AU2017272273B2 - Assay systems for determination of source contribution in a sample - Google Patents

Abstract

The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.

Description

ASSAY SYSTEMS FOR DETERMINATION OF SOURCE CONTRIBUTION IN A SAMPLE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of Australian Patent Application No.

2015201176, which in turn is a divisional of Australian Patent Application No.

2011285477, which in turn claims priority to U.S. Ser. No. 13/013,732,

filed January 25, 2011, which claims priority to U.S. Ser. No. 61/371,605, filed

August 6, 2010, each of which applications are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to assay systems for identifying copy number variation in mixed samples in a single assay.

BACKGROUND OF THE INVENTION

[0003] In the following discussion certain articles and methods will be described

for background and introductory purposes. Nothing contained herein is to be

construed as an "admission" of prior art. Applicant expressly reserves the right to

demonstrate, where appropriate, that the articles and methods referenced herein do

not constitute prior art under the applicable statutory provisions. Recent advances in

diagnostics have focused on less invasive mechanisms for determining disease risk,

presence and prognosis. Diagnostic processes for determining genetic anomalies

have become standard techniques for identifying specific diseases and disorders, as

well as providing valuable information on disease source and treatment options.

[0004] The identification of cell free nucleic acids in biological samples such as

blood and plasma allow less invasive techniques such as blood extraction to be used in making clinical decisions. For example, cell free DNA from malignant solid tumors has been found in the peripheral blood of cancer patients; individuals who have undergone transplantation have cell free DNA from the transplanted organ present in their bloodstream; and cell-free fetal

DNA and RNA have been found in the blood and plasma of pregnant women.

In addition, detection of nucleic acids from infectious organisms, such as

detection of viral load or genetic identification of specific strains of a viral or

bacterial pathogen, provides important diagnostic and prognostic indicators.

Cell free nucleic acids from a source separate from the patient's own normal

cells can thus provide important medical information, e.g., about treatment

options, diagnosis, prognosis and the like.

[0005] The sensitivity of such testing is often dependent upon the

identification of the amount of nucleic acid from the different sources, and in

particular identification of a low level of nucleic acid from one source in the

background of a higher level of nucleic acids from a second source. Detecting

the contribution of the minor nucleic acid species to cell free nucleic acids

present in the biological sample can provide accurate statistical interpretation

of the resulting data.

[0006] There is thus a need for processes for calculating copy number

variation (CNV) in one or more genomic regions in a biological sample using

information on contribution of nucleic acids in the sample. The present

invention addresses this need.

SUMMARY OF THE INVENTION

[0007] This Summary is provided to introduce a selection of concepts in a

simplified form that are further described below in the Detailed Description.

This Summary is not intended to identify key or essential features of the

claimed subject matter, nor is it intended to be used to limit the scope of the

claimed subject matter. Other features, details, utilities, and advantages of the

claimed subject matter will be apparent from the following written Detailed

Description including those aspects illustrated in the accompanying drawings

and defined in the appended claims.

[0008] The methods of the invention comprise a single assay system with the

ability to determine contribution of a major source and/or a minor source

within a sample from an individual and determining copy number information

for one or more genomic regions within a single source to determine a value

difference for the genomic region compared to the contribution of the source of

the loci in the mixed sample. The present invention utilizes a single assay

system that utilizes both non-polymorphic and polymorphic detection to

determine source contribution and copy number variations (CNVs) from a

single source within the mixed sample. Determination of contribution of the

major and/or minor source within the sample can provide information on the

presence of sufficient genetic material from both sources to allow adequate

identification of genomic regions for determination of CNV in the mixed

samples.

[0009] In one aspect, the assay system utilizes amplification and detection of

selected loci in a mixed sample from an individual to calculate source

contribution and to identify the copy number of one or more genomic regions.

In one specific aspect, the invention provides single assay systems with the

ability to determine the contribution of nucleic acids from a major and/or

minor source in the mixed sample and the presence or absence of CNVs at one or more genomic regions from a single source in a mixed sample. The assay system can specifically detect copy number of genomic regions present in two or more sources within the mixed sample. For determination of contribution, the selected loci from one source are distinguished from the selected loci of at least one other source in the mixed sample. For determination of copy number variation of a genomic region, the selected loci can but do not need to be distinguished as to source contribution, as copy number variation can be detected by comparison of the levels of two or more genomic regions within a mixed sample. Preferably, the cell free nucleic acids analyzed in the assay system are cell free DNA (cfDNA).

[00010] Thus in a first implementation, the invention provides a single assay

system for 1) determining the contribution of a major source and/or a minor

source in a mixed sample using frequency data derived from two or more

informative loci; 2) determining the frequency of one or more genomic

regions in the major and minor source; and 3) identifying the presence or

absence of a CNV for one or more genomic regions in the major and/or minor

source. Preferably, the CNV identification is based on the comparison of the

copy number of two or more genomic regions from the major and/or minor

source in the mixed sample.

[00011] Preferably, the nucleic acids analyzed using the systems of the invention

are cell free nucleic acids. More preferably, the nucleic acids analyzed in the

assay system comprise cell free DNA (cfDNA).

[00012] In another specific aspect, the invention provides an assay system for

calculation of source contribution and detection of the presence or absence of

CNVs in one or more genomic regions within a mixed sample, the assay comprising the steps of introducing a first set of fixed sequence oligonucleotides to a mixed sample under conditions that allow the fixed oligonucleotides to specifically hybridize to complementary regions on one or more loci in or associated with a genomic region; introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed oligonucleotides to specifically hybridize to complementary regions on two or more informative loci; ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the selected loci; amplifying the contiguous ligation products to create amplification products; and detecting the amplification products. The detection of the amplification products is used to calculate source contribution and the copy number of one or more genomic regions in the mixed sample.

[00013] The sets of fixed oligonucleotides comprise two or more

oligonucleotides that hybridize to contiguous regions of the genomic regions

or the informative loci. In some preferred aspects, sets of loci are interrogated

for CNV, and are indicative of an amplification of a larger genomic region,

e.g., all or part of a chromosome. Preferably, the assay systems can

distinguish the copy number of these loci between a major source and a minor

source within a mixed sample. Levels of selected loci can be determined for a

genomic region of interest and compared to the quantities of loci of one or

more other genomic regions of interest and/or one or more reference genomic

regions to detect potential CNVs based on loci frequencies in the mixed

sample.

[00014] Detection of chromosomal abnormality in a sample can be based on

detection of CNV for multiple selected loci located on or associated with a single chromosome from a minor and/or major source. Thus, in another specific aspect, the invention provides a single assay system for 1) determining the contribution of a major source and/or a minor source in a mixed sample using frequency data derived from two or more informative loci; 2) determining the frequency of one or more genomic regions in the major and minor source; and 3) identifying the presence or absence of a chromosomal abnormality in the major and/or minor source in the mixed sample.

[00015] Thus, the invention provides an assay system for calculation of source

contribution and detection of the presence or absence of a chromosomal

abnormality in a mixed sample using a single assay, the assay comprising the

steps of: introducing a first set of fixed sequence oligonucleotides to a mixed

sample under conditions that allow the fixed sequence oligonucleotides to

specifically hybridize to complementary regions on two or more loci

corresponding to a first chromosome; introducing a second set of fixed

sequence oligonucleotides to the mixed sample under conditions that allow the

fixed oligonucleotides to specifically hybridize to complementary regions on

two or more loci corresponding to a second chromosome; introducing a third

set of fixed sequence oligonucleotides to the mixed sample under conditions

that allow the fixed oligonucleotides to specifically hybridize to

complementary regions on two or more informative loci; ligating the

hybridized oligonucleotides to create contiguous ligation products

complementary to the nucleic acids; amplifying the contiguous ligation

products to create amplification products; and detecting the amplification

products. The detection of amplification products can be used for calculation of source contribution as well as identification of chromosomal abnormalities in the mixed sample.

[00016] In a more specific aspect the assay systems of the invention are used to

identify percent fetal contribution and chromosomal abnormalities in a

maternal sample. The invention provides an assay system comprising the

steps of: introducing a first set of fixed sequence oligonucleotides to a

maternal sample under conditions that allow the fixed oligonucleotides to

specifically hybridize to complementary regions on two or more loci

corresponding to a first chromosome; introducing a second set of fixed

sequence oligonucleotides to the maternal sample under conditions that allow

the fixed oligonucleotides to specifically hybridize to complementary regions

on two or more loci corresponding to a second chromosome; introducing a

third set of fixed sequence oligonucleotides to the maternal sample under

conditions that allow the fixed oligonucleotides to specifically hybridize to

complementary regions on two or more informative loci; ligating the

hybridized oligonucleotides to create contiguous ligation products

complementary to the nucleic acids; amplifying the contiguous ligation

products to create amplification products; and detecting the amplification

products. The detection of amplification products can be used for calculation

of fetal contribution as well as identification of chromosomal abnormalities in

the fetal nucleic acids in the maternal sample.

[00017] In certain aspects of the invention, the fixed oligonucleotides are

hybridized immediately adjacent in the contiguous region, so that they are

ligated directly during the ligation step of the assay. In other aspects,

however, there may be a gap of one or more nucleotides between the ends of the fixed oligonucleotides following hybridization in the contiguous region.

The fixed oligonucleotides are joined by a combination of, e.g., primer

extension using a polymerase and ligation.

[00018] Each set of fixed sequence nucleic acids is designed to hybridize to at

least two separate regions in a selected locus. In preferred aspects, two or

more separate oligos are used in a set to hybridize to these regions to provide

adjacent nucleic acids complementary to the selected loci. In some aspects,

however, a set can comprise a single probe with two or more distinct non

adjacent regions that are complementary to the selected loci (e.g., padlock

probes), as described in more detail herein. The sets of fixed sequence oligos

can be provided in the assay sequentially or simultaneously in the assay.

[00019] In certain preferred aspects, bridging oligos are used to increase

specificity of the oligo sets and / or fill a gap between fixed sequence

oligonucleotides. Accordingly, another specific aspect of the invention

provides an assay system for calculation of source contribution and detection

of the presence or absence of CNVs in one or more genomic regions within a

mixed sample, the assay comprising the steps of introducing a first set of fixed

sequence oligonucleotides to a mixed sample under conditions that allow the

fixed oligonucleotides to specifically hybridize to complementary regions on

one or more loci in or associated with a genomic region; introducing a second

set of fixed sequence oligonucleotides to the mixed sample under conditions

that allow the fixed oligonucleotides to specifically hybridize to

complementary regions on at least one informative loci; introducing one or

more bridging oligonucleotides under conditions that allow the bridging

oligonucleotides to specifically hybridize to complementary regions in the loci, wherein one or more bridging oligonucleotides are complementary to a region of the loci between and immediately adjacent to the regions complementary to the fixed sequence oligonucleotides of each set; ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the nucleic acids; amplifying the contiguous ligation products to create amplification products; and detecting the amplification products. Detection of the amplification products is used to calculate source contribution and the copy number of one or more genomic regions in the mixed sample.

[00020] Another specific aspect of the invention provides an assay system using

bridging oligonucleotides to calculate source contribution and identify

chromosomal abnormalities in a mixed sample. This assay comprises the steps

of: introducing a first set of fixed sequence oligonucleotides to a mixed sample

under conditions that allow the fixed oligonucleotides to specifically hybridize

to complementary regions on two or more loci corresponding to a first

chromosome; introducing a second set of fixed sequence oligonucleotides to

the mixed sample under conditions that allow the fixed oligonucleotides to

specifically hybridize to complementary regions on two or more loci

corresponding to a second chromosome; introducing a third set of fixed

sequence oligonucleotides to the mixed sample under conditions that allow the

fixed oligonucleotides to specifically hybridize to complementary regions on

two or more informative loci; introducing one or more bridging

oligonucleotides under conditions that allow the bridging oligonucleotides to

specifically hybridize to complementary regions in the loci, wherein one or

more bridging oligonucleotides are complementary to a region of the loci between and immediately adjacent to the regions complementary to the fixed sequence oligonucleotides of each set; introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the loci, wherein one or more bridging oligonucleotides are complementary to a region of the loci between and immediately adjacent to the regions complementary to the fixed sequence oligonucleotides of each set; ligating the hybridized oligonucleotides to create contiguous ligation products complementary to the nucleic acids; amplifying the contiguous ligation products to create amplification products; and detecting the amplification products. The detection of amplification products can be used for calculation of source contribution (such as fetal contribution in a maternal sample), as well as identification of chromosomal abnormalities in the mixed sample.

[00021] In certain aspects of the invention, the fixed oligonucleotides are

hybridized immediately adjacent to the bridging oligos, so that they are all

ligated directly during the ligation step of the assay. In other aspects,

however, there may be a gap of one or more nucleotides between the end of

one or both of the fixed oligonucleotides and the bridging oligo following

hybridization of the bridging oligo. The fixed oligonucleotides and the

bridging oligo are joined by a combination of, e.g., primer extension using a

polymerase and ligation.

[00022] It is an important feature that the multiplexed assays of the invention

allow the analysis of 5 or more, preferably 10 or more, preferably 16 or more,

preferably 20 or more, preferably 30 or more, preferably 32 or more,

preferably 40 or more, preferably 48 or more, preferably 50 or more, preferably 60 or more, preferably 70 or more, preferably 80 or more, preferably 90 or more, and more preferably 96 or more selected loci simultaneously. These selected loci may be different loci from a single sample, or they may be loci from two or more mixed samples. In the latter case, at least one of the two fixed sequence oligonucleotides used for analysis of a selected locus will comprise a sample identifier (e.g., a "sample index") that will allow the locus to be associated with a particular sample.

Alternatively, a sample index may be added during amplification of the

ligation product by using a primer comprising the sample index.

[00023] In preferred aspects, the interrogation of these loci utilizes universal

amplification techniques that allow amplification of multiple loci in a single

amplification reaction. The selected nucleic acids for contribution calculation

and detection of CNV and/or chromosomal abnormalities in the assay system

of the invention can be amplified using universal amplification methods

following the initial selective amplification from the mixed sample. The use

of universal amplification allows multiple nucleic acids regions from a single

or multiple samples to be amplified using a single or limited number of

amplification primers, and is especially useful in amplifying multiple selected

regions in a single reaction. In a preferred aspect, the universal primer regions

are used in sequence determination of the amplification products. In another

preferred aspect, the same universal primer regions are used in the fixed

sequence oligonucleotides used for detection of genomic regions and the fixed

sequence oligonucleotides used for detection of polymorphisms.

[00024] Thus, in a specific aspect of the invention, sequences complementary to

primers for use in universal amplification are introduced to the selected loci during or following selective amplification. Preferably such sequences are introduced to the ends of such selected nucleic acids, although they may be introduced in any location that allows identification of the amplification product from the universal amplification procedure.

[00025] In certain preferred aspects, one or both of the primers used comprise a

sample index or other identifier. In a specific aspect, a sample index is

included in one or more of the universal primers. The sample index is

incorporated into the amplification products, and amplification products from

different samples may then be combined. The sample index is detected

concurrently with the detection of the CNV or chromosomal abnormality and

the detection of polymorphism such that the CNV and polymorphism may be

properly assigned to the sample of origin.

[00026] In certain aspects, the assay system multiplexes loci interrogation using

one or more common bridging oligonucleotides that are complementary to

regions in two or more interrogated loci, i.e. a single bridging oligo can be

used for two or more fixed oligonucleotide sets. This allows the number of

bridging oligonucleotides used in the multiplexed assay system to be less than

the number of loci interrogated in the assay. In certain specific aspects, the

assay system uses a pool of bridging oligonucleotides that are each designed to

be compatible with two or more loci interrogated using the assays system of

the invention.

[00027] Frequencies of selected loci can be determined for a genomic region of

interest and compared to the frequencies of loci of one or more other genomic

regions of interest and/or one or more reference genomic regions to detect

potential CNVs based on loci frequencies in the mixed sample.

[00028] In some instances, the chromosomal abnormality detected using is

associated with gene amplification or loci expansion on a chromosome of

interest. In other instances, the chromosomal abnormality is associated with a

translocation resulting in the presence of an extra portion of a chromosome in

the genome. In yet other instances, the chromosomal abnormality is a

deletion.

[00029] In certain preferred aspects, the chromosomal abnormality is associated

with aneuploidy of a chromosome of interest. For example, the most common

chromosomal abnormalities in the fetus are trisomy 21, 18, 13, X and/or Y or

monosomy X. In specific preferred aspects, the assay systems of the invention

are used to detect such common chromosomal aneuploidies in the fetal DNA

of a maternal sample.

[00030] In the assay systems of the invention, the amplification products are

optionally isolated prior to detection. When isolated, they are preferably

isolated as individual molecules to assist in subsequent detection. Following

isolation, the amplification products can be further amplified to create

identical copies of all or a portion of the individual amplification products

prior to detection. Alternatively, the isolated amplification products can be

further amplified to create identical copies of molecules complementary to all

or a portion of the individual amplification products prior to detection.

[00031] Various methods of detection of CNVs can be employed in conjunction

with the detection of the polymorphisms in the assay systems of the invention.

In one general aspect, the assay system employs a method for determination of

a CNV in one or more loci in a mixed sample, comprising the steps of

amplifying one or more selected nucleic acids from a first genomic region of interest in a mixed sample; amplifying one or more selected nucleic acids from a second locus of interest in the mixed sample, determining the relative frequency of the selected loci, comparing the relative frequency of the selected loci, and identifying the presence or absence of a CNV based on the compared relative frequencies of the selected nucleic acids from the first and second loci.

Preferably, the assay method amplifies two or more selected loci from

different genomic regions, although the loci may be located in the same

general genomic region for confirmation of CNVs arising from chromosomal

abnormalities rather than CNVs from a single locus.

[00032] More preferably, the unhybridized fixed sequence oligonucleotides are

removed prior to introduction of the bridging oligonucleotides. In some

aspects, the bridging oligonucleotides are introduced simultaneously with the

ligation mixture. In other aspects, the hybridization products of the fixed

sequence oligonucleotides and the locus are isolated prior to introduction of the

bridging oligonucleotides.

[00033] In certain specific aspects, the assay system uses a pool of bridging

oligonucleotides that are each designed to be compatible with two or more loci

interrogated using the assay system of the invention. In these aspects, the

bridging oligonucleotides used in the multiplexed assay are preferably

designed to have a Tm in a range of±5C, more preferably in a range of±2C.

[00034] In certain aspects, the bridging oligonucleotides of are between 2-45

nucleotides in length. In a specific aspect, the bridging oligonucleotides are

between 3-9 nucleotides in length. In yet another specific aspect, the

oligonucleotides are between 10-30 nucleotides in length.

[00035] The loci interrogated for CNV can in some instances be indicative of a

amplification of a larger genomic region, e.g., all or part of a chromosome.

Preferably, the assay systems can distinguish the copy number of these loci

between a major source and a minor source within a mixed sample.

[00036] In another aspect, the present invention utilizes techniques that allow the

identification of both CNVs and infectious agents in a mixed sample. This

may be especially helpful to monitor patients in which the clinical outcome

may be compromised by the presence of an infectious agent. For example, a

patient that has undergone a transplant will likely be taking

immunosuppressant medication, and so more prone to infection in general.

Similarly, pregnant women have changes in their immune system and thus

may be more susceptible to infection with pathogens that may have an adverse

effect on the mother and/or fetus. Also, certain types of cancer are associated

with infectious agents (e.g., liver cancer associated with hepatitis B and C

infections, cervical cancer associated with human papilloma virus infection),

and identification of the infectious agents may be informative in predicting

clinical outcome or determining the preferred course of medical treatment for

the patient.

[00037] Thus, in certain aspects, the invention provides an assay system for

calculation of source contribution, detection of the presence or absence of

CNV of a genomic region, and the presence or absence of an infectious agent

in a mixed sample using a single assay, the assay comprising the steps of: of

introducing a first set of fixed sequence oligonucleotides to a mixed sample

under conditions that allow the fixed oligonucleotides to specifically hybridize

to complementary regions on one or more loci in or associated with a genomic region; introducing a second set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed oligonucleotides to specifically hybridize to complementary regions on at least one informative loci; introducing a third set of fixed sequence oligonucleotides to the mixed sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions on loci indicative of an infectious agent; ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the loci; amplifying the contiguous ligation product to create amplification products; and detecting the amplification products. The detection of the amplification products correlates to copy number of the genomic region and the presence or absence of an infectious agent in the mixed sample.

[00038] In another general aspect, the assay system employs a method for

determining the presence or absence of a chromosomal abnormality associated

with CNV in a genomic region, comprising the steps of amplifying one or

more selected loci from a first chromosome of interest in a mixed sample;

amplifying one or more selected loci from a second chromosome of interest in

the mixed sample, determining the relative frequency of the selected regions

from the first and second chromosomes of interest, comparing the relative

frequency of the selected regions from the first and second chromosomes of

interest, and identifying the presence or absence of an abnormality based on

the compared relative frequencies of the selected regions. Preferably, two or

more nucleic acids regions are selected from each chromosome, and more

preferably five or more loci are selected from each chromosome.

[00039] In yet another general aspect, the assay system employs a method for

determination of the presence or absence of an aneuploidy in a mixed sample

from an individual, comprising the steps of amplifying two or more selected

loci in the cfDNA corresponding to a first chromosome of interest in a mixed

sample; amplifying two or more selected loci in the cfDNA corresponding to a

second chromosome of interest in the mixed sample, determining the relative

frequency of the selected regions from the first and second chromosomes of

interest, comparing the relative frequency of the selected regions from the first

and second chromosomes of interest, and identifying the presence or absence

of an aneuploidy based on the compared relative frequencies of the selected

regions. In a specific aspect, the loci of the first and second chromosomes are

amplified in a single reaction, and preferably in a single reaction contained

within a single vessel.

