HK1176094A - Comparative genomic hybridization array method for preimplantation genetic screening - Google Patents

Description

Comparative genomic hybridization array method for pre-implantation genetic screening

Cross Reference to Related Applications

This application claims the benefit of U.S. patent application 12/724,865 filed 3, 16, 2010, the entire contents of which are incorporated herein by reference.

FIELD

The present invention relates to methods for detecting genetic abnormalities in cells of an embryo, oocyte, polar body or related biopsy.

Background

In the field of IVF (in vitro fertilization), it is desirable to identify chromosome numbers and chromosome sets in embryonic cells prior to embryo implantation. There is increasing evidence that one of the most important factors affecting embryo survival is chromosomal imbalance, including copy number increase/loss and aneuploidy of the entire chromosome (chromosomal number abnormalities).

Current testing methods first involve the isolation of genetic material representing the embryo being tested. The samples currently used in aneuploidy analysis are slices of polar bodies associated with oocytes, single cells from blastomere slices (associated with day 3 embryos), or blastocyst trophoblast cells (associated with day 5 embryos or blastocysts). But in some cases samples taken at other points or points in the process have proven to be more effective. The polar bodies or cells are then tested by a variety of methods to detect imbalance in copy number. Although the term often found in the literature is PGD, for the purposes of this application, such test methods are referred to simply as pre-embryo implantation genetic screening (PGS). The term PGS shall also include testing polar bodies to assess the quality of oocytes to, for example, ensure informative egg pool services.

Comparative Genomic Hybridization (CGH) techniques have been used to detect the presence of amplified or deleted sequences (corresponding to so-called copy number changes) in genomic DNA and their location. Generally, genomic DNA is isolated from normal reference cells, as well as from test cells. The two nucleic acid samples are differentially labeled and then hybridized in situ to metaphase chromosomes of the reference cell. Repetitive sequences in the reference and test DNAs are removed or their hybridization ability is reduced by some means. By detecting regions of altered signal ratios from the two DNAs, chromosomal regions of increased or decreased copy number in the test cell can be identified. The detection of such regions of copy number variation is particularly important for the diagnosis of genetic diseases.

Metaphase CGH as described above has been adopted and has the ability to screen for abnormalities in all chromosomes. To apply the CGH assay to a PGS environment, the entire genome needs to be amplified prior to the assay to increase the amount of DNA from a single cell (5-10pg) to a level suitable for metaphase CGH (1. mu.g). Commonly used amplification methods include DOP-PCR (Telenius et al, 1992), or more recently, whole genome amplification kits such as GENOMEPLEX (Rubicon genetics) and REPLI-G (Qiagen). The main problem with using metaphase CGH in a clinical setting is that it may take about 4 days to complete, which is incompatible with the time frame required before embryo implantation in IVF without embryo freezing and implantation in subsequent cycles. Furthermore, the method is technically rather challenging and requires a high level of expertise to implement and analyze. These difficulties limit the widespread use of intermediate CGH in PGS.

Pinkel et al, 1998 and 2003, disclose well-known techniques for array comparative genomic hybridization, hereinafter referred to as arrayCGH. In 1998, Solinas-Toldo et al described a similar "matrix-based comparative genomic hybridization" approach.

The arrayCGH technique relies on similar analytical principles as CGH in terms of binding specificity using double stranded DNA. In arrayCGH, the metaphase chromosome of the reference cell is replaced by a collection of unlabeled target nucleic acids (probes) that may be thousands of solid support-bound, e.g., an array of clones that have been placed in specific locations on the chromosome. ArrayCGH is therefore a type of comparative technique for high throughput detection of copy number differences between two DNA samples hybridized to the same hybridization region. It has the advantage over CGH that it can achieve higher resolution and can be applied to the detection and diagnosis of genetic diseases induced by copy number changes, in addition to other areas where copy number detection is important. Although the details vary, a variety of different probe types can be used, including those encountered in oligonucleotide, PAC and Bacterial Artificial Chromosome (BAC) arrays.

ArrayCGH is currently used in somatocytogenetics to assist clinical staff in studying genomic imbalances and is increasingly used in cancer research. These applications have incredible requirements that require the production standards for microarrays designed for these applications to be much more stringent than those for academic or preclinical research applications.

ArrayCGH has advantages over medium-term CGH in that interpretation is simpler and automation is easier to achieve; furthermore, the time required for a complete analysis is shorter. ArrayCGH can be used to detect aneuploidy in single cells and has been successfully applied to PGS. For this technique, it is necessary to expand the single cells using the same methods as those used for metaphase CGH. ArrayCGH allowed complete analysis of the entire genome to be completed within 48 hours, which allowed aneuploidy screening without the need for cell cryopreservation in PGS.

For optimal analytical results, arrayCGH requires that the test and reference samples match well in terms of mass and concentration. In the case of PGS, the starting point for any analysis is genetic material representing a fertilized embryo or oocyte for the egg pool. Currently, it is possible to examine the genetic material contained within the polar body or blastomere, single cells extracted from an 8-cell embryo, or alternatively a small number of cells from a blastocyst or related biopsy. Since only limited amounts of DNA are available from such materials, most downstream analyses require the use of DNA amplification procedures to produce large copy numbers of the starting material. It will be appreciated that when the fertilization process begins, there are two types of pole bodies (PB1 and PB2) that are expelled. This process is not so simple. As used herein, the term "polar body" may include a polar body that is expelled or sliced from a primary or secondary oocyte.

Although unamplified genomic reference material may be used, the corresponding arrayCGH results may show high noise levels due to poor matching of the amplification test to the unamplified reference. Thus, the reference material used in this case is often a collection of "normal" DNA samples diluted to contain a substantially similar amount of DNA as a few individual cells. This diluted reference material was then amplified as the test sample using the same method. Even if these steps are taken to fit the nature of the test and reference samples, this is not always valid and the results may vary in clarity. This may be due to a number of reasons, including small errors in the quantification of the starting DNA and consequent variations in the amount of DNA in the diluted sample; changes due to the random nature of the amplification process; amplification of impurities in the test sample that are not present in the reference; increased non-specific amplification due to low sample DNA "mass"; the amount and type of reagents used in sample extraction and preservation vary. In all cases, differences in the amplified sample and reference may alter and obscure the true amplification results, leading to altered arrayCGH properties and often increased noise and suppressed dynamic range.

PGS is a diagnostic application and the standard practice for each experiment is to include an internal control to demonstrate the successful operation of the experiment and to be able to assess the variation in dynamic range between experiments due to, for example, the amplification problem described above. When arrayCGH is used, the most common method to solve this problem is to use a reference sample whose copy number is known to increase/decrease relative to the test sample. These can then be used to measure the performance of each individual test.

