US0093 09565B2

(12) United States Patent (10) Patent No.: US 9,309,565 B2 BrOOmer et al. (45) Date of Patent: Apr. 12, 2016

(54) KARYOTYPING ASSAY 4,889,818. A 12/1989 Gelfand et al. 4,965,188 A 10, 1990 Mullis et al. (75) Inventors: Adam Broomer, Carlsbad, CA (US); 5.835 A RE E. tal Kelly Li, San Jose, CA (US), Andreas 3.66: A 56, RI. R. Tobler, Fremont, CA (US); Caifu 5.436,134. A 7/1995 Haugland et al. Chen, Palo Alto, CA (US); David N. 5,487.972 A 1/1996 Gelfand et al. Keys, Jr., Oakland, CA (US) 5,538,848. A 7/1996 Livak et al. s s 5,618,711 A 4/1997 Gelfand et al. (73) Assignee: Life Technologies Corporation, 5,658,7515,677,152 A 10/19978, 1997 Yues et. al. al. Carlsbad, CA (US) 5,723,591 A 3/1998 Livak et al. 5,773.258 A 6/1998 Birch et al. (*) Notice: Subject to any disclaimer, the term of this 5,789,224 A 8, 1998 Gelfand et al. patent is extended or adjusted under 35 38. A 3. 3. E. al U.S.C. 154(b) by 998 days. 5,854,033.sy w A 12/1998 LizardiCaO (ca. 5,876,930 A 3, 1999 Livak et al. (21) Appl. No.: 13/107,786 5,994,056. A 1 1/1999 Higuchi (22) Filed: May 13, 2011 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2011 FO281755A1 Nov. 17, 2011 EP OOTO685 A2 1, 1983 WO WO-2006/081222 8, 2006 Related U.S. Application Data (Continued) (60) Provisional application No. 61/334,658, filed on May OTHER PUBLICATIONS 14, 2010. Kim et al., Disruption of Neurexin 1 Associated with Autism Spec 51) Int. C trum Disorder, The American Journal of Human Genetics 82, 199 (51) Int. Cl. 207, Jan. 2008.* CI2O I/68 (2006.01) (52) U.S. Cl. (Continued) CPC ...... CI2O I/6844 (2013.01) (58) Field of Classification Search Primary Examiner — Reza Ghafoorian CPC ...... C12Q 1/6844 (74) Attorney, Agent, or Firm — Life Technologies See application file for complete search history. Corporation (56) References Cited (57) ABSTRACT This disclosure relates to methods and kits for karyotyping in U.S. PATENT DOCUMENTS which chromosomes are interrogated by amplifying loci that 4,257,774 A 3/1981 Richardson et al. are not within copy number variable regions thereof. 4,683, 195 A 7, 1987 Mullis et al. 4,683.202 A 7, 1987 Mullis 21 Claims, 8 Drawing Sheets

Chronosome 2 Taq Man CNV assay 7900 – 10 uL – 10 ng dry gDNA

20090612 HsO4692736 2p23.3 GTF3C2 CC BC US 9,309,565 B2 Page 2

(56) References Cited French, D.J., “HyBeaconTM Probes: A New Tool for DNA Sequence Detection and Allele Discrimination'. Molecular and Cellular U.S. PATENT DOCUMENTS Probes, vol. 15, 2001, 363-374. Jain, S. C., "Stereochemistry of Actinomycin Binding to DNA'. 6,030,787 A 2/2000 Livak et al. Journal of Molecular Biology, vol. 68, 1972, 1-20. 6,084,102 A 7/2000 Kutyavin et al. Johnston, Brian H., "Characterization of the Photoreaction Between 6,127,155 A 10, 2000 Gelfand et al. DNA and Aminomethyl-Trimethylpsoralen Using Absorption and 6,171,785 B1 1/2001 Higuchi 6,180.349 B1* 1/2001 Ginzinger et al...... 435/6.11 Fluorescence Spectroscopy”. Photochemistry and Photobiology, vol. 6.214.979 B1 4/2001 Gelfand et al. 33, 1981, 785-791. 6,258,569 B1 7/2001 Livak et al. Kapuscinski, Jan, “Interactions of 4", 6-diamidine-2-phenylindole 6,814,934 B1 1 1/2004 Higuchi with Synthetic Polynucleotides'. Nucl. Acids Res., vol. 6, No. 11, 6,821,727 B1 1 1/2004 Livak et al. 1979, 3519-3534. RE39,007 E 3/2006 Dattagupta et al. Li, Qingge, “A New Class of Homogeneous Nucleic Acid Probes 7,141,377 B2 11/2006 Gelfand et al. Based on Specific Displacement Hybridization.” Nucleic Acids 7,387,887 B2 6, 2008. Wittwer et al. Research, vol. 30, No. 2, e5, 2002, 1-9. 7,445,900 B2 11/2008 Gelfand et al. Little, Michael C. "Strand Displacement Amplification and Homo 2004/0265897 A1 12, 2004 Lizard geneous Real-Time Detection Incorporated in a Second-Generation 2008/01 18925 A1* 5/2008 Cuppens et al...... 435/6 2008/0286783 A1* 11/2008 Hosono et al...... 435/6 DNA Probe System, BDProbeTechET, Clinical Chemistry, vol. 2009/O1972.54 A1 8, 2009 Lee 45(6), 1999, 777-784. 2010. 031791.6 A1 12, 2010 Scott et al. Livak, Kenneth J., “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-AAcTMethod.” Methods, vol. 25, 2001, 402-408. FOREIGN PATENT DOCUMENTS Lizardi. “Mutation Detection and Single-Molecule Counting Using WO WO-2006/087574 8, 2006 Isothermal Rolling-Circle Amplification', Nature Genetics vol. 19. WO 2006/128195 11, 2006 Jul. 1998, 225-232. WO 2009,153568 12/2009 Morrison, Larry E., "Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization'. OTHER PUBLICATIONS Anal. Biochem., vol. 183, No. 2, 1989, 231-244. Nazarenko, Irina A., “A Closed Tube Format for Amplification and Berggren et al., Detecting Homozygous Deletions in the Detection of DNA Based on Energy Transfer”. Nucleic Acids CDKN2A(p16INK4a)/ARF(p14ARF) Gene in Urinary Bladder Research, vol. 25, No. 12, Oxford University Press, 1997, 2516 2521. Cancer Using Real-Time Quantitative PCR. Clinical Cancer Nazarenko, Irina, “Multiplex quantitative PCR using self quenched Research, vol. 9, 235-242, Jan. 2003.* primers labeled with a single fluorophore”. Nucleic Acids Research, Schmittgen et al., Analyzing real-time PCR data by the comparative vol. 30, No. 9, e37 2002, 1-7. CT method, Nature Protocols, vol. 3, No. 6, 2008, 1101-1108.* Nutiu, Razvan, “Tripartite Molecular Beacon.” Nucleic Acids Bubner, B. et al., “Use of Real-Time PCR for Determining Copy Research, vol. 30, e94, 2002, 1-9. Number and Zygosity in Transgenic Plants'. Plant Cell Reports, vol. Oser, Andreas, “Nonradioactive Assay of DNA Hybridization by 23(5), 2004, 263-271. DNA-Template-Mediated Formation of a Ternary Tbill Complex in Gouas, L. et al., “Gene Dosage Methods as Diagnostic Tools for the Pure Liquid Phase'. Angewandte Chemie International Edition in Identification of Chromosome Abnormalities”. Pathologie-Biologie, English, vol. 29, No. 10, 1990, 1167-1169. vol. 56(6), 2008, 345-353. Pellestor, Franck, “The Peptide Nucleic Acids (PNAs), Powerful Jeon, J. P. et al., “A Comprehensive Profile of DNA Copy Number Tools for Molecular Genetics and Cytogenetics”, European Journal Variations in a Korean Population: Identification of Copy Number of Human Genetics, vol. 12, 2004, 694-700. Invariant Regions Among Koreans'. Experimental & Molecular Redon, Richard, “Global Variation in Copy Number in the Human Medicine, vol. 41(9), 2009, 618-628. Genome', Nature, vol. 444, 2006, 444-454. International Search Report along with the Written Opinion for Inter Searle, Mark S., “Sequence-specific interaction of Hoescht 33258 national Application No. PCT/US2011/036516 date mailed Feb. 9, with the minor groove of an adenine-tract DNA duplex studied in 2012. solution by 1H NMR spectroscopy”. Nucl. Acids. Res., vol. 18, No. 13, Oxford University Press, 1990, 3753-3762. Nature Genetics, vol.39, Jul. 2007, pp. S1-S54, entire issue. Sharp, Andrew J., “Structural Variation of the Human Genome.” Afonina, I. A., “Minor Groove Binder-Conjugated DNA Probes for Annual Rev. Genomics & Human Genet., vol. 7, 2006, 407-442. Quantitative DNA Detection by Hybridization-Triggered Fluores Singh, S., “LNA (locked nucleic acids): synthesis and high-affinity cence”, BioTechniques, vol. 32, 2002, 940-949. nucleic acid recognition'. Chem. Commun., vol. 4, 1998, 455-456. Baner, Johan, “Signal Amplification of PadlockProbes by Rolling Speicher, Michael R. “The New Cytogenetics: Blurring the Bound Circle Replication.” Nucleic Acids Research, vol. 26, No. 22 1998, aries with Molecular Biology.” Nature Reviews Genetics, vol. 6, 5O73-5078. 2005, 782-792. Barany, “Genetic Disease Detection and DNA Amplification Using Svanvik, Nicke, “Light-Up Probes: Thiazole Orange-Conjugated Cloned Thermostable Ligase.” Proceedings of the National Academy Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homo of Sciences (PNAS) vol. 88, Issue 1 1991, 189-193. geneous Solution'. Analytical Biochemistry vol. 281, 2000, 26-35. Cardullo, Richard A., “Detection of Nucleic Acid Hybridization by Todd, Alison V., “DzyNA-PCR: Use of DNAZymes to Detect and Non Radiative Fluorescence Resonance Energy Transfer”. Proceed Quantify Nucleic Acid Sequences in a Real-Time Fluorescent For ings of the National Academy of Sciences (PNAS), vol. 85 1988, mat”, Clinical Chemistry, vol. 46, No. 5, 2000, 625-630. 8790-8794. Trask, Barbara J., “Human Cytogenetics: 46 Chromosomes, 46 Years Cikos, Stefan, “Transformation of Real-Time PCR Fluorescence and Counting.” Nature Reviews Genetics, vol. 3, 2002, 769-778. Data to Target Gene Quantity.” Analytical Biochemistry, vol. 384. Tyagi, Sanjay, “Molecular Beacons: Probes that Fluoresce upon 2009, 1-10. Hybridization', Nature Biotechnology, vol. 14, Mar. 1996, 303-308. Feuk, Lars, "Structural Variation in the Human Genome.' Nature Whitcombe, David, "Detection of PCR products using self-probing Reviews Genetics, vol. 7(2), 2006, 85-97. amplicons and fluorescence'. Nature Biotechnology, vol. 17. Aug. Fiandaca, Mark J., “Self-Reporting PNA/DNA Primers for PCR 1999, 804-807. Analysis”. Genome Research, vol. 11, 2001, 609-613. Wu, Dan Y., “The Ligation Amplification Reaction (LAR)-Amplifi Freeman, Jennifer L., “Copy Number Variation: New Insights in cation of Specific DNA Sequences Using Sequential Rounds of Tem Genome Diversity'. Genome Research, vol. 16, 2006, 949-961. plate-Dependent Ligation.” Genomics, vol. 4, 1989, 560-569. US 9,309,565 B2 Page 3

