Methods for non-invasive prenatal ploidy calling

• US 9,334,541 B2
• Filed: 07/29/2014
• Issued: 05/10/2016
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Rabinowitz, Matthew, Gemelos, George, Banjevic, Milena, Ryan, Allison, Demko, Zachary, Hill, Matthew, Zimmermann, Bernhard, Baner, Johan
• Primary Examiner(s): Riggs, II, Larry D
• Application Number:
  US14/446,232
• Publication Number:
  US 20140336060A1
• Time in Patent Office: 651 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes
• 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 20/40 Population genetics; Linkage disequilibrium
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search
• CPC Class Codes: G16B 5/00 : ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
• CPC Class Codes: G16B 5/20 : Probabilistic models
• CPC Class Codes: G16H 10/40 : for data related to laboratory analysis, e.g. patient specimen analysis
• CPC Class Codes: G16H 50/30 : for calculating health indices; for individual health risk assessment

Abstract

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a sample of DNA from the mother of the fetus and from the fetus, and from genotypic data from the mother and optionally also from the father. The ploidy state is determined by using a joint distribution model to create a set of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias.

Claims

1. A method for determining the number of copies of a chromosome or chromosome segment of interest in the genome of at least one gestating fetus, the method comprising:

sequencing DNA from a first fraction of each mixed sample in a plurality of mixed samples from a plurality of pregnant mothers to obtain a first set of measured genetic data at a plurality of loci on a chromosome or chromosome segment of interest;

wherein each mixed sample comprises DNA from a fetus and DNA from the mother of the fetus;

generating a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses;

determining, on a computer, a first probability for each of the hypotheses using the first set of measured genetic data;

sequencing DNA from a second fraction of a least one of the mixed samples for which the first probability of an aneuploid number of copies of the chromosome or chromosome segment of interest is above a threshold value to obtain a second set of measured genetic data at the plurality of loci on the chromosome or chromosome segment of interest;

determining, on a computer, a second probability for each of the hypotheses using the second set of measured genetic data and optionally the first set of measured genetic data;

selecting the hypothesis with the greatest probability for the second probability determination; and

outputting the hypothesis with the greatest probability for the second probability determination as an indication of the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses.

2. The method of claim 1, comprising shotgun sequencing of randomly selected DNA fragments.

3. The method of claim 1, comprising sequencing non-polymorphic loci.

4. The method of claim 1, comprising sequencing polymorphic loci.

5. The method of claim 1, wherein the method further comprises amplifying the DNA prior to sequencing the DNA.

6. The method of claim 1, comprising:

determining, on a computer, allele ratios at polymorphic loci from the measured genetic data;

creating, on a computer, a joint distribution model for expected allele ratios of each polymorphic locus for each hypothesis using genetic data from one or both biological parents of at least one of the fetuses;

determining, on a computer, the probability of each of the hypotheses using the joint distribution model and the calculated allele ratios;

selecting the hypothesis with the greatest probability; and

outputting the hypothesis with the greatest probability for the second probability determination as an indication of the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses.

7. The method of claim 1, comprising aggregating the measured genetic data on the chromosome or chromosome segment of interest.

8. The method of claim 1, comprising

determining, on a computer, allele ratios at polymorphic loci from the measured genetic data;

aggregating the allele ratios;

determining, on a computer, the probability of each of the hypotheses using the aggregated allele ratios; and

selecting the hypothesis with the greatest probability; and

outputting the hypothesis with the greatest probability for the second probability determination as an indication of the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses.

9. The method of claim 7, wherein determining the number of copies of the chromosome or chromosome segment of interest comprises comparing the aggregated value for the chromosome or chromosome segment of interest to the aggregated value for one or more chromosomes expected to be disomic.

10. The method of claim 1, wherein determining the number of copies of the chromosome or chromosome segment of interest comprises comparing the total number of sequence reads that map to the chromosome or chromosome segment of interest to the total number of sequence reads that map to one or more other chromosomes.

11. The method of claim 10, wherein the total number of sequence reads that map to the chromosome or chromosome segment of interest or the total number of sequence reads that map to one or more other chromosomes is adjusted for bias.

12. The method of claim 1, wherein determining the number of copies of the chromosome or chromosome segment of interest comprises comparing the mean value of sequence reads that map to the chromosome or chromosome segment of interest to the mean value of sequence reads that map to one or more other chromosomes.

13. The method of claim 1, wherein determining the number of copies of the chromosome or chromosome segment of interest comprises comparing the mean of the allele ratios for the chromosome or chromosome segment of interest to the mean of the allele ratios for one or more other chromosomes.

14. The method of claim 1, comprising for at least one of the mixed samples:

distributing the mixed sample into a plurality of reaction samples to randomly provide individual reaction samples that contain a sequence from the chromosome or chromosome segment of interest and individual reaction samples that do not contain a sequence from the chromosome or chromosome segment of interest;

sequencing the DNA in the individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome or chromosome segment in the reaction samples and a second number of binary results representing presence or absence of a possibly aneuploid fetal chromosome or chromosome segment of interest in the reaction samples;

determining an expected distribution of a number of binary results for a presumably euploid fetal chromosome or chromosome segment in the reaction samples using the first number;

determining an expected distribution of a number of binary results for a presumably aneuploid fetal chromosome or chromosome segment in the reaction samples using the first number and the ratio of fetal to maternal DNA found in the mixed sample; and

using a maximum likelihood estimation to select the hypothesis with the greatest probability, thereby determining the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses.