The reference sample is often not matched to the sex of the test sample, so that the log2 ratio for the X and Y chromosome tests relative to the reference sample is shifted, and therefore the dynamic range measurements are also shifted. Although many cases apply, it is generally not possible to know the sex of the sample in advance for the case of PGS, and in particular for aneuploidy screening of blastomere or blastocyst section samples, either sex may be the case. Therefore, using a single reference as an internal control is not reliable. Furthermore, it is generally not possible to select a single appropriate reference for the test sample, and the imbalance in copy number of the reference in regions outside the sex chromosome is known, because the degree of copy number variation in embryos/oocytes is very high, and current studies indicate that no region is predictably stable. In some embodiments, embryos for implantation may be selected based on aneuploidy status. In other embodiments, the selection is based on minor genetic aberrations.

An alternative approach is to use a reference comprising a non-human control sequence. However, this approach is not ideal because it is difficult to select non-human sequences that can accurately mimic the behavior of human sequences. In any event, the use of non-human control sequences, or subject to the same amplification bias and other biases, is challenging to select as a single reference.

To overcome this problem with PGS, it is necessary to perform two conventional arrayCGH hybridizations to a single test sample, one against a male reference sample and one against a female reference sample to ensure that the assays are functioning correctly. However, the associated cost of such a strategy is too high for this application.

When two or more cells are removed from the embryo (e.g., blastocyst/trophectoderm), the probability of mosaicism of the test sample occurring becomes high in the case of PGS, because embryos are often chimeric. Further complicating the problem, the number of cells removed from an embryo may be unknown due to the imprecision of the biopsy method. Although arrayCGH can detect mosaics, it does not provide a means to directly quantify such mosaics due to the lack of sufficiently complex internal controls, and for the same reason, experimental noise may be mistaken for mosaics. In this case, the dependence of ArrayCGH on a single reference sample is also problematic.

ArrayCGH requires a contrast fluorescent dye to label test and reference samples. Commonly used dye pairs, Cy3 and Cy5, are often used for arrayCGH. Cy5 dye is susceptible to degradation by ozone in the environment, especially in combination with high humidity, which can have an effect on the quality of the analysis that can result in the loss of experimental data. The use of ArrayCGH in which two fluorescently labeled samples compete for hybridization with the same hybridization region allows the relative increase or decrease in genetic material to be determined by a ratio comparison. Typically, one sample is a test sample of unknown genetic composition and one sample is a reference sample known to have a normal copy number, where normal is defined by the particular application. ArrayCGH is a powerful and reliable technology, but PGS applications pose unique technical challenges. In some embodiments, the assessment of the chromosomal content of the embryo may be performed by extracting the cells directly after fertilization, or indirectly by assessing the polar body and the oocyte from which the embryo was produced. In some embodiments, there is an application where the only purpose is to assess the contents of an oocyte, and no embryo production is required. This is referred to herein as an egg bank.

Buffart et al (2008) proposed an improved arrayCGH technique, named "across arrayCGH" (aaCGH), as an improvement over the current art. AaCGH is similar to arrayCGH, however, this technique compares test and reference samples from separate hybridization regions, as compared to hybridization of both test and reference samples to a single hybridization region. This method, developed autonomously by the authors of the present invention, offers advantages in terms of cost and possibly data quality, since it eliminates any noise due to dye bias. The quality of the information obtained using the aaCGH is reported to be comparable to or even superior to that obtained using the conventional two-channel arrayCGH. It is described that using a multi-format array, the reference and test samples are hybridized simultaneously on the same slide, and the test and reference are labeled with the same fluorescent dye. They compare a single test sample with a single reference sample. This approach does not overcome the unique challenges faced by PGS.

SNP array technology, as distinguished from arrayCGH, can also be used to determine copy number in DNA samples, and has also been applied in PGS applications. SNP arrays provide screening for all chromosomes and allow for parallel genotyping. The mechanism used is quite different from that of arrayCGH, since the technique is not compared. The method of determining copy number without using a reference sample and without simultaneous hybridization in this technique relies on quantification of individual alleles and subsequent ratiometric analysis, whereas arrayCGH does not evaluate individual alleles. Disadvantages of SNP arrays include increased noise levels, longer protocols, complex data interpretation and ethical issues, and potentially lower applicability to haploid samples.

Molecular cytogenetic technique FISH (fluorescence in situ hybridization) uses chromosome-specific DNA probes, often applied to PGS, to give detectable signals in interphase nuclei. Although no amplification step is required, one significant disadvantage of this technique is that only a limited number of chromosomes can be evaluated simultaneously, limited by the number of different colors available to label the DNA probes. The most comprehensive FISH method for routine embryo screening currently evaluates only half of the chromosomes and, therefore, misses some chromosomal abnormalities. Other disadvantages of FISH include overlapping signals that are difficult to score.

SUMMARY

It is a feature of the present invention to provide a method for determining the copy number imbalance of genomic DNA present in a test DNA sample that reduces the risk of analysis failure due to mismatch between test and reference samples associated with conventional arrayCGH. This method can improve the quality, accuracy and yield of analysis.

It is another feature of the present invention to provide a method for determining the copy number imbalance of genomic DNA present in a test DNA sample by measuring the hybridization of a single test sample to one hybridization array and the hybridization of a set of reference samples to one or more other hybridization arrays, respectively.

It is still another feature of the present invention to provide a method for determining the presence of copy number imbalances in genomic DNA comprising selecting the best match between the test DNA and the reference DNA sample.

The present invention provides a method for determining the presence of a copy number imbalance in genomic DNA of a test sample. The sample may comprise a sample genomic DNA from the test sample or an amplification product thereof labeled to form a labeled test DNA; hybridizing the labeled DNA to be detected with a first hybridization array; labeling a first reference genomic DNA from a reference sample or an amplification product thereof to form a labeled first reference DNA; hybridizing the labeled first reference DNA to a second hybridization array; labeling a second reference genomic DNA from a second reference sample or an amplification product thereof to form a labeled second reference DNA; the labeled second reference DNA is hybridized to the third hybridization array. The method may include analyzing the first hybridization array to determine the signal intensity resulting from hybridization of the labeled test DNA after hybridization of the labeled test DNA; after hybridization of the labeled first reference DNA, analyzing the second hybridization array to determine the signal intensity resulting from hybridization of the labeled first reference DNA; and analyzing the third hybridization array after hybridization of the labeled second reference DNA to determine the signal intensity resulting from hybridization of the labeled second reference DNA.