[00040] Preferably, the assay system detects the presence or absence of loci in

samples that can be easily obtained from a subject, such as blood, plasma,

serum and the like. In one general aspect, the assay system utilizes detection

of selected regions in cfDNA in a mixed sample. In one more specific aspect,

the assay system utilizes detection of selected regions in cfDNA of a mixed

sample from an individual to identify the presence or absence of CNVs in a

genomic region and the presence or absence of a polymorphism in one or

more loci. Copy number within a genomic region can be determined based on

detection of quantities of selected loci and comparison to the quantities of

selected loci from another genomic region and/or to the quantities of selected

loci from a reference genomic region. In a particular aspect, the ratio of the

frequencies of the nucleic acid are compared to a reference mean ratio that has been determined for a statistically significant population of genetically

"normal" subjects, i.e. subjects that do not have a CNV associated with the

particular loci interrogated in the assay system.

[00041] In a preferred aspect of the invention, the amplification products

corresponding to the selected nucleic acids are isolated as individual

molecules for analysis of the selected loci. These individual amplification

products are isolated from one another, and preferably physically isolated

(e.g., on a substrate or in individual vessels). The individual molecules may

be further amplified following isolation to make multiple, identical copies of

the amplification product, a portion thereof, or a nucleic acid complementary

to the amplification product or a portion thereof.

[00042] In a preferred aspect, the individual amplification products are analyzed

through sequence determination. In other aspects, the individual amplification

products are analyzed using hybridization techniques.

[00043] It is a feature of the present invention that copy number of the selected

loci can be detected using non-polymorphic detection methods, i.e., detection

methods that are not dependent upon the presence or absence of a particular

polymorphism to identify the selected nucleic acid region. In a preferred

aspect, the assay detection systems utilize non-polymorphic detection methods

to "count" the relative numbers of selected loci present in a mixed sample.

These numbers can be utilized to determine if, statistically, a mixed sample is

likely to have a CNV in a genomic region in a major and/or minor source

within the mixed sample. Similarly, these numbers can be utilized to

determine, if statistically, nucleic acids from the major source and/or minor

source has one or more polymorphisms. Such information can be used to identify a particular pathology or genetic disorder, to confirm a diagnosis or recurrence of a disease or disorder, to determine the prognosis of a disease or disorder, to assist in determining potential treatment options, etc.

[00044] In some aspects, the methods for determination of aneuploidy used by

the assay system measure the copy number variation of multiple selected loci

from two or more chromosomes in a sample. The levels of the different

selected loci corresponding to specific chromosomes can be individually

quantified and compared to determine the presence or absence of a

chromosomal aneuploidy in one or more cell source in a mixed sample. The

individually quantified regions may undergo a normalization calculation or the

data may be subjected to outlier exclusion prior to comparison to determine

the presence or absence of an aneuploidy in a mixed sample.

[00045] In other aspects, the relative frequencies of the selected loci are used to

determine a chromosome frequency of the first and second chromosomes of

interest, and the presence or absence of an aneuploidy is based on the

compared chromosome frequencies of the first and second chromosomes of

interest.

[00046] In yet other aspects, the relative frequencies of the selected loci are used

to determine a chromosome frequency of a chromosome of interest and a

reference chromosome, and the presence or absence of an aneuploidy is based

on the compared chromosome frequencies of the chromosome of interest and

the reference chromosome.

[00047] As the assay system of the invention is preferably configured as a highly

multiplexed system, multiple loci from a single or multiple chromosomes

within an individual sample and/or multiple samples can be analyzed simultaneously. In such multiplexed systems, the samples can be analyzed separately, or they may be initially pooled into groups of two or more for analysis of larger numbers of samples. When pooled data is obtained, such data is preferably identified for the different samples prior to analysis of aneuploidy. In some aspects, however, the pooled data may be analyzed for potential CNVs, and individual samples from the group subsequently analyzed if initial results indicates that a potential aneuploidy is detected within the pooled group.

[00048] In certain aspects, the assay systems utilize one or more indices that

provide information on specific samples. For example, an index can be used

in selective or universal amplification that is indicative of a sample from

which the nucleic acid was amplified.

[00049] In one particular aspect, the selected loci are isolated prior to detection.

The selected loci can be isolated from the mixed sample using any means that

selectively isolate the particular nucleic acids present in the mixed sample for

analysis, e.g., hybridization, amplification or other form of sequence-based

isolation of the nucleic acids from the mixed sample. Following isolation, the

selected nucleic acids are individually distributed in a suitable detection

format, e.g., on a microarray or in a flow cell, for determination of the

sequence and/or relative quantities of each selected nucleic acid in the mixed

sample. The relative quantities of the detected nucleic acids are indicative of

the number of copies of chromosomes that correspond to the selected nucleic

acids present in the mixed sample.

[00050] Following isolation and distribution of the selected nucleic acids in a

suitable format, the selected sequences are identified, e.g., through sequence

determination of the selected sequence.

[00051] In one specific aspect, the invention provides an assay system for

detection of the presence or absence of a fetal aneuploidy, comprising the

steps of providing a mixed sample comprising maternal and fetal cfDNA,

amplifying two or more selected loci from a first and second chromosome of

interest in the mixed sample, amplifying two or more selected loci from the

first and second chromosome of interest in the mixed sample, determining the

relative frequency of the selected regions from the chromosomes of interest,

comparing the relative frequency of the selected loci from the first and second

chromosomes of interest, and identifying the presence or absence of a fetal

aneuploidy based on the compared relative frequencies of the selected loci.

[00052] In some specific aspects, the relative frequencies of the loci from a

genomic region are individually calculated, and the relative frequencies of the

individual loci are compared to determine the presence or absence of a

chromosomal abnormality. In other specific aspects, the relative frequencies of

the selected loci are used to determine a chromosome frequency of a first and

second chromosome of interest and a reference chromosome, and the copy

number variation for the chromosome or a genomic region of the chromosome

is based on the compared chromosome frequencies of the first and second

chromosomes of interest.

[00053] The mixed sample used for analysis can be obtained or derived from any

sample which contains the nucleic acid of interest to be analyzed using the

assay system of the invention. For example, a mixed sample may be from any maternal fluid which comprises both maternal and fetal cell free nucleic acids, including but not limited to maternal plasma, maternal serum, or maternal blood. A mixed sample from a transplant patient would be any fluid or tissue which contains cell free nucleic acids from both the donor cells and the cells of the patient. A mixed sample from a patient with a malignancy would contain cell free nucleic acids from the patient's normal, healthy tissue as well as cell free nucleic acids from the cancerous cells.

[00054] Although preferably the assay system is used to detect cfDNA in a

mixed sample, in certain aspects the DNA of interest to be analyzed using the

assay system of the invention comprises DNA directly from the different cell

types rather than from a mixed sample containing DNA from the major and

minor cell types. Such samples can be obtained from various sources

depending upon the target DNA. For example, fetal cells for analysis can be

derived from samples such as amniotic fluid, placenta (e.g., the chorionic

villi), and the like. Samples of donor organs can be obtained in an individual

by biopsy. Infectious organisms can be isolated directly from an individual

and analyzed following isolation. DNA can be extracted from cancerous cells

or tissues and used for analysis.

[00055] It is another feature of the invention that the substantial majority of the

nucleic acids isolated from the mixed sample and detected in the assay system

provide information relevant to the presence, quantity and/or polymorphic

nature of a particular locus in the mixed sample. This ensures that the

majority of nucleic acids analyzed in the assay system of the invention are

informative.

[00056] In some aspects, a set of multiple selected loci are interrogated for each

genomic region, and the quantity of the set of selected regions present in the

mixed sample are individually summed to determine the relative frequency of

a genomic region in a mixed sample. This includes determination of the

frequency of the locus for the combined maternal and fetal DNA present in the

mixed sample. Preferably, the determination does not require a distinction

between the DNA from separate sources, although in certain aspects this

information may be obtained in addition to the information of relative

frequencies in the sample as a whole.

[00057] In preferred aspects, selected nucleic acids corresponding to informative

loci are detected and summed to determine the relative frequency of a genomic

region in the mixed sample. Frequencies that are higher than expected for loci

from a first genomic region when compared to the loci from a second locus in

a mixed sample is indicative of a CNV of the first genomic region in the

mixed sample.

[00058] Comparison of genomic regions can be a comparison of part or all of a

chromosome. For example, the genomic region detected for CNV may be an

entire chromosome in the fetus (e.g., chromosomes 18 and 21), where the

likelihood of both being aneuploid is minimal. This can also be a comparison

of chromosomes where one is putatively aneuploid (e.g., chromosome 21) and

the other acts as a reference chromosome (e.g. an autosome such as

chromosome 2). In yet other aspects, the comparison may utilize two or more

chromosomes that are putatively aneuploid and one or more reference

chromosomes.

[00059] In one aspect, the assay system of the invention analyzes multiple

nucleic acids representing selected loci on chromosomes of interest, and the

relative frequency of each selected locus from the sample is analyzed to

determine a relative chromosome frequency for each particular chromosome

of interest in the sample. The chromosomal frequency of two or more

chromosomes or portions thereof is then compared to statistically determine

whether a chromosomal abnormality exists.

[00060] In another aspect, the assay system of the invention analyzes multiple

copies of a set of selected loci on chromosomes of interest, and the relative

frequency of each of the selected loci from the sample is analyzed and

independently quantified to determine a frquency for each selected locus in the

sample. The sum of the loci in the sample is compared to statistically

determine whether a CNV exists for one or more loci in a genomic region of

one source in a mixed sample.

[00061] In another aspect, subsets of loci on each chromosome are analyzed to

determine whether a chromosomal abnormality exists. The loci frequency can

be summed for a particular chromosome, and the summations of the loci used

to determine aneuploidy. This aspect of the invention sums the frequencies of

the individual loci in each genomic region and then compares the sum of the

loci on a genomic region of one chromosome against a genomic region of

another chromosome to determine whether a chromosomal abnormality exists.

The subsets of loci can be chosen randomly but with sufficient numbers of loci

to yield a statistically significant result in determining whether a chromosomal

abnormality exists. Multiple analyses of different subsets of loci can be

performed within a mixed sample to yield more statistical power. In another aspect, particular loci can be selected that are known to have less variation between samples, or by limiting the data used for determination of chromosomal frequency, e.g., by ignoring the data from loci with very high or very low frequency within a sample.

[00062] In a particular aspect, the measured quantities of one or more particular

loci are normalized to account for differences in loci quantity in the sample.

This can be done by normalizing for known variation from sources such as the

assay system (e.g., temperature, reagent lot differences), underlying biology of

the sample (e.g., nucleic acid content), operator differences, or any other

variables.

[00063] In certain specific aspects, determining the relative percentage of nucleic

acids from the minor source in a mixed sample may be beneficial in

performing the assay system, as it will provide important information on the

relative statistical presence of loci that may be indicative of copy number

variation within the minor source in that sample. Determining the loci

contributed to the mixed sample from the minor source can provide

information used to calculate the statistically significant differences in

frequencies for genomic regions of interest. Such loci could thus provide two

forms of information in the assay - allelic information can be used for

determining the percent minor cell contribution in a mixed sample and a

summation of the allelic information can be used to determine the relative

overall frequency of that locus in a mixed sample. The allelic information is

not needed to determine the relative overall frequency of that locus.

[00064] In another specific aspect, the assay system of the invention can be

utilized to determine potential mosaicism in a cell population, and whether further confirmatory tests should be undertaken to confirm the identification of mosaicism in the major and/or minor source. In certain instances, determination of the percent nucleic acids from the minor source in a mixed sample could assist in quantification of the estimated level of mosaicism.

Mosaicism could be subsequently confirmed using other testing methods that

could distinguish mosaic full or partial aneuploidy in specific cells or tissue.

[00065] In yet another specific aspect, the assay system of the invention can be

utilized to determine contamination in a sample, with the minor species

representing a contaminant species.

[00066] In another aspect, the oligonucleotides for a given selected nucleic acid

can be connected at the non-sequence specific ends such that a circular or

unimolecular probe may bind thereto. In this aspect, the 3' end and the 5' end

of the circular probe binds to the selected locus and at least one universal

amplification region is present in the non-selected specific sequence of the

circular probe.

[00067] It is an important feature of the assay that the amplification products

may be analyzed directly without the need for enrichment of polymorphic

regions from the initial mixed sample. Thus, the current invention allows

detection of both CNV and polymorphisms from a maternal sample without an

intervening polymorphic enrichment step prior to sequence determination of

the selected loci.

[00068] It is another important feature of the assay that both CNV and source

contribution are determined using a targeted approach of selected amplification

and detection. This allows the majority of information gathered in the assay to

be useful for the determination of the CNV and/or source contribution, and obviates the need to generate sequence reads that must be aligned with a reference sequence.

[00069] These and other aspects, features and advantages will be provided in

more detail as described herein.

BRIEF DESCRIPTION OF THE FIGURES

[00070] HG. 1 is a simplified flow chart of the general steps utilized in the assay

systems of the invention.

[00071] HG. 2 illustrates a first general schematic for a ligation-based assay

system of the invention.

[00072] HG. 3 illustrates a second general schematic for a ligation-based assay

system of the invention.

[00073] FIG. 4 is a third general schematic for a ligation-based assay system of

the invention.

[00074] HG. 5 illustrates the genotyping performance that was obtained using

one assay system of the invention.

[00075] HG. 6 is a graph illustrating results from a determination of percent fetal

using an assay of the invention.

[00076] HG. 7 illustrates the elements used for a detection of aneuploidy and

polymorphism for two cohorts of maternal samples.

[00077] HG. 8 is a summary of patient and sample information and data for a

subset of a second cohort of pregnant subjects.

[00078] HG. 9 illustrates the chromosome 21 aneuploidy detection achieved

using one aspect of the invention for a first cohort.

[00079] HG. 10 illustrates the chromosome 18 aneuploidy detection achieved

using one aspect of the invention for a first cohort.

[00080] HG. 11 illustrates the chromosome 21 aneuploidy detection achieved

using one aspect of the invention for a second cohort.

[00081] HG. 12 illustrates the chromosome 18 aneuploidy detection achieved

using one aspect of the invention for a second cohort.

DEFINITIONS

[00082] The terms used herein are intended to have the plain and ordinary

meaning as understood by those of ordinary skill in the art. The following

definitions are intended to aid the reader in understanding the present

invention, but are not intended to vary or otherwise limit the meaning of such

terms unless specifically indicated.

[00083] The term "amplified nucleic acid" is any nucleic acid molecule whose

amount has been increased at least two fold by any nucleic acid amplification

or replication method performed in vitro as compared to its starting amount in a

mixed sample.

[00084] The term "amplification product" as used herein refers to the product

resulting from an amplification reaction using the contiguous ligation product

as a template, or the product resulting from an amplification reaction using a

molecule complementary to the contiguous ligation product as a template.

[00085] The term "chromosomal abnormality" refers to any genetic variation

that affects all or part of a chromosome larger than a single locus. The genetic

variants may include but not be limited to any CNV such as amplifications or

deletions, translocations, inversions, and mutations. Examples of chromosomal

abnormalities include, but are not limited to, Down Syndrome (Trisomy 21),

Edwards Syndrome (Trisomy 18), Patau Syndrome (Trisomy 13), Klinefelter's

Syndrome (XXY), Triple X syndrome, XYY syndrome, Trisomy 8, Trisomy

16, Turner Syndrome, Robertsonian translocation, DiGeorge Syndrome and

Wolf-Hirschhorn Syndrome.

[00086] The terms "complementary" or "complementarity" are used in reference

to nucleic acid molecules (i.e., a sequence of nucleotides) that are related by

base-pairing rules. Complementary nucleotides are, generally, A and T (or A

and U), or C and G. Two single stranded RNA or DNA molecules are said to

be substantially complementary when the nucleotides of one strand, optimally

aligned and with appropriate nucleotide insertions or deletions, pair with at

least about 90% to about 95% complementarity, and more preferably from

about 98% to about 100% complementarity, and even more preferably with

100% complementarity. Alternatively, substantial complementarity exists

when an RNA or DNA strand will hybridize under selective hybridization

conditions to its complement. Selective hybridization conditions include, but

are not limited to, stringent hybridization conditions. Stringent hybridization

conditions will typically include salt concentrations of less than about 1 M,

more usually less than about 500 mM and preferably less than about 200 mM.

Hybridization temperatures are generally at least about 2°C to about 6°C lower

than melting temperatures (Tm).

[00087] The term "copy number variation" or "CNV" as used interchangeably

herein are alterations of the DNA of a genome that results in a cell having an

abnormal number of copies of one or more loci in the DNA. CNVs that are

clinically relevant can be limited to a single gene or include a contiguous set of

genes. A CNV can also correspond to relatively large regions of the genome

that have been deleted, inverted or duplicated on certain chromosomes, up to an including one or more additional copies of a complete chromosome. The term

CNV as used herein does not refer to any sequence-related information, but

rather to quantity or "counts" of genetic regions present in a sample.

[00088] The term "correction index" refers to an index that may contain

additional nucleotides that allow for identification and correction of

amplification, sequencing or other experimental errors including the detection

of deletion, substitution, or insertion of one or more bases during sequencing as

well as nucleotide changes that may occur outside of sequencing such as oligo

synthesis, amplification, and any other aspect of the assay. These correction

indices may be stand-alone indices that are separate sequences, or they may be

embedded within other regions to assist in confirming accuracy of the

experimental techniques used, e.g., a correction index may be a subset of

sequences used for universal amplification or a subset of nucleotides of a

sample locus.

[00089] The term "diagnostic tool" as used herein refers to any composition or

assay of the invention used in combination as, for example, in a system in order

to carry out a diagnostic test or assay on a patient sample.

[00090] The term "disease trait" refers to a monogenic or polygenic trait

associated with a pathological condition, e.g., a disease, disorder, syndrome or

predisposition.

[00091] The term "genomic region" as used herein refers to any region of one

or more loci that are normally found in a contiguous fashion in a genome. A

genomic region may vary in size up to and including an entire chromosome.

[00092] The term "hybridization" generally means the reaction by which the

pairing of complementary strands of nucleic acid occurs. DNA is usually

double-stranded, and when the strands are separated they will re-hybridize

under the appropriate conditions. Hybrids can form between DNA-DNA,

DNA-RNA or RNA-RNA. They can form between a short strand and a long

strand containing a region complementary to the short one. Imperfect hybrids

can also form, but the more imperfect they are, the less stable they will be (and

the less likely to form).

[00093] The term "informative locus" as used herein refers to a locus that is

homozygous for one cell source and heterozygous for a second cell source on a

particular chromosome or portion of a chromosome interrogated for purposes

of determining a CNV of all or part of that chromosome. Informative loci for

use in the assay system of the invention include loci used for interrogation of a

reference chromosome as well as loci used for interrogation of a chromosome

that is putatively aneuploid in a cell source. Informative loci can also

distinguish copy number of loci in cell sources from different individuals

within a single individual (e.g., detection of transplant donor cells in a

transplant recipient or detection of a fetal DNA within a maternal mixed

sample).

[00094] The terms "locus" and "loci" as used herein refer to a locus of known

location in a genome.

[00095] The term "major source" refers to a source of nucleic acids in a sample

from an individual that is representative of the predominant genomic material

in that individual.

[00096] The term "maternal sample" as used herein refers to any sample taken

from a pregnant mammal which comprises both fetal and maternal cell free

genomic material (e.g., DNA). Preferably, maternal samples for use in the

invention are obtained through relatively non-invasive means, e.g., phlebotomy

or other standard techniques for extracting peripheral samples from a subject.

[00097] The term "melting temperature" or Tm is commonly defined as the

temperature at which a population of double-stranded nucleic acid molecules

becomes half dissociated into single strands. The equation for calculating the

Tm of nucleic acids is well known in the art. As indicated by standard

references, a simple estimate of the Tm value may be calculated by the

equation: Tm = 81.5+16.6(logO[Na+])0.41(%[G+C])-675/n-1.0m, when a

nucleic acid is in aqueous solution having cation concentrations of 0.5 M or

less, the (G+C) content is between 30% and 70%, n is the number of bases, and

m is the percentage of base pair mismatches (see, e.g., Sambrook J et al.,

Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor

Laboratory Press (2001)). Other references include more sophisticated

computations, which take structural as well as sequence characteristics into

account for the calculation of Tm.

[00098] "Microarray" or "array" refers to a solid phase support having a surface,

preferably but not exclusively a planar or substantially planar surface, which

carries an array of sites containing nucleic acids such that each site of the array

comprises substantially identical or identical copies of oligonucleotides or

polynucleotides and is spatially defined and not overlapping with other member

sites of the array; that is, the sites are spatially discrete. The array or microarray

can also comprise a non-planar interrogatable structure with a surface such as a bead or a well. The oligonucleotides or polynucleotides of the array may be covalently bound to the solid support, or may be non-covalently bound.

Conventional microarray technology is reviewed in, e.g., Schena, Ed.,

Microarrays: A Practical Approach, IRL Press, Oxford (2000). "Array

analysis", "analysis by array" or "analysis by microarray" refers to analysis,

such as, e.g., sequence analysis, of one or more biological molecules using a

microarray.

[00099] The term "minor source" refers to a source of nucleic acids within an

individual that is present in limited amounts and which is distinguishable from

the major source due to differences in its genomic makeup and/or expression.

Examples of minor sources include, but are not limited to, fetal cells in a

pregnant female, cancerous cells in a patient with a malignancy, cells from a

donor organ in a transplant patient, nucleic acids from an infectious organism

in an infected host, and the like.

