interest is determined. In an embodiment, the maternal serum is measured using a single-nucleotide polymorphism (SNP) microarray, along with parental genomic data, and the determination of the chromosome copy number is used to make clinical decisions pertaining to the fetus.

Claims

1. A method of determining at least a portion of the genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, and the father having a paternal genome with paternal haplotypes and the mother having a maternal genome with maternal haplotypes, the method comprising:

measuring at least 1,000 SNP loci, comprising amplifying the SNP loci from a biological sample obtained from the pregnant female containing a mixture of free floating maternal and fetal nucleic acid molecules;

analyzing a plurality of the nucleic acid molecules, comprising;

identifying a location of the nucleic acid molecule in the human genome; and

determining a respective allele of the nucleic acid molecule;

determining a paternal allele inherited by the fetus from the father at each of a first plurality of loci, wherein the maternal genome is heterozygous at the first plurality of loci;

determining each of two maternal haplotypes of the first plurality of loci;

based on the determined alleles of the nucleic acid molecules, determining, with a computer system, amounts of respective alleles at each of the first plurality of loci;

comparing relative amounts of the respective alleles of the nucleic acid molecules at more than one locus of the first plurality of loci; and

based on the comparison, determining which of the two maternal haplotypes is inherited by the unborn fetus from the mother at the portion of the genome covered by the first plurality of loci.

2. The method of claim 1, wherein the relative amounts include a size distribution of the nucleic acid molecules.

3. The method of claim 1, wherein determining each of the two maternal haplotypes of the first plurality of loci is based on the analysis of the plurality of nucleic acid molecules from a biological sample.

4. The method of claim 1, wherein determining the allele inherited from the father at each of the first plurality of loci includes:

determining a second plurality of loci of the paternal genome that are heterozygous, and wherein the maternal genome is homozygous at the second plurality of loci;

identifying, in the plurality of nucleic acid molecules, alleles that are present in the paternal genome at respective ones of the second plurality of loci and absent in the maternal genome;

identifying the inherited paternal haplotype as the haplotype with the identified alleles; and

using the inherited paternal haplotype to determine the allele inherited from the father at the first plurality of loci.

5. The method of claim 1, wherein determining each of the two maternal haplotypes of the first plurality of loci includes:

identifying the alleles of the maternal genome at one or more of the first plurality of loci based on the amounts of the determined respective alleles of the nucleic acid molecules at a respective locus;

identifying a plurality of reference haplotypes; and

comparing the identified alleles of the maternal genome to the alleles in the corresponding loci of the plurality of reference haplotypes to identify the two maternal haplotypes.

6. The method of claim 5, wherein determining each of the two maternal haplotypes of the first plurality of loci further includes:

repeatedly comparing an identified allele of the maternal genome to the plurality of reference haplotypes until each of the two maternal haplotypes are uniquely identified.

7. The method of claim 1, wherein determining the allele inherited from the father at each of the first plurality of loci is based on the analysis of the plurality of nucleic acid molecules from a biological sample, and wherein determining the allele inherited from the father at each of the first plurality of loci includes:

determining a second plurality of loci at which the fetal genome is heterozygous and the maternal genome is homozygous;

determining the allele inherited from the father at each of the second plurality of loci by;

determining relative amounts of the determined respective alleles of the nucleic acid molecules at the respective locus of the second plurality; and

identifying the allele having the least relative amount as being the inherited allele at the respective locus;

identifying a plurality of reference haplotypes;

using the alleles inherited from the father at each of the second plurality of loci to determine which of the reference haplotypes is inherited from the father, the determined haplotype including the first plurality of loci; and

determining the alleles inherited from the father at the first plurality of loci from the haplotype determined to be inherited from the father.

8. The method of claim 7, wherein determining which of the reference haplotypes is inherited from the father includes:

repeatedly comparing the alleles determined to be inherited from the father at each of the second plurality of loci to the alleles in the corresponding loci of the plurality of reference haplotypes until the reference haplotype inherited from the father is uniquely identified.