The present invention provides a method of assessing the copy number of at least one region in sample genomic DNA by comparing the signal intensity of a test hybridization array to the signal intensity of at least one of two or more reference hybridization arrays.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

Brief Description of Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate some embodiments of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is a flow chart demonstrating an exemplary method for determining copy number in a test DNA sample according to the present invention.

FIG. 2 is a flow chart showing an exemplary method for preparing a set of reference DNA samples according to the present invention.

FIG. 3 is a flow chart showing how the copy number of a DNA sample to be tested is determined according to the present invention.

FIG. 4 is a flowchart showing how to determine a region where the copy number of a DNA sample to be tested has changed on the basis of two pieces of ratio information according to the present invention.

Figure 5 shows a pair of graphs, where the upper graph shows the sample compared to a male reference and the lower graph shows the sample compared to a female reference.

Detailed Description

The present invention relates to a method for detecting the presence of minor copy number imbalances or minor imbalances in aneuploidy or genomic DNA. According to the present invention, methods of detecting aneuploidy or copy number imbalance ("detection methods") may be used to analyze oocytes prior to egg banking; or for pre-embryo implantation genetic screening (PGS) to identify chromosome number and chromosome set within embryo cells prior to implantation using in vitro fertilization procedures. The detection method can identify a chromosomal region of genomic DNA that can represent a test embryo ("test DNA") that contains an increased or decreased copy number. The detection method may comprise comparing genomic hybridization using a single channel array ("single channel arrayCGH"), whereby the test DNA hybridizes to the hybridization array, and one or more DNA molecules of known copy number ("reference DNA") hybridize to one or more different hybridization arrays. Imbalance in copy number in the test DNA can be identified by detecting regions where the signal intensity resulting from hybridization of the reference DNA differs from that resulting from hybridization of the test DNA. Various properties of the present invention may include genomic hybridization methods, devices, and kits described in WO96/17958 and U.S. patent application 12/609,156 filed 10/30/2009, which are incorporated herein by reference in their entirety.

The detection method may include labeling a test sample DNA obtained from a test sample to form a test DNA, and hybridizing the labeled test DNA to a first hybridization array. The test sample DNA may be labeled so as to enable detection and/or measurement of hybridization of the test sample DNA to the first hybridization array. The signal generated by hybridization of the labeled test DNA can be detected and analyzed to determine the intensity of the signal generated by the first hybridization array, thereby obtaining a test hybridization result. The test hybridization results can be compared to reference hybridization results, or the signal intensities generated by one or more reference DNAs can be compared to one or more other hybridization arrays different from the first hybridization array. For example, the signal intensity of the reference DNA can be determined by labeling the reference DNA and hybridizing the labeled reference DNA to the second hybridization array. The signal generated by hybridization of the labeled reference DNA can be detected and analyzed to determine the signal intensity of the reference DNA. The copy number imbalance present in the test DNA can be determined by identifying one or more regions in the first hybridization array that have a signal intensity that is different from the signal intensity produced by one or more corresponding regions in the second hybridization array.

The determination of the signal intensity produced by hybridization of the labeled reference DNA can be performed before or after the determination of the signal intensity produced by hybridization of the labeled test DNA. If the signal intensity produced by hybridization of the labeled reference DNA is determined before the signal intensity produced by hybridization of the labeled test DNA is determined, the reference hybridization results can be recorded and stored as historical reference hybridization results. The test hybridization results for the test sample DNA obtained thereafter can then be compared to historical reference hybridization combinations to determine whether a copy number imbalance exists in the test DNA. The use of historical reference hybridization results avoids the need to actually hybridize to a reference sample each time a comparison to a particular test hybridization result is required.

The detection method may use one or more reference DNA or a plurality of reference DNA samples. For example, after determining the signal intensity of a reference DNA, the signal intensity of a second reference DNA can be determined. The signal intensity of the second reference DNA can be determined by labeling the second reference DNA and hybridizing to a third hybridization array. The signal generated by hybridization of the labeled second reference DNA is detected and analyzed to determine the intensity of the signal generated by hybridization of the second reference DNA. The presence of copy number imbalance can be determined by identifying one or more regions in the first hybridization array that have a signal intensity that is different from the signal intensity produced by one or more corresponding regions in the second and/or third hybridization array.

A method for determining the presence of copy number imbalance in genomic DNA of a test sample can comprise: labeling sample genomic DNA from a test sample or an amplification product thereof to form labeled DNA to be tested; hybridizing the labeled DNA to be detected with a first hybridization array; labeling a first reference genomic DNA from a reference sample or an amplification product thereof to form a labeled first reference DNA; hybridizing the labeled first reference DNA to a second hybridization array; labeling a second reference genomic DNA from a second reference sample or an amplification product thereof to form a labeled second reference DNA; the labeled second reference DNA is hybridized to the third hybridization array. The method may include analyzing the first hybridization array to determine the signal intensity resulting from hybridization of the labeled test DNA after hybridization of the labeled test DNA; after hybridization of the labeled first reference DNA, analyzing the second hybridization array to determine the signal intensity resulting from hybridization of the labeled first reference DNA; and analyzing the third hybridization array after hybridization of the labeled second reference DNA to determine the signal intensity resulting from hybridization of the labeled second reference DNA. By comparing the signal intensity of the first hybridization array to the signal intensity of at least one of the second hybridization array and the third hybridization array, the copy number can be assessed for at least one region of the sample genomic DNA.

The labeled first reference DNA may comprise at least one copy number change in one or more pre-designated regions of the genome relative to the labeled test DNA. The second labeled reference DNA may contain at least one pre-designated region that has a different copy number variation from the labeled test DNA as the first labeled reference DNA. In some cases, the signal intensity resulting from hybridization of the labeled test DNA is compared to the signal intensity resulting from hybridization of the labeled first reference DNA at the one or more pre-designated regions; comparing the intensity of the signal generated by hybridization of the labeled test DNA with the intensity of the signal generated by hybridization of the labeled second reference DNA at the one or more pre-designated regions; and the method further comprises determining a dynamic range of the method based on the expected copy number. The labeled first reference DNA may be from a male animal of a first species (e.g., from a mammal such as a human); the labeled second reference DNA may be from a female animal of the first species. The labeled first reference DNA and the labeled second reference DNA may comprise a mixture of DNA from males and females of the same animal species. The first, labeled reference DNA may comprise a triploid and the second reference DNA may comprise a monosomy. In some embodiments, the first reference DNA may comprise a small amplification on any chromosome and the second reference DNA may exclude such a small amplification.