[000100] The term "mixed sample" as used herein refers to any sample

comprising cell free genomic material (e.g., DNA) from two or more cell types

of interest, one being a major source and the other being a minor source within

a single individual. Exemplary mixed samples include a maternal sample (e.g.,

maternal blood, serum or plasma comprising both maternal and fetal DNA),

and a peripherally-derived somatic sample (e.g., blood, serum or plasma

comprising different cell types, e.g., hematopoietic cells, mesenchymal cells,

and circulating cells from other organ systems). Mixed samples include

samples with genomic material from both a major and a minor source in an

individual, which may be e.g., normal and atypical somatic cells, or cells that

comprise genomes from two different individuals, e.g., a sample with both maternal and fetal genomic material or a sample from a transplant patient that comprises cells from both the donor and recipient.

[000101] The term "monogenic trait" as used herein refers to any trait, normal or

pathological, that is associated with a mutation or polymorphism in a single

gene. Such traits include traits associated with a disease, disorder, or

predisposition caused by a dysfunction in a single gene. Traits also include

non-pathological characteristics (e.g., presence or absence of cell surface

molecules on a specific cell type).

[000102] The term "non-maternal" allele means an allele with a polymorphism

and/or mutation that is found in a fetal allele (e.g., an allele with a de novo SNP

or mutation) and/or a paternal allele, but which is not found in the maternal

allele.

[000103] By "non-polymorphic", when used with respect to detection of selected

loci, is meant a detection of such locus, which may contain one or more

polymorphisms, but in which the detection is not reliant on detection of the

specific polymorphism within the region. Thus a selected locus may contain a

polymorphism, but detection of the region using the assay system of the

invention is based on occurrence of the region rather than the presence or

absence of a particular polymorphism in that region.

[000104] As used herein "nucleotide" refers to a base-sugar-phosphate

combination. Nucleotides are monomeric units of a nucleic acid sequence

(DNA and RNA). The term nucleotide includes ribonucleoside triphosphates

ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,

dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

[000105] According to the present invention, a "nucleotide" may be unlabeled or

detectably labeled by well known techniques. Fluorescent labels and their

attachment to oligonucleotides are described in many reviews, including

Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Ed.,

Molecular Probes, Inc., Eugene OR (2002); Keller and Manak, DNA Probes,

2nd Ed., Stockton Press, New York (1993); Eckstein, Ed., Oligonucleotides

and Analogues: A Practical Approach, IRL Press, Oxford (1991); Wetmur,

Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991);

and the like. Other methodologies applicable to the invention are disclosed in

the following sample of references: Fung et al., U.S. Pat. No. 4,757,141;

Hobbs, Jr., et al., U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No.

5,091,519; Menchen et al., U.S. Pat. No. 5,188,934; Begot et al., U.S. Pat. No.

5,366,860; Lee et al., U.S. Pat. No. 5,847,162; Khanna et al., U.S. Pat. No.

4,318,846; Lee et al., U.S. Pat. No. 5,800,996; Lee et al., U.S. Pat. No.

5,066,580: Mathies et al., U.S. Pat. No. 5,688,648; and the like. Labeling can

also be carried out with quantum dots, as disclosed in the following patents and

patent publications: U.S. Pat. Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303;

6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002/0045045; and

2003/0017264. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6 carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N' tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX),

4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue,

Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-1

sulfonic acid (EDANS). Specific examples of fluroescently labeled nucleotides

include [R6G]dUTP, [TAMRA]dUTP, [Ri1O]dCTP, [R6G]dCTP,

[TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [Ri1O]ddCTP,

[TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRI10]ddCTP,

[dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster

City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink

Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, and FluoroLink

Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein-15

dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP,

Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2'-dATP

available from Boehringer Mannheim, Indianapolis, IN; and Chromosomee

Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY

TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY

TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12

UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5

UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,

tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and

Texas Red-12-dUTP available from Molecular Probes, Eugene, OR.

[000106] The terms "oligonucleotides" or "oligos" as used herein refer to linear

oligomers of natural or modified nucleic acid monomers, including

deoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptide nucleic

acid monomers (PNAs), locked nucleotide acid monomers (LNA), and the like,

or a combination thereof, capable of specifically binding to a single-stranded

polynucleotide by way of a regular pattern of monomer-to-monomer

interactions, such as Watson-Crick type of base pairing, base stacking,

Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually

monomers are linked by phosphodiester bonds or analogs thereof to form

oligonucleotides ranging in size from a few monomeric units, e.g., 8-12, to

several tens of monomeric units, e.g., 100-200 or more. Suitable nucleic acid

molecules may be prepared by the phosphoramidite method described by

Beaucage and Carruthers (Tetrahedron Lett., 22:1859-1862 (1981)), or by the

triester method according to Matteucci, et al. (J. Am. Chem. Soc., 103:3185

(1981)), both incorporated herein by reference, or by other chemical methods

such as using a commercial automated oligonucleotide synthesizer.

[000107] The term "polygenic trait" as used herein refers to any trait, normal or

pathological, that is associated with a mutation or polymorphism in more than a

single gene. Such traits include traits associated with a disease, disorder,

syndrome or predisposition caused by a dysfunction in two or more genes.

Traits also include non-pathological characteristics associated with the

interaction of two or more genes.

[000108] As used herein the term "polymerase" refers to an enzyme that links

individual nucleotides together into a long strand, using another strand as a

template. There are two general types of polymerase-DNA polymerases, which synthesize DNA, and RNA polymerases, which synthesize RNA.

Within these two classes, there are numerous sub-types of polymerases,

depending on what type of nucleic acid can function as template and what type

of nucleic acid is formed.

[000109] As used herein "polymerase chain reaction" or "PCR" refers to a

technique for replicating a specific piece of selected DNA in vitro, even in the

presence of excess non-specific DNA. Primers are added to the selected DNA,

where the primers initiate the copying of the selected DNA using nucleotides

and, typically, Taq polymerase or the like. By cycling the temperature, the

selected DNA is repetitively denatured and copied. A single copy of the

selected DNA, even if mixed in with other, random DNA, can be amplified to

obtain billions of replicates. The polymerase chain reaction can be used to

detect and measure very small amounts of DNA and to create customized

pieces of DNA. In some instances, linear amplification methods may be used

as an alternative to PCR.

[000110] The term "polymorphism" as used herein refers to any genetic changes

or sequence variants in a locus, including but not limited to single nucleotide

polymorphisms (SNPs), methylation differences, short tandem repeats (STRs),

single gene polymorphisms, point mutations, trinucleotide repeats, indels and

the like.

[000111] Generally, a "primer" is an oligonucleotide used to, e.g., prime DNA

extension, ligation and/or synthesis, such as in the synthesis step of the

polymerase chain reaction or in the primer extension techniques used in certain

sequencing reactions. A primer may also be used in hybridization techniques as a means to provide complementarity of a locus to a capture oligonucleotide for detection of a specific locus.

[000112] The term "research tool" as used herein refers to any composition or

assay of the invention used for scientific enquiry, academic or commercial in

nature, including the development of pharmaceutical and/or biological

therapeutics. The research tools of the invention are not intended to be

therapeutic or to be subject to regulatory approval; rather, the research tools of

the invention are intended to facilitate research and aid in such development

activities, including any activities performed with the intention to produce

information to support a regulatory submission.

[000113] The term "sample index" refers generally to a series of unique

nucleotides (i.e., each sample index is unique to a sample in a multiplexed

assay system for analysis of multiple samples). The sample index can thus be

used to assist in locus identification for multiplexing of different samples in a

single reaction vessel, such that each sample can be identified based on its

sample index. In a preferred aspect, there is a unique sample index for each

sample in a set of samples, and the samples are pooled during sequencing. For

example, if twelve samples are pooled into a single sequencing reaction, there

are at least twelve unique sample indexes such that each sample is labeled

uniquely.

[000114] The term "selected locus" as used herein refers to a locus corresponding

to a loci interrogated, e.g., for copy number, the presence or absence of one or

more polymorphism, presence or absence of an infectious organism, etc. Such

selected loci may be directly isolated and amplified from the sample for

detection, e.g., based on hybridization and/or other sequence-based techniques, or they may be amplified using the sample as a template prior to detection of the sequence. Nucleic acids regions for use in the assay systems of the present invention may be selected on the basis of DNA level variation between individuals, based upon specificity for a particular chromosome, based on CG content and/or required amplification conditions of the selected loci, or other characteristics that will be apparent to one skilled in the art upon reading the present disclosure.

[000115] The terms "sequencing", "sequence determination" and the like as used

herein refers generally to any and all biochemical methods that may be used to

determine the order of nucleotide bases in a nucleic acid.

[000116] The term "source contribution" as used herein refers to the relative

contribution of two or more sources of nucleic acids within an individual. The

contribution from a single is generally determined as a percent of the nucleic

aciods from a sample, although any relative measurement can be used.

[000117] The term "specifically binds", "specific binding" and the like as used

herein, when referring to a binding partner (e.g., a nucleic acid probe or primer,

antibody, etc.) that results in the generation of a statistically significant positive

signal under the designated assay conditions. Typically the interaction will

subsequently result in a detectable signal that is at least twice the standard

deviation of any signal generated as a result of undesired interactions

(background).

[000118] The term "status" as used herein in relationship to a gene refers to the

sequence status of the alleles of a particular gene, including the coding regions

and the non-coding regions that affect the translation and/or protein expression

from that gene. The status of a gene associated with an autosomal dominant disease such as achondroplasia (e.g., the gene encoding the fibroblast growth factor receptor) or Huntington's disease (e.g., the Huntingtin gene), or for an

X-linked disease in the case of a male fetus, can be classified as affected i.e.,

one allele possesses mutation(s) that is causative of the diseases or disorder, or

non-affected, i.e. both alleles lack such mutations(s). The status of a gene

associated with an autosomal recessive disease or a maternal gene associated

with an X-linked recessive disorder, may be classified as affected, i.e., both

alleles possess mutation(s) causative of the diseases or disorder; carrier, i.e. one

allele possesses mutation(s) causative of the diseases or disorder; or non

affected, i.e. both alleles lack such mutations(s). The status of a gene may also

indicate the presence or absence of a particular allele associated with a risk of

developing a polygenic disease, e.g., a polymorphism that is protective against

a particular disease or disorder or a polymorphism associated with an enhanced

risk for a particular disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

[000119] The assay systems and methods described herein may employ, unless

otherwise indicated, conventional techniques and descriptions of molecular

biology (including recombinant techniques), cell biology, biochemistry,

microarray and sequencing technology, which are within the skill of those who

practice in the art. Such conventional techniques include polymer array

synthesis, hybridization and ligation of oligonucleotides, sequencing of

oligonucleotides, and detection of hybridization using a label. Specific

illustrations of suitable techniques can be had by reference to the examples

herein. However, equivalent conventional procedures can, of course, also be

used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds., Genome Analysis: A Laboratory

Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds., Genetic Variation: A

Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A

Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A

Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and

Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from

Molecular Cloning: A LaboratoryManual (2006); and Sambrook and Russell,

Molecular Cloning: A LaboratoryManual (2002) (all from Cold Spring Harbor

Laboratory Press); Stryer, L., Biochemistry (4th Ed.) W.H. Freeman, New York

(1995); Gait, "Oligonucleotide Synthesis: A PracticalApproach" IRL Press,

London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd

Ed., W. H. Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5f

Ed., W.H. Freeman Pub., New York (2002), all of which are herein

incorporated by reference in their entirety for all purposes. Before the present

compositions, research tools and methods are described, it is to be understood

that this invention is not limited to the specific methods, compositions, targets

and uses described, as such may, of course, vary. It is also to be understood that

the terminology used herein is for the purpose of describing particular aspects

only and is not intended to limit the scope of the present invention, which will

be limited only by appended claims.

[000120] It should be noted that as used herein and in the appended claims, the

singular forms "a," "an," and "the" include plural referents unless the context

clearly dictates otherwise. Thus, for example, reference to "a locus" refers to

one, more than one, or mixtures of such regions, and reference to "an assay" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[000121] Where a range of values is provided, it is to be understood that each

intervening value between the upper and lower limit of that range-and any

other stated or intervening value in that stated range-is encompassed within the

invention. Where the stated range includes upper and lower limits, ranges

excluding either of those included limits are also included in the invention.

[000122] Unless expressly stated, the terms used herein are intended to have the

plain and ordinary meaning as understood by those of ordinary skill in the art.

The following definitions are intended to aid the reader in understanding the

present invention, but are not intended to vary or otherwise limit the meaning

of such terms unless specifically indicated. All publications mentioned herein

are incorporated by reference for the purpose of describing and disclosing the

formulations and methodologies that are described in the publication and which

might be used in connection with the presently described invention.

[000123] In the following description, numerous specific details are set forth to

provide a more thorough understanding of the present invention. However, it

will be apparent to one of skill in the art that the present invention may be

practiced without one or more of these specific details. In other instances,

well-known features and procedures well known to those skilled in the art have

not been described in order to avoid obscuring the invention.

The Invention in General

[000124] The present invention provides single assay systems with the ability to

detect copy number variations, polymorphisms and nucleic acids associated with disease states (for example, nucleic acids from pathogens, or associated with cancer, diabetes, Alzheimers Disease, and the like) in a mixed sample from a single individual. The assay allows the identification of genetic variation in one or more minor sources in a mixed sample using information about the copy number of selected loci in the sample and information about the percentage contribution of nucleic acids from the major and the one or more minor sources in the sample. These methods are useful for any mixed sample containing genomic material (e.g., DNA) from a major and a minor source that are present in a single individual.

[000125] The use of selected loci in the assay methods of the invention provides

direct detection of loci for determination of copy number variation in one or

more sources within the mixed sample. A distinct advantage of the invention is

that the selected loci corresponding to copy number variation and/or

polymorphisms can be further analyzed using a variety of detection and

quantification techniques, including but not limited to hybridization techniques,

digital PCR and high throughput sequencing determination techniques. Probes

can be designed against any number of selected loci for any chromosome.

Although amplification prior to the identification and quantification of the

selected loci is not mandatory, limited amplification of the mixed sample prior

to detection may be used to expand the overall number of nucleic acids within

the starting materials.

[000126] Figure 1 is a simplified flow chart of the general steps utilized in the

assay systems of the invention. Figure 1 shows method 100, where in a first

step 110, a mixed nucleic acid sample is provided for analysis. The mixed

sample can be prepared from virtually any sample as such techniques are known to those of skill in the art (see, e.g., Tietz Textbood of Clinical

Chemistry and Molecular Diagnostics, 4th Ed., Chapter 2, Burtis, C. Ashwood

E. and Bruns, D, eds. (2006); Chemical Weapons Convention Chemicals

Analysis: Sample Collection, Preparation and Analytical Methods, Mesilaakso,

M., ed., (2005); Pawliszyn, J., Sampling and Sample Preparation for Field and

Laboratory, (2002); Venkatesh Iyengar, G., et al., Element Analysis of

Biological Samples: Priniciples and Practices (1998); Drielak, S., Hot Zone

Forensics: Chemical, Biological, and Radiological Evidence Collection (2004);

Wells, D., High Throughput Bioanalytical Sample Preparation (Progress in

Pharmaceutical and Biomedical Analysis) (2002)), each of which is

incorporated by reference). Depending on the type of mixed sample chosen,

additional processing and/or purification steps may be performed to obtain

nucleic acid fragments of a desired purity or size, using processing methods

including but not limited to sonication, nebulization, gel purification, PCR

purification systems, nuclease cleavage, or a combination of these methods. In

a preferred aspect, samples comprising cell-free DNA (cfDNA) are used.

[000127] At step 120, a first set of fixed sequence oligonucleotides are introduced

to the mixed nucleic acid sample, under conditions that allow the first set of

fixed sequence oligonucleotides to hybridize to the mixed nucleic acid sample.

The first set of fixed sequence oligonucleotides comprise nucleic acid

sequences that are complementary to one or more selected loci in the mixed

sample, which as will be described in detail herein are useful in determining

copy number variations and/or chromosomal abnormalities. The nucleic acid

sequences capable of determining copy number variations and/or chromosomal

abnormalities include sequences that allow for identification of chromosomal abnormalities such as amplifications or deletions, aneuploidies, translocations, or inversions.

[000128] At step 130, a second set of fixed sequence oligonucleotides are

introduced to the mixed nucleic acid sample and first set of fixed sequence

oligonucleotides under conditions that allow the second set of fixed sequence

oligonucleotides to hybridize to the mixed nucleic acid sample. The second set

of fixed sequence oligonucleotides comprise nucleic acid sequences that are

complementary to one or more selected loci in the mixed sample, able to detect

polymorphisms. Washing steps optionally may be included between steps 120

and130,and130and140.

[000129] At step 140, the first and second sets of fixed sequence oligonucleotides

that have hybridized to adjacent regions of the selected loci in the mixed

sample are ligated, and at step 150, the ligated oligonucleotides are amplified.

The ligated and amplified oligonucleotides are then detected and analyzed,

which allows for determination of copy number variations or chromosomal

abnormalities and identification of polymorphisms at step 160.

[000130] The sets of fixed sequence nucleic acids are designed to hybridize to at

least two separate regions in a selected locus. In preferred aspects, two or more

separate oligos are used to hybridize to these regions to provide adjacent

nucleic acids complementary to the selected locus. In some aspects, however, a

single probe can be used which comprises two or more distinct non-adjacent

regions that are complementary to the selected loci including precircular probes

such as so-called "padlock probes" or "molecular inversion probes (MIPs)".

[000131] The present invention provides an improved system over more random

techniques such as massively parallel sequencing, shotgun sequencing, and the use of random digital PCR which have been used by others to detect CNVs.

These aforementioned approaches rely upon sequencing of all or a statistically

significant population of DNA fragments in a sample, followed by mapping of

these fragments or otherwise associating the fragments to their appropriate

chromosomes. The identified fragments are then compared against each other

or against some other reference (e.g., normal chromosomal makeup) to

determine CNVs on particular chromosomes. These methods are inherently

inefficient as compared to the present invention, as the primary chromosomes

of interest only constitute a minority of data that is generated from the detection

of such DNA fragments in the mixed samples.

[000132] The assays of the present invention provide targeted detection of

selected loci, which provides information on both the content of the selected

locus (i.e., presence of a polymorphic region) and information on the frequency

of the selected locus in a sample (with or without detecting any putative

polymorphisms in that region). This key feature provides the ability to detect

both copy number of selected loci and the presence or absence of

polymorphisms in selected loci as a single data set from performance of a

multiplexed assay of the invention.

[000133] Techniques that are dependent upon a very broad sampling of DNA in a

sample are providing a very broad coverage of the DNA analyzed, but in fact

are sampling the DNA contained within a sample on a 1X or less basis (i.e.,

subsampling). In contrast, the amplification of selected loci used in the present

assays provides depth of coverage of particular loci of interest, and provides a

"super-sampling" of such selected loci with an average sequence coverage of

preferably 2X or more, more preferably sequence coverage of 1OOX of more, even more preferably sequence coverage of 1000X or more of the selected loci

(including from the one or more minor sources) present in the initial mixed

sample.

[000134] A distinct advantage of the invention is that the amplification products

resulting from the assays can be analyzed using a variety of detection and

quantification techniques, including but not limited to hybridization techniques,

digital PCR and high throughput sequencing determination techniques.

[000135] The methods of the invention provide a more efficient and economical

use of data, and the substantial majority of sequences analyzed following

sample amplification result in affirmative information about the sequence

identity and frequency of selected loci in the mixed sample. Thus, unlike

techniques relying on massively parallel sequencing or random digital

"counting" of chromosome regions and subsequent identification of relevant

data from such counts, the assay system of the invention provides a much more

efficient use of data collection than the random approaches taught by others in

the art.

Assay Methods

[000136] The assay systems of the invention utilize a general scheme as described

above, though many different configurations and variations can be employed, a

few of which are described below and more of which are exemplified in US

Ser. No. 61/371605 filed August 6, 2010, incorporated by reference herein in

its entirety.

[000137] Figure 2 illustrates a first general schematic for a ligation-based assay

system of the invention. The fixed sequence oligonucleotides 201, 203 comprise universal primer regions 209 and 211, respectively, and regions complementary to the selected locus 205 and 207, respectively. However, in addition, the assay system in Figure 2 employs a sample index region 221 on the first fixed sequence oligonucleotide 201. In certain aspects, all or a portion of the sequences of the selected loci are directly detected using the described techniques, e.g., by sequence determination or hybridization techniques. In the example of Figure 2, a sample index is associated with the first fixed sequence oligonucleotide 201. The detection of the indices can identify a sequence from a specific sample in a highly multiplexed assay system.

[000138] At step 202, the fixed sequence oligonucleotides 201, 203 are introduced

in step 202 to the mixed sample 200 and allowed to specifically bind to the

selected locus 215. Following hybridization, the unhybridized fixed sequence

oligonucleotides are preferably separated from the remainder of the genetic

sample (by, e.g., washing -- not shown). A bridging oligo is then introduced

and allowed to hybridize in step 204 to the region of the locus 215 between the

first 201 and second 203 fixed sequence oligonucleotides. The bound

oligonucleotides are ligated at step 206 to create a contiguous nucleic acid

spanning and complementary to the locus of interest. In certain aspects of the

invention, the bridging oligonucleotides of are between 2-45 nucleotides in

length. In a specific aspect, the bridging oligonucleotides are between 3-9

nucleotides in length. In yet another specific aspect, the oligonucleotides are

between 10-30 nucleotides in length.