9. The method of claim 7, wherein determining a specific locus to be one of the second plurality of loci at which the fetal genome is heterozygous and the maternal genome is homozygous includes:

determining a cutoff value for a number of predicted counts of an allele at the specific locus, the cutoff value predicting whether the maternal genome is homozygous and the fetal genome is heterozygous, wherein the cutoff value is determined based on a statistical distribution of numbers of counts for different combinations of homozygosity and heterozygosity at the specific locus;

based on the analysis of the nucleic acid molecules from the biological sample, detecting a first allele and a second allele at the specific locus;

determining a number of actual counts of the first allele based on the sequencing of the plurality of nucleic acid molecules from the biological sample; and

determining the fetal genome is heterozygous for the first allele and a second allele and the maternal genome is homozygous for the second allele when the number of actual counts is less than the cutoff value.

10. The method of claim 9, wherein the statistical distribution is dependent on a fractional concentration of nucleic acid molecules from the biological sample that are derived from the fetus.

11. The method of claim 10, wherein the statistical distribution is further dependent on the number of the plurality of nucleic acid molecules corresponding to the specific locus.

12. The method of claim 1, wherein determining the allele inherited from the father at each of the first plurality of loci includes:

determining a second plurality of loci of the paternal genome that are homozygous by analyzing the paternal genome, wherein the first plurality of loci is the second plurality of loci;

determining the allele of the paternal genome at each the first plurality of loci; and

assigning the respective alleles at the first plurality of loci to be the alleles inherited from the father.

13. The method of claim 1, wherein analyzing a nucleic acid molecule includes implementing on at least a portion of the nucleic acid molecules at least one technique selected from the group consisting of massively parallel sequencing, microarray, hybridization, PCR, digital PCR, and mass spectrometry.

14. The method of claim 1, further comprising:

for each of a first subset of neighboring loci of the first plurality of loci, determining which haplotype is inherited by the unborn fetus from the mother for a first genomic section including the first subset of neighboring loci, wherein determining which haplotype includes;

(a) determining a first amount of determined respective alleles of the nucleic acid molecules that match one of the two maternal haplotypes for the first subset of consecutive loci;

(b) determining a second amount of determined respective alleles of the nucleic acid molecules that match the other of the two maternal haplotypes for the first subset of consecutive loci; and

(c) determining the inherited haplotype for the first genomic section based on a comparison of the first amount to the second amount.

15. The method of claim 14, wherein the comparison of the first amount to the second amount uses the sequential probability ratio test.

16. The method of claim 14, wherein determining the first amount and the second amount are both performed sequentially with respect to the locations of the first subset of neighboring loci.

17. The method of claim 14, wherein the first subset of neighboring loci is further divided into two subgroups, wherein the first subgroup consists of loci for which the genotypes of the father match the constituent genotypes of a first haplotype of the mother, and the second subgroup consists of loci for which the genotypes of the father match the constituent genotypes of a second haplotype of the mother;

and wherein (a)-(c) are performed individually for the two subgroups, the method further comprising;

determining the inherited haplotype for the first genomic section based on results of (c) for these two subgroups.

18. The method of claim 1, further comprising:

determining that the fetus has inherited a mutation from the mother by;

analyzing the haplotype of the mother that was inherited by the fetus; and

identifying the mutation in the inherited haplotype.

19. The method of claim 1, wherein analyzing a plurality of nucleic acid molecules from the biological sample includes:

enriching the biological sample for nucleic acids in a target region of a genome; and

/or preferentially sequencing nucleic acids in the target region, and wherein a first plurality of loci are in the target region.

20. The method of claim 19, wherein the target region is identified as containing a high number of informative loci.

28. The method of claim 19, wherein the sequencing only sequences nucleic acids in the target region.

21. A method of determining at least a portion of the genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, and the father having a paternal genome with paternal haplotypes and the mother having a maternal genome with maternal haplotypes, the method comprising:

measuring at least 1,000 SNP loci, comprising amplifying the SNP loci from a biological sample obtained from the pregnant female containing a mixture of free floating maternal and fetal nucleic acid molecules;

analyzing a plurality of the nucleic acid molecules, comprising;

identifying a location of the nucleic acid molecule in the human genome; and

determining a respective allele of the nucleic acid molecule;

determining a first plurality of loci of the paternal genome that are heterozygous, wherein the paternal genome is obtained from the father of the unborn fetus, and wherein the maternal genome is homozygous at the first plurality of loci; and

based on the determined respective alleles at the first plurality of loci, determining, with a computer system, the haplotype that is inherited by the unborn fetus from the father at the portion of the genome covered by the first plurality of loci.