The signal intensity resulting from hybridization of the labeled test DNA may be compared to the signal intensity resulting from hybridization of the labeled first reference DNA in one or more pre-designated regions to determine a first estimate of copy number; the signal intensities resulting from hybridization of the labeled test DNA may be compared with the signal intensities resulting from hybridization of the labeled second reference DNA in one or more pre-designated regions to determine a second estimate of copy number, and the first and second estimates of copy number may be combined to obtain an overall estimate of copy number. In some embodiments, the signal intensity is normalized prior to assessing copy number.

In some cases, the first reference genomic DNA from the reference sample or an amplification product thereof can comprise an amplification product produced by the first amplification technique; the second reference genomic DNA from the reference sample or an amplification product thereof may comprise an amplification product produced by the same first amplification technique. The first reference genomic DNA or an amplification product thereof from the reference sample may comprise a plurality of different amplification products, each formed by amplification of a different initial concentration of the same first reference genomic DNA. The method may include determining the aneuploidy status of a human polar body or embryo based on the copy number assessment. The method may further comprise using copy number information, such as aneuploidy status, to select embryos for implantation in an IVF procedure. The method may include isolating genomic DNA from a test sample to form sample genomic DNA or an amplification product thereof.

The test sample may comprise at least one cell from an embryo. The first genomic reference DNA may comprise DNA obtained from a tissue or cell of an animal having a chromosomal abnormality. In some embodiments, the first genomic reference DNA comprises DNA obtained from a mosaic tissue or cell. In some cases, the labeled first reference DNA has a first DNA concentration, comprising normal male DNA; the labeled second reference DNA has a second DNA concentration diluted relative to the first concentration and comprises the same normal male DNA as the labeled first reference DNA. DNA from females may be used in place of, or in addition to, DNA from males. The labeled first reference DNA may comprise a pool of genomic DNA extracted from blood samples of at least two individuals.

According to the present invention, there is provided a method for determining the presence of copy number imbalance in genomic DNA of a test sample, the method comprising labeling test DNA to form labeled test DNA; hybridizing the labeled DNA to be detected with a first hybridization array; analyzing the first hybridization array after hybridization to obtain a first hybridization result; and comparing the first hybridization result to a historical reference hybridization result, the historical reference hybridization result resulting from the hybridization of the labeled first reference DNA to the second hybridization array. The method may further comprise comparing the first hybridization result to a historical reference hybridization result resulting from the hybridization of the labeled second reference DNA to the third hybridization array; and determining the presence of a copy number imbalance by identifying one or more regions in the first hybridization array that have a signal intensity that is different from the signal intensity produced by one or more corresponding regions of at least one of the second hybridization array and the third hybridization array. The first reference DNA that is labeled may be from a male animal of the first species and the second reference DNA that is labeled may be from a female animal of the first species.

The invention also provides a library of reference array datasets stored in a processor. Each reference array data set may comprise data collected from a respective reference array during a copy number hybridization analysis performed on the respective reference array, wherein (1) each reference array from which the respective data set is collected comprises elements in common with each other reference array from which data is collected, and (2) each copy number hybridization analysis from which the respective data set is collected is performed under one or more different conditions than each other copy number hybridization analysis from which data is collected. The at least two reference array datasets in the library may be different from each other. In some embodiments, some reference data sets are generated under the same conditions to assess the variability of the technique. Each reference array data set may include fluorescence signal intensity data.

The invention also provides a method comprising comparing a test array dataset collected from a test array during a copy number hybridization analysis with a reference array dataset in a library; and determining, with the signal processor, a ratio between the test array dataset and the dataset in the library. A best fit data set in the library can be determined and is the reference array data set that maximizes the SNR ratio spectrum determined by the processor.

Also provided according to the invention is a kit comprising a first copy number hybridization array; a second copy number hybridization array identical to the first copy number hybridization array; a third copy number hybridization array identical to the first copy number hybridization array; a first reference genomic DNA; a second reference genomic DNA; and instructions directing how to compare test results produced by a hybridization assay performed on the first copy number hybridization array to test results produced by a hybridization assay performed on the second copy number hybridization array using the first reference genomic DNA. The instructions may also relate to how to compare the test results from the hybridization analysis performed on the first copy number hybridization array to the test results from the hybridization analysis performed on the third copy number hybridization array using the second reference genomic DNA. In some cases, the first reference genomic DNA may comprise an amplification product of the reference genomic DNA. The invention also provides a kit comprising a copy number hybridization array; an electronic storage medium containing a plurality of reference array data sets stored thereon; and instructions directing how to compare a data set corresponding to a test result from a hybridization assay performed on the copy number hybridization array to the plurality of reference array data sets.

Since the measurement of the test and reference DNA samples can be performed on separate hybridization arrays, the detection method does not require the use of a strong dye for labeling. In addition, the test DNA can be compared to an infinite number of reference samples, rather than a reference that is simply co-hybridized. In this way as many comparisons as possible can be made in order to determine the best analysis result for the test sample.

The use of more than one reference DNA also avoids the risk of analysis failure due to poor matching of test and reference DNAs and allows the selection of a single optimal pairing of test and reference DNAs. The plurality of reference DNAs may include reference DNAs obtained from single cell DNA amplification that match well with the test DNAs. In other words, a single reference DNA that gives the best comparison with the test DNA can be selected from the series of reference DNAs generated. For example, a series of reference DNA samples can be generated with minor modifications to the amplification protocol to yield a reference DNA that matches well with the DNA to be tested. This small change in the amplification protocol can result in a series of technical variations. In addition, the reference DNA generated may have specific known biological properties. For example, the reference DNA generated may be from a mosaic individual or from an individual of a particular sex. The reference DNA generated may contain one or more chromosomal abnormalities. The reference DNA may be derived from damaged cells in order to match the condition of the test sample. The reference DNA may be derived from an individual that is biologically related to the test sample.

Hybridization arrays as used herein can comprise microarrays, or collections of unlabeled target nucleic acids (probes) bound to a solid support, e.g., a clone array that has been mapped to a chromosomal location. Hybridization arrays can comprise a plurality of probes or target nucleic acid molecules, such as at least two target nucleic acid molecules bound to a solid support or surface. The target nucleic acid molecules can be organized at pre-designated locations on the solid surface, with each probe occupying a discrete location. The target nucleic acid molecules bound to the solid surface can be a plurality of the same target nucleic acid molecule, a plurality of different nucleic acid molecules, or a combination of both. For example, in embodiments where it is desired to be able to multiplex detection assays (i.e., detect more than one nucleic acid molecule at a time), a plurality of different target nucleic acid molecules that bind to different nucleic acid molecules can be used. The solid surface may be any surface suitable for arrayCGH, including flexible and rigid surfaces. The flexible surface may include, but is not limited to, a nylon membrane. The rigid surface may include, but is not limited to, a glass slide. The solid surface may also include a three-dimensional matrix or a plurality of beads thereon. Any suitable method can be used to immobilize the target nucleic acid on the solid surface.