(56) References Cited litus'. BMC Genomics, vol. 11, No. 426, downloaded from http:// www.biomedcentral.com/1471-2164/11/426, 2010, 10 pages. OTHER PUBLICATIONS PCT/US2011/036516; International Preliminary Report on Patent ability mailed Nov. 20, 2012. EP 11781377.4: Extended European Search Report mailed Mar. 12, 2014. Jeon, et al., “Copy Number variation at leptin receptor gene locus associated with metabolic traits and the risk of type 2 diabetes mel * cited by examiner U.S. Patent Apr. 12, 2016 Sheet 1 of 8 US 9,309,565 B2

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US 9,309,565 B2 1. 2 KARYOTYPNG ASSAY outside of CNVRs. In addition, the methods described herein provide for simultaneous interrogation of multiple chromo CROSS-REFERENCE TO RELATED Somes using a single, or multiple reporting labels. Using these APPLICATIONS methods, karyotypes may be rapidly and accurately deter mined. These and other advantages may be drawn from the This application claims the benefit of priority under 35 description provided below. U.S.C. S 119(e) to U.S. Provisional Patent Application No. 61/334,658, filed May 14, 2010, which is hereinincorporated BRIEF DESCRIPTION OF THE DRAWINGS by reference in its entirety. FIG.1. Results of exemplary assay of chromosome 2. 10 FIG 2. Results of exemplary assay of chromosome 3. FIELD FIG 3. Results of exemplary assay of chromosome 4. FIG. 4. Results of exemplary assay of chromosome 5. This disclosure relates to methods and kits for karyotyping FIG.S. Results of exemplary assay of chromosome 6. in which at least one chromosome is interrogated by ampli FIG. 6. Results of exemplary assay of chromosome 20. fying one or more loci that are not within copy number vari 15 FIG. 7. Results of additional exemplary assay of chromo able regions (CNVR) thereof. some 20. FIG. 8. Results of exemplary assay of chromosome 22. BACKGROUND INFORMATION For FIGS. 1-8, the genomic DNA (gDNA) samples on the x-axis from left-to-right are as follows: The human haploid genome is a deoxyribonucleic acid NA 10859 1, NA10859 2, NA 10859 3, NA 10859 4, N (DNA) sequence and consists of approximately three billion 14474, NA 14476, NA 17102, NA 17103, NA 17104, base pairs grouped into 23 chromosomes. However, the 17105, NA 17106, NA 17107, NA 17108, NA 17109, human genome is diploid and consists of approximately six 17110, NA 17111, NA 17112, NA 17113, NA 17114, billion base pairs grouped into 46 chromosomes. Hence, two 17115, NA 17116, NA 17117, NA 17118, NA 17119, copies of each genomic segment and two copies of each 25 17120, NA 17121, NA 17122M NA 17123, NA 17124, chromosome are represented in most of the human genome. 17125, NA 17126, NA 17127, NA 17128, NA 17129, The exception is the male human, which has only one copy 17130, NA 17131, NA 17132, NA 17134, NA 17136, each of chromosome X and chromosome Y. Nevertheless, 17137, NA 17139, NA 17140, NA 17144, NA 17147, variable copy numbers (i.e. not two copies) of genomic seg 17148, NA 17149, NA 17155, NA 17188, NA 17194, ments and chromosomes are observed in individual genomic 30 17201, NA 17202, NA 17203, NA 17204, NA 17205, DNA (gDNA) samples. Such copy number variable regions 17206, NA 17207, NA 17208, NA 17209, NA 17210, (CNVR) that are typically greater than one kilobase in length 17211, NA 17212, NA 17213, NA 17214, NA 17215, and generally occurataminor frequency of equal to or greater 17216, NA 17217, NA 17220, NA 17221, NA 17223, than 1% in the population are termed "copy number variants’ 17225, NA 17226, NA 17227, NA 17228, NA 17230, (CNVs; see, e.g., Feuk, et al. Nat. Rev. Genet. 7:85-97 35 17231, NA 17232, NA 17235, NA 17237, NA 17239, (2006)). Copy number variation (CNV) and its mechanisms 17240, NA 17241, NA 17242, NA 17245, NA 17247, of formation, associations with phenotype, and methods of 17251, NA 17253, NA 17254, NA 17255, NA 17258, analysis have been extensively reviewed (Feuk, supra; Free 17259, NA 17260, NA 17261, NA17262, NA17263. man, et al. Genome Res. 16:949-961 (2006); Sharp, et al. Annu. Rev. Genomics Hum. Genet. 7:407-442 (2006): 40 SUMMARY Nature Genetics 39:S1-S54 (2007), entire issue). Currently, approximately 8600 CNVs have been identified and cover Disclosed herein are methods for karyotype analysis and about 5-10% of the human genome (Redon, et al. Nature for determining the copy number of a test locus on a chromo 444:444-454 (2006); Conrad, et al. Nature 464:704-712 Some in a test biological sample, the test locus being located (2010)). Continuing studies toward finer mapping of the CNV 45 outside of a copy number variable region (CNVR) of the map and interrogating more diverse g|DNA samples are chromosome, by amplifying a nucleic acid sequence corre tracked at the Database of Genomic Variants (http://project sponding to the test locus to produce an amplification prod S. tcag.ca/variation?). Aberrations in the normal complement uct, or amplicon, and quantifying the relative copy number of of 46 human chromosomes have been identified by cytoge the chromosome in the test biological sample relative to a netic analysis. Cytogenetics and its history, linking of chro 50 control biological sample. Karyotyping methods are also pro mosomal defects and disease, and methods of analysis have vided here that do not require comparison to a reference assay been extensively reviewed (Speicher, et al. Nat. Rev. Genet. or reference sample. Such methods include those using Vir 6:782-792 (2005); Trask, Nat. Rev. Genet. 3:769-778 tual reference assays or virtual calibrator samples. (2002)). More recently, polymerase chain reaction (PCR)- Embodiments of the methods disclosed herein can be per based assays have been used to identify such aberrations. 55 formed on one or multiple samples with or without a calibra There is a need in the art for methods that provide fast, tor sample or reference assay. In other embodiments, the accurate, easy-to-use and reliable karyotype information. target copy number assays can serve as the copy number Karyotype includes an analysis of chromosome number, type, reference assay herein denoted as a “virtual copy number shape, and banding. Currently available methods for deter reference assay'. mining karyotype and chromosome number lack accuracy 60 Variations of these methods, as well as reagents, kits, due to the presence of CNVRs, are labor-intensive and do not arrays and devices for carrying out the same are also pro provide for simultaneous interrogation of multiple chromo vided. Somes without requiring multiple reporting labels. Unless chromosomes are interrogated outside of CNVRs, an inaccu DETAILED DESCRIPTION rate karyotype or chromosome number determination may 65 result. Accordingly, this disclosure provides such methods by This disclosure relates to methods for measuring the copy using as target sequences only those targets known to be number of one, more than one, or all chromosomes of a US 9,309,565 B2 3 4 genome using real-time amplification methods (i.e., "karyo chromosomal copy number or detrimentally affect the assay typing'). The term “karyotype” refers to the interrogation of by making it less reliable or accurate. Typically, a “universal one, more than one, or all chromosomes of a particular bio set of karyotyping assays (i.e., applicable to all samples or logical sample, Such as a nucleic acid preparation including, persons) will not target CNVRs. However, it is important to but not limited to, deoxyribonucleic acid (DNA, including but note that CNVRs may vary between individuals or popula not limited to, genomic (gDNA) and mitochondrial DNA tions of individuals. For instance, a duplication observed in an (mtDNA)), ribonucleic acid (RNA), proteins, cells, tissues, individual or population of individuals may not be observed and organisms. in another individual or population of individuals. As such, To “interrogate' one or more chromosomes means to per the methods described herein may target loci that are within a form an assay, including the methods described herein, to 10 CNVR in one individual or population of individuals but not obtain information about the one or more chromosomes con within a CNVR or another individual or population of indi