[000139] Following ligation, the ligation product is eluted from the gDNA

template. Universal primers 217, 219 are introduced in step 208 to amplify the

ligated first and second fixed sequence oligonucleotides to create 210 amplification products 223 that comprise the sequence of the locus of interest.

These products 223 are isolated, detected, identified and quantified to provide

information regarding the presence and amount of the selected loci in the

mixed sample. Preferably, the amplification products are detected and

quantified through sequence determination. In specific aspects, it is desirable

to determine the sequences of both the sample index and the amplification

products, for example, to provide identification of the sample as well as the

locus. The indices such as the sample index shown here envisioned in the

invention may be associated with the first fixed sequence oligonucleotides, the

second fixed sequence oligonucleotides or both. Alternatively or in addition,

indices may be associated with primers that are used to amplify the ligated first

and second fixed sequence oligonucleotides, which also serves to incorporate

indices into the amplification products.

[000140] In preferred aspects and as shown in Figure 2, indices representative of

the mixed sample from which a nucleic acid may be isolated are used to

identify the source of the selected loci in a multiplexed assay system. In such

aspects, the nucleic acids are uniquely identified with the sample index.

Uniquely identified oligonucleotides may then be combined into a single

reaction vessel with nucleic acids from other mixed samples prior to

sequencing. In such a case, the sequencing data is segregated by the unique

sample index to determine the frequency of each target locus for each mixed

sample and to determine whether there is a chromosomal abnormality in an

individual sample.

[000141] In aspects of the invention using sample indices, the fixed sequence

oligonucleotides preferably are designed so that sample indices comprising identifying information are located between the universal primer regions 209 and 211 and the regions complementary to the selected loci in the sample 205 and 207. Alternatively, the indices and universal amplification sequences can be added to the ligated first and second fixed sequence oligos (and the bridging oligo, if present) by including these indices in the primers used to amplify the ligation products for separate samples. In either case, preferably the indices are encoded upstream of the locus-specific sequences but downstream of the universal primers so that they are preserved upon amplification.

[000142] Figure 3 exemplifies methods of the assay system in which one or more

bridging olignucleotides are employed and exemplifies how polymorphisms

may be detected and identified. In Figure 3, two fixed sets of sequence

oligonucleotides are used which comprise substantially the same universal

primers 309, 311 and sequence-specific regions 305, 307, but comprise

different sample indices, 321, 323 on the fixed sequence oligonucleotides of the

set where the different indices correspond to different base sequences for the

single nucleotide polymorphism present in a particular sample. The ligation

reactions are carried out with material from the same mixed sample 300, but in

separate tubes with the different allele-specific oligo sets. Bridging

oligonucleotides corresponding to two possible nucleotides for this SNP in the

selected loci 313, 333 are used to detect of the selected locus in each ligation

reaction. Two allele indices 321, 323 that are indicative of the particular

polymorphic alleles are incorporated into the amplification products so that

sequence determination of the actual sequence of the ligated first, second and

bridging oligonucleotides are not necessarily needed, although the sequences of the entire ligation products may still be determined to identify polymorphisms and/or provide confirmation.

[000143] Each of the fixed sequence oligonucleotides comprises a region

complementary to the selected locus 305, 307, and universal primer regions

309, 311 used to amplify the different selected loci following initial selection

and/or isolation of the selected loci from the mixed sample. The universal

primer regions are located at the ends of the fixed sequence oligonucleotides

301, 303, and 323 flanking the indices and the regions complementary to the

nucleic acid of interest, thus preserving the nucleic acid-specific sequences and

the sample indices in the products of any universal amplification methods. The

fixed sequence oligonucleotides 301, 303, 323 are introduced at step 302 to an

aliquot of the genetic sample 300 and allowed to specifically bind to the

selected loci 315 or 325. Following hybridization, the unhybridized fixed

sequence oligonucleotides are preferably separated from the remainder of the

genetic sample by, e.g., washing (not shown).

[000144] The bridging oligos corresponding to an A/T SNP 313 or a G/C SNP

333 are introduced and allowed to bind in step 304 to the region of the selected

locus 315 or 325 between the first 305 and second 307 nucleic acid

complementary regions of the fixed sequence oligonucleotides. Alternatively,

the bridging oligos 313, 333 can be introduced to the sample simultaneously

with the fixed sequence oligonucleotides. The bound oligonucleotides are

ligated in step 306 in the single reaction mixture to create a contiguous nucleic

acid spanning and complementary to the selected locus.

[000145] Following ligation, the separate reactions may preferably be combined

for the universal amplification and detection steps. Universal primers 317, 319 are introduced to the combined reactions at step 308 to amplify the ligated template regions and create at step 310 ligated first and second fixed sequence oligos and bridging oligo products 327, 329 that comprise the sequence of the selected locus representing both SNPs in the selected locus. These ligation products 327, 329 are detected and quantified through sequence determination of the ligation product, through the sample index and/or the region of the product containing the SNP in the selected locus.

[000146] In an alternative configuration of the methods of the assay systems of

the invention, the bridging oligo may hybridize to a region that is not directly

adjacent to the region complementary to one or both of the fixed sequence

oligos, and an intermediate step requiring extension of one or more of the

oligos is necessary prior to ligation. For example, as illustrated in Figure 4,

each set of oligonucleotides preferably contains two oligonucleotides 401, 403

of fixed sequence and one or more bridging oligonucleotides 413. Each of the

fixed sequence oligonucleotides comprises a region complementary to the

selected locus 405, 407, and primer sequences, preferably universal primer

sequences, 409, 411, i.e., oligo regions complementary to universal primers.

The primer sequences 409, 411 are located at or near the ends of the fixed

sequence oligonucleotides 401, 403, and thus preserve the nucleic acid-specific

sequences in the products of any universal amplification methods. The fixed

sequence oligonucleotides 401, 403 are introduced at step 402 to the mixed

sample 400 and allowed to specifically bind to the complementary portions of

the locus of interest 415. Following hybridization, the unhybridized fixed

sequence oligonucleotides are preferably separated from the remainder of the

genetic sample (not shown).

[000147]The bridging oligonucleotide is then introduced at step 404 and allowed

to bind to the region of the selected locus 415 between the first 401 and

second 403 fixed sequence oligonucleotides. Alternatively, the bridging oligo

can be introduced simultaneously with the fixed sequence oligonucleotides. In

this exemplary aspect, the bridging oligo hybridizes to a region directly

adjacent to the first fixed sequence oligo region 405, but is separated by one or

more nucleotides from the complementary region of the second fixed sequence

oligonucleotide 407. Following hybridization of the fixed sequence and

bridging oligos, the bridging oligo 413 is extended at step 406, e.g., using a

polymerase and dNTPs, to fill the gap between the bridging oligo 413 and the

second fixed sequence oligo 403. Following extension, the bound

oligonucleotides are ligated at step 408 to create a contiguous nucleic acid

spanning and complementary to the locus of interest 415. After ligation,

universal primers 417, 419 are introduced at step 410 to amplify the ligated

first, second and bridging oligos to create at step 412 amplification products

423 that comprise the sequence of the selected locus of interest. Amplification

products 423 are optionally isolated, detected, and quantified to provide

information on the presence and amount of the selected locus(s) in the mixed

sample.

Detecting Copy Number Variations

[000148] The present invention provides methods for identifying copy number

variation at one or more loci and the presence or absence of one or more

polymorphisms. This can be performed using amplification methods for identification of loci corresponding to specific chromosomes of interest loci corresponding to single gene sequences.

[000149] The assay systems utilize nucleic acid probes designed to identify, and

preferably to isolate, selected loci in a mixed sample. Certain of the probes

identify sequences of interest in selected loci interrogated for copy number (i.e.

loci frequency), and other probes identify sequences that correspond to

polymorphisms of interest (i.e. loci content) in nucleic acids corresponding to a

major source or minor source in a mixed sample.

[000150] In specific aspects, the assay systems of the invention employ one or

more selective amplification steps (e.g., using one or more primers that

specifically hybridize to a selected locus) for isolating, amplifying or analyzing

substantially all of the selected loci analyzed. This is in direct contrast to the

random amplification approach used by others employing, e.g., massively

parallel sequencing, as such amplification techniques generally involve random

amplification of all or a substantial portion of the genome. In addition, the

initial sample can optionally be enriched using methods such as general

amplification to increase the copy number of nucleic acids in the mixed sample.

Preferably the hybridization, ligation, and amplification steps used to identify

the loci of interest are performed directly on the mixed sample.

[000151] In a general aspect, the user of the invention analyzes multiple selected

loci on different chromosomes. When multiple loci are analyzed for a sample,

a preferred embodiment is to amplify all of the selected loci for each sample in

one reaction vessel. The frequencies of the multiple selected loci are used to

determine whether a chromosomal abnormality exists, and optionally to

identify polymorphisms in the selected loci.

[000152] In preferred aspects, multiple selected loci from two or more samples

may be amplified in a single reaction vessel, and the information

simultaneously analyzed in a single data set, e.g., through sequence

determination. The resulting data is then analyzed to separate the results for

the different sample and used to determine the presence of absence of CNV and

the source contribution in individual samples.

[000153] In one aspect, chromosomal abnormalities are identified in the assay

system of the invention using multiple selected loci on multiple chromosomes,

and the frequency of the selected loci on the multiple chromosomes compared

to identify an increase likelihood of aneuploidy based on the ratios frequencies

of the multiple loci on the chromosomes. Normalization or standardization of

the frequencies can be performed for one or more selected loci.

[000154] In another aspect, the assay system sums the frequencies of the selected

loci on two or more chromosomes and then compares the sum of the selected

loci on one chromosome against another chromosome to determine whether a

chromosomal aneuploidy exists. In another aspect, the assay system analyzes

subsets of selected loci frequencies on two or more chromosomes to determine

whether a chromosomal aneuploidy exists for one of the two chromosomes.

The comparison can be made either within the same or different chromosomes.

[000155] In certain aspects, the data used to determine the frequency of the

selected loci may exclude outlier data that appear to be due to experimental

error, or that have elevated or depressed levels based on an idiopathic genetic

bias within a particular sample. In one example, the data used for summation

may exclude DNA regions with a particularly elevated frequency in one or

more samples. In another example, the data used for summation may exclude selected loci that are found in a particularly low abundance in one or more samples.

[000156] In another aspect subsets of selected loci can be chosen to yield a

statistically significant result when determining whether a chromosomal

abnormality exists. Multiple analyses of different subsets of selected loci can

be performed within a mixed sample to yield more statistical power. For

example, if there are 100 selected loci for chromosome 21 and 100 selected loci

for chromosome 18, a series of analyses could be performed that evaluate fewer

than 100 regions for each of the chromosomes. In this example, selected loci

are not being selectively excluded.

[000157] The quantity of different nucleic acids detectable on certain

chromosomes may vary depending upon a number of factors, including general

representation of loci in different cell sources in mixed samples, degradation

rates of the different nucleic acids representing different loci in mixed samples,

sample preparation methods, and the like. Thus, in another aspect, the quantity

of particular loci on a chromosome is summed to determine the loci quantity

for different chromosomes in the sample. The loci frequencies are summed for

a particular chromosome, and the sum of the loci are used to determine

aneuploidy. This aspect of the invention sums the frequencies of the individual

loci on each chromosome and then compares the sum of the loci on one

chromosome against one or more other chromosomes to determine whether a

chromosomal abnormality exists.

[000158] The nucleic acids analyzed using the assay systems of the invention are

preferably selectively amplified and optionally isolated from the mixed sample

using primers specific to the locus of interest (e.g., to a locus of interest in a mixed sample). The primers for selective amplification may be chosen for various reasons, but are preferably designed to 1) efficiently amplify a region from the chromosome of interest; 2) have a predictable range of expression from maternal and/or fetal sources in different mixed samples; and 3) be distinctive to the particular chromosome, i.e., not amplify homologous regions on other chromosomes. The following are exemplary techniques that may be employed in the assay system or the invention.

[000159] The assay system of the invention detects both aneuploidies and

specific chromosomal abnormalities through identification and quantification

of specific loci of interest. Such chromosomal abnormalities include, but are

not limited to, deletion mutations, insertion mutations, copy number

polymorphisms, copy number variants, chromosome 22q11 deletion syndrome,

1lq deletion syndrome on chromosome 11, 8p deletion syndrome on

chromosome 8, and the like. Generally, at least two selected loci present on the

same or separate chromosomes are analyzed, and at least one of the selected

loci is associated with the fetal allelic abnormality. The sequences of the two

selected loci and number of copies of the two selected loci are then compared

to determine whether the chromosomal abnormality is present, and if so, the

nature of the abnormality.

[000160] While much of the description contained herein describes detecting

aneuploidy by counting the abundance of selected loci on one or more putative

aneuploid chromosomes and the abundance of selected loci on one or more

normal chromosomes, the same techniques may be used to detect copy number

variations where such copy number variation occurs on only a portion of a

chromosome. In detection of copy number variations, multiple selected loci within the putative copy number variation location are compared to multiple selected loci outside of the putative copy number variation location. For instance, one may detect a chromosome 22q11 deletion syndrome in a fetus in a maternal sample by selecting two or more loci within the 22q11 deletion and two or more loci outside of the 22q11 deletion. The loci outside of the 22q11 deletion may be on another region of Chromosome 22 or may be on a completely different chromosome. The abundance of each loci is determined by the methods described in this application.

[000161] In some aspects a universal amplification may be used for amplifying

the selected loci. In some aspects, the selected loci for each sample are assayed

in a single reaction in a single vessel. In other aspects, loci from multiple

samples can be assayed in a single reaction in a single vessel.

[000162] Certain aspects of the invention can detect a deletion, including the

boundaries of such deletions. In some aspects, at least 24 selected loci may be

used within the region of the putative deletion and at least 24 selected loci may

be used outside of the region of the putative deletion. In another aspect at least

48 selected loci may be used within the region of the putative deletion and at

least 48 selected loci may be used outside of the region of the putative deletion.

In another aspect at least 48 selected loci may be used within the region of the

putative deletion and at least 96 selected loci may be used outside of the region

of the putative deletion. In another aspect at least 48 selected loci may be used

within the region of the putative deletion and at least 192 selected loci may be

used outside of the region of the putative deletion. In a preferred aspect at least

384 selected loci may be used within the region of the putative deletion and at

least 384 selected loci may be used outside of the region of the putative deletion. The selected loci within the region of the putative and the selected loci outside of the region of the putative deletion are summed. These sums are then compared to each other to determine the presence or absence of a deletion.

Optionally, a ratio of the sums is computed and that ratio may be compared to

an average ratio created from a normal population. When the ratio for one or

more selected loci falls statistically outside of an expected ratio, a deletion is

detected. The threshold for positively identifying a deletion may be twice or

more, preferably four or more times the variation calculated in the normal

population. When a plurality of selected loci are sued within and outside the

region of the putative deletion, boundaries of the deletion may be identified.

Polymorphisms Associated with Diseases or Predispositions

[000163] The assay systems of the invention can also be utilized to detect

polymorphisms, such as those associated with an autosomal dominant or

recessive disease or mutation. Given the multiplexed nature of the assay

systems of the invention, detection takes place in the same assay as the

detection of chromosomal abnormalities. Thus a single assay system can

provide diagnostic information on different classes of genetic mutations.

Accordingly, as the preferred assay systems of the invention are highly

multiplexed and able to interrogate hundreds or even thousands of selected loci

within a mixed sample, in certain aspects it is desirable to interrogate the

sample for marker loci within the mixed sample, e.g., loci associated with

genetic risk or that identify the presence or absence of infectious organisms.

Thus, the assay systems provide detection of such marker loci in conjunction with the detection of selected loci for copy number determination in a mixed sample.

[000164] Thus, the assay system of the invention can be used to detect

polymorphisms in a mixed sample, where such polymorphisms are associated

with genes associated with autosomal recessive disorders, mutations associated

with autosomal dominant disorders; polymorphisms associated with risk of

developing a disease and/or disease progression (e.g., metastasis) and prognosis

indicators.

[000165] In other specific aspects, the assay system of the invention can be used

to detect fetal mutations or polymorphisms in a maternal sample, where such

mutations or polymorphisms are associated with polygenic disorders such as

coronary heart disease, diabetes, hypertension, congenital heart defects, and

epilepsy. Examples include mutations in genes associated with predispositions

such as mutations in cancer susceptibility genes, (e.g. mutations ion BRCAI or

II or in p53); polymorphisms associated with increased risk of developing later

onset diseases, such as the apoE3 gene polymorphism associated with

Alzheimer's risk,

[000166] In addition to detection of chromosomal abnormalities and single gene

mutations or polymorphisms associated with monogenic or polygenic disease,

disorders or predispositions, the assay systems of the invention may identify

infectious agents in the mixed sample.

Selected amplification

[000167] Numerous selective amplification methods can be used to provide the

amplified nucleic acids that are analyzed in the assay systems of the invention,

and such methods are preferably used to increase the copy numbers of selected

loci in a mixed sample in a manner that allows preservation of information

concerning the initial content of the selected loci in the mixed sample.

Although not all combinations of amplification and analysis are described

herein in detail, it is well within the skill of those in the art to utilize different

amplification methods and/or analytic tools to isolate and/or analyze the

nucleic acids of region consistent with this specification, and such variations

will be apparent to one skilled in the art upon reading the present disclosure.

[000168] Such amplification methods include but are not limited to, polymerase

chain reaction (PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCR

Technology: Principles and Applications for DNA Amplification, ed. H. A.

Erlich, Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wu and

Wallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077, 1988),

strand displacement amplification (SDA) (U.S. Pat. Nos. 5,270,184; and

5,422,252), transcription-mediated amplification (TMA) (U.S. Pat. No.

5,399,491), linked linear amplification (LLA) (U.S. Pat. No. 6,027,923), and

the like, self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad.

Sci. USA, 87, 1874 (1990) and W090/06995), selective amplification of target

polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence

primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),

arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,

413,909, 5,861,245) and nucleic acid based sequence amplification (NASBA).

(See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used include: Qbeta Replicase, described in PCT Patent Application No.

PCT/US87/00880, isothermal amplification methods such as SDA, described in

Walker et al., Nucleic Acids Res. 20(7):1691-6 (1992), and rolling circle

amplification, described in U.S. Pat. No. 5,648,245. Other amplification

methods that may be used are described in, U.S. Pat. Nos. 5,242,794,

5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317 and US Pub. No.

20030143599, each of which is incorporated herein by reference. In some

aspects DNA is amplified by multiplex locus-specific PCR. In a preferred

aspect the DNA is amplified using adaptor-ligation and single primer PCR.

Other available methods of amplification, such as balanced PCR

(Makrigiorgos, et al., Nature Biotechnol, 20:936-9 (2002)) and isothermal

amplification methods such as nucleic acid sequence based amplification

(NASBA) and self-sustained sequence replication (Guatelli et al., PNAS USA

87:1874 (1990)). Based on such methodologies, a person skilled in the art

readily can design primers in any suitable regions 5' and 3' to a locus of

interest. Such primers may be used to amplify DNA of any length so long that

the DNA comprises the selected loci of interest.

[000169] The length of an amplified selected locus from a genomic region of

interest is long enough to provide enough sequence information to distinguish

the amplified locus from other loci that are amplified and/or selected.

Generally, an amplified nucleic acid corresponding to a selected locus is at

least about 16 nucleotides in length, and more typically, an amplified nucleic

acid corresponding to a selected locus is at least about 20 nucleotides in length.

In a preferred aspect of the invention, an amplified nucleic acid corresponding to a selected locus is at least about 30 nucleotides in length. In a more preferred aspect of the invention, an amplified nucleic acid corresponding to a selected locus is at least about 32, 40, 45, 50, or 60 nucleotides in length. In other aspects of the invention, an amplified nucleic acid corresponding to a selected locus can be about 100, 150 or up to 200 in length.

[000170] In certain aspects, selective amplification comprises an initial linear

amplification step, which can be particularly useful if the starting amount of

DNA from the mixed sample is quite limited, e.g., where the cell-free DNA in

a sample is available in limited quantities. This mechanism increases the

amount of DNA molecules that are representative of the original DNA content,

and helps to reduce sampling error where accurate quantification of the DNA or

a fraction of the DNA (e.g., fetal DNA contribution in a maternal sample) is

needed.

[000171] Thus, in one aspect, a limited number of cycles of sequence-specific

linear amplification are performed on the starting mixed sample comprising

cfDNA. The number of cycles is generally less than that used for a typical

PCR amplification, e.g., 5-30 cycles or fewer. Primers or probes may be

designed to amplify specific genomic segments or regions comprising selected

loci. The primers or probes may be modified with an end label at the 5' end

(e.g. with biotin) or elsewhere along the primer or probe such that the

amplification products can be purified or attached to a solid substrate (e.g.,

bead or array) for further isolation or analysis. In a preferred aspect, the

primers are multiplexed such that a single reaction yields multiple DNA

fragments from different regions. Amplification products from the linear amplification could then be further amplified with standard PCR methods or with additional linear amplification.

[000172] For example, cfDNA can be isolated from blood, plasma, or serum

from a pregnant woman, and incubated with primers against a set number of

selected loci that correspond to chromosomes of interest. Preferably, the

number of primers used for initial linear amplification will be 12 or more, more

preferably 24 or more, more preferably 36 or more, even more preferably 48 or

more, and even more preferably 96 or more. Each of the primers corresponds

to a single selected locus, and is optionally tagged for identification and/or

isolation. A limited number of cycles, preferably 10 or fewer, are performed

with linear amplification. The amplification products are subsequently

isolated, e.g., when the primers are linked to a biotin molecule the

amplification products can be isolated via binding to avidin or streptavidin on a

solid substrate. The amplification products are then subjected to further

biochemical processes such as further amplification with other primers and/or

detection techniques such as sequence determination and hybridization.