It should be understood that although DNA hybridization is described herein, any kind of nucleic acid, such as RNA, DNA or cDNA, may be used. Likewise, the target nucleic acid molecule or probe can be, for example, RNA, DNA, or cDNA. The nucleic acid may be derived from any organism. The probes may be synthetic oligonucleotides or may be derived from cloned DNA or PCR products. Oligonucleotides may be synthesized in situ, or synthesized and then translocated. The cloned DNA may be a Bacterial Artificial Chromosome (BAC) clone or a PI-derived artificial chromosome (PAC). The sequence of the nucleic acid molecule may be derived from a chromosomal location known to be associated with a disease, may be selected to represent the chromosomal region to be determined as being associated with a disease, or may correspond to the gene whose transcription is to be analyzed.

The reference DNA can be labeled and hybridized to a hybridization array. The hybridization array is washed to remove any non-specifically bound labels. The hybridization array can then be scanned and the signal intensity of the reference DNA recorded and stored as historical reference hybridization results for comparison with the test hybridization results of the test DNA sample. Similarly, a set of historical reference hybridization results can be generated and recorded for a plurality of different reference DNAs. The plurality of reference DNA samples may be individually labeled and hybridized to separate hybridization arrays having the same array design. The hybridization array may be scanned and the scanned data converted to historical reference hybridization results and stored for later use. Since the historical reference hybridization results can be recorded, it is not necessary to hybridize a plurality of reference DNAs more than once. The historical reference hybridization results can be used repeatedly for subsequent analysis using one or more different test DNAs. The DNA to be tested may be labelled for hybridisation to a hybridisation array having the same array design as the hybridisation array used to obtain the historical reference hybridisation result. The historical reference hybridization results may be communicated to the end user electronically or in an electronic storage medium.

It is to be understood that "scanning" as used herein refers to any conventional method performed by a scanner that allows for the detection of hybridization of a sample to a hybridization array. Scanning may include, for example, emitting light from a light source of a scanner, receiving at a detector of the scanner emitted light reflected from various locations of the hybridization array. In some embodiments, scanning may include, for example, exciting a fluorescent dye on a microarray; and measuring the intensity of the emitted fluorescence at the detector of the scanner. Scanning is further described, for example, in WO96/17958 and in U.S. patent application 12/609,156 filed on 30/10/2009, each of which is hereby incorporated by reference in its entirety.

FIG. 1 is a flow chart illustrating one method of determining the copy number of a DNA sample to be tested. As shown in FIG. 1, the labeled DNA sample to be tested can be hybridized to the hybridization region A. The signal intensity or amplitude generated by hybridization can be measured to construct an amplitude profile (amplitude profile) of the test DNA sample. The amplitude profile of the test DNA sample may be normalized. A set of reference DNA samples can be selected and hybridized with hybridization regions other than the hybridization region A, respectively. The signal intensity or amplitude generated by hybridization of each reference DNA can be measured to construct a reference DNA amplitude profile. The reference DNA amplitude profile may be normalized. The copy number of the DNA sample to be tested can be determined by comparing the amplitude profile of the DNA sample to be tested with the amplitude profile of the reference DNA. In some embodiments, the same array means that the array contains many of the same contents, for example at least 90% of the same contents, but other contents may be different. Also, slight variations in the amplification process are possible, as long as they are still considered to be the same.

In some embodiments, each reference DNA will have an associated amplitude distribution. An initial estimate of the copy number is then determined by taking the ratio of the test amplitude to the reference amplitude (or amplitudes), and possibly various normalizations. This copy number estimate is naturally noisy, and other steps may be used to assess whether an estimated copy number is likely to correspond to a true change in the biological copy number, or simply due to noise (and thus zero copy number).

The plurality of different reference DNAs may comprise reference DNAs having specific known biological properties. For example, the reference DNA may be from a male or female sample. The reference DNA may be obtained from a cell line having the desired chromosomal abnormality. The reference DNA may be obtained from a mixed sample. Synthetic mosaic reference samples can be constructed to replicate mosaic karyotype patterns by pooling cells or extracted DNA with different but known karyotypes. This incorporation can occur at any stage of the preparation of the reference DNA, or subsequent labeling.

The reference DNA may be derived from damaged cells in order to match the condition of the test sample. The reference DNA may be from an individual that is biologically related to the individual from which the test sample was taken.

The test DNA may be prepared from a test sample, such as a test cell, cell population, or tissue under investigation. The test DNA may be isolated from one or more test cells. The test DNA may be obtained from polar bodies, where one-half of the genome of the ovum is expelled before fertilization. The test DNA may be obtained from blastomeres, single cells extracted from 8-cell embryos or a small number of cells from blastocysts or related biopsies (e.g., blastocyst). The test cell can comprise at least one cell from an embryo. To generate large copy numbers of the DNA to be tested, a DNA amplification procedure may be used.

The reference DNA may be prepared from a reference cell, group of cells or tissue. The reference cells may be normal, non-diseased cells, or they may be from a diseased tissue sample, as a standard for other aspects of disease. The reference DNA is genomic material for which the copy number of the target gene or nucleic acid molecule is known.

The reference DNA may be generated using a variety of starting materials. Examples of starting material may include one or more individually donated tissues, such as blood. Other sources of starting material may include single cells. The reference DNA may be isolated from the appropriate tissue or cell using standard procedures. The reference DNA or starting material may be selected from individuals having normal chromosomes and/or individuals having chromosomal abnormalities, such as an increase or loss of one or more chromosomes or an increase or loss of one or more chromatids. The single cells may be derived from an in vitro cell culture or may be ex vivo human cells, of the same or different type as the sample to be tested. Single cells may be selected because their chromatin structure is similar or dissimilar to that of the sample to be tested, e.g.sperm cells with compact chromatin may be selected. Also, cells at different stages of the cell cycle can be selected. Alternatively, a concentrated genomic reference DNA of high quality may be extracted from a cell culture or blood and diluted to a level comparable to the concentration obtained from a single cell after extraction.