tained in the sample. Typically, information relating to "copy viduals. number (CN) is desired. The term copy number generally The difference in copy number between samples is typi refers to the number of amplicons resulting from polymerase cally determined by “relative quantitation’. Relative quanti chain reaction (PCR) amplification, the number of genes or 15 tation is used to report the copy number of a target locus or regions on a chromosome, the number of chromosomes in a chromosome on which the target locus is found in a test genome, and the like. The term “amplicon refers to a piece of sample (i.e., “test locus' or “unknown locus') relative to one DNA formed as the product of an amplification reaction, such or more reference loci or “calibrator samples’. With reference as PCR, ligase chain reaction (LCR) or natural gene duplica to the use of a calibrator sample, the term “relative to” means tion. The skilled artisan will understand that, within this dis compared to the copy number of a target locus or chromo closure, any such molecules can be referred to as target or Some on which the target locus is foundina calibrator sample. reference loci depending on the particular assay system. For A “calibrator sample' is a biological sample, often of known example, a genome can be queried using as the biological karyotype (i.e., a “control g|DNA), that is interrogated sample a gCNA that preferably, but not necessarily, contains before, after or simultaneously with the test sample. The at least one of each chromosome that the user desires to 25 number of copies in the test sample can then be determined interrogate. In some embodiments, the g|DNA sample can relative to the calibrator sample. One or more reference loci or contain a pair of each of chromosomes 1-22 and either two X calibrator samples can be used as desired by the user. The one chromosomes, or an X and a Y chromosome. However, it is or more reference loci can be the same or different and can be understood that variations and abnormalities occur Such that present in the test sample(s) or calibrator sample(s). Typi the number or composition of chromosomes among g|DNA 30 cally, the measure of relative quantitation is reported using the samples can vary. In fact, the methods described herein are term “fold change', which refers to the amount of amplified directed to detecting such variations and abnormalities. In product (which relates to the copy number) in a test sample addition, the user may desire to interrogate only a Subset of relative to a reference or calibrator sample. Fold change can chromosomes (i.e., one or more) present in a genome and it is be quantified using any of several available methods, includ therefore necessary that only those chromosomes of interest 35 ing but not limited to those described by Livak, et al. (Meth be present. Furthermore, the interrogation typically includes ods, 25:402-408 (2001)) or commercially available products a reference locus (i.e., a “reference locus' or “endogenous such as CopyCallerTM (Applied Biosystems, see below). The control') which is a locus different from the test locus and is methods described herein can be performed on one or more not found within a CNVR. The copy number of the reference than one sample with or without a calibrator sample or refer locus is typically, but not necessarily, known. A reference 40 ence assay. Various embodiments of Such methods are locus can reside on a chromosome of interest or on another described herein. chromosome and can be interrogated to normalize sample The real-time polymerase chain reaction (RT-PCR) is a input or to determine relative copy number (i.e., an "endog conventional tool which can be used to amplify and quantify enous control'). a target nucleic acid molecule, including but not limited to, The methods and assays described herein, which include 45 DNA and RNA, in a sample. The amount of target nucleic the determination of the copy number of target loci, are acid can be determined as an absolute copy number or as a referred to herein as “copy number assays”. The methods and relative amount. Specifically, the use of RT-PCR to quantify assays described herein for interrogating reference loci are gene expression using the comparative C-method is knownto referred to herein as "copy number reference assays”. Typi one of skill in the art (Livak, et al. (Supra)). In general, the cally, the reference locus is present in all samples and is used 50 threshold cycle (C) for a given genetic locus can be deter to normalize for sample input variation and determine relative mined by arbitrarily setting a signal intensity threshold that copy number. However, the test locus can be present or falls within the linear range of amplification of real-time PCR absent. For example, a Y chromosome test assay may inter data. Previous application of this calculation has been used, rogate as “present in a normal male sample, but “absent in for example, to normalize the amplification product of a target a normal female sample. Existing assay systems, such as the 55 gene to the amplification product of an endogenous control TaqMan(R) Copy Number Assays (Applied Biosystems), have gene and then to normalize the data to a calibrator sample, been used in some instances to interrogate target and refer Such as an untreated reference sample, to determine the ence loci. However, interrogation of test loci within copy expression of the target gene. number variable regions (CNVRs) has been found to lead to Certain currently available assay systems, such as PCR inaccurate karyotype determinations. 60 based TaqMan(R) systems, can be useful for relative quantifi An important feature of the methods described herein is cation of nucleic acids amplified from a biological sample to that the test loci are outside of (i.e., not within) CNVRs. As determine gene or chromosome copy number (see, e.g., the described herein, a CNVR includes, but is not limited to, any TaqMan(R) assays described in the following Applied Biosys type of structural variation of a target locus that can affect tems publications: “Product Bulletin: TaqMan(R) Copy Num copy number determination including, but not limited to, 65 ber Assays and Custom TaqMan(R) Copy Number Assays, duplications, deletions, insertions, repeats, Substitutions, and “Protocol: TaqMan(R) Copy Number Assays” (PN 4397.425 the like. Certain variations can imitate or mimic changes in Rev. C), “Quick Reference Card: TaqMan(R) Copy Number US 9,309,565 B2 5 6 Assays” (PN4397424 Rev. B)). The TaqMan(R) Copy Number the same or different. In certain embodimetns, the reference Assay and the TaqMan(R) Copy Number Reference Assay assay is used to normalize for sample input variation between have been used in tandem to interrogate chromosomes to samples (i.e., between multiple test samples or between the amplify target and control loci, respectively. Usually, the testand calibrator Samples). Thus, in Such assays, one or more assays are carried out as a single step, “two-plex real-time 5 reference loci can be amplified from the test or control PCR assay (i.e., two separate assays within the same reaction samples and the copy number (i.e., fold change) is calculated well or container). In this type of assay, target and control loci as described herein as a copy number reference assay. Varia are detected using differently labeled probes. Often, the tions of such embodiments are also contemplated as would be sample interrogated by Such PCR-based assays is human understood by one of skill in the art. genomic DNA (gDNA) and, typically a minimum of two 10 The methods described herein can also be conducted using human g|NA samples are interrogated, including a testanda a calibrator sample that, as described above, is typically a calibrator sample. Currently, the TaqMan(R) Copy Number biological sample of known karyotype, while the karyotype Assay interrogates test loci, which are located throughout the of the test sample is unknown. The relative difference in copy genome and may reside within a copy number variable region number between the calibrator sample and the test sample can (CNVR) and provides copy number variation (CNV) infor- 15 be calculated as is known in the art or described herein. mation. The TaqMan(R) Copy Number Reference Assay inter Typically, the “test assay” and “calibrator assay” (i.e., refer rogates one or more reference loci which, as defined above, ence or endogenous control assay) are both used for relative are found outside of CNVRs (e.g., Ribonuclease P RNA quantification of all samples in order to normalize for sample component H1 (H1RNA) gene (RPPH1) on chromosome 14, input variation between samples prior to comparing the dif cytoband 