Resolving genome fractions using polymorphism counts

Patent metadata

Field	Value
Publication number	US-9447453-B2
Application number	US-201213445778-A
Country	US
Kind code	B2
Filing date	Apr 12, 2012
Priority date	Apr 12, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

Methods of reliably estimating genomic fraction (e.g., fetal fraction) from polymorphisms such as small base variations or insertions-deletions are disclosed. Sequenced data from a multigenomic source is used to determine allele counts for one or more of the polymorphisms. For one or more of the polymorphisms, zygosity is assigned, and genomic fraction is determined from the zygosity and allele counts. Certain embodiments employ SNPs as the relevant polymorphism. The disclosed methods can be applied as part of an intentional, pre-designed re-sequencing study targeted against known polymorphisms or can be used in a retrospective analysis of variations found by coincidence in overlapping sequences generated from maternal plasma (or any other setting where a mixture of DNA from several people are present).

First claim

What is claimed is:

1. A method of estimating a fraction of fetal DNA in DNA obtained from a bodily fluid of a pregnant individual carrying a fetus, the method comprising:
(a) receiving a sample of the bodily fluid;
(b) extracting DNA from the sample under conditions that extract DNA of both a maternal genome and a fetal genome present in the bodily fluid;
(c) sequencing the extracted DNA with a nucleic acid sequencer under conditions that produce DNA sequence reads containing a plurality of polymorphisms;
(d) mapping the DNA sequence reads to a plurality of polymorphism sites on a reference sequence corresponding to the plurality of polymorphisms, wherein the mapping is performed using a computational apparatus programmed to map nucleic acid sequences to polymorphism sites of the reference sequence;
(e) determining allele frequencies of the mapped DNA sequence reads for the plurality of polymorphisms; and
(f) estimating the fraction of fetal DNA in the extracted DNA, wherein the estimating comprises performing a calculation or calculations using the allele frequencies determined in (e) for polymorphisms of two or more of the following zygosity cases: (i) the pregnant individual is homozygous and the fetus is homozygous, (ii) the pregnant individual is homozygous and the fetus is heterozygous, (iii) the pregnant individual is heterozygous and the fetus is homozygous, and (iv) the pregnant individual is heterozygous and the fetus is heterozygous;
wherein (e)-(f) are performed on one or more processors running under program instructions for the determining and estimating.

2. The method of claim 1 , wherein the allele frequencies for polymorphisms of two or more of the four zygosity cases in (f) comprise allele frequencies of polymorphisms in case (ii).

3. The method of claim 2 , wherein the estimating of (f) comprises combining data of case (ii) and (iii) to calculate the fraction of fetal DNA.

4. The method of claim 3 , wherein combining data of case (ii) and (iii) comprises transforming data of case (iii) to data of case (ii).

5. The method of claim 4 , wherein combining data of case (ii) and (iii) to calculate the fraction of fetal DNA comprises applying regression techniques to the data of case (ii) and the transformed data of case (iii).

6. The method of claim 5 , wherein the fraction of fetal DNA is estimated as twice the slope of a regression line of a linear regression model.

7. The method of claim 4 , wherein transforming data of case (iii) to data of case (ii) comprises transforming data (D, A) to (D1, A1) as:
A 1=0.5 D−A
D 1 =D
wherein D is the coverage of the polymorphism and A is the minor allele count of the polymorphism.

8. The method of claim 4 , wherein transforming data of case (iii) to data of case (ii) comprises trigonometric transformation or use of rotation matrices.

9. The method of claim 3 , wherein the fraction of fetal DNA is estimated as 2 A/D for case (ii) and as 1−2 A/D for case (iii), wherein A is the minor allele frequency and D is the coverage for the polymorphism in question.

10. The method of claim 1 , wherein the estimating of (f) further comprises filtering the plurality of polymorphisms to remove from consideration any polymorphism among the plurality of polymorphisms having a minor allele frequency of greater than a defined threshold.

11. The method of claim 1 , wherein the estimating of (f) further comprises filtering the plurality of polymorphisms to remove from consideration any polymorphism among the plurality of polymorphisms having a minor allele frequency of less than a defined threshold.

12. The method of claim 1 , further comprising applying a threshold to the allele frequencies determined in (e) before the estimating in (f).

13. The method of claim 1 , wherein the estimating of (f) further comprises applying the allele frequencies determined in (e) to a mixture model.

14. The method of claim 13 , wherein the mixture model employs factorial moments to combine data from two or more of the four zygosity cases to calculate the fraction of fetal DNA.

15. The method of claim 14 , wherein the factorial moments are calculated as:

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Assignees

• Rava Richard P
• Rhees Brian K
• Burke John P
• Verinata Health Inc

Inventors

• Rava Richard P
• Rhees Brian K
• Burke John P

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• G16B20/00

  ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• G16B45/00

  ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks · CPC title