[000173] Efficiencies of linear amplification may vary between sites and

between cycles so that in certain systems normalization may be used to ensure

that the products from the linear amplification are representative in frequency

and sequence of the nucleic acids in the mixed sample. One practicing the

assay system of the invention can utilize information from multiple aliquots of

a sample to determine variation in the quantity of different amplification

products representing the selected loci, including variation in different selected

loci and/or between selected loci following the limited initial linear

amplification. Such information can be used to determine the initial levels of selected loci in the sample DNA content, allowing for normalization of the frequency of selected loci.

Universal amplification

[000174] In preferred aspects of the invention, the selectively amplified loci are

preferably further amplified through universal amplification of all or

substantially all of the various selected loci using the assay systems of the

invention. Universal primer regions are added to the fixed sequence

oligonucleotides so that the selectively amplified loci may be further amplified

in a single universal amplification reaction. These universal primer sequences

may be added to the nucleic acids regions during the selective amplification

process, i.e., the primers for selective amplification comprise universal primer

sequences. Alternatively, adapters comprising universal amplification

sequences can be added to the ends of the selectively amplified selected loci as

adapters following initial amplification and after isolation of the selectively

amplified selected loci from the mixed sample.

[000175] In one exemplary aspect, selected lociare initially amplified from a

mixed sample using primers complementary to selected loci of interest,

followed by a universal amplification step to increase the number of loci for

analysis. Introduction of primer regions to the initial amplification products

from a mixed sample allows subsequent controlled universal amplification of

all or a portion of selected nucleic acids prior to or during analysis, e.g.

sequence determination.

[000176] Bias and variability can be introduced during DNA amplification, such

as that seen during polymerase chain reaction (PCR). In cases where an amplification reaction is multiplexed, there is the potential that different selected loci will amplify at different rates or efficiency. This may be due in part to some primers in a multiplex reaction having better efficiency (i.e. more favorable hybridization kinetics), due to experimental conditions that favor some primers over others such as sequence content of the primer and template

DNA, buffer conditions, and other conditions. Universal DNA amplification in

a multiplexed assay system generally introduces less bias and variability

between amplified loci.

[000177] Accordingly, in a one aspect, a small number (e.g., 1-10, preferably 3

5) of cycles of selected amplification using loci specific sequences is

performed, followed by universal amplification using universal primers. The

number of cycles using universal primers will vary, but will preferably be at

least 10 cycles, more preferably at least 5 cycles, even more preferably 20

cycles or more. By moving to universal amplification following a lower

number of amplification cycles, the bias of having certain loci amplify at

greater rates than others is reduced.

[000178] Optionally, the assay system will include a step between the selected

amplification process and universal amplification process to remove any

nucleic acids that are not selectively amplified in the selective amplification

reaction.

[000179] The whole product or an aliquot of the product from the selected

amplification may be used for the universal amplification reaction. The same

or different conditions (e.g., polymerase, buffers, and the like) may be used in

the amplification steps, e.g., to ensure that bias and variability are not

inadvertently introduced due to experimental conditions. In addition, variations in primer concentrations may be used to differentially limit the number of sequence-specific amplification cycles for some selected loci as compared to other selected loci.

[000180] In certain aspects, the universal primer regions of the primers or

adapters used in the assay system are designed to be compatible with

conventional multiplexed assay methods that utilize general priming

mechanisms to analyze large numbers of nucleic acids simultaneously in one

reaction in one vessel. Such "universal" priming methods allow for efficient,

high volume analysis of the quantity of selected loci present in a mixed sample,

and allow for comprehensive quantification of the presence of selected loci

within such a mixed sample for the determination of aneuploidy.

[000181] Examples of such assay methods include, but are not limited to,

multiplexing methods used to amplify and/or genotype a variety of samples

simultaneously, such as those described in Oliphant et al., US Pat. No.

7,582,420.

[000182] Some aspects utilize coupled reactions for multiplex detection of

nucleic acid sequences where oligonucleotides from an early phase of each

process contain sequences which may be used by oligonucleotides from a later

phase of the process. Exemplary processes for amplifying and/or detecting

nucleic acids in samples can be used, alone or in combination, including but not

limited to the methods described below, each of which are incorporated by

reference in their entirety.

[000183] In certain aspects, the assay system of the invention utilizes one of the

following combined selective and universal amplification techniques: (1) ligase

detection reaction ("LDR") coupled with polymerase chain reaction ("PCR");

(2) primary PCR coupled to secondary PCR coupled to LDR; and (3) primary

PCR coupled to secondary PCR. Each of these aspects of the invention has

particular applicability in detecting certain nucleic acid characteristics.

However, each requires the use of coupled reactions for multiplex detection of

nucleic acid sequence differences where oligonucleotides from an early phase

of each process contain sequences which may be used by oligonucleotides from

a later phase of the process.

[000184] Barany et al., US Pat Nos. 6,852,487, 6,797,470, 6,576,453, 6,534,293,

6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889, 5,830,711, 5,494,810,

describe the use of the ligase chain reaction (LCR) assay for the detection of

specific sequences of nucleotides in a variety of nucleic acid samples.

[000185] Barany et al., US Pat Nos. 7,807,431, 7,455,965, 7,429,453, 7,364,858,

7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831, 7,198,894, 7,166,434,

7,097,980, 7,083,917, 7,014,994, 6,949,370, 6,852,487, 6,797,470, 6,576,453,

6,534,293, 6,506,594, 6,312,892, and 6,268,148 describe LDR coupled PCR

for nucleic acid detection.

[000186] Barany et al., US Pat No. 7,556,924 and 6,858,412, describe the use of

precircle probes (also called "padlock probes" or "multi-inversion probes")

with coupled LDR and polymerase chain reaction ("PCR") for nucleic acid

detection.

[000187] Barany et al., US Pat Nos. 7,807,431, 7,709,201, and 7,198, 814

describe the use of combined endonuclease cleavage and ligation reactions for

the detection of nucleic acid sequences.

[000188] Willis et al., US Pat Nos. 7,700,323 and 6,858,412, describe the use of

precircle probes in multiplexed nucleic acid amplification, detection and

genotyping.

[000189] Ronaghi et al., US Pat. No. 7,622,281 describes amplification

techniques for labeling and amplifying a nucleic acid using an adapter

comprising a unique primer and a barcode.

[000190] In a preferred aspect, the amplification products are multiplexed, as

described previously. In a preferred aspect, the multiplex amplification

products are quantified by analysis of the amplification products. In a preferred

aspect, a representational sample of individual molecules from the

amplification processes is isolated from the rest of the sample for further

analysis. To obtain a representational sample of individual molecules, the

average number of molecules per locus must exceed the sampling noise created

by the multiplexed reaction. In one aspect, the average number per locus is

greater than 100. In another aspect, the average number per locus is greater

than 500. In another aspect the average number per locus is greater than 1000.

[000191] Individual molecules from the amplification product are preferably

isolated physically from the other molecules in a manner that allows the

different amplification products to be distinguished from one another in

analysis. In a preferred aspect, this isolation occurs on a solid substrate. Each

isolated molecule may be associated with a particular identifiable or physical

address either prior to analysis, or the address may become known for the

particular amplification products based on the outcome of the analysis. The

substrate may be a planar surface or three-dimensional surface such as a bead.

[000192] Once isolated, the individual amplification product may be further

amplified to make multiple identical copies of that molecule at the same known

or identifiable location. The amplification may occur before or after that

location becomes an identifiable or physical address. The amplification

product and/or its copies (which may be identical or complementary to the

amplification product) are then analyzed based on the sequence of the

amplification product or its copies to identify the particular locus and/or allele

it represents.

[000193] In a preferred aspect, the entire length of the amplification product or a

portion of the amplification product may be analyzed using sequence

determination. The number of bases that need to be determined must be

sufficient to uniquely identify the amplification product as belonging to a

specific locus and/or allele. In one preferred aspect, the locus is analyzed

through sequence determination of the amplification product.

[000194] Numerous methods of sequence determination are compatible with the

assay systems of the inventions. Exemplary methods for sequence

determination include, but are not limited to, including, but not limited to,

hybridization-based methods, such as disclosed in Drmanac, U.S. Pat. Nos.

6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patent

publication 2005/0191656, which are incorporated by reference, sequencing by

synthesis methods, e.g., Nyren et al, U.S. Pat. No. 7,648,824, 7,459,311 and

6,210,891; Balasubramanian, U.S. Pat. Nos. 7,232,656 and 6,833,246; Quake,

U.S. Pat. No. 6,911,345; Li et al, Proc. Natl. Acad. Sci., 100: 414-419 (2003);

pyrophosphate sequencing as described in Ronaghi et al., U.S. Pat. Nos.

7,648,824, 7,459,311, 6,828,100, and 6,210,891; and ligation-based sequencing determination methods, e.g., Drmanac et al., U.S. Pat. Appln No.

20100105052, and Church et al, U.S. Pat. Appln Nos. 20070207482 and

20090018024.

[000195] Sequence information may be determined using methods that

determine many (typically thousands to billions) of nucleic acid sequences in

an intrinsically parallel manner, where many sequences are read out preferably

in parallel using a high throughput serial process. Such methods include but are

not limited to pyrosequencing (for example, as commercialized by 454 Life

Sciences, Inc., Branford, CT); sequencing by ligation (for example, as

commercialized in the SOLiDTM technology, Life Technology, Inc., Carlsbad,

CA); sequencing by synthesis using modified nucleotides (such as

commercialized in TruSeqTM and HiSeqTM technology by Illumina, Inc., San

Diego, CA, HeliScopeTM by Helicos Biosciences Corporation, Cambridge, MA,

and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, CA),

sequencing by ion detection technologies (Ion Torrent, Inc., South San

Francisco, CA); sequencing of DNA nanoballs (Complete Genomics, Inc.,

Mountain View, CA); nanopore-based sequencing technologies (for example,

as developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and like

highly parallelized sequencing methods.

[000196] Alternatively, in another aspect, the entire length of the amplification

product or a portion of the amplification product may be analyzed using

hybridization techniques. Methods for conducting polynucleotide

hybridization assays for detection of have been well developed in the art.

Hybridization assay procedures and conditions will vary depending on the

application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A

Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger and

Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning

Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davis,

P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and

controlled hybridization reactions have been described in U.S. Pat. Nos.

5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are

incorporated herein by reference.

[000197] The present invention also contemplates signal detection of

hybridization between ligands in certain preferred aspects. See U.S. Pat. Nos.

5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601;

6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent

application 60/364,731 and in PCT Application PCT/US99/06097 (published as

W099/47964), each of which also is hereby incorporated by reference in its

entirety for all purposes.

[000198] Methods and apparatus for signal detection and processing of intensity

data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,

5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324,

5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803;

and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application

PCT/US99/06097 (published as W099/47964), each of which also is hereby

incorporated by reference in its entirety for all purposes.

Variation Minimization Within and Between Samples

[000199] One challenge with the detection of chromosomal abnormalities by

detection in a mixed sample is that the nucleic acids from the minor source

may be present in much lower abundance than the nucleic acids from normal

the major source. In the case of a maternal sample containing fetal and

maternal cfDNA, the cell free fetal DNA as a percentage of the total cfDNA

may vary from less than one to forty percent, and most commonly is present at

or below twenty percent and frequently at or below ten percent. In the

detection of an aneuploidy such as Trisomy 21 (Down Syndrome) in the fetal

DNA of such maternal sample, the relative increase in Chromosome 21 is 50%

in the fetal DNA and thus as a percentage of the total DNA in a maternal

sample where, as an example, the fetal DNA is 5% of the total, the increase in

Chromosome 21 as a percentage of the total is 2.5%. If one is to detect this

difference robustly through the methods described herein, the variation in the

measurement of Chromosome 21 has to be much less than the percent increase

of Chromosome 21 contributed by a third chromosome 21 from the fetal

DNA.

[000200] The variation between levels found between samples and/or for loci

within a sample may be minimized by using a combination of analytical

methods, many of which are described in this application. For instance,

variation is lessened by using an internal reference in the assay. An example of

an internal reference is the use of a chromosome present in a "normal"

abundance (e.g., disomy for an autosome) to compare against a chromosome

present in putatively abnormal abundance, such as aneuploidy, in the same

sample. While the use of one such "normal" chromosome as a reference

chromosome may be sufficient, it is also possible to use two to many normal chromosomes as the internal reference chromosomes to increase the statistical power of the quantification.

[000201] One method of using an internal reference is to calculate a ratio of

abundance of the putatively abnormal chromosomes to the abundance of the

normal chromosomes in a sample, called a chromosomal ratio. In calculating

the chromosomal ratio, the abundance or counts of each of the selected loci for

each chromosome are summed together to calculate the total counts for each

chromosome. The total counts for one chromosome are then divided by the

total counts for a different chromosome to create a chromosomal ratio for those

two chromosomes.

[000202] Alternatively, a chromosomal ratio for each chromosome may be

calculated by first summing the counts of each of the selected loci for each

chromosome, and then dividing the sum for one chromosome by the total sum

for two or more chromosomes. Once calculated, the chromosomal ratio is then

compared to the average chromosomal ratio from a normal population.

[000203] The average may be the mean, median, mode or other average, with or

without normalization and exclusion of outlier data. In a preferred aspect, the

mean is used. In developing the data set for the chromosomal ratio from the

normal population, the normal variation of the measured chromosomes is

calculated. This variation may be expressed a number of ways, most typically

as the coefficient of variation, or CV. When the chromosomal ratio from the

sample is compared to the average chromosomal ratio from a normal

population, if the chromosomal ratio for the sample falls statistically outside of

the average chromosomal ratio for the normal population, the sample contains an aneuploidy. The criteria for setting the statistical threshold to declare an aneuploidy depend upon the variation in the measurement of the chromosomal ratio and the acceptable false positive and false negative rates for the assay. In general, this threshold may be a multiple of the variation observed in the chromosomal ratio. In one example, this threshold is three or more times the variation of the chromosomal ratio. In another example, it is four or more times the variation of the chromosomal ratio. In another example it is five or more times the variation of the chromosomal ratio. In another example it is six or more times the variation of the chromosomal ratio. In the example above, the chromosomal ratio is determined by summing the counts of selected loci by chromosome. Typically, the same number of selected loci for each chromosome is used. An alternative method for generating the chromosomal ratio would be to calculate the average counts for the loci for each chromosome. The average may be any estimate of the mean, median or mode, although typically an average is used. The average may be the mean of all counts or some variation such as a trimmed or weighted average. Once the average counts for each chromosome have been calculated, the average counts for each chromosome may be divided by the other to obtain a chromosomal ratio between two chromosomes, the average counts for each chromosome may be divided by the sum of the averages for all measured chromosomes to obtain a chromosomal ratio for each chromosome as described above. As highlighted above, the ability to detect an aneuploidy in a maternal sample where the putative DNA is in low relative abundance depends greatly on the variation in the measurements of different selected loci in the assay. Numerous analytical methods can be used which reduce this variation and thus improve the sensitivity of this method to detect aneuploidy. One method for reducing variability of the assay is to increase the number of selected loci used to calculate the abundance of the chromosomes. In general, if the measured variation of a single selected locus of a chromosome is X% and Y different selected loci are measured on the same chromosome, the variation of the measurement of the chromosomal abundance calculated by summing or averaging the abundance of each selected locus on that chromosome will be approximately X% divided by YA1/2. Stated differently, the variation of the measurement of the chromosome abundance would be approximately the average variation of the measurement of each selected locus' abundance divided by the square root of the number of loci.

[000204] In a preferred aspect of this invention, the number of selected loci

measured for each chromosome is at least 24. In another preferred aspect of

this invention, the number of selected loci measured for each chromosome is at

least 48. In another preferred aspect of this invention, the number of selected

loci measured for each chromosome is at least 100. In another preferred aspect

of this invention the number of selected loci measured for each chromosome is

at least 200. There is incremental cost to measuring each locus and thus it is

important to minimize the number of selected loci. In a preferred aspect of this

invention, the number of selected loci measured for each chromosome is less

than 2000. In a preferred aspect of this invention, the number of selected loci

measured for each chromosome is less than 1000. In a most preferred aspect of

this invention, the number of selected loci measured for each chromosome is at

least 48 and less than 1000. In one aspect, following the measurement of

abundance for each selected locus, a subset of the selected loci may be used to determine the presence or absence of aneuploidy. There are many standard methods for choosing the subset of selected loci. These methods include outlier exclusion, where the selected loci with detected levels below and/or above a certain percentile are discarded from the analysis. In one aspect, the percentile may be the lowest and highest 5% as measured by abundance. In another aspect, the percentile may be the lowest and highest 10% as measured by abundance. In another aspect, the percentile may be the lowest and highest

25% as measured by abundance.

[000205] Another method for choosing the subset of selected loci includes the

elimination of regions that fall outside of some statistical limit. For instance,

selected loci that fall outside of one or more standard deviations of the mean

abundance may be removed from the analysis. Another method for choosing

the subset of selected loci may be to compare the relative abundance of a

selected locus to the expected abundance of the same selected locus in a

healthy population and discard any selected loci that fail the expectation test.

To further minimize the variation in the assay, the number of times each

selected locus is measured may be increased. As discussed, in contrast to the

random methods of detecting aneuploidy where the genome is measured on

average less than once, the assay systems of the present invention intentionally

measures each selected locus multiple times. In general, when counting events,

the variation in the counting is determined by Poisson statistics, and the

counting variation is typically equal to one divided by the square root of the

number of counts. In a preferred aspect of the invention, the selected loci are

each measured on average at least 100 times. In a preferred aspect to the

invention, the selected loci are each measured on average at least 500 times. In a preferred aspect to the invention, the selected loci are each measured on average at least 1000 times. In a preferred aspect to the invention, the selected loci are each measured on average at least 2000 times. In a preferred aspect to the invention, the selected loci are each measured on average at least 5000 times.

[000206] In another aspect, subsets of loci can be chosen randomly but with

sufficient numbers of selected loci to yield a statistically significant result in

determining whether a chromosomal abnormality exists. Multiple analyses of

different subsets of loci can be performed within a mixed sample to yield more

statistical power. In this example, it may or may not be necessary to remove or

eliminate any selected loci prior to the random analysis. For example, if there

are 100 selected loci for chromosome 21 and 100 selected loci for chromosome

18, a series of analyses could be performed that evaluate fewer than 100 loci

for each of the chromosomes.

[000207] In addition to the methods above for reducing variation in the assay,

other analytical techniques, many of which are described earlier in this

application, may be used in combination. In general, the variation in the assay

may be reduced when all of the selected loci for each sample are interrogated in

a single reaction in a single vessel. Similarly, the variation in the assay may be

reduced when a universal amplification system is used. Furthermore, the

variation of the assay may be reduced when the number of cycles of

amplification is limited.

Use of Assay Systems for Detection in Mixed Samples from CancerPatients

[000208] The assay system allow the detection of quantitative and qualitative

tumor-specific alterations of cfDNA, such as DNA strand integrity, frequency

of mutations, abnormalities of microsatellites, and methylation of genes, as

diagnostic, prognostic, and monitoring markers in cancer patients. The ability

to combine such detection of single gene alterations (including point mutations,

indels and copy number variation) with CNV detection provides a powerful

method for assisting with clinical diagnosis, treatments, outcome prediction and

progression monitoring in patients with or suspected of having a malignancy.

[000209] In some aspects, the assay system of the invention is used for

diagnostic purposes e.g., to detect the presence and/or nature of a malignancy

in a patient or to provide a quantitative estimate of tumor load in a patient.

Circulating tumor DNA and microRNAs have been associated with certain

cancers, such as lung cancer (Roth C et al., Mol Oncol. 2011 Jun;5(3):281- 91.

Epub 2011 Feb 24). Copy number variations have also been detected in certain

cancers, such as amplified HER2 and estrogen receptor in the cfDNA in breast

cancer patients. (Page K., Br J Cancer. 2011 Apr 12;104(8):1342-8. Epub 2011

Mar 22).

[000210] In other aspects of the invention, the assay system is used in cancer

patients to monitor a response to treatment and/or to follow progress of the

disease, e.g., to measure single gene alterations and cfDNA in patients

receiving chemoradiotherapy (CRT). For certain cancers, it has been shown

that cfDNA integrity index can be significantly and independently associated

with tumor response to treatment. Agostini M et al., Ann Surg Oncol. 2011 Mar

17. Also, the presence or absence of certain genetic alterations and/or differences in copy number variation has been associated with response to chemotherapy and/or prognosis of a disease. See, e.g., Savas S., Acta Oncol.

2010 Nov;49(8):1217-26. Epub 2010 Jul 29, which describes useful genetic

variations for determination of treatment response and survival in cancer. For

example, the detection of cfDNA levels combined with detection of mutations

in the K-RAS gene and/or the p53 gene provide a powerful, relatively non

invasive tool in measuring the prognosis of various cancers, including ovarian

cancer, endometrial cancer and lymphomas. Dobrzycka B et al., Ann Oncol.

2011 May;22(5):1133-40. Epub 2010 Nov 23; Dobrzycka B et al., Int J Cancer.

2010 Aug 1;127(3):612-21; Hosny G et al., Cancer Lett. 2009 Mar

18;275(2):234-9. Epub 2008 Nov 28. Such analysis can be further assisted

using tools such as Varietas, a functional database portal for identification of

genetic variation and association with treatment outcomes and prognosis.

Paananen J et al., Database (Oxford). 2010 Jul 29;2010:baqO16.