15. The method of claim 1, further comprising determining the ratio of fetal to maternal DNA in each of the mixed samples by calculating a maximum likelihood estimate of the ratio of fetal to maternal DNA.

16. The method of claim 1, further comprising determining the ratio of fetal to maternal DNA in each of the mixed samples by:

identifying polymorphic loci where the father of the fetus has an allele that is not present in the mother of the fetus; and

using the amount of the allele possessed by the father but not by the mother in each of the mixed samples for each of the identified polymorphic loci to determine the ratio of fetal to maternal DNA in each of the mixed samples.

17. The method of claim 1, comprising measuring the difference in methylation between one or more maternal and fetal alleles.

18. The method of claim 1, wherein determining the number of copies of the chromosome or chromosome segment of interest comprises determining the presence or absence of a deletion or duplication.

19. The method of claim 1, wherein the mixed samples are isolated from maternal blood.

20. The method of claim 1, wherein determining the probability of each of the hypotheses comprises comparing the measured genetic data to either (i) a threshold value or (ii) a value for a disomic chromosome or chromosome segment.

21. A method for determining the number of copies of a chromosome or chromosome segment of interest in the genome of at least one gestating fetus, the method comprising:

measuring DNA from a first fraction of each mixed sample in a plurality of mixed samples from a plurality of pregnant mothers to obtain a first set of measured genetic data at a plurality of loci on a chromosome or chromosome segment of interest;

wherein each mixed sample comprises DNA from a fetus and DNA from the mother of the fetus;

determining a parameter by analyzing the first set of measured genetic data;

sequencing DNA from a second fraction of a least one of the mixed samples for which the parameter is above or below a threshold value to obtain a second set of measured genetic data at the plurality of loci on the chromosome or chromosome segment of interest;

determining, on a computer, a z-score for at least one possible number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses using the second set of measured genetic data and optionally the first set of measured genetic data;

selecting the number of copies of the chromosome or chromosome segment of interest that is most likely to be correct based on the z-score; and

outputting the selected number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses.

22. The method of claim 21, comprising determining a number of sequence reads to be obtained from at least one of the second fractions using the parameter.

23. The method of claim 21, wherein the analyzing the first set of measured genetic data comprises:

creating a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment of interest in the genome of at least one of the fetuses; and

determining, on a computer, a first probability for each of the hypotheses using the first set of measured genetic data;

wherein the parameter is the probability of the hypothesis with the greatest probability.

24. The method of claim 21, wherein the parameter is a Z-score.

25. The method of claim 21, wherein the parameter is a ratio of fetal to maternal DNA in the mixed sample.

26. The method of claim 21, wherein the parameter is the number of loci in the plurality of loci.

27. A method for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a cancer in at least one individual, the method comprising:

sequencing DNA from a first fraction of each mixed sample in a plurality of mixed samples from a plurality of individuals to obtain a first set of measured genetic data at a plurality of loci on a chromosome or chromosome segment of interest;

wherein each mixed sample comprises DNA from a cancer and DNA not from a cancer;

generating a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment of interest in the genome of at cancer in least one of the individuals;

determining, on a computer, a first probability for each of the hypotheses using the first set of measured genetic data;

sequencing DNA from a second fraction of a least one of the mixed samples for which the first probability of an aneuploid number of copies of the chromosome or chromosome segment of interest is above a threshold value to obtain a second set of measured genetic data at the plurality of loci on the chromosome or chromosome segment of interest;

determining, on a computer, a second probability for each of the hypotheses using the second set of measured genetic data and optionally the first set of measured genetic data; and

selecting the hypothesis with the greatest probability for the second probability determination; and

outputting the hypothesis with the greatest probability for the second probability determination as an indication of the number of copies of the chromosome or chromosome segment of interest in the genome of a cancer in at least one individual.

28. The method of claim 27, comprising sequencing polymorphic loci.

29. A method for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a cancer in at least one individual, the method comprising:

measuring DNA from a first fraction of each mixed sample in a plurality of mixed samples from a plurality of individuals to obtain a first set of measured genetic data at a plurality of loci on a chromosome or chromosome segment of interest;

wherein each mixed sample comprises DNA from a cancer and DNA not from a cancer;

determining a parameter by analyzing the first set of measured genetic data;

sequencing DNA from a second fraction of a least one of the mixed samples for which the parameter is above or below a threshold value to obtain a second set of measured genetic data at the plurality of loci on the chromosome or chromosome segment of interest;

determining, on a computer, a z-score for at least one possible number of copies of the chromosome or chromosome segment of interest in the genome of a cancer in at least one individual using the second set of measured genetic data and optionally the first set of measured genetic data;

selecting the number of copies of the chromosome or chromosome segment of interest that is most likely to be correct based on the z-score; and

outputting the selected number of copies of the chromosome or chromosome segment of interest in the genome of a cancer in at least one individual.

30. The method of claim 29, wherein the parameter is a Z-score.