Although there may be a family relationship between the donors of the materials used to generate the reference DNA and the test sample, such a relationship is not necessary. To enable direct comparison between the test sample and the parent sample, reference material may be obtained from one or both parents.

Various conditions can be used to generate a plurality of reference DNAs of varying quality. The reference DNA may be produced from cells of different integrity, for example, a reference sample produced from damaged cells. The reference DNA may be processed after sample collection, such as DNA extracted from formalin fixed paraffin embedded tissue. The reference DNA may be subjected to physical treatment, such as heating or sonication. Chemical treatments such as enzymatic or protease digestion may also be used. Other treatments that stimulate the condition of the test sample during the IVF procedure may also be performed. Such treatments may include the incorporation of mineral oil into and into the culture medium in order to normalize the contribution of any factors that contribute to analytical differences between the test sample and the reference sample.

The preparation of the reference DNA involves the use of whole genome amplification. The whole genome amplification protocol may be modified to introduce variations in the amplified DNA product. For amplification, SURPLEX amplification, or other convenient amplification, may be used. The exact nature of the DNA amplification used is not critical to the present invention. Although unamplified genomic reference DNA may be used, high noise levels may result because the amplified test DNA does not match well with the unamplified reference DNA. Thus, the reference material used may be a "normal" DNA sample collection that, upon dilution, contains approximately similar amounts of DNA to a small number of single cells. This diluted reference material can then be amplified using the same method as the test sample. To compensate for differences between the amplification of the test and reference DNA, the reference sample set can be carefully constructed to span the range of variation that causes poor matching. This strategy may reduce the risk of analysis failure due to poor matching between test and reference samples associated with conventional arrayCGH. As described herein, separating the measurement of the test and reference samples allows the measurement of a single test sample to be compared to the measurement of a series of reference samples. In this way, a single optimal pairing of test and reference samples can be found, or alternatively, combinations from multiple comparisons can be combined and compared to the test DNA.

FIG. 2 is a flow chart illustrating an exemplary method of preparing a set of reference DNA samples. As shown in FIG. 2, variations can be introduced into the reference DNA by varying the amplification protocol that produces a set of reference DNA samples. Multiple identical pairs of reference DNA samples can be constructed. Each reference DNA sample pair may comprise a normal male reference DNA and a normal female reference DNA. Each sample pair may undergo a different degree of dilution resulting in a gradient dilution. Each reference DNA within a sample pair can be diluted to the same extent. Each reference DNA of each sample pair can be amplified using the same amplification method used to amplify the DNA to be tested.

The test DNA and the reference DNA may be labeled to allow detection of the hybridization complex. The particular label attached to the DNA is not a critical aspect of the invention, so long as the label does not significantly interfere with the hybridization of the DNA to the target nucleic acid molecule. The label may be any material having a detectable physical or chemical property. The label may include, for example, a fluorescent dye, a radioactive label, or an enzyme. Generally, fluorescent labels commonly used in arrayCGH, such as Cy3 and Cy5, are preferred. For example, CYTOCHIP from BLUEGNOME may be used. Standard detection and analysis methods can be used for the signal generated by the label. For fluorescent labeling, standard methods commonly used in array comparative genomic hybridization ("arrayCGH") can be used. The hybridization array can be imaged in a fluorescence microscope with a polychromatic beam splitter. Images of different colors can be acquired with a CCD camera, a laser scanner, a combination thereof, etc., and the digitized images stored in a computer. The signals generated by the array can then be analyzed using a computer program.

The selection of a single optimal pairing of test and reference DNA and the determination of copy number imbalances in the test DNA can be automated to simplify the analysis and interpretation of the data and/or improve reproducibility. A set of algorithms may be provided to enable automation of reference data selection and scoring of the analysis. These algorithms may simplify the analysis, interpretation and/or improve the repeatability of the data. The algorithm may include a reference selection algorithm and an access algorithm (calibrating algorithm). Wherein the reference selection algorithm compares the test hybridization results of the test DNA with a corresponding set of historical reference hybridization results and determines which of the plurality of reference DNAs produced the best comparison with the test DNA.

In some cases, detection methods may suffer from spatial noise due to hybridization variability within the array and/or hybridization of the sample at different times. Such detection methods may include methods for spatial bias correction or methods for spatial correction of hybridization between arrays. Any spatial bias due to hybridization differences between the test DNA and the reference DNA can be detected and removed by methods known in the art, such as the method described in U.S. patent application 12/609,156 filed 10/30/2009, the contents of which are incorporated herein by reference in their entirety.

The reference selection algorithm may characterize the results of each test/reference comparison with a metric. The metric may be, for example, signal to noise ratio. The signal component is defined as the difference between selected chromosomes in the hybridization array to the median of the log2 ratios between the test and reference. The noise component can be obtained by taking a set of target nucleic acids or probes in the hybridization array for each chromosome and subtracting the median log2 ratio of chromosomes from the log2 ratio for each individual probe. Once chromosome biases (chromosometrends) are removed, noise can be determined by calculating the interquartile range of all probes.

The reference selection algorithm can select a reference DNA that maximizes the SNR of the ratio data (representing the number of copies in the test DNA). The test-reference pairing can then be automatically presented to the access algorithm. The access algorithm can be applied to identify regions of copy number imbalance between test and reference samples. The access algorithm may compare the observed imbalance pattern to an expected imbalance pattern. Since the karyotype of the reference sample is known, then the karyotype of the test sample can be inferred. The final classification of the sample may be "euploid" (no copy number imbalance) or "aneuploid" (copy number imbalance). In some cases, the test data may be of poor quality, such that any results obtained are unreliable. In these cases, the access algorithm may classify the result as "no result".

FIG. 3 is a flow chart showing how the copy number of a DNA sample to be tested is determined. As shown in fig. 3, a set of virtual ratio spectra can be constructed by dividing the test DNA sample amplitude spectra by the reference DNA amplitude spectra of each reference DNA. The "noise" and "dynamic range" of each virtual ratio spectrum can be calculated. The dynamic range may be calculated on the basis of the ratio of the X/Y chromosomes. A pair of ratio spectra are selected, corresponding to the reference sample pair, with the best combination of low noise and expected dynamic range. In other words, the best "amplified" match of the test and reference DNA pairs can be selected. The region of copy number variation in the DNA to be tested can be determined on the basis of the ratio spectrum pair by applying an access algorithm.