Use of Assay Systems for Detection of Mixed Samples from Transplant

Patients

[000211] The assay systems of the invention can be used to monitor organ health

in a transplant patient using a combination of detection ofcfDNA and detection

of SNPs or mutations in one or more single genes. Transplanted organs have

genomes that are distinct from the genome of a recipient patient, and organ

health can be detected using assay system. For example, acute cellular

rejection has been shown to be associated with significantly increased levels of

cell-free DNA from the donor genome in heart transplant recipients. Snyder

TM et al., Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):6229-34. Epub

2011 Mar 28. In addition, chemokines and adhesion molecules mediate

allograft rejection by recruiting leukocytes into the allograft, and SNPs located

in interleukin (IL)-8, CXCR1, CXCR2, have been shown to correlate with

allograft outcomes. Ro H. et al., Transplantation. 2011 Jan 15;91(1):57-64.

Thus, the assay systems of the invention can provide noninvasive tests for

monitoring solid organ transplant recipients, and can aid in identification of

early signs of rejection without the necessity of organ biopsies or other more

onerous diagnostic or prognostic techniques.

Use of Assay Systems for Detection in MaternalSamples

[000212] In certain specific aspects, determining the relative percentage of fetal

DNA in a maternal sample may be beneficial in analyzing the amplification

products, as the percentage of fetal DNA in the sample provides important

information on the expected statistical presence of chromosomes, and variation

from that expectation may be indicative of fetal aneuploidy. This may be

especially helpful in circumstances where the level of fetal DNA in a maternal

sample is low, as the percent fetal contribution can be used in determining the

quantitative statistical significance in the variations of levels of identified

selected loci in a maternal sample. In other aspects, the determining of the

relative percent fetal cfDNA in a maternal sample may be beneficial in

estimating the level of certainty or power in detecting a fetal aneuploidy.

[000213] In some specific aspects, the relative fetal contribution of maternal

DNA at the allele of interest can be compared to the paternal contribution at

that allele to determine approximate fetal DNA concentration in the sample. In other specific aspects, the relative quantity of solely paternally-derived sequences (e.g., Y-chromosome sequences or paternally-specific polymorphisms) can be used to determine the relative concentration of fetal

DNA in a maternal sample.

[000214] Another exemplary approach to determining the percent fetal

contribution in a maternal sample is through the analysis of DNA fragments

with different patterns of DNA methylation between fetal and maternal DNA.

In a preferred aspect, the amplified DNA from cell-free DNA is by

polymerase chain reaction (PCR). Other mechanisms for amplification can be

used as well, including those described in more detail herein, as will be

apparent to one skilled in the art upon reading the present disclosure.

[000215] In circumstances where the fetus is male, percent fetal DNA in a

sample can be determined through detection of Y-specific loci and

comparison to calculated maternal DNA content. Quantities of an amplified

Y-specific locus, such as a region from the sex-determining region Y gene

(SRY), which is located on the Y chromosome and is thus representative of

fetal DNA, can be determined from the sample and compared to one or more

amplified selected loci which are present in both maternal DNA and fetal

DNA and which are preferably not from a chromosome believed to

potentially be aneuploid in the fetus, e.g., an autosomal region that is not on

chromosome 21 or 18. Preferably, this amplification step is performed in

parallel with the selective amplification step, although it may be performed

either before or after the selective amplification depending on the nature of

the multiplexed assay.

[000216] In particular aspects, the percentage of cell-free fetal DNA in a

maternal sample can determined by PCR using serially diluted DNA isolated

from the maternal sample, which can accurately quantify the number of

genomes comprising the amplified genes. PCR using serially diluted DNA

isolated from the maternal sample may be preferred when determining

percent fetal DNA with a male fetus. For example, if the blood sample

contains 100% male fetal DNA, and 1:2 serial dilutions are performed, then

on average the Y-linked signal will disappear 1 dilution before the autosomal

signal, since there is 1 copy of the Y-linked gene and 2 copies of the

autosomal gene.

[000217] In a specific aspect, the percentage of free fetal DNA in maternal

plasma is calculated for a male fetus using the following formula: percentage

of free fetal DNA = (No. of copies of Y-linked gene x 2 x 100)/(No. of copies

of autosomal gene), where the number of copies of each gene is determined

by observing the highest serial dilution in which the gene was detected. The

formula contains a multiplication factor of 2, which is used to normalize for

the fact that there is only 1 copy of the Y-linked gene compared to two copies

of the autosomal gene in each genome, fetal or maternal.

Determinationof Minor source DNA Content in a Mixed Sample

[000218] In certain aspects of the invention, determination of the contribution of

DNA form a minor source may be useful in determining copy number variation

of loci in those samples. For example, in each maternally-derived sample, the

DNA from a fetus will inherit approximately 50% of genetic loci inherited

from the mother and 50% of genetic loci from the father. Determining the loci

contributed to the fetus from non-maternal sources allows the estimation of fetal DNA in a maternal sample, and thus provides information used to calculate the statistically significant differences in chromosomal frequencies for chromosomes of interest.

[000219] In certain aspects, the determination of minor source polymorphisms

requires targeted SNP and/or mutation analysis to identify the presence of the

minor source DNA in a mixed sample. The information needed for this

calculation can be provided using the assay of the invention. In some aspects,

the use of prior genotyping is helpful, e.g., genotyping of the donor of a

transplant, genotyping of the father and mother in a maternal sample. But

generally this information pertaining to the prior genotyping is not necessary

prior to performing the assay, and the genotyping is performed simultaneously

with the determination of copy number of selected loci within a mixed sample.

[000220] In one preferred aspect, the percent minor source nucleic acids in a

mixed sample can be quantified using multiplexed SNP detection without using

prior genotypic knowledge. In this aspect, two or more selected polymorphic

loci with a known SNP in each region are used. In a preferred aspect, the

selected polymorphic loci are loci amplified. In a preferred aspect, the

amplification is universal. In a preferred embodiment, the selected

polymorphic loci are amplified in one reaction in one vessel. Each allele of the

selected polymorphic loci in the maternal sample is determined and quantified.

In a preferred aspect, high throughput sequencing is used for such

determination and quantification. Loci are identified where the major and

minor source genotypes are different, e.g., the donor genotype is homozygous

and the recipient genotype is heterozygous. This identification is done by

observing a high relative frequency of one allele (>80%) and a low relative frequency (<20% and >0.15%) of the other allele for a particular selected locus.

The use of multiple loci is particularly advantageous as it reduces the amount

of variation in the measurement of the abundance of the alleles. All or a subset

of the loci that meet this requirement are used to determine minor source

nucleic acid concentration through statistical analysis. In one aspect,

concentration is determined by summing the low frequency alleles from two or

more loci together, dividing by the sum of the high frequency alleles and

multiplying by two. In another aspect, the percent minor source nucleic acid is

determined by averaging the low frequency alleles from two or more loci,

dividing by the average of the high frequency alleles and multiplying by two.

[000221] For many alleles, major and minor source nucleic acid sequences may

be homozygous and identical, and as this information is not distinguishing it is

not useful in the determination of minor source nucleic acid in a mixed sample.

The present invention utilizes allelic information where there is a

distinguishable difference between the cell sources (e.g., a fetal allele

containing at least one allele that differs from the maternal allele) in

calculations of minor source nucleic acid percentages. Data pertaining to

allelic regions that are the same for the major and minor source are thus not

selected for analysis, or are removed from the pertinent data prior to

determination of percentage so as not to swamp out the useful data.

[000222] Exemplary methods for quantifying fetal DNA in maternal plasma can

be found, e.g., in Chu et al., Prenat Diagn 2010; 30:1226-1229, which is

incorporated herein by reference.

[000223] In one aspect, selected loci may be excluded if the amount or

frequency of the region appears to be an outlier due to experimental error, or from idiopathic genetic bias within a particular sample. In another aspect, selected nucleic acids may undergo statistical or mathematical adjustment such as normalization, standardization, clustering, or transformation prior to summation or averaging. In another aspect, selected nucleic acids may undergo both normalization and data experimental error exclusion prior to summation or averaging.

[000224] In a preferred aspect, 12 or more loci are used for the analysis. In

another preferred aspect, 24 or more loci are used for the analysis. In another

preferred aspect, 48 or more loci are used for the analysis. In another aspect,

one or more indices are used to identify the sample

[000225] In a specific aspect, minor source contribution can be quantified using

tandem SNP detection. Techniques for identifying tandem SNPs in DNA

extracted from, e.g., a maternal sample are disclosed in Mitchell et al, US Pat.

No. 7,799,531 and U.S. Pat App Nos. 12/581,070, 12/581,083, 12/689,924, and

12/850,588. These describe the differentiation of fetal and maternal loci

through detection of at least one tandem single nucleotide polymorphism (SNP)

in a maternal sample that has a different haplotype between the fetal and

maternal genome. Identification and quantification of these haplotypes can be

performed directly on the maternal sample, as described in the Mitchell et al.

disclosures, and used to determine the percent fetal contribution in the maternal

sample.

[000226] As a sample which has not previously been tested for fetal sex has an

approximate 50% likelihood that the fetus will be male, Y-specific sequences

will only be applicable in half of such samples. In a specific aspect, the polymorphic loci used for determination of fetal source contribution are not on the Y chromosome.

[000227] Once the percent cfDNA has been calculated for the minor source, this

data may be combined with methods for aneuploidy detection to determine the

likelihood that a mixed sample may contain an aneuploidy. In one aspect, an

aneuploidy detection method that utilizes analysis of random DNA segments is

used, such as that described in, e.g., Quake, US Pat App No. 11/701,686;

Shoemaker et al., US Pat App No. 12/230,628. In a preferred aspect,

aneuploidy detection methods that utilize analysis of selected loci in a mixed

sample include both regions for determination of minor source DNA content as

well as non-polymorphic regions from two or more chromosomes to detect a

chromosomal abnormality in a single reaction. The single reaction helps to

minimize the risk of contamination or bias that may be introduced during

various steps in the assay system which may otherwise skew results when

utilizing minor source DNA content to help determine the presence or absence

of a chromosomal abnormality. In other aspects, a selected locus or regions

may be utilized both for determination of minor source DNA content as well as

detection of minor source chromosomal abnormalities. Utilizing the same

regions for both DNA content and detection of chromosomal abnormalities

may further help minimize any bias due to experimental error or contamination.

EXAMPLES

[000228] The following examples are put forth so as to provide those of ordinary

skill in the art with a complete disclosure and description of how to make and use

the present invention, and are not intended to limit the scope of what the inventors

regard as their invention, nor are they intended to represent or imply that the

experiments below are all of or the only experiments performed. It will be

appreciated by persons skilled in the art that numerous variations and/or

modifications may be made to the invention as shown in the specific aspects

without departing from the spirit or scope of the invention as broadly described.

The present aspects are, therefore, to be considered in all respects as illustrative

and not restrictive.

[000229] Efforts have been made to ensure accuracy with respect to numbers

used (e.g., amounts, temperature, etc.) but some experimental errors and

deviations should be accounted for. Unless indicated otherwise, parts are parts by

weight, molecular weight is weight average molecular weight, temperature is in

degrees centigrade, and pressure is at or near atmospheric.

Example 1: General Aspects of the Assay Systems of the Invention

[000230] A number of assay formats were tested to demonstrate the ability to

perform selective amplification and detection of independent loci to demonstrate

multiplexed, ligation-based detection of a large number (e.g., 96 or more) of loci

of interest. These loci included loci that were indicative of the presence of a

particular chromosome or the presence or absence of a mutation or polymorphism

in a particular allele.

[000231] These assays were designed based on human genomic sequences, and

each interrogation consisted of two fixed sequence oligos per selected locus

interrogated in the assay. The first oligo, complementary to the 3' region of a

genomic region, comprised the following sequential (5' to 3') oligo elements: a

universal PCR priming sequence common to all assays:

TACACCGGCGTTATGCGTCGAGAC (SEQ ID NO:1); a nine nucleotide

identification index specific to the selected locus; a 9 base locus- or locus/allele

specific sequence that acts as a locus index in the first SNP-independent set and a

locus/allele index in the polymorphism-specific second set; a hybridization

breaking nucleotide which is different from the corresponding base in the genomic

locus; and a 20-24 bp sequence complementary to the selected genomic locus. In

cases where a SNP or mutation was detected in this portion of the selected

genomic locus, the allele-specific interrogation set consisted of two first fixed

sequence tandem ligation primers, each with a different locus/allele index and a

different allele-specific base at the SNP position. These first oligos were designed

for each selected nucleic acid to provide a predicted uniform Tm with a two degree

variation across all interrogations in the assay set.

[000232] The second fixed sequence oligo, complementary to the 5' region of

the genomic loci, comprised the following sequential (5' to 3') elements: a 20-24b

sequence complimentary to the 5' region in the genomic locus; a hybridization

breaking nucleotide different from the corresponding base in the genomic locus;

and a universal PCR priming sequence which was common to all third oligos in

the assay set: ATTGCGGGGACCGATGATCGCGTC (SEQ ID NO:2).

[000233] In cases where a SNP or mutation was detected in the selected genomic

locus, the allele-specific interrogation set consisted of two tandem ligation primers, each with a different locus/allele index and a different allele-specific base at the mutation/SNP position. This second fixed sequence oligo was designed for each selected nucleic acid to provide a predicted uniform Tm with a two degree variation across all interrogations in the assay set that was substantially the same Tmrange as the first oligo set.

[000234] In certain tested aspects, one or more bridging oligos were used that

were complementary to the genomic locus sequence between the region

complementary to the first and second fixed sequence oligos used for each

selected locus. In specific aspects tested, more than one bridging oligo was

used to span the gap between the fixed sequence oligonucleotides, and the one

or more bridging oligo may optionally be designed to identify one or more

mutations or SNPs in the sequence. The length of the bridging

oligonucleotides used in the assay systems varied from 5 to 36 base pairs.

[000235] All oligonucleotides used in the tandem ligation formats were

synthesized using conventional solid-phase chemistry. The second fixed

sequence oligos and the bridging oligonucleotides were synthesized with 5'

phosphate moieties to enable ligation to 3' hydroxyl termini of adjacent

oligonucleotides.

Example 2: Preparation of DNA for Use in Tandem Ligation Procedures

[000236] Genomic DNA from a Caucasian male (NA12801) or a Caucasian

female (NA11995) was obtained from Coriell Cell Repositories (Camden, New

Jersey) and fragmented by acoustic shearing (Covaris, Woburn, MA) to a mean

fragment size of approximately 200bp.

[000237] The Coriell DNA was biotinylated using standard procedures. Briefly,

the Covaris fragmented DNA was end-repaired by generating the following reaction in a 1.5 ml microtube: 5pg DNA, 12 pI loX T4 ligase buffer

(Enzymatics, Beverly MA), 50 U T4 polynucleotide kinase (Enzymatics, Beverly

MA), and H 2 0 to 120 pl. This was incubated at 37°C for 30 minutes. The DNA

was diluted using 10 mM Tris 1mM EDTA pH 8.5 to desired final concentration

of -2 ng/pl.

[000238] 5 pl DNA was placed in each well of a 96-well plate, and the plate

sealed with an adhesive plate sealer and spun for 10 seconds at 250 x g. The plate

was then incubated at 95°C for 3 minutes, cooled to 25°C, and spun again for 10

seconds at 250 x g. A biotinylation master mix was prepared in a 1.5ml microtube

to final concentration of: IX TdT buffer (Enzymatics, Beverly, MA), 8U TdT

(Enzymatics, Beverly, MA), 250 PM CoCl 2 , 0.01 nmol/pl biotin-16-dUTP

(Roche, Nutley NJ), and H 2 0 to 1.5 ml. 15 pl of the master mix was aliquoted into

each well of a 96 well plate, and the plate sealed with adhesive plate sealer. The

plate was spun for 10 seconds at 250 x g and incubated for 37°C for 60 minutes.

Following incubation, the plate was spun again for 10 seconds at 250 x g, and 7.5

Il precipitation mix (1 g/pl Dextran Blue, 3mM NaOAC) was added to each

well.

[000239] The plate was sealed with an adhesive plate sealer and mixed using an

IKA plate vortexer for 2 minutes at 3000 rpm. 27.5 pl of isopropanol was added

into each well, the plate sealed with adhesive plate sealer, and vortexed for 5

minutes at 3000 rpm. The plate was spun for 20 minutes at 3000 x g, the

supernatant was decanted, and the plate inverted and centrifuged at 10 x g for 1

minute onto an absorbent wipe. The plate was air-dried for 5 minutes, and the

pellet resuspended in 30 pl10mM Tris pH8.0, 1mM EDTA.

Example 3: Exemplary Assay Formats using Tandem Ligation

[000240] Numerous tandem ligation assay formats using the biotinylated DNA

were tested to illustrate proof of concept for the assay systems of the invention,

and demonstrated the ability to perform highly multiplexed, targeted detection of a

large number of independent loci using the series of different assay formats. The

exemplary assay systems of the invention were designed to comprise 96 or more

interrogations per loci in a genetic sample, and in cases where SNPs were detected

the assay formats utilized 192 or more separate interrogations, each utilizing the

detection of different alleles per 96 loci in genetic samples. The examples

described for each assay format utilized two different sets of fixed sequence

oligonucleotides and/or bridging oligos (as described in Example 1), comprising a

total 96 or 192 interrogation reactions for the selected loci depending upon

whether or not SNPs were identified.

[000241] A first exemplary assay format used locus-specific fixed sequence

oligos and bridging oligos, where there was a one base gap between the first fixed

sequence oligo and the bridging oligos, and a second one base gap between the

bridging oligos and the second fixed sequence oligo. Each of the two gaps

encompassed two different SNPs. In this format, a DNA polymerase was used to

incorporate each of the SNP bases, and ligase was used to seal the nicks formed

thereby. SNP base discrimination derived from the fidelity of base incorporation

by the polymerase, and in the event of mis-incorporation, the tendency of ligase to

not seal nicks adjacent to mismatched bases.

[000242] The second exemplary assay format used two locus-specific fixed

sequence oligonucleotides without a bridging oligo, where there was a -15-35

base gap between the fixed sequence oligos, and where the gap spanned one or

more SNPs. In this format, a polymerase was used to incorporate the missing bases of the gap, and a ligase was used to seal the nick formed thereby. SNP base discrimination derived from the fidelity of base incorporation by the polymerase, and in the event of misincorporation, the tendency of ligase to not seal nicks adjacent to mismatched bases.

[000243] A third exemplary assay format used allele-specific first and second

fixed sequence oligos without a bridging oligo, where there was a -15-35 base

gap between the first and second fixed sequence oligos, and where the gap

spanned one or more SNPs. Two separate allele-specific first fixed sequence

oligos and two separate allele-specific second fixed sequence oligos were used. A

polymerase was used to incorporate the missing bases, and a ligase was used to

seal the nick formed thereby. SNP base discrimination derived from hybridization

specificity, the tendency of non-proofreading polymerase to not extend annealed

primers with mismatches near the 3' end, and the tendency of the ligase to not seal

nicks adjacent to mismatched bases.

[000244] A fourth exemplary format used allele-specific fixed sequence oligos

and a locus-specific bridging oligo. In this format, two separate fixed sequence

oligos complementary to the 3'end of the loci of interest, the first with a 3' base

specific for one allele of the targeted SNP, and the second with a 3' base specific

for the other allele of the targeted SNP. Similarly, two separate second fixed

sequence oligos were used, the first with a 5' base specific for one allele of a

second targeted SNP, and the second with a 5' base specific for the other allele of

the second targeted SNP. The bridging oligos were complementary to the region

directly adjacent to the locus regions complementary to the first and second fixed

sequence oligos, and thus no polymerase was needed prior to ligation. Ligase was

used to seal the nicks between the fixed sequence oligos and the bridging oligo.

SNP base discrimination in this assay format derived from hybridization

specificity and the tendency of the ligase to not seal nicks adjacent to mismatched

bases. This exemplary format was tested using either T4 ligase or Taq ligase for

creation of the contiguous template, and both were proved effective in the reaction

as described below.

[000245] A fifth exemplary format used locus-specific fixed sequence oligos

that were complementary to adjacent regions on the nucleic acid of interest, and

thus no gap was created by hybridization of these oligos. In this format, no

polymerase was required, and a ligase was used to seal the single nick between the

oligos.

[000246] A sixth exemplary format used allele-specific fixed sequence oligos

and locus-specific bridging oligos, where there was a short base gap of five bases

between the loci region complementary to the fixed sequence oligos. The locus

specific bridging oligo in this example was a 5mer complementary to the regions

directly adjacent to the regions complementary to the first and second fixed

sequence oligos. In this format, no polymerase was required, and a ligase was

used to seal the two nicks between the oligos.

[000247] A seventh exemplary format used locus-specific fixed sequence oligos

and a locus-specific bridging oligo, where there was a shorter base gap of five

bases containing a SNP in the region complementary to the bridging oligo.

Allele-specific bridging oligos corresponding to the possible SNPs were included

in the hybridization and ligation reaction. In this format, no polymerase was

required, and a ligase was used to seal the two nicks between the oligos. SNP

base discrimination in this assay format derived from hybridization specificity and

the tendency of the ligase to not seal nicks adjacent to mismatched bases.

[000248] An eighth exemplary format used locus-specific fixed sequence oligos

and two adjacent locus-specific bridging oligos, where there was a 10 base gap

between the regions complementary to the first and second fixed sequence oligos.

Locus-specific bridging oligos were included in the ligation reaction, with the gap

requiring two contiguous 5mers to bridge the gap. In this format, no polymerase

was required, and a ligase was used to seal the three nicks between the oligos.