22. The method of claim 21, wherein determining the haplotype that is inherited by the unborn fetus from the father includes:

identifying, in the plurality of nucleic acid molecules, alleles that are present in the paternal genome at respective ones of the first plurality of loci and absent in the maternal genome; and

identifying the inherited paternal haplotype as the haplotype with the identified alleles.

23. The method of claim 21, further comprising:

determining that the fetus has inherited a mutation from the father by;

analyzing the haplotype of the father that was inherited by the fetus; and

identifying the mutation in the inherited haplotype.

24. A method of determining at least a portion of the genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, and the father having a paternal genome with paternal haplotypes and the mother having a maternal genome with maternal haplotypes, the method comprising:

determining a first plurality of loci of the paternal genome that are heterozygous, wherein the paternal genome is obtained from the father of the unborn fetus, and wherein the maternal genome, obtained from the mother of the unborn fetus, is also heterozygous at the first plurality of loci, and wherein each of two paternal haplotypes and each of two maternal haplotypes at the first plurality of loci are known;

determining one or more second loci of the paternal genome that are heterozygous, wherein the maternal genome is homozygous at the second loci, and wherein the first plurality of loci and the second loci are on the same chromosome;

measuring at least 1,000 SNP loci, comprising amplifying the SNP loci from a biological sample obtained from the pregnant female containing a mixture of free floating maternal and fetal nucleic acid molecules;

analyzing a plurality of the nucleic acid molecules, comprising;

identifying a location of the nucleic acid molecule in the human genome; and

determining a respective allele of the nucleic acid molecule;

determining which of the two paternal haplotypes has been inherited by the fetus by analyzing the determined respective alleles of the plurality of nucleic acid molecules from the biological sample at least one of the second loci;

comparing, with a computer system, relative amounts of the determined respective alleles of the nucleic acid molecules at more than one locus of the first plurality of loci; and

based on the paternal haplotype determined to be inherited by the fetus and based on the comparison of the relative amounts, determining the haplotype that is inherited by the unborn fetus from the mother at the portion of the genome covered by the first plurality of loci.

25. A method of determining at least a portion of the genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, and the father having a paternal genome with paternal haplotypes and the mother having a maternal genome with maternal haplotypes, the method comprising:

measuring at least 1,000 SNP loci, comprising amplifying the SNP loci from a biological sample obtained from the pregnant female containing a mixture of free floating maternal and fetal nucleic acid molecules;

analyzing a plurality of the nucleic acid molecules, comprising;

identifying a location of the nucleic acid molecule in the human genome; and

determining a respective allele of the nucleic acid molecule;

determining a first plurality of loci at which the fetal genome is heterozygous and the maternal genome is homozygous;

determining, with a computer system, an allele inherited from the father at each of the first plurality of loci by;

determining relative amounts of the determined respective alleles of the nucleic acid molecules at the respective locus of the first plurality; and

identifying the allele having the least relative amount as being the inherited allele at the respective locus;

identifying a plurality of reference haplotypes; and

using the alleles inherited from the father at each of the first plurality of loci to determine which of the reference haplotypes is inherited from the father at the portion of the genome covered by the first plurality of loci.

26. The method of claim 25, wherein determining which of the reference haplotypes is inherited from the father includes:

repeatedly comparing the alleles determined to be inherited from the father at each of the first plurality of loci to the alleles in the corresponding loci of the plurality of reference haplotypes until the reference haplotype inherited from the father is uniquely identified.

27. The method of claim 25, wherein determining a specific locus to be one of the first plurality of loci at which the fetal genome is heterozygous and the maternal genome is homozygous includes:

determining a cutoff value for a number of predicted counts of an allele at the specific locus, the cutoff value predicting whether the maternal genome is homozygous and the fetal genome is heterozygous, wherein the cutoff value is determined based on a statistical distribution of numbers of counts for different combinations of homozygosity and heterozygosity at the specific locus;

based on the analysis of the nucleic acid molecules from the biological sample, detecting a first allele and a second allele at the specific locus;

determining a number of actual counts of a first allele based on the sequencing of the plurality of nucleic acid molecules from the biological sample; and

determining the fetal genome is heterozygous for the first allele and a second allele and the maternal genome is homozygous for the second allele when the number of actual counts is less than the cutoff value.