It will be appreciated that if the test sample from which the test DNA is isolated is from a first polar body, an optimal pairing of reference DNA and test DNA may be formed, although for reasons outlined elsewhere it may still be necessary to select a second pairing. If the DNA to be tested is isolated from a blastomere section of which the sex is not known beforehand, the two best matches can be selected by reference to the selection algorithm. In some embodiments, an unlimited number of pairs may be selected. If the test DNA is isolated from a blastomere slice, the reference DNA may comprise different masses of male genomic DNA and female genomic DNA. Two optimal pairings may include test DNA with male reference DNA, and test DNA with female reference DNA. The access algorithm may then identify a copy number imbalance present in one or both of the pairs. These imbalances may be compared to expected imbalance patterns. Since the karyotype of both reference samples is known, the karyotype of the test sample can be inferred.

FIG. 4 is a flow chart showing how to determine regions having copy number variations in the DNA to be tested on the basis of two ratio spectra. As shown in FIG. 4, the copy number imbalance was determined by comparing the signal intensity of the hybridized normal test DNA sample with that of the reference DNA of the normal male woman. The test DNA sample may be of the same sex as the reference DNA having the same copy number as the test DNA. The normal test DNA and the normal male reference DNA, and the ratio spectrum of the test DNA and the normal female reference DNA can be obtained. For each ratio spectrum, an algorithm can be used to determine regions of potential significance in the DNA under test that are aberrant, in other words, regions of the spectrum that are significantly more noisy than the baseline. By considering the X/Y ratios in the ratio spectrum corresponding to the test DNA sample and its gender-mismatched reference DNA, a significant ratio level consistent with true copy number variation can be determined. Using the significance level obtained in the previous stage, it can be determined whether each abnormal region is consistent with the true copy number variation. The copy number access values of the individual ratio spectra can be combined, for example by averaging, to form a single copy number access value for the DNA to be tested. It will be appreciated, however, that X and Y chromosome region access values are prioritized based on a ratio profile that is consistent with a gender match between the sample and the reference.

More than one DNA to be tested can hybridize to the same hybridization region. For example, two or more DNA samples to be tested may be labeled with different dyes and hybridized to the same hybridization region.

In some embodiments, the test sample is male, the first reference is female, and the second reference is male. In some cases, the test sample is female, the first reference is male, and the second reference is female. In both cases, the first reference can be used to establish a dynamic range for detecting all chromosomes, while the second reference can be used to access X/Y. To address the difficulties presented by not knowing the sex of the sample, if the test sample is a polar body, both female and male references can be used so that one reference matches the sample and the other provides information about the dynamic range.

It is understood that "copy number" as used herein is relative to a reference genome. For example, if the reference is a mixture of male and female DNA, the copy number is not necessarily an integer. In some cases, the triploid reference may be used as normal, i.e., there may be different copy numbers.

According to the invention, the first reference is an amplification product of the sample comprising a copy number change in the first defined reference. In some embodiments, the first reference is an amplification product of a first predetermined region and the second reference has a deletion within the predetermined region, but not a change within the predetermined region.

According to the invention, the first, second, third and any further hybridization regions may be identical if different labels are used.

Although the methods described herein are designed for IVF, the methods are also applicable to prenatal, oncology, and/or stem cell contexts. The detection method also allows for a number of references representing different degrees of mosaicism, using different amplification conditions.

Even if the gender of a sample is known, it is generally useful to be able to analyze the sample against gender-matched and gender-mismatched references. For example, in the case of detecting an electrode, it is known that the sample is female, and therefore, in order to obtain an internal dynamic range control, a male reference may be selected as the actual reference. However, such side effects are complicated by the interpretation of the polar X and Y chromosomes. It is therefore still beneficial to calculate the dynamic range using gender mismatches and provide access values for chromosomes 1-22, while accessing all chromosomes using gender matches.

In some cases, at least one reference sample differs in copy number from the test sample within the predetermined area. This copy number difference can be used as a "control" to effectively represent the dynamic range of the experiment (which depends on the individual test samples and hybridization conditions specifically discussed). For example, if the copy number difference between a particular predetermined region in the test and reference is expected to be "1", for experimental reasons the difference is only "0.25", which gives information about the dynamic range of the assay, so 0.25 in a particular assay may be significant. This estimate of dynamic range or "saliency" can then be used to estimate copy number variation in other reference datasets or in different chromosomes of the same dataset.

Examples

A normal female sample is provided that contains chromosomes 13 and 19 that are missing one copy. Fig. 5 shows a pair of curves, where the upper curve of each graph shows the comparison of a sample to a male reference and the bottom curve shows the comparison of a sample to a female reference. In the upper curve of fig. 5, the sample is compared to the male reference and the X chromosome shows a gain because there are two copies of X in women and only one copy in the male reference; likewise the Y chromosome shows a loss, since there is no Y in women, one in the male reference. These expected X/Y changes provide an indication as to what is important. Chromosomes 13 and 19 are clearly missing.

The bottom curve of figure 5 shows the same sample compared to a female reference. In this case, the number of X and Y chromosomes is expected to be the same in both the sample and the reference. Also, loss of chromosomes 13 and 19 can be seen, particularly in conjunction with the information about dynamic range obtained from the X/Y comparison in the upper curve.

All references mentioned in this disclosure are incorporated herein by reference in their entirety. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from all upper (or preferred) values and all lower (or preferred) values, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. When defining ranges, the scope of the present invention is not intended to be limited to the specific values recited.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary, with a true scope and spirit of the teachings being indicated by the following claims and their equivalents.

Claims (30)

1. A method for determining the presence of a copy number imbalance in genomic DNA of a test sample, the method comprising:

a) labeling sample genomic DNA from a test sample or an amplification product thereof to form labeled DNA to be tested;

b) hybridizing the labeled DNA to be detected with a first hybridization array;

c) labeling a first reference genomic DNA from a reference sample or an amplification product thereof to form a labeled first reference DNA;

d) hybridizing the labeled first reference DNA to a second hybridization array;

e) labeling a second reference genomic DNA from a second reference sample or an amplification product thereof to form a labeled second reference DNA;

f) hybridizing the labeled second reference DNA to a third hybridization array;

g) after hybridization of the labeled test DNA, analyzing the first hybridization array to determine the signal intensity resulting from hybridization of the labeled test DNA;

h) after hybridization of the labeled first reference DNA, analyzing the second hybridization array to determine the signal intensity resulting from hybridization of the labeled first reference DNA;

i) after hybridization of the labeled second reference DNA, analyzing the third hybridization array to determine the signal intensity resulting from hybridization of the labeled second reference DNA; and

j) evaluating the copy number of the at least one region in the sample genomic DNA by comparing the signal intensity of the first hybridization array to the signal intensity of at least one of the second hybridization array and the third hybridization array.