[000249] For each of the above-described assay formats, an equimolar pool (40

nM each) of sets of first and second loci- or allele-specific fixed sequence

oligonucleotides was created from the oligos prepared as set forth in Example 2. A

separate equimolar pool (20 pM each) of bridging oligonucleotides was likewise

created for the assay processes based on the sequences of the selected genomic

loci.

[000250] 100pg of strepavidin beads were transferred into the wells of a 96 well

plate, and the supernatant was removed. 60 pl BB2 buffer (100mM Tris pH 8.0,

10mM EDTA, 500mM NaCl 2 , 58% formamide, 0.17% Tween-80), 10 pL 40 nM

fixed sequence oligo pool and 30 pL of the biotinylated template DNA prepared

in Example 2 were added to the beads. The plate was sealed with an adhesive

plate sealer and vortexed at 3000 rpm until beads were resuspended. The oligos

were annealed to the template DNA by incubation at 70°C for 5 minutes, followed

by slow cooling to room temperature.

[000251] The plate was placed on a raised bar magnetic plate for 2 minutes to

pull the magnetic beads and associated DNA to the side of the wells. The

supernatant was removed by pipetting, and was replaced with 50pl of 60% BB2

(v/v in water). The beads were resuspended by vortexing, placed on the magnet

again, and the supernatant was removed. This bead wash procedure was repeated once using 50 pl 60% BB2, and repeated twice more using 50 pl wash buffer

(10mM Tris pH 8.0, 1mM EDTA, 50mM NaCl 2 ).

[000252] The beads were resuspended in 37 pl ligation reaction mix consisting

of IX Taq ligase buffer (Enzymatics, Beverly, MA), 1U Taq ligase, and 2 pM

bridging oligo pool (depending on the assay format), and incubated at 37°C for

one hour. Where appropriate, and depending on the assay format, a non

proofreading thermostable polymerase plus 200nM each dNTP was included in

this mixture. The plate was placed on a raised bar magnetic plate for 2 minutes to

pull the magnetic beads and associated DNA to the side of the wells. The

supernatant was removed by pipetting, and was replaced with 50pLwash buffer.

The beads were resuspended by vortexing, placed on the magnet again, and the

supernatant was removed. The wash procedure was repeated once.

[000253] To elute the products from the strepavidin beads, 30 pl of 10mM Tris

1mM EDTA, pH 8.0 was added to each well of 96-well plate. The plate was

sealed and mixed using an IKA vortexer for 2 minutes at 3000 rpm to resuspend

the beads. The plate was incubated at 95°C for 1 minute, and the supernatant

aspirated using an 8-channel pipetter. 25 pl of supernatant from each well was

transferred into a fresh 96-well plate for universal amplification.

Example 4: Universal Amplification of Tandem Ligated Products

[000254] The polymerized and/or ligated nucleic acids were amplified using

universal PCR primers complementary to the universal sequences present in the

first and second fixed sequence oligos hybridized to the loci of interest. 25 pl of

each of the reaction mixtures of Example 3 were used in each amplification

reaction. A 50 pl universal PCR reaction consisting of 25 pl eluted ligation product plus IX Pfusion buffer (Finnzymes, Finland), IM Betaine, 400nM each dNTP, 1 U Pfusion error-correcting thermostable DNA polymerase, and the following primer pairs:

TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGA

(SEQ ID NO:3) and

TCAAGCAGAAGACGGCATACGAGATXAAACGACGCGATCATCGGTCC

CCGCAA (SEQ ID NO:4), where X represents one of 96 different sample indices

used to uniquely identify individual samples prior to pooling and sequencing. The

PCR was carried out under stringent conditions using a BioRad TetradTM

thermocycler.

[000255] 10 pl of universal PCR product from each of the samples were pooled

and the pooled PCR product was purified using AMPureXPTM SPRI beads

(Beckman-Coulter, Danvers, MA), and quantified using Quant-iTTM PicoGreen,

(Invitrogen, Carlsbad, CA).

Example 5: Detection and Analysis of Selected Loci

[000256] The purified PCR products of each assay format were sequenced on a

single lane of a slide on an Illumina HiSeqTM 2000 (Illumina, San Diego, CA).

Sequencing runs typically give rise to -100M raw reads, of which -85M (85%)

mapped to expected assay structures. This translated to an average of -885K

reads/sample across the experiment, and (in the case of an experiment using 96

loci) 9.2K reads/replicate/locus across 96 loci. The mapped reads were parsed into

replicate/locus/allele counts, and various metrics were computed for each

condition,including:

[000257] Yield: a metric of the proportion of input DNA that was queried in

sequencing, computed as the average number of unique reads per locus (only

counting unique identification index reads per replicate/locus) divided by the total

number of genomic equivalents contained in the input DNA.

[000258] 80 percentile locus frequency range: a metric of the locus frequency

variability in the sequencing data, interpreted as the fold range that encompasses

80% of the loci. It was computed on the distribution of total reads per locus,

across all loci, as the 90h percentile of total reads per locus divided by the 10t

percentile of the total reads per locus.

[000259] SNP error rate: a metric of the error rate at the SNP position, and

computed as the proportion of reads containing a discordant base at the SNP

position.

[000260] These results are summarized in Table 1:

Table 1:Results Summary of Tandem Ligation Assay Formats

HIXED BRDIG80% SN ASSAY SEQUENCE BROLGING ENZYME YIELD NPERR FORMAT OLIGO (1 and/or USED FREQ 2 d) USED RANGE RATE L OCUS-SPECIFIC Locu pol+lig 9.5% 5.3 0.18% specific 2 LOCUS-SPECIFIC No pol+lig 1.4% 58.3 0.19%

3 APEFI No pol+lig 0.4% 61.7 1.00% 4 ALLELE- Locus Ta lig 5.0% 5.9 0.92% SPECIFIC specific 4 ALLELE- Locus T4 lig 5.3% 4.4 0.95% SPECIFIC specific 5 LOCUS-SPECIFIC No Taq lig 22.5% 1.7 N/A 6 LOCUS-SPECIFIC Locus Taq lig 12.5 2.9 N/A specific

7 LOCUS-SPECIFIC Allele Taq lig 14.3 2.8 0.20% specific 8 LOCUS-SPECIFIC 2 Locus Taq lig 18.5% 2.8 N/A specific

[000261] Table 1 indicates that the locus-specific tandem ligation assay using a

bridging oligo converted template DNA into targeted product with high yield

(-10%), with a high proportion of product derived from targeted loci (15% of

reads did not contain expected assay structures), with limited locus bias (80% of

loci fall within a -5-fold concentration range), and with high SNP accuracy (0.2%

SNP error rate). The locus-specific tandem ligation assay without the use of a

bridging oligo produced reduced yields and substantial locus bias, but still

produced high accuracy SNP genotyping data. The allele-specific tandem ligation

assay with a bridging oligo produced intermediate yields compared to the locus

specific assay using both T4 and Taq ligase, but still produced limited locus bias

and high accuracy SNP genotyping data. The allele-specific tandem ligation assay

without a bridging produced reduced yields and substantial locus bias, but still

produced high accuracy SNP genotyping data.

[000262] Assay formats six through eight showed that template DNA can be

converted into targeted product with high yield (12-18%), with a high proportion

of product derived from targeted loci (-76% of reads contained expected assay

structures), and with limited locus bias (80% of loci fall within a 2-3-fold

concentration range). Figure 5 illustrates the genotyping performance that was

obtained using assay format seven, comparing the sequence counts for the two

alleles of all polymorphic assays observed in a single sample. Note the clear

separation of the homozygous and heterozygous clusters, as well as the low

background counts observed amongst the homozygous clusters.

Example 6: Determination of Percent Fetal DNA using Tandem Ligation

[000263] One exemplary assay system of the invention was designed to

determine percent fetal DNA concentration in a genetic sample as well as to

provide counts for selected loci within the sample. This exemplary assay

comprised 480 separate interrogations, each utilizing the detection of different

loci in a maternal sample. The initial example utilized a determination of

percent fetal DNA in subjects carrying a male fetus, and so loci on the Y

chromosome were utilized as well as loci containing a paternally-inherited fetal

SNP that is different from the maternal sequence.

[000264] Specifically, 480 selected nucleic acids were interrogated using the

assay system. The 480 selected nucleic acids comprised 48 sequence-specific

interrogations of nucleic acids corresponding to loci on chromosome Y, 192

sequence-specific interrogations of nucleic acids corresponding to loci on

chromosome 21, 192 sequence-specific interrogations of selected nucleic acids

corresponding to loci on chromosome 18, and 144 sequence-specific

interrogations of selected nucleic acids corresponding to polymorphic loci on

chromosomes 1-16 which. These assays were designed based on human

genomic sequences, and each interrogation used three oligos per selected

nucleic acid interrogated in the assay.

[000265] The first oligo used for each interrogation was complementary to the 3'

region of the selected genomic region, and comprised the following sequential

(5' to 3') oligo elements: a universal PCR priming sequence common to all

assays: TACACCGGCGTTATGCGTCGAGAC (SEQ ID NO:1); an

identification index specific to the selected loci comprising nine nucleotides;

and a 20-24 bp sequence complementary to the selected genomic locus. This

first oligo was designed for each selected nucleic acid to provide a predicted uniform Tm with a two degree variation across all interrogations in the 480 assay set.

[000266] The second oligo used for each interrogation was a bridging oligo

complementary to the genomic locus sequence directly adjacent to the genomic

region complementary to the first oligonucleotide. Based on the selected

nucleic acids of interest, the bridging oligos were designed to allow utilization

of a total of 12 oligonucleotide sequences that could serve as bridging oligos

for all of the 480 interrogations in the assay set.

[000267] The third oligo used for each interrogation was complementary to the

5' region of the selected genomic locus, comprised the following sequential (5'

to 3') elements: a 20-24b sequence complimentary to the 5' region in the

genomic locus; a hybridization breaking nucleotide which was different from

the corresponding base in the genomic locus; and a universal PCR priming

sequence which was common to all third oligos in the assay set:

ATTGCGGGGACCGATGATCGCGTC (SEQ ID NO:2). This third oligo was

designed for each selected nucleic acid to provide a predicted uniform Tm with

a two degree variation across all interrogations in the 480 assay set, and the Tm

range was substantially the same as the Tmrange as the first oligo set.

[000268] All oligonucleotides were synthesized using conventional solid-phase

chemistry. The first and bridging oligonucleotides were synthesized with 5'

phosphate moieties to enable ligation to 3' hydroxyl termini of adjacent

oligonucleotides. An equimolar pool of sets of the first and third

oligonucleotides used for all interrogations in the multiplexed assay was

created, and a separate equimolar pool of all bridging oligonucleotides was

created to allow for separate hybridization reactions.

[000269] Genomic DNA was isolated from 5mL plasma using the Dynal Silane

viral NA kit (Invitrogen, Carlsbad, CA). Approximately 12ng DNA was

processed from each of 37 females, including 7 non-pregnant female subjects,

10 female subjects pregnant with males, and 22 female subjects pregnant with

females. The DNA was biotinylated using standard procedures, and the

biotinylated DNA was immobilized on a solid surface coated with streptavidin

to allow retention of the genomic DNA in subsequent assay steps.

[000270] The immobilized DNA was hybridized to the first pool comprising the

first and third oligos for each interrogated sequences under stringent

hybridization conditions. The unhybridized oligos in the pool were then

washed from the surface of the solid support, and the immobilized DNA was

hybridized to the pool comprising the bridging oligonucleotides under stringent

hybridization conditions. Once the bridging oligonucleotides were allowed to

hybridize to the immobilized DNA, the remaining unbound oligos were washed

from the surface and the three hybridized oligos bound to the selected loci were

ligated using T4 ligase to provide a contiguous DNA template for

amplification.

[000271] The ligated DNA was amplified from the solid substrate using an error

correcting thermostable DNA polymerase, a first universal PCR primer

TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGA

GA (SEQ ID NO:3) and a second universal PCR primer

TCAAGCAGAAGACGGCATACGAGATXAAACGACGCGATCATCGGTC

CCCGCAA (SEQ ID NO:4), where X represents one of 96 different sample

indices used to uniquely identify individual samples prior to pooling and

sequencing. 10pL of universal PCR product from each of the 37 samples described above were and the pooled PCR product was purified using

AMPureTM SPRI beads (Beckman-Coulter, Danvers, MA), and quantified using

Quant-iTTM PicoGreen, (Invitrogen, Carlsbad, CA).

[000272] The purified PCR product was sequenced on 6 lanes of a single slide

on an Illumina HiSeqTM 2000. The sequencing run gave rise to 384M raw

reads, of which 343M (89%) mapped to expected genomic loci, resulting in an

average of 3.8M reads per sample across the 37 samples, and 8K reads per

sample per locus across the 480 loci. The mapped reads were parsed into

sample and locus counts, and two separate metrics of percent fetal DNA were

computed as follows.

[000273] Percent male DNA detected by chromosome Y loci corresponds to the

relative proportion of reads derived from chromosome Y locus interrogations

versus the relative proportion of reads derived from autosomal locus

interrogations, and was computed as (number of chromosome Y reads in a test

subject/number of autosome reads in test subject)/(number of reads in male

control subject/number of autosome reads in the male control subject). This

metric was used as a measure of percent fetal DNA in the case of a male fetus

using the relative reads of chromosome Y.

[000274] Percent fetal DNA detected by polymorphic loci corresponds to the

proportion of reads derived from non-maternal versus maternal alleles at loci

where such a distinction can be made. First, for each identified locus, the

number of reads for the allele with the fewest counts (the low frequency allele)

was divided by the total number of reads to provide a minor allele frequency

(MAF) for each locus. Then, loci with an MAF between 0.075% and 15%

were identified as informative loci. The estimated percent fetal DNA for the sample was calculated as the mean of the minor allele frequency of the informative loci multiplied by two, i.e. computed as 2X average (MAF) occurrence where 0.075%<MAF<15%.

[000275] Figure 6 demonstrates the results from these computations. As shown

in Figure 6, the percent male loci determined using the above-described

chromosome Y metrics (grey circles) can separate pregnancies involving male

fetuses from pregnancies involving female fetuses (grey diamonds) and non

pregnant samples (black circles). In addition, computation of the percent fetal

amount in a sample by polymorphic loci metric can distinguish pregnant

samples from non-pregnant samples. Finally, there was a correlation between

the percent fetal DNA estimates for a sample obtained from chromosome Y

and polymorphic loci in pregnancies involving male fetuses. This correlation

persists down to quite low percent fetal values.

Example 7: Detection of Aneuploidy in a Maternal Sample

[000276] The assay systems of the invention were used in the detection of

polymorphisms and chromosomal abnormalities in two separate cohorts of

pregnant females. A first cohort of 190 normal, 36 T21, and 8 T18 pregnancies

and a second cohort of 126 normal, 36 T21, and 8 T18 pregnancies were tested

for fetal aneuploidy. The chromosomal aneuploidies were detected using 576

chromosome 21 and 576 chromosome 18 assays, pooled together and assayed

in a single reaction, as set forth below.

[000277] The elements used in the aneuploidy detection assays are illustrated in

FIG. 7. The cfDNA 701 isolated from maternal samples was used as a template

for hybridization, ligation, and amplification of multiple selected loci from both chromosome 21 and chromosome 18 in each maternal sample. Three oligonucleotides were hybridized to each selected locus to create ligation products for amplification and detection. The left (or first) fixed sequence oligonucleotide comprised a region complementary to a selected locus 709 and a first universal primer region 711. The right (or second) fixed sequence oligonucleotide 705 comprised a second region complementary to the selected locus 713 and a second universal primer region 715. The bridging oligonucleotides 707 used were designed so that each would hybridize to bridging regions of two or more selected loci used in the aneuploidy detection assay. When the fixed sequence oligonucleotides 703, 705 and the bridging oligonucleotide 707 hybridized to the complementary region on the cfDNA

701, their termini formed two nicks. Upon ligation of the hybridized

oligonucleotides to the cfDNA, a ligation product was created for each selected

locus comprising 703, 705 and 707 which was used as a template for

amplification primers 719, 721.

[000278] Two amplification primers 719, 721 comprising regions

complementary to the first and second universal primer regions, respectively,

were then used to amplify the ligation product. This amplification product

comprised the sequence of the selected locus. The right amplification primer

also comprised a sample index 717 to identify the particular sample from which

the locus was obtained in the multiplexed assay. Amplification with 96 distinct

right amplification primers 729 enabled pooling and simultaneous sequencing

of 96 different amplification products on a single lane.

[000279] The amplification primers 719, 721 also contained a left cluster

sequence 723 (TAATGATACGGCGACCACCGA)(SEQ ID NO:7) and a right cluster sequence 725 (ATCTCGTATGCCGTCTTCTGCTTGA)(SEQ ID

NO:8) that supported cluster amplification for sequencing using the Illumina

HiSeqTM2000 system (Illumina, San Diego, CA). A sequencing primer 727

comprising the first universal primer sequence was used to determine the

sequence of the amplification product, and a second sequencing primer 729

was used to determine the sample index 717 of the amplification product.

[000280] Briefly, approximately 1OmL peripheral blood was collected from each

patient into a BCT tube (Streck, Omaha, NE), which was shipped via overnight

courier to Tandem Diagnostics. Plasma was isolated from BCT tubes within

72h of blood collection by centrifugation at 1600g for 10m. The plasma was

transferred to a second tube and centrifuged at 16000g for 10m to remove any

remaining cells. cfDNA was isolated from 4-5mL plasma per patient.

Approximately 15ng cfDNA was isolated from each patient sample and arrayed

into individual wells of a 96 well plate. All subsequent processing occurred on

multiplexed batches of up to 96 cfDNA patient samples per array system

method.

[000281] cfDNA isolated from the maternal samples in each well was

biotinylated precipitated and resuspended in 30uL TE as in Example 3 above.

The biotinylated template DNA was mixed with100ug MyOneC1 streptavidin

coated magnetic beads (Life Technologies, Carlsbad, CA), 60 pl BB2 buffer

(100mM Tris pH 8.0, 10mM EDTA, 500mM NaC 2, 58% formamide, 0.17%

Tween-80), and10 pL of pooled 40 nM left 703 and right 705 fixed sequence

oligonucleotides.. The mixture was heated to70°C, and cooled 2 hours. The

beads were then magnetically immobilized to the side of the well, washed twice

with 50uL 60% BB2 (v/v with H20), washed twice more with 50 pl wash buffer (10mM Tris pH 8.0, 1mM EDTA, 50mM NaCl2), and then resuspended in a 50pL reaction containing 1U Taq ligase (Enzymatics, Beverly MA), IX

Taq ligase buffer (Enzymatics), and 1OuM of a 5'-phosphorylated 5mer

bridging oligonucleotide 707. The mixture was incubated at 37C for 1 hour.

The beads were again magnetically immobilized to the side of the well, washed

twice with 50uL wash buffer and then resuspended in 30pL TE.

[000282] The ligation products were eluted from the immobilized beads by

incubation at 95°C for 3 minutes. The eluted ligation products were amplified

by 26 cycles of PCR in a 50uL reaction containing 1U Pfusion polymerase

(Thermo Fisher, Waltham MA), IM Betaine, IX Pfusion buffer, and 400nM

left and right amplification primers (719, 721 respectively). The right primer

contained a 7 base sample index (717) that enabled 96 sample multiplexed

sequencing on the HiSeq2000 (Illumina, San Diego, CA). The sequence of the

left fixed sequence oligo was:

TAATGATACGGCGACCACCGAGATCTACACCGGCGTTATGCGTCGAGA C

(SEQ ID NO: 5)

[000283] And the sequence of the right fixed sequence oligo was:

TCAAGCAGAAGACGGCATACGAGATNNNNNNNAAACGACGCGATCATCG

GTCCCCGCAAT (SEQ ID NO:6)

[000284] Amplification products from a single 96 well plate were pooled in

equal volume, and the pooled amplification products were purified with

AMPureXPTM SPRI beads (Beckman-Coulter, Danvers, MA) according to the

manufacturer's instructions. Each purified pooled library was used as template

for cluster amplification on an Illumina TruSeq v2 SR cluster kit flow cell

(Illumina, San Diego, CA) according to manufacturer's protocols. The slide

was processed on an Illumina HiSeqTM 2000 (Illumina, San Diego, CA) to

produce 56 bases of locus-specific sequence from a left sequence primer 723

and a separate read of 8 bases of sample specific sequence was obtained from

the second sequence primer 725. An average of 903K raw reads per sample

were collected. An average of 876K (97%) of the reads was assigned to

expected assay structures.

[000285] FIG. 8 shows exemplary data for a subset of the patient samples from

the second cohort, which were all analyzed in one multiplexed assay on a

single lane of a sequencing run. Initially 96 different samples were run in this

particular run, but -six samples were later excluded from this analytical set as

not meeting sample quality control thresholds. A trimmed mean was calculated

for each chromosome 18 and chromosome 21 for the samples based on reads

produced in the assay. The trimmed mean was computed by removing 10% of

high and low counts for each chromosome by sample. The detected

amplification products corresponding to the various selected loci were used to

compute a chromosome 21 proportion metric and a chromosome 18 proportion

metric for each sample. For chromosome 21 proportion, this was calculated as

the trimmed mean of counts in the 384 chromosome 21 selected loci divided by

the sum of trimmed means of counts for all 576 chromosome 21 loci and 576

chromosome 18 loci for each sample.

[000286] On average 834 read counts were observed per selected locus in the

maternal samples of the first cohort, and 664 read counts were observed per

selected locus from the second cohort. These counts were used to compute

chromosome proportion z-scores for chromosome 21 and chromosome 18.

