Detecting and classifying copy number variation

Patent metadata

Field	Value
Publication number	US-9260745-B2
Application number	US-201213555037-A
Country	US
Kind code	B2
Filing date	Jul 20, 2012
Priority date	Jan 19, 2010
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.

First claim

What is claimed is:

1. A method for determining the presence or absence of a fetal chromosomal aneuploidy in a maternal test sample comprising fetal and maternal nucleic acids, said method comprising:
(a) providing sequence reads from fetal and maternal nucleic acids in the maternal test sample, wherein the sequence reads are provided in an electronic format;
(b) aligning the sequence reads to one or more chromosome reference sequences using a computing apparatus and thereby providing sequence tags corresponding to the sequence reads;
(c) computationally identifying a number of those sequence tags that are from one or more chromosomes of interest or chromosome segments of interest selected from chromosomes 1-22, X, and Y and segments thereof and computationally identifying a number of those sequence tags that are from at least one normalizing chromosome sequence or normalizing chromosome segment sequence for each of the one or more chromosomes of interest or chromosome segments of interest,
wherein the at least one normalizing chromosome sequence or normalizing chromosome segment sequence for a chromosome or segment of interest has been selected by (i) providing a plurality of unaffected samples lacking a fetal chromosomal aneuploidy of the chromosome or segment of interest and/or one or more affected samples having said fetal chromosomal aneuploidy; (ii) calculating a chromosome or segment dose for the chromosome or chromosome segment of interest for each of the samples provided in (i) using multiple different potential normalizing chromosome sequences or normalizing chromosome segment sequences; and (iii) selecting the normalizing chromosome sequence or normalizing chromosome segment sequence, alone or in a combination with one or more other potential normalizing chromosome sequences or normalizing chromosome segment sequences, giving chromosome or segment doses for the chromosome or segment of interest having: (1) the smallest variability among a plurality of unaffected samples, (2) the greatest differentiability between one or more unaffected samples and one or more affected samples, (3) the smallest variability and the greatest differentiability, or (4) an optimal combination of small variability and large differentiability;
(d) calculating, using said number of sequence tags identified for each of said one or more chromosomes of interest or chromosome segments of interest and said number of sequence tags identified for each said normalizing chromosome sequence or normalizing chromosome segment sequence, a single chromosome or segment dose for each of said one or more chromosomes of interest or chromosome segments of interest; and
(e) comparing, using said computing apparatus, each of said single chromosome doses for each of one or more chromosomes of interest or chromosome segments of interest to a corresponding threshold value for each of said one or more chromosomes of interest or chromosome segments of interest, and thereby determining the presence or absence of at least one fetal aneuploidy in said test sample.

2. The method of claim 1 , further comprising:
determining whether a read under consideration aligns to a site on a chromosome reference sequence where another read from the test sample previous aligned; and
determining whether to include the read under consideration in the number of sequence tags for a chromosome of interest or a chromosome segment of interest, wherein both determining operations are performed with the computing apparatus.

3. The method of claim 1 , further comprising storing in a computer readable medium, at least temporarily, sequence information for said fetal and maternal nucleic acids in said sample.

4. The method of claim 1 , wherein step (d) comprises calculating a chromosome or segment dose for a selected one of said chromosomes or segments of interest as the ratio of the number of sequence tags identified for said selected chromosome or segments of interest and the number of sequence tags identified for a corresponding at least one normalizing chromosome sequence or normalizing chromosome segment sequence for the selected chromosome or segment of interest.

5. The method of claim 1 , wherein said one or more chromosomes of interest or chromosome segments of interest selected from chromosomes 1-22, X, and Y comprise at least four chromosomes selected from the group consisting of chromosomes 1-22, X, and Y, and wherein the presence or absence of at least four different fetal chromosomal aneuploidies is determined.

6. The method of claim 5 , wherein said at least four chromosomes of interest selected from chromosomes 1-22, X, and Y is all of chromosomes 1-22, X, and Y, and wherein the presence or absence of complete fetal chromosomal aneuploidies of all of chromosomes 1-22, X, and Y is determined.

7. The method of claim 1 , wherein said at least one normalizing chromosome sequence is a group of chromosomes selected from the group consisting of chromosomes 1-22, X, and Y.

8. The method of claim 1 , further comprising calculating a normalized segment value (NSV), wherein said NSV relates said segment dose to the mean of the corresponding segment dose in a set of qualified samples as:

NSV
ij

=

x
ij

-

μ
^

j

σ
^

j

where {circumflex over (μ)} j and {circumflex over (σ)} j are the estimated mean and standard deviation, respectively, for the j-th segment dose in a set of qualified samples, and x ij is the segment dose calculated in (c) for the segment of interest.

9. The method of claim 1 , further comprising calculating a normalized chromosome value (NCV), wherein said NCV relates said chromosome dose to the mean of the corresponding chromosome dose in a set of qualified samples as:

NCV
ij

=

x
ij

-

μ
^

j

σ
^

j

where {circumflex over (μ)} j and {circumflex over (σ)} j are the estimated mean and standard deviation, respectively, for the j-th chromosome dose in a set of qualified samples, and x ij is the chromosome dose calculated in (c) for the chromosome of interest.

10. The method of claim 1 , wherein said normalizing segment sequence is a single segment of any one or more of chromosomes 1-22, X, and Y.

11. The method of claim 1 , wherein said normalizing segment sequence is a group of segments of any one or more of chromosomes 1-22, X, and Y.

12. The method of claim 1 , wherein said fetal chromosomal aneuploidy is selected from the group consisting of partial duplications, partial

Assignees

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

Inventors

• Rava Richard P
• Rhees Brian K

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title