2. The method of claim 1, wherein the labeled first reference DNA comprises at least one copy number change in one or more pre-designated regions of the genome relative to the labeled test DNA.

3. The method of claim 2, wherein the labeled second reference DNA comprises at least one pre-designated region having a different copy number variation from the labeled test DNA as the labeled first reference DNA.

4. The method of claim 2, wherein the signal intensity resulting from hybridization of the labeled test DNA is compared to the signal intensity resulting from hybridization of the labeled first reference DNA at the one or more pre-designated regions; comparing the intensity of the signal generated by hybridization of the labeled test DNA with the intensity of the signal generated by hybridization of the labeled second reference DNA at the one or more pre-designated regions; the method also includes determining a dynamic range of the method based on the expected copy number.

5. The method of claim 1, wherein the first labeled reference DNA is from a male animal of a first species and the second labeled reference DNA is from a female animal of the first species.

6. The method of claim 1, wherein the labeled first reference DNA and labeled second reference DNA comprise a mixture of DNA from a male and a female of the same animal species.

7. The method of claim 1, wherein the signal intensity resulting from hybridization of the labeled test DNA is compared to the signal intensity resulting from hybridization of the labeled first reference DNA in one or more pre-designated regions to determine a first estimate of copy number; comparing the signal intensity resulting from hybridization of the labeled test DNA with the signal intensity resulting from hybridization of the labeled second reference DNA in the one or more pre-designated regions to determine a second estimated copy number value, and combining the first and second estimated copy number values to obtain an overall estimated copy number value.

8. The method of claim 1, wherein the signal intensity is normalized prior to assessing copy number.

9. The method of claim 1, wherein the first reference genomic DNA from the reference sample or an amplification product thereof comprises an amplification product produced by a first amplification technique; the second reference genomic DNA from the reference sample or an amplification product thereof comprises an amplification product produced by the same first amplification technique.

10. The method of claim 1, wherein the first reference genomic DNA or amplification product thereof from the reference sample comprises a plurality of different references at different concentrations.

11. The method of claim 1, further comprising:

hybridizing the labeled third reference DNA to a fourth hybridization array;

hybridizing the labeled fourth reference DNA to the fifth hybridization array;

hybridizing the labeled fifth reference DNA to a sixth hybridization array,

wherein evaluating the copy number comprises comparing the signal intensity of the first hybridization array to the signal intensity of each of the second, third, fourth, fifth, and sixth hybridization arrays.

12. The method of claim 1, further comprising determining the aneuploidy status of a human polar body or embryo based on the copy number assessment.

13. The method of claim 12, further comprising implanting an embryo based on the aneuploidy status determined in the IVF procedure.

14. The method of claim 1, further comprising isolating genomic DNA from the test sample to form sample genomic DNA or an amplification product thereof.

15. The method of claim 1, wherein the test sample comprises at least one cell from an embryo or associated biopsy.

16. The method of claim 1, wherein the first genomic reference DNA comprises DNA obtained from a tissue or cell of an animal having a chromosomal abnormality.

17. The method of claim 1, wherein the first genomic reference DNA comprises DNA obtained from a chimeric tissue or cell.

18. The method of claim 1, wherein the labeled first reference DNA has a first DNA concentration and comprises an amplification product of the first DNA at the first concentration; the labeled second reference DNA comprises an amplification product of the first DNA produced upon dilution of the first DNA at a first concentration.

19. The method of claim 1, wherein the labeled first reference DNA comprises a pool of genomic DNA extracted from blood samples of at least two individuals.

20. A method for determining the presence of a copy number imbalance in genomic DNA of a test sample, the method comprising:

a) labeling the test DNA to form a labeled test DNA;

b) hybridizing the labeled DNA to be detected with a first hybridization array;

c) analyzing the first hybridization array after hybridization to obtain a first hybridization result;

d) comparing the first hybridization result to reference data comprising at least one of historical reference hybridization results from hybridization of the labeled first reference DNA to the second hybridization array, or data synthetically generated using a mathematical model;

d) comparing the first hybridization result with a historical reference hybridization result, the historical reference hybridization result being obtained by hybridizing the labeled second reference DNA with the third hybridization array; and

e) the presence of a copy number imbalance is determined by identifying one or more regions in the first hybridization array that have a signal intensity that is different from the signal intensity produced by one or more corresponding regions of at least one of the second hybridization array and the third hybridization array.

21. The method of claim 20, wherein the first labeled reference DNA is from a male animal of a first species and the second labeled reference DNA is from a female animal of the first species.

22. A library of reference array data sets stored in a processor, each reference array data set comprising data collected from a relevant reference array during a copy number hybridization analysis performed on the relevant reference array, wherein

(1) Each reference array from which a relevant data set is collected is substantially the same or identical to each further reference array from which data is collected;

(2) each copy number hybridization assay from which the relevant data set is collected is performed under the same conditions, or one or more different conditions, as each additional copy number hybridization assay from which data is collected; and

(3) at least two reference array datasets in the library are distinct from each other.

23. The library of claim 22, wherein each reference array dataset comprises fluorescence signal intensity data.

24. The library of claim 22, wherein each reference array dataset is calculated without hybridization.

25. The method comprises the following steps:

comparing a test array dataset collected from a test array during copy number hybridization analysis with a reference array dataset in the library of claim 22; and

a ratio between the test array dataset and the dataset of the library is determined using a signal processor.

26. The method of claim 25, wherein the best fit data set in the library is the reference array data set that maximizes the SNR ratio spectrum determined by the processor.

27. A kit, comprising:

a first copy number hybridization array;

a second copy number hybridization array identical to the first copy number hybridization array;

a third copy number hybridization array identical to the first copy number hybridization array;

a first reference genomic DNA;

a second reference genomic DNA; and

instructions that (1) compare test results from a hybridization assay performed on a first copy number hybridization array to test results from a hybridization assay performed on a second copy number hybridization array using a first reference genomic DNA, and (2) compare test results from a hybridization assay performed on the first copy number hybridization array to test results from a hybridization assay performed on a third copy number hybridization array using a second reference genomic DNA.

28. The kit of claim 27, wherein the first reference genomic DNA comprises an amplification product of a reference genomic DNA.

29. A kit, comprising:

a copy number hybridization array;

a plurality of reference array data sets; and

instructions for comparing a data set corresponding to a test result from a hybridization assay performed on the copy number hybridization array to the plurality of reference array data sets.

30. The kit of claim 29, further comprising an electronic storage medium, wherein the plurality of reference array datasets are stored on the electronic storage medium.