[000287] Briefly, the z-scores were calculated by scaling the median per locus

count to a common value (e.g., 1000) for each sample, and the scaled counts

were transformed by log base 2. An RMA log linear modeling and median

polish were performed (Bolstad, B.Met al. (2003) Bioinformatics 19(2):185

193; Rafael. A. (2003) Nucleic Acids Research 31(4):e15; Irizarry, RA et al.

(2003) Biostatistics4(2):249-64) to estimate chromosome effects, locus effects,

sample effects, and residuals. The estimated chromosome effects were set to a

common value, e.g., 0, and 2A(chromosome effect + sample effect + residual)

was calculated for each locus to create normalized counts. The Z scores were

scaled using iterative censoring so that they had a mean of 0 and a standard

deviation of 1.

[000288] Data obtained from the first cohort of samples was used to determine

first cohort z-scores for chromosome 21 and chromosome 18 are illustrated in

FIGs. 9 and 10, respectively. The normal samples are shown as dark grey

diamonds, and the samples with a trisomy are shown as light grey diamonds.

179/180 (99.4%) normal samples (dark grey diamonds) had z-scores <3; one

normal sample had a chromosome 21 z-score of 3.4 and a chromosome 18 z

score of 3.0. 35/35 (100%) T21 and 7/7 (100%) T18 samples had chromosome

proportion z-scores >3. The mean T18 z-score was 8.5, and the range was 5.8

10.9. The mean T21 z-score was 11.5, and the range was 6.1-19.8.

[000289] The data provided in HG. 8 was combined with data from the

remaining samples of the second cohort to determine z-scores for chromosome

21 and chromosome 18 are illustrated in FIGs. 11 and 12, respectively. The

normal samples are shown as dark grey diamonds, and the samples with a

trisomy are shown as light grey diamonds. 125/125 normal samples had z scores <3, 36/36 (100%) T21 and 8/8 (100%) T18 samples had z-scores >3.

The mean T18 z-score was 9.5 and the range was 5.1-19.8. The mean T21 z

score was 11.4 and the range was 3.4-21.8.

[000290] In addition to the detection of aneuploidy in these cohorts, specific

polymorphisms were also used to determine percent fetal contribution to the

maternal samples. The general methodology used for determination of these

fetal contribution percentages is described in US Ser No. 61/509,188 filed July

19, 2011, which is incorporated by reference in its entirety.

[000291] Briefly, the sequencing of certain loci having detectable

polymorphisms identified these loci as informative. The counts of the

identified loci having fetal polymorphic regions different from the maternal

polymorphic regions were used to calculate the approximate fetal contribution

for the maternal sample. Each of the loci used in the calculation of percent

fetal contribution to the maternal sample had a minimum of 256 counts.

Exemplary SNP data sets for this calculation are illustrated below in Tables 2

and 3. The data corresponding to the identified informative loci from these sets

were used in the calculation of percent contribution. The informative loci are

shown in each table in bolded text.

Table 2: SNP Detection and Calculated Percent Fetal for Maternal Sample 1

SNP_1 SNP_2 Total Calculated Chromosome/Locus Counts Percent Counts Counts Fetal ChO1_Lc067487 294 26 320 0.210138 ChO1_LcO67489 187 167 354 ChOl_Lc067490 389 1 390 ChO1_LcO67491 233 113 346 ChOl_Lc067492 0 267 267 ChO1_LcO67493 145 132 277 ChO1_LcO67495 106 172 278 ChO1_Lc067496 308 28 336 ChOl_Lc067497 298 0 298 ChOl_Lc067498 310 1 311 ChOl_Lc067499 256 1 257 ChO1_Lc067501 26 273 299 ChOl_Lc067503 296 0 296 ChOl_Lc067504 134 149 283 Ch02_Lc067508 0 337 337 Ch02_Lc067510 37 324 361 Ch02_Lc067511 138 147 285 Ch02_Lc067512 180 251 431 Ch02_Lc067514 0 383 383 Ch02_Lc067515 316 31 347 Ch02_Lc067516 276 2 278 Ch02_Lc067519 42 276 318 Ch02_Lc067521 312 47 359 Ch02_Lc067522 158 170 328 Ch02_Lc067523 38 328 366 Ch02_Lc067524 177 127 304 Ch02_Lc067525 292 0 292 Ch02_Lc067526 361 0 361 Ch02_Lc067527 261 26 287 Ch02_Lc067529 140 146 286 Ch03_Lc067530 0 268 268 Ch03_Lc067531 217 178 395 Ch03_Lc067532 245 153 398 Ch03_Lc067533 1 286 287 Ch03_Lc067534 384 38 422 Ch03_Lc067535 192 114 306 Ch03_Lc067537 32 276 308 Ch03_Lc067538 243 15 258 Ch03_Lc067539 132 247 379 Ch03_Lc067540 162 105 267 Ch03_Lc067541 239 35 274

Ch03_Lc067542 3 406 409 Ch03_Lc067544 2 271 273 Ch03_Lc067545 373 0 373 Ch03_Lc067546 354 0 354 Ch03_Lc067547 1 256 257 Ch03_Lc067548 365 0 365 Ch03_LcO67549 187 111 298 Ch04_Lc067550 33 312 345 Ch04_Lc067552 323 1 324 Ch04_Lc067553 217 119 336 Ch04_Lc067557 35 236 271 Ch04_Lc067558 184 166 350 Ch04_Lc067559 295 32 327 Ch04_Lc067560 140 141 281 Ch04_Lc067561 160 123 283 Ch04_Lc067562 313 2 315 Ch04_Lc067566 142 191 333 Ch04_Lc067569 117 206 323 Ch05_Lc067570 0 403 403 Ch05_Lc067571 229 219 448 Ch05_Lc067572 185 134 319 Ch05_Lc067573 271 22 293 Ch05_Lc067575 261 142 403 Ch05_Lc067578 0 399 399 Ch05_Lc067579 307 46 353 Ch05_Lc067581 189 109 298 Ch05_Lc067582 0 268 268 Ch05_Lc067583 167 203 370 Ch05_Lc067585 209 119 328 Ch05_Lc067586 3 327 330 Ch05_Lc067587 321 0 321 Ch06_Lc067589 286 0 286 Ch06_Lc067590 2 344 346 Ch06_Lc067591 124 179 303 Ch06_Lc067592 0 330 330 Ch06_Lc067593 0 286 286 Ch06_Lc067594 396 2 398 Ch06_Lc067595 349 0 349 Ch06_Lc067597 340 1 341 Ch06_Lc067598 0 412 412 Ch06_Lc067599 182 93 275 Ch06_Lc067600 44 307 351 Ch06_Lc067601 43 324 367 Ch06_Lc067602 358 1 359 Ch07_Lc067603 160 141 301 Ch07_Lc067604 302 0 302

Ch07_Lc067605 37 414 451 Ch07_Lc067606 269 290 559 Ch07_Lc067607 166 159 325 Ch07_Lc067609 1 396 397 Ch07_LcO67610 225 134 359 Ch07_Lc067611 48 391 439 Ch07_LcO67612 2 333 335 Ch07_Lc067614 200 246 446 Ch07_Lc067615 188 184 372 Ch07_Lc067616 167 116 283 Ch07_Lc067617 204 186 390 Ch07_Lc067618 281 28 309 Ch07_Lc067619 44 297 341 Ch07_Lc067620 336 0 336 Ch07_Lc067621 48 342 390 ChO8_Lc067622 313 1 314 ChO8_Lc067623 414 0 414 Ch08_Lc067624 230 142 372 Ch08_Lc067625 0 377 377 Ch08_Lc067626 41 357 398 ChO8_Lc067627 133 258 391 ChO8_Lc067628 388 1 389 Ch08_Lc067629 348 0 348 Ch08_Lc067630 37 314 351 ChO8_Lc067631 185 129 314 Ch08_Lc067632 49 308 357 ChO8_Lc067633 186 195 381 ChO8_Lc067634 174 217 391 ChO8_Lc067635 161 152 313 ChO8_Lc067637 0 284 284 Ch08_Lc067638 343 50 393 Ch09_Lc067639 164 99 263 Ch09_Lc067640 185 186 371 Ch09_Lc067641 344 0 344 Ch09_Lc067642 294 0 294 Ch09_Lc067643 36 336 372 Ch09_Lc067644 221 144 365 Ch09_Lc067645 315 36 351 Ch09_Lc067646 141 143 284 Ch09_Lc067647 33 270 303 Ch09_Lc067648 43 349 392 Ch09_Lc067649 147 152 299 Ch09_Lc067650 201 187 388 Ch09_Lc067651 176 151 327 ChlO_Lc067652 29 277 306 ChlO_Lc067653 134 157 291

Chl0_Lc067654 174 196 370 Chl0_Lc067655 189 181 370 Chl0_Lc067656 125 174 299 Chl0_Lc067657 375 2 377 Chl0_Lc067658 0 345 345 Chl0_Lc067659 204 174 378 Chl0_Lc067661 236 271 507 Ch1O_Lc067662 39 325 364 Ch1l_Lc067663 2 378 380 Chi1_Lc067664 42 298 340 Chl1_Lc067666 196 200 396 Ch1l_Lc067667 220 164 384 Chi1_Lc067668 20 290 310 Chl1_Lc067670 1 356 357 Chl2_Lc067671 212 195 407 Chl2_Lc067673 1 298 299 Chl2_Lc067674 242 30 272 Chl2_Lc067675 2 292 294 Chl2_Lc067676 1 381 382 Chl2_Lc067677 139 179 318

Table 3: Sample 2 SNP Detection and Calculated Percent Fetal

SNP 1 SNP 2 Total Calculate Chromosome/Locus SNts SNts Tots d Percent Counts Counts Counts Fea Fetal ChOl_Lc067487 181 134 315 0.096075 ChO1_Lc067489 18 337 355 ChO1_Lc067490 17 333 350 ChOl_Lc067491 356 0 356 ChO1_Lc067492 12 264 276 ChO1_Lc067493 7 385 392 ChOl_Lc067494 140 118 258 ChOl_Lc067495 163 118 281 ChOl_Lc067496 198 172 370 ChO1_Lc067498 15 301 316 ChOl_Lc067499 162 170 332 ChO1_Lc067501 11 252 263 ChOl_Lc067502 129 130 259 ChOl_Lc067503 148 172 320 ChOl_Lc067504 157 146 303 Ch02_Lc067508 188 196 384 Ch02_Lc067510 1 356 357

Ch02_LcO67511 3 308 311 Ch02_LcO67512 262 193 455 Ch02_LcO67513 318 0 318 Ch02_Lc067514 1 440 441 Ch02_LcO67515 169 166 335 Ch02_LcO67516 189 149 338 Ch02_LcO67519 218 133 351 Ch02_Lc067521 153 180 333 Ch02_Lc067522 14 326 340 Ch02_Lc067523 180 178 358 Ch02_Lc067524 330 1 331 Ch02_Lc067526 202 185 387 Ch02_Lc067527 149 192 341 Ch02_Lc067529 140 160 300 Ch03_Lc067530 132 128 260 Ch03_Lc067531 20 392 412 Ch03_Lc067532 202 270 472 Ch03_Lc067533 328 0 328 Ch03_Lc067534 224 223 447 Ch03_Lc067535 188 142 330 Ch03_Lc067537 315 1 316 Ch03_Lc067538 265 0 265 Ch03_Lc067539 191 214 405 Ch03_Lc067540 166 124 290 Ch03_Lc067542 256 208 464 Ch03_Lc067543 139 123 262 Ch03_Lc067544 190 180 370 Ch03_Lc067545 378 12 390 Ch03_Lc067546 22 352 374 Ch03_Lc067547 184 162 346 Ch03_Lc067548 363 0 363 Ch03_Lc067549 0 312 312 Ch04_Lc067550 162 156 318 Ch04_Lc067552 331 0 331 Ch04_Lc067553 225 155 380 Ch04_Lc067555 121 146 267 Ch04_Lc067557 284 13 297 Ch04_Lc067558 229 208 437 Ch04_Lc067559 7 311 318 Ch04_Lc067560 154 136 290 Ch04_Lc067561 12 258 270 Ch04_Lc067562 410 1 411 Ch04_Lc067566 320 18 338 Ch04_Lc067569 0 289 289 Ch05_Lc067570 19 444 463 Ch05_Lc067571 498 0 498

Ch05_LcO67572 169 182 351 Ch05_Lc067573 294 0 294 Ch05_Lc067575 422 0 422 Ch05_Lc067578 18 388 406 Ch05_Lc067579 17 304 321 Ch05_Lc067580 156 149 305 Ch05_Lc067581 303 19 322 Ch05_Lc067583 23 347 370 Ch05_Lc067585 22 293 315 Ch05_Lc067586 391 0 391 Ch05_Lc067587 434 1 435 Ch05_LcO67588 157 129 286 Ch06_Lc067589 274 0 274 Ch06_Lc067590 23 320 343 Ch06_Lc067591 10 342 352 Ch06_Lc067592 181 177 358 Ch06_Lc067593 0 296 296 Ch06_Lc067594 267 200 467 Ch06_Lc067595 212 201 413 Ch06_Lc067596 329 12 341 Ch06_Lc067597 319 1 320 Ch06_Lc067598 243 186 429 Ch06_Lc067600 0 341 341 Ch06_Lc067601 417 0 417 Ch06_Lc067602 1 340 341 Ch07_Lc067603 168 185 353 Ch07_Lc067604 333 11 344 Ch07_Lc067605 211 287 498 Ch07_Lc067606 2 542 544 Ch07_Lc067607 310 0 310 Ch07_Lc067609 178 189 367 Ch07_Lc067610 425 0 425 Ch07_Lc067611 1 449 450 Ch07_Lc067612 169 149 318 Ch07_Lc067614 0 446 446 Ch07_Lc067615 0 427 427 Ch07_Lc067616 13 278 291 Ch07_Lc067617 239 246 485 Ch07_Lc067618 17 267 284 Ch07_Lc067619 0 335 335 Ch07_Lc067620 319 0 319 Ch07_Lc067621 0 354 354 Ch08_Lc067622 25 232 257 ChO8_Lc067623 470 0 470 ChO8_Lc067624 0 376 376 ChO8_Lc067625 212 202 414

Ch08_Lc067626 377 0 377 Ch08_Lc067627 379 16 395 ChO8_LcO67628 189 210 399 Ch08_Lc067629 16 338 354 Ch08_Lc067630 152 153 305 Ch08_Lc067631 0 379 379 Ch08_LcO67632 25 355 380 Ch08_Lc067633 186 236 422 Ch08_Lc067634 2 375 377 Ch08_Lc067635 169 159 328 Ch08_Lc067636 13 274 287 Ch08_Lc067637 373 11 384 Ch08_Lc067638 28 431 459 Ch09_Lc067640 367 0 367 Ch09_Lc067641 359 0 359 Ch09_Lc067642 307 1 308 Ch09_Lc067643 350 32 382 Ch09_Lc067644 168 241 409 Ch09_Lc067645 174 179 353 Ch09_Lc067646 133 177 310 Ch09_Lc067647 199 188 387 Ch09_Lc067648 24 450 474 Ch09_Lc067649 340 0 340 Ch09_Lc067650 22 348 370 Ch09_Lc067651 20 365 385 ChlO_Lc067652 0 292 292 ChlO_Lc067653 0 279 279 ChlO_Lc067654 0 396 396 ChlO_Lc067655 160 170 330 ChlO_Lc067656 175 121 296 ChlO_Lc067657 212 188 400 ChlO_Lc067658 0 356 356 ChlO_Lc067659 400 13 413 ChlO_Lc067661 263 268 531 ChlO_Lc067662 20 324 344 Chi1_Lc067663 12 357 369 Chl1_Lc067664 142 179 321 Chl_Lc067665 278 0 278 Chl_Lc067666 180 232 412 Chit_Lc067667 13 438 451 Chl_Lc067668 0 365 365 Ch11_Lc067669 15 263 278 Chl_Lc067670 0 374 374 Chl2_Lc067671 233 183 416 Chl2_Lc067672 0 269 269 Chl2_Lc067673 412 11 423

Ch12_LeO67676 37 436 473 Chl2_LcO67677 168 197 365

[000292] The data for these polymorphisms was obtained in the same data set as

the aneuploidy data illustrated in FIGs. 11 and 12. Thus, a single assay

demonstrated the ability to identify fetal aneuploidy, and the polymorphic

differences between fetal and maternal loci allowed the identification of

informative loci and calculation of estimated percent fetal cfDNA in the sample

based on the informative loci.

Claims (23)

The claims defining the invention are as follows:

1. A method for determining percent fetal contribution in a maternal sample, comprising the

steps of:

(a) introducing at least two first sets of first and second fixed sequence oligonucleotides to

the maternal sample under conditions that allow the first sets of fixed sequence oligonucleotides to

specifically hybridize to complementary regions in nucleic acid regions of interest, wherein the

nucleic acid regions comprise informative loci;

(b) ligating the hybridized oligonucleotides to create ligation products complementary to the

nucleic acid regions of interest;

(c) amplifying the ligation products to create amplification products; and

(d) detecting and quantifying the amplification products from the first sets of fixed sequence

oligonucleotides to determine the percent fetal contribution in the maternal sample.

2. The method of claim 1 wherein the first and second fixed sequence oligonucleotides in each of the

first sets hybridize to adjacent complementary regions in the nucleic acid regions of interest.

3. The method of claim 1, wherein the first and second fixed sequence oligonucleotides in each of the

first sets hybridize to nonadjacent complementary regions in the nucleic acid regions of interest.

4. The method of claim 3, wherein the method further comprises extending the hybridized first and/or

second fixed sequence oligonucleotides of the first sets using a primer extension reaction to create

adjacently-hybridized oligonucleotides.

5. The method of claim 3, wherein the method further comprises introducing one or more bridging oligonucleotides to the maternal sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest to create adjacently or nonadjacently-hybridized oligonucleotides, wherein the one or more bridging oligonucleotides are complementary to a region between the regions of the nucleic acid regions of interest complementary to the first and second fixed sequence oligonucleotides of the first sets.

6. The method of any one of claims 1 to 5 further comprising

(e) introducing at least two second sets of first and second fixed sequence oligonucleotides to

the maternal sample under conditions that allow the second sets of fixed sequence oligonucleotides to

specifically hybridize to complementary regions in nucleic acid regions of interest in a first

chromosome:

(f) introducing at least two third sets of first and second fixed sequence oligonucleotides to

the maternal sample under conditions that allow the third sets of fixed sequence oligonucleotides to

specifically hybridize to complementary regions in nucleic acid regions of interest in a second

chromosome, wherein the second chromosome is different from the first chromosome;

(g) ligating the hybridized oligonucleotides to create ligation products complementary to the

nucleic acid regions of interest in the first chromosome and the second chromosome;

(h) amplifying the ligation products to create amplification products; and

(i) detecting and quantifying the amplification products from the second and third sets of

fixed sequence oligonucleotides to detect the presence or absence of copy number variations in the

nucleic acid regions of interest in the first chromosome and the second chromosome.

7. The method of any one of claims 1 to 6, wherein one or both of the first or second fixed

sequence oligonucleotides of the first, second and third sets of fixed sequence oligonucleotides comprise universal primer regions.

8. The method of claim any one of claims 1 to 7, wherein the hybridization products of the first,

second and third sets of fixed sequence oligonucleotides and the nucleic acid regions of interest to

which they hybridize are isolated prior to amplification.

9. The method of any one of claims 1 to 8, wherein the amplification products are quantified by

next generation sequencing.

10. The method of any one of claims 1 to 9, wherein the first or second fixed sequence

oligonucleotide of each set of fixed sequence oligonucleotides comprises one or more indices.

11. The method of claim 10, wherein the amplification product is detected and quantified by next

generation sequencing of the one or more indices.

12. The method of claim 10, wherein the first or second fixed sequence oligonucleotide of the

first sets of fixed sequence oligonucleotides comprises an allele index and the first or second fixed

sequence oligonucleotide of the second and third sets of fixed sequence oligonucleotides comprises a

locusindex.

13. The method of claim 12, wherein the amplification products are detected and quantified by

hybridization of the locus index or allele index to an array.

14. The method of claim 12, wherein the amplification products are detected and quantified by next generation sequencing of the locus index or allele index.

15. The method of any one of claims 1 to 14, wherein the amplification products are isolated

prior to the detecting and quantifying step.

16. The method of any one of claims 6 to 15, wherein the amplification products from the first

sets of fixed sequence oligonucleotides are used with the second and third sets of fixed sequence

oligonucleotides to determine the presence or absence of copy number variations of a genomic

region.

17. The method of any one of claims 6 to 16, wherein the copy number variations are

chromosomal aneuploidies.

18. The method of claim 17, wherein the chromosomal aneupolidies are trisomy 13, trisomy 18

or trisomy 21.

19. The method of any one of claims 6 to 18, wherein the second and third sets of fixed sequence

oligonucleotides comprise fixed sequence oligonucleotides that hybridize to at least 96 nucleic acid

regions.

20. The method of any one of claims 1 to 8, 10, 12, 13, and 15 to 19, wherein the amplification

products are detected and quantified by hybridization to an array.

21. The method of any one of claims 6 to 20, wherein any one of the first, second or third sets of first and second fixed sequence oligonucleotides comprise precircle probes.

22. The method of any one of claims 1 to 21, wherein the informative loci are those loci that are

homozygous in the mother and heterozygous in the fetus

23. The method of any one of claims 1 to 21, wherein the informative loci are those loci that are

homozygous in the fetus and heterozygous in the mother.