14q11.2, also known as “RNase P, and the telom- 20 ferent samples themselves. The copy number of the target loci erase reverse transcriptase (TERT) gene located on chromo of the test samples is calculated by dividing the fold change of some 5, cytoband 5p15.33). The TaqMan(R) Copy Number the target copy number in the test sample by that of the Reference Assay thereby serves as the endogenous control by calibrator Sample and then multiplying by the target copy interrogating a non-CNVR sequence of interest to normalize number of the calibrator sample. This calculation is used in for sample input variation by relative quantification (RQ). For 25 the analysis of the currently and commercially available Taq example, the TaqMan(R) Copy Number Assay can utilize an Man(R) assay systems and similar systems. The test and cali FAMTM dye-labeled minor groove binder (MGB) probe and brator assays are, at least in part, directed to the same target loci, but the target loci can also be different. In some embodi unlabeled PCR primers directed at the one or more test loci in ments, the methods can include amplifying a target locus the test sample or calibrator sample, which are typically from outside (i.e., not within) a CNVR from a test biological assayed separately. The TaqMan(R) Copy Number Reference 30 sample (such as a gCNA sample in which the number of Assay can use VIC dye-labeled TAMRATM probes directed at copies of a particular chromosome is not known), amplifying the one or more reference loci. By comparing the products of the same target loci from a calibrator biological sample, and these reactions, the user normalizes for sample amount and calculating the copy number of the target loci in the test determines the relative number of copies of the target loci in sample relative to the control sample. Variations of Such the sample. However, as described above, interrogation of test 35 embodiments, including those that do not use a reference loci within copy number variable regions (CNVRs) has been assay or calibrator sample, are also contemplated as would be found to lead to inaccurate karyotype determinations. Thus, it understood by one of skill in the art. is important that the target loci are found outside of (e.g., not As discussed above, many embodiments of the methods within) CNVRs. described herein are contemplated as would be understood by The methods described herein can utilize a copy number 40 one of skill in the art. Exemplary assay formats using refer reference assay. In certain embodiments, the target loci in the ence or calibrator samples are illustrated in Table 1 and test and calibrator sample(s) of for example, gDNA, can be described below. TABLE 1. Exemplary Karyotyping Assay Design Formats when an Unknown Sample is Tested Relative to a Calibrator Sample Reaction #1 Reaction #2

No. Detectable Detectable No. CN Reactions Plexy Detectable Label Detectable Label Format No. CN Reference (per Per Label (CN (CN Ref. Label (CN (CN Ref. No. Assays Assays sample) Reaction Assay) Assay) Assay) Assay) 1 1 1 1 2 Dye #1 * Dye #2 2 1 1 2 1 Dye #1 Dye #1 3 1 1 2 1 Dye #1 Dye #2 4 1 1 2 1 DNA DNA Binding Binding Dyes: Dye 5 e2 1 e3 1 Dye #1 Dye #1 6 e2 1 e3 1 Dye #1 Dye #2 7 e2 1 e3 1 DNA DNA Binding Binding Dye Dye 8 1 e2 e3 1 Dye #1 Dye #1 9 1 e2 e3 1 Dye #1 Dye #2 10 1 e2 e3 1 DNA DNA Binding Binding Dye Dye US 9,309,565 B2 7 8 TABLE 1-continued Exemplary Karyotyping Assay Design Formats when an Unknown Sample is Tested Relative to a Calibrator Sample Reaction #1 Reaction #2

No. Detectable Detectable No. CN Reactions Plexy Detectable Label Detectable Label Format No. CN Reference (per Per Label (CN (CN Ref. Label (CN (CN Ref. No. Assays Assays sample) Reaction Assay) Assay) Assay) Assay) 11 e2 e2 e4 1 Dye #1 Dye #1 12 e2 e2 e4 1 Dye #1 Dye #2 13 e2 e2 e4 1 DNA DNA Binding Binding Dye Dye 14 e2 O e2 1 Dye #1 Dye #1 15 e2 O e2 1 Dye #1 Dye #2 16 e2 O e2 1 DNA DNA Binding Binding Dye Dye Dyes are typically conjugated to primers or probes, **DNA binding dyes are typically not conjugated to primers or probes,

NOTES (Modified) permutations of different formats are also pos sible (e.g. format 14 with multiple Copy Number Assays The number of reactions is per replicate per sample. Dye #1 25 per chromosome via incorporating aspects of format 5). and Dye #2 represent different fluorophores conjugated Detectable entities other than fluorophores may also be to probes or primers of the Copy Number Assay and employed. Copy Number Reference Assay and provide differential In certain formats outlined in Table 1, the detectable label fluorescence as the reaction generates amplification can be the same (e.g., formats 2, 4, 5, 7, 8, 10, 11, 13, 14, and products. 30 16). In other formats, the detectable labels can be different DNA Binding Dye represents a fluorophore that binds to (e.g., Dye #1, Dye #2, and DNA binding dye as in formats 1, amplification products and provides fluorescence as the 3, 6, 9, 12, or 15) and are conjugated to probes or primers to reaction generates amplification products. provide a differential value as the reaction generates ampli Format 1 is one 2-plex reaction (i.e. a multiplex reaction). fication products. One or more DNA binding dyes can be used Formats 2, 3, and 4 are variations of two 1-plex reactions 35 to detect amplification products generated during a reaction (i.e., a single-plex reaction). (such as real-time PCR) in a multiplex reaction or in separate Formats 5, 6, and 7 are variations of 1-plex reactions that 1-plex reactions. Typically, the DNA binding dyes are not combine (via average, median, etc.) C values from mul- conjugated to a probe or primer. The skilled artisan will also tiple Copy Number Assays. note the following from Table 1: Formats 8,9, and 10 are variations of 1-plex reactions that 40 1) format 1 is a 2-plex reaction (i.e., multiplex reaction) and combine (via average, median, etc.) C values from mul- uses different detectable labels for each reaction; tiple Copy Number Reference Assays. 2) formats 2, 3, and 4 are variations of two 1-plex reactions Formats 11, 12, and 13 are variations of 1-plex reactions (i.e., single-plex reaction) and use the same or different that combine (via average, median, etc.) C values from detectable labels in each reaction; multiple Copy Number Assays and combine (via aver- 45 3) formats 5, 6, and 7 are variations of 1-plex reactions that age, median, etc.) C values from multiple Copy Num- combine C values from multiple copy number assays ber Reference Assays. (via average, median, etc.), typically target the same Formats 14, 15, and 16 are variations of 1-plex reactions chromosome, and can use the same or different detect that provide multiple analyses via each Copy Number able labels in each reaction; Assay, in turn, being compared to one or more of the 50 4) formats 8, 9, and 10 are variations of 1-plex reactions remaining virtual Copy Number Reference Assays. that combine C values from multiple copy number ref Copy Number Assays of format 5, 6, or 7 target the same erence assays (via average, median, etc.), target the same chromosome. chromosome, and use the same or different detectable Copy Number Reference Assays of format 8, 9, or 10 target labels in each reaction; the same chromosome. 55 5) formats 11, 12, and 13 are variations of 1-plex reactions Copy Number Assays of format 11, 12, or 13 target the that combine C values from multiple copy number same chromosome. assays (via average, median, etc.) and combine C val Copy Number Reference Assays of format 11, 12, or 13 ues from multiple copy number reference assays (via target the same chromosome. average, median, etc.), target the same chromosome, and Copy Number Assays of format 14, 15, or 16 target differ- 60 use the same or different detectable labels in each reac ent chromosomes. tion; Formats 5, 6,7,8,9, 10, 11, 12, 13, 14, 15, and 16 may also 6) formats 14, 15, and 16 are variations of 1-plex reactions permute and combine (via average, median, etc.) AC that provide multiple analyses in which each copy num values. ber assay is compared to one or more of the remaining Formats 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 65 virtual copy number reference assays, target different may also be multiplexed (even though the multiplexed chromosomes, and use the same or different detectable assays may not provide AC value). labels in each reaction. US 9,309,565 B2 9 10 Formats 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 can also to the probe or primer; 2) the use of previously incompatible permute and combine (e.g., via average, median, etc.) AC assays that cannot be multiplexed due to assay interaction values. issues or PCR bias of one assay over another; and 3) the use The AC value is defined herein as the C of the test copy of a DNA binding dye in order to minimize reagent costs. It is number assay minus the C of the copy number reference possible to use this assay format, or modified versions assay. The AAC value is defined herein as: the C of the test thereof, to interrogate a single chromosome or multiple chro assay minus the C of the reference assay of each test sample mosomes. Each chromosome is interrogated with both a copy minus the C of the test assay minus the C of the reference number assay and a copy number reference assay and typi assay of the calibrator sample. In some embodiments, the cally, but not necessarily, each chromosome is assayed sepa AAC values can also be permuted and combined. However, 10 because the AC of the calibrator sample is subtracted from rately from one another. each AC of the test sample, the values being permuted and In some embodiments, the karyotyping assay uses one or combined now (AAC vs. AC) are of a different scale, and more copy number assays specific for the chromosome of track in a “parallel manner (e.g., ACS of 2, 4, 5 vs. AACS of interest, and one or more copy number reference assays. In 1, 3, 4 where a value of 1 is subtracted from each). Formats 2, 15 some embodiments, the average or median C. values can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 can also be used. An average average (or "double average') C value is multiplexed even though the multiplexed assays may not calculated by averaging the multiple average C values provide a AC value. Modified permutations of different for derived from the replicates of each copy number assay when mats are also possible (for example, formats 5 and 14 can be more than one copy number assay is used. One or more copy combined for analysis of multiple copy number assays per number assays against a chromosome or chromosomes dif chromosome). ferent from the test sample can be used as controls. These In one embodiment, karyotyping assay format 1 of Table 1 control copy number assays fill the role served by copy num uses a 2-plex real-time PCR reaction comprised of a copy ber reference assay in karyotyping assay formats 1-4 number assay (or test assay) and a copy number reference described above. A “double' average C value is calculated assay wherein target loci outside of CNVRs are interrogated. 25 by averaging the multiple average C. values derived from the Generally, the detectable labels used by each component of replicates of each copy number assay. The “double' average this karyotyping assay are different because the amplified AC value can then be calculated from the two “double' products are contained within the same reaction. Subsequent average C values. Subsequent copy number analysis by the copy number analysis by the 2^^ method is the same as 2^* method is carried out as previously described herein. that used in the TaqMan(R) Copy Number Assay. In this cal 30 Karyotyping assay formats 5, 6, 7, 8, 9, 10, 11, 12, and 13 culation, AAC, determined for each test copy number assay provide the following advantages: 1) allow one to use previ serves as the negative exponent of 2 in order to calculate the ously incompatible assays that cannot be multiplexed due to fold change for each test copy number assay relative to its assay interaction issues or PCR bias of one assay over corresponding copy number reference assay. This assay for another; 2) allow one to use a DNA binding dye (e.g., SYBR mat provides for multiplexing of the copy number assay and 35 Green I) in order to minimize reagent costs; 3) allow one to the copy number reference assay, which minimizes the error eliminate the use of a defined copy number reference assay; of the AC calculation and minimizes the use of reagents and and 4) allow one to minimize aberrant copy number observed consumables. This karyotyping assay format can be used to from a minority of copy number assays by diluting the aber interrogate a single chromosome or multiple chromosomes. rant average C. values in a "double' average C. value. Karyo In certain embodiments, the test copy number assay provides 40 typing assay formats 5,6,7,8,9, 10, 11, 12, or 13, or modified the copy number of the target locus of the test sample, as well versions thereof, can be used to interrogate a single chromo as the copy number of the chromosome containing the target Some or multiple chromosomes. locus. Different samples having a particular target locus In other embodiments, the copy number assays can serve as known to be outside of a CNVR can be used for the reference a copy number reference assay herein denoted as a “virtual assay. To properly normalize for sample input variation, the 45 copy number reference assay'. In these embodiments, a mul target locus or chromosomal copy number for the reference tiplicity of copy number assays are performed and each indi assay should be the same for all samples. In Such embodi vidual copy number assay is compared to one or more of the ments, the copy number assay and copy number reference remaining copy number assays, which then serve as the copy assay can target different chromosomes. Alternatively, or in number reference assays. For example, if ten copy number addition, each chromosome can be interrogated with both a 50 assays are performed, copy number assay no. 1 is compared to copy number assay and a copy number reference assay. Typi each of copy number assays nos. 2-9, copy number assay no. cally, but not necessarily, each chromosome is assayed sepa 2 is compared to each of copy number assays nos. 1 and 3-10, rately from one another. and so forth, for a possible total number of 90 different copy Karyotyping assay formats 2, 3, and 4 of Table 1 each use number calculations. For each copy number determination, two single-plex real-time PCR reactions and the same, two or 55 any two or more of the other nine virtual copy number refer three different, detectable label(s). One real-time PCR reac ence assays can be combined (e.g., via average, median, etc.) tion is the copy number assay and the other real-time PCR toward a total number often different analyses. This combin reaction is the copy number reference assay. The detectable ing takes place at the virtual copy number reference assay C. label used in each assay can be the same or different. Subse level or AC level with one copy number assay and up to nine quent copy number analysis by the 2^* method is per 60 virtual copy number reference assays. These karyotyping formed as previously described hereinabove, with the excep assay formats provide the following advantages: 1) the use of tion that the replicate C values of the copy number assay and incompatible assays that cannot be multiplexed due to assay copy number reference assay are typically, but not necessar interaction issues or PCR bias of one assay over another; 2) ily, averaged separately prior to calculating the average AC, the use of a DNA binding dye in order to minimize reagent value. These karyotyping assay designs provide the following 65 costs; 3) elimination of a properly defined copy number ref advantages: 1) the use of copy number assay and copy number erence assay; 4) confirmation of copy number using each reference assay utilizing the same detectable label conjugated copy number assay as a virtual copy number reference assay; US 9,309,565 B2 11 12 and 5) for troubleshooting of a copy number assay by the use virtual calibrator assay no. 1). Typically, in these karyotyping of each copy number assay as a virtual copy number reference assays, one or more copy number assays targeting one or assay. more test chromosomes, one or more copy number reference It is also possible to further modify these karyotyping assay assays targeting one or more reference chromosomes, and formats. For example, in some embodiments, at least ten two or more test samples (e.g., gDNA) are utilized. In these different copy number analyses can be used in karyotyping assays, the copy number of the reference chromosome may or assay formats 14, 15, or 16, and the ten different average AC may not be previously known. An average C. value is calcu values derived from the ten different copy number analyses lated from the replicate C values of copy number assay. If are then averaged toward a "double' average AC value. Sub more than one copy number assay is used, the average C. sequent copy number analysis by the 2^^ method is per 10 values of each copy number assay are averaged. This calcu formed as previously described herein. This copy number lation is performed for each of the two or more test samples, analysis mirrors that of the other assays with the exception and for the one or more reference assay(s). The average C. that average AC values, not average C values, are being values or “double' average C values can then be further averaged. In other embodiments, assay formats 14, 15 or 16 averaged across the two or more test samples. This “double' can be combined with assay formats 5, 6, 7, 11, 12 or 13 by 15 or “triple average C value is representative of a “virtual averaging the average C. values from multiple copy number calibrator sample. If appropriate, other calculations, such as assays prior to calculating AC values. For example, a karyo the median, can be used in lieu of the average. The calculated typing assay with 10 copy number assays in which five target C. values from the copy number assay(s) and copy number chromosome 1 and five target chromosome 2, the replicate C, reference assay(s) can then be used to calculate the AC value values are averaged prior to averaging the five assays target for each of the test samples. Subsequent copy number analy ing each chromosome. Accordingly, only two "double' aver sis by the 2^' method (see below) is similar to that as age C values exist, one for each chromosome. A variety of previously described herein. More accurate results will typi Subsequent calculations of AC are possible. These karyotyp cally result when the test samples are normalized to the same ing assay formats with modified copy number analyses pro concentration prior to chromosomal copy number analysis vide the following advantages: 1) the use of previously 25 and when additional reference assays are utilized. Even in the incompatible assays that can not be multiplexed due to assay presence of equal numbers of target loci, different reference interaction issues or PCR bias of one assay over another; 2) assays may provide different C values due to differences in the use of a DNA binding dye in order to minimize reagent assay performance. Therefore, the use of additional reference costs; 3) do not rely on the use of a properly defined copy assays can provide a more accurate C representation of each number reference assay; and 4) minimize aberrant copy num 30 test gldNA sample. Likewise, the use of additional test ber results observed in some copy number assays by diluting samples can provide a more accurate C representation of the the aberrant average AC values in a "double' average AC “virtual” calibrator sample. value. Such assay formats can be used to interrogate a single In another of these embodiments, a modified “virtual cali chromosome or multiple chromosomes. It should also be brator sample” (i.e., virtual calibrator assay no. 2) can be used noted that the real-time PCR reactions can utilize increased 35 in lieu of an actual calibrator sample as described in the multiplexing. immediately preceding paragraph. However, after averaging Karyotyping methods that use a virtual copy number ref replicate C values, the average C values from one copy erence assay (i.e., those that do not utilize either a reference number reference assay can be averaged across the multiple assay or calibrator sample) are also provided. Situations in test samples. This “double' average C value represents the which a reference assay is not used include the lack of a 40 C. value from the virtual calibrator sample. If multiple copy Suitable reference genomic sequence, non-identification of a number reference assays are used, average C values from control assay locus from a reference genomic sequence, assay replicates can be averaged across multiple copy number ref unavailability, cost reduction, or to facilitate ease of use. erence assays and then averaged across multiple test samples Situations in which a calibrator sample is not used include toward a “triple average C value representing the virtual unavailability of Such a sample or a reference sequence, to 45 calibrator sample. Multiple copy number assays can be reduce costs, or facilitate ease of use. Such assays are treated separately or combined. described below. In yet other embodiments, multiple copy number assays In the following two embodiments, the copy number assay with virtual calibrator assays are used, for example, multiple C. values and copy number reference assay C. values for the copy number assays targeting one or more test loci on a virtual calibrator sample are calculated. The copy number 50 chromosome (virtual calibrator assay no. 1 targeting the test assay C values can be calculated by averaging the replicate sample); multiple copy number assays targeting one or more C for each copy number assay and then averaging these test locus on a reference chromosome (virtual calibrator assay values across the multiple test samples. Alternatively, the no. 2 targeting the virtual calibrator sample); multiple copy copy number assay C. values can be calculated by averaging number reference assays targeting a reference chromosome replicate C values of each copy number assay, averaging 55 (virtual calibrator assay no. 3, copy number reference assay these values across multiple copy number assays (those tar targeting the test sample and the virtual calibrator sample). In geting the same chromosome), and averaging these values these embodiments, the copy number of the reference chro across the multiple test samples. These methods can also be mosome may or may not be previously known. The copy applied to the copy number reference assay C values. number of the targeted chromosome(s) in virtual calibrator Depending upon the number of copy number assays and copy 60 assays no. 2 and no. 3 need not be the same. In these embodi number reference assays used, up to four formats can be used ments, a calibrator sample is not required. "Double' average to perform this calculation. Furthermore, the copy number C. values are calculated from each of the three assay groups. assays and the copy number reference assays for the test The "double' average C values from virtual calibrator assays samples can be treated separately or in combination as out no. 1 and no. 3 are then used to calculate the AC value for the lined in Table 1. 65 test sample. If appropriate, other calculations, such as the In the first of these embodiments, a “virtual calibrator median, can be used in lieu of the average. The “double' sample' can be used in lieu of an actual calibrator sample (i.e., average C. values from virtual calibrator assays no. 2 and no. US 9,309,565 B2 13 14 3 are used to calculate the AC value for the “virtual calibra The data generated using the methods described hereincan tor sample. Subsequent copy number analysis by the 2^ also be analyzed using CopyCallerTM software available from method is similar to that previously described herein. It is Applied Biosystems. CopyCallerTM software is based on the noted that the use of additional test and reference assays can 2^* method described by Livak, et al. (supra). In one provide a more accurate C representation of the test gldNA method, the user is required to select one sample as the sample and “virtual calibrator gldNA sample. calibrator sample and to identify the copy number thereof. Yet another embodiment is similar to that described in the For example, if the target loci in the test sample (such as immediately preceding paragraph except that virtual calibra gDNA) is detected using VICTM dye and the target loci in the tor assay #3 is omitted. Because the test sample and “virtual calibrator sample is detected using FAMTM dye, the AC value calibrator sample are derived from the same biological 10 is calculated for each reaction well by subtracting the VICTM C. value from the FAMTM C value. The calculation of the sample, there is no need to normalize for input variation by AC value normalizes for Sample input variation between using one or more reference assays. Therefore, the reference wells. Because multiple replicates are typically assayed, the assays can be removed from the workflow. Upon calculating AC values from each replicate for each sample are averaged the “double' average C. values from virtual calibrator assays 15 toward an average AC value for each sample. The AAC, no. 1 and no. 2, the “double' average C value from virtual value is calculated for each sample by Subtracting the average calibrator assay no. 2, representing the “virtual calibrator AC value of the calibrator sample from the average AC, sample, is subtracted from the “double' average C value value of each test sample. The calculation of the AAC value from assay group no. 1, representing the test sample, to cal provides for determining the copy number of a test sample culate a AC value. If appropriate, other calculations, such as relative to the calibrator sample of known copy number. The the median, can be used in lieu of the average. Because the AAC values are converted to a relative quantity (RQ) by the reference assays are not used, calculation of relative quantity equation of RQ-2^^. The RQ value of each sample is then via the equation of 2^* is replaced with calculation of multiplied by the copy number of the calibrator sample to relative quantity via the equation of the 2^. Subsequent obtain the copy number determination of each sample. copy number analysis is similar to that previously described 25 Another method of copy number analysis by CopyCallerTM herein. utilizes proprietary algorithms for determining copy number As described above, data generated using the karyotyping without selection of a calibrator sample. The user is only methods described herein (with or without reference or cali required to identify the most common copy number to be brator Samples) can be analyzed using any of several well provided by the samples. Both methods of copy number known methods or algorithms. As an example, relative copy 30 analysis by CopyCallerTM employ outlier removal strategies number can be calculated using assay format 1 (of Table 1) as for all C, and AC values. CopyCallerTM can also provide shown in Table 2: confidence and absolute Z-score values for the predicted copy number values. It is also noted that copy number analysis TABLE 2 methods other than the 2^^ method can be utilized (e.g., 35 Cikos, et al. Anal Biochem. 384:1-10 (2009)). Other methods Karyotyping Assay Format 1 Performed Without Replicates ofanalysis can also be used, as would be understood by one of skill in the art. Test or Cali Unknown brator While many of the embodiments described herein relate to Step Description Sample Sample PCR, other amplification or product detection methods or 40 reagents can also be utilized. For example, such methods or 1 Determine copy number of calibrator sample TBD 2 reagents may include any of several methods that can be used Collect C value from copy number assay 23 2O 3 Collect C value from copy number reference 23 21 to amplify the target nucleic acid from the sample. The term assay “amplifying which typically refers to an “exponential 4 Calculate AC value O -1 increase in target nucleic acid is used herein to describe both (C. from CN Assay - C from CN Ref Assay) 45 linear and exponential increases in the numbers of a select 5 Calculate AAC value 1 O (AC from each sample - AC from calibrator target sequence of nucleic acid. The term “amplification reac sample) tion mixture” refers to an aqueous solution comprising the 6 Calculate relative quantity (RQ = 2^) O.S 1 various reagents used to amplify a target nucleic acid. These 7 Calculate copy number 1 2 include enzymes, including, but not limited to, polymerases (CN = RQx CN of calibrator sample) 50 (such as DNA polymerase, RNA polymerase and reverse transcriptase), aqueous buffers, salts, amplification primers, As understood by those of skill in the art, a one cycle target nucleic acid, and nucleoside triphosphates. Depending difference is theoretically a two-fold difference in quantity of upon the context, the mixture can be either a complete or the target sample. The skilled artisan can derive from step 2 incomplete amplification reaction mixture. The method used that the Test or Unknown Sample (C. value=23) can have 55 to amplify the target nucleic acid can be any method available eight-fold fewer copies than the Calibrator Sample (C. to one of skill in the art. Any in vitro means for multiplying or value=20). However, because the copy number reference amplifying the copies of a target sequence of nucleic acid can assay targets the same number of copies in all samples, the be utilized. These include linear, logarithmic, or any other test sample (C. value=23) actually contains four-fold less amplification method. Exemplary methods include poly sample input the calibrator sample (C. value-21). Because of 60 merase chain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683, the normalization of sample input variation via the copy num 202; 4,683, 195; 4.965, 188; and 5,035,996), isothermal pro ber reference assay, it can be determined that the test sample cedures (using one or more RNA polymerases (see, e.g., WO has two-fold fewer copies than the calibrator sample (i.e., 2006/081222), strand displacement (see, e.g., U.S. Pat. No. 4-fold variation is accounted for as sample input variation RE39,007), partial destruction of primer molecules (see, e.g., from the initial 8-fold variation) indicating that the actual 65 WO 2006/087574), ligase chain reaction (LCR) (see, e.g., copy number variation between the test and calibrator Wu, et al. Genomics 4:560-569 (1989) and Barany, Proc. samples is two-fold. Natl. Acad. Sci. USA 88:189-193 (1991)), QB RNA replicase US 9,309,565 B2 15 16 systems, RNA transcription-based systems (e.g., TAS, 3SR), ing nucleic acid amplification on a target polynucleotide rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. using a nucleic acid polymerase having 5'-to-3' nuclease 5,854,033: U.S. Pub. No. 2004/0265897; Lizardi, et al. Nat. activity, a primer capable of hybridizing to said target poly Genet. 19: 225-232 (1998); and Barter, et al. Nucleic Acid nucleotide, and an oligonucleotide probe capable of hybrid Res. 26:5073-5078 (1998)), and strand displacement ampli 5 izing to said target polynucleotide 3' relative to said primer. fication (SDA) (Little, et al. Clin. Chem. 45:777-784 (1999)), The oligonucleotide probe typically includes a detectable among others. Many systems are suitable for use in amplify label (e.g., a fluorescent reporter molecule) and a quencher ing target nucleic acids and are contemplated hereinas would molecule capable of quenching the fluorescence of said be understood by one of skill in the art. reporter molecule. The oligonucleotide probe typically exists Any of several methods can be used to detect amplified 10 in at least one single-stranded conformation when unhybrid target nucleic acids using primers or probes. Many different ized where said quencher molecule quenches the fluores reagents, systems, or detectable labels can be used in the cence of said reporter molecule. When hybridized to a target methods described herein. These include, for example, Taq nucleic acid, the probe exhibits at least one other conforma Man(R) systems, detectable nucleic acid intercalating agents, tion in which the fluorescence of the reporter molecule is such as ethidium bromide and SYBR dyes, detectable label 15 unduenched. Typically, the detectable label and quencher quencher systems (e.g., FRET. salicylate/DTPA ligand sys molecule are part of a single probe. As amplification pro tems (see, e.g., Oser et al. Angew. Chem. Int. Ed. Engl. ceeds, the polymerase digests the probe to separate the detect 29:1167-1169 (1990)), displacement hybridization, homolo able label from the quencher molecule. The detectable label gous probes, assays described in EP 070685), molecular bea (e.g., fluorescence) is monitored during the reaction, where cons (e.g., NASBA), Scorpion, locked nucleic acid (LNA) detection of the label corresponds to the occurrence of nucleic bases (Singh, et al. Chem. Commun. 4:455-456 (1998)), pep acid amplification (e.g., the higher the signal the greater the tide nucleic acid (PNA) probes (Pellestor, et al. Eur. J. Hum. amount of amplification). Variations of TaqMan(R) assays Gen. 12:694-700 (2004)), Eclipse probes (Afonina, et al. (e.g., LNATM spiked TaqMan(R) assay) are known in the art Biotechniques 32:940-949 (2002)), light-up probes (Svanvik, and would be suitable foruse in the methods described herein. et al. Anal. Biochem. 281:26-35 (2000)), molecular beacons 25 Another exemplary system utilizes double-stranded probes (Tyagi, et al. Nat. Biotechnol. 14:303-308 (1996)), tripartite in displacement hybridization methods (see, e.g., Morrison, molecular beacons (Nutiu, Nucleic Acids Res. 30:E94 et al. Anal. Biochem. 183:231-244 (1989); and Li, et al., (2002)), QuantiProbes (www.qiagen.com), HyBeacons (Supra)). In Such methods, the probe typically includes two (French, et al. Mol. Cell. Probes 15:363-374 (2001)), dis complementary oligonucleotides of different lengths where placement probes (Li, et al. Nucleic Acids Res. 30:E5 30 one includes a detectable label and the other includes a (2002)), HybProbes (Cardullo, et al. Proc. Natl. Acad. Sci. quencher molecule. When not bound to a target nucleic acid, USA 85:8790-8794 (1988)), MGB Alert(R) probes (www. the quencher suppresses the signal from the detectable label. nanogen.com), Q-PNA (Fiandaca, et al. Genome Res. The probe becomes detectable upon displacement hybridiza 11:609-613 (2001)), Plexor (www.Promega.com), LUX tion with a target nucleic acid. Multiple probes can be used, primers (Nazarenko, et al. Nucleic Acids Res. 30:E37 35 each containing different detectable labels, such that multiple (2002)), Scorpion primers (Whitcombe, et al. Nat. Biotech target nucleic acids may be queried in a single reaction. nol. 17:804-807 (1999)), AmpliFluor R. (Sunrise) primers Additional exemplary methods for amplifying and detect (Nazarenko, et al. Nucleic Acids Res. 25:2516-2521 (1997)), ing target nucleic acids involve "molecular beacons', which DzyNA primers (Todd, et al. Clin. Chem. 46:625-630 are single-stranded hairpin shaped oligonucleotide probes. In (2000)), and the like. In each of these assays, the generation of 40 the presence of the target sequence, the probe unfolds, binds amplification products can be monitored while the reaction is and emits a signal (e.g., fluoresces). A molecular beacon in progress. An apparatus for detecting the signal generated typically includes at least four components: 1) the "loop', an by the detectable label can be used to detect, measure, and 18-30 nucleotide region which is complementary to the target quantify the signal before, during, or after amplification. The sequence; 2) two 5-7 nucleotide “stems’ found on either end particular type of signal may dictate the choice of detection 45 of the loop and being complementary to one another; 3) at the method. For example, in Some embodiments, fluorescent 5' end, a detectable label; and 4) at the 3' end, a quencher dye dyes are used to label probes or amplified products. The that prevents the detectable label from emitting a signal when probes bind to single-stranded or double-stranded amplified the probe is in the closed loop shape (i.e., not bound to a target products, or the dyes intercalate into the double-stranded nucleic acid). Thus, in the presence of a complementary tar amplified products, and consequently, the resulting fluores 50 get, the 'stemportion of the beacon separates out resulting in cence increases as the amount of amplified product increases. the probe hybridizing to the target. Other types of molecular In some embodiments, the T is ascertained by observing a beacons are also known and may be suitable for use in the fluorescence decrease as the double-stranded amplified prod methods described herein. Molecular beacons can be used in uct dissociates and the intercalating dye is released therefrom. a variety of assay systems. One Such system is nucleic acid The amount of fluorescence can be quantitated using standard 55 sequence-based amplification (NASBAR), a single step iso equipment such as a spectra-fluorometer, for example. The thermal process for amplifying RNA to double stranded DNA use of other methods or reagents is also contemplated herein without temperature cycling. A NASBAR reaction typically as would be understood by one of skill in the art. requires avian myeloblastosis virus (AMV), reverse tran One exemplary method for amplifying and detecting target scriptase (RT), T7 RNA polymerase, RNase H, and two oli nucleic acids is the TaqMan(R) system described above (see, 60 gonucleotide primers. After amplification, the amplified tar e.g., U.S. Pat. Nos. 4,889,818; 5,079,352: 5,210,015; 5,436, get nucleic acid can be detected using a molecular beacon. 134:5,487.972:5,658,751:5,538,848; 5,618,711; 5,677,152; Other uses for molecular beacons are known in the art and 5,723,591; 5,773.258; 5,789,224; 5,801,155; 5,804,375; would be suitable for use in the methods described herein. 5,876,930; 5,994,056; 6,030,787; 6,084,102: 6,127,155; The Scorpion system is another exemplary assay format 6,171,785; 6,214,979; 6,258,569; 6,814,934; 6,821,727: 65 that can be used in the methods described herein. Scorpion 7,141,377; and 7,445,900). As described herein and else primers are bi-functional molecules in which a primer is where, TaqMan Rassays are typically carried out by perform covalently linked to the probe, along with a detectable label US 9,309,565 B2 17 18 (e.g., a fluorophore) and a quencher. In the presence of a target fluorescent protein (e.g., GFP EGFP), blue fluorescent pro nucleic acid, the detectable label and the quencher separate tein (e.g., BFP EBFP, EBFP2, AZurite, mKalamal), cyan which leads to an increase in signal emitted from the detect fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow able label. Typically, a primer used in the amplification reac fluorescent protein (e.g., YFP Citrine, Venus, YPet), FRET tion includes a probe element at the 5' end along with a “PCR donor/acceptor pairs (e.g., fluorescein?tetramethyl blocker' element (e.g., HEG monomer) at the start of the rhodamine, IAEDANS/fluorescein, EDANS/dabcyl, fluores hairpin loop. The probe typically includes a self-complemen cein/fluorescein, BODIPY FL/BODIPY FL, Fluorescein/ tary stem sequence with a detectable label at one end and a QSY7 and QSY9), LysoTracker and LysoSensor (e.g., quencher at the other. In the initial amplification cycles (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, PCR), the primer hybridizes to the target and extension occurs 10 LysoTrackeryellow HCK-123, LysoTracker Green DND-26, due to the action of the polymerase. The Scorpion system can LysoTracker Red DND-99, LysoSensor Blue DND-167, be used to examine and identify point mutations using mul LysoSensor Green DND-189, LysoSensor Green DND-153, tiple probes that have different tags to distinguish between the LysoSensorYellow/Blue DND-160, LysoSensorYellow/Blue probes. Using PCR as an example, after one extension cycle 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, is complete, the newly synthesized target region will be 15 514); rhodamines (e.g., 110, 123, B, B200, BB, BG, B extra, attached to the same Strand as the probe. Following the second 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, cycle of denaturation and annealing, the probe and the target 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine hybridize. The hairpin sequence then hybridizes to a part of B, Phallicidine, Phalloidine, Red, Rhod-2,5-ROX (carboxy the newly produced PCR product. This results in the separa X-rhodamine), Sulphorhodamine B can C. Sulphorhodamine tion of the detectable label from the quencher and causes G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, emission of the signal. Other uses for molecular beacons are Texas Red-X, VIC and other labels described in, e.g., US Pub. known in the art and would be suitable for use in the methods No. 2009/0197254), among others as would be known to described herein. those of skill in the art. One or more detectable labels or quenching agents are The amplified target nucleic acid can also be detected using typically attached to a primer or probe. The detectable label 25 a detectable nucleic acid binding agent (see, e.g., Table 1) can emit a signal when free or when bound to the target which can be, for example, an intercalating agent or a non nucleic acid. The detectable label can also emit a signal when intercalating agent. As used herein, an intercalating agent is in proximity to another detectable label. Detectable labels can an agent or moiety capable of non-covalent insertion between also be used with quencher molecules Such that the signal is stacked base pairs of a double-stranded nucleic acid mol only detectable when not in sufficiently close proximity to the 30 ecule. A non-intercalating agent is one that does not insert quencher molecule. For instance, in Some embodiments, the into the double-stranded nucleic acid molecule. The nucleic assay system can cause the detectable label to be liberated acid binding agent can produce a detectable signal directly or from the quenching molecule. Any of several detectable indirectly. The signal can be detectable directly using, for labels can be used to label the primers and probes used in the example, fluorescence or absorbance, or indirectly using, for methods described herein. 35 example, any moiety or ligand that is detectably affected by As mentioned above, in some embodiments the detectable its proximity to double-stranded nucleic acid is suitable, for label can be attached to a probe, which can be incorporated example a Substituted label moiety or binding ligand attached into a primer, or may otherwise bind to amplified target to the nucleic acid binding agent. It is typically necessary for nucleic acid (e.g., a detectable nucleic acid binding agent the nucleic acid binding agent to produce a detectable signal Such as an intercalating or non-intercalating dye). When 40 when bound to a double-stranded nucleic acid that is distin using more than one detectable label, each should differ in guishable from the signal produced when that same agentis in their spectral properties such that the labels can be distin Solution or bound to a single-stranded nucleic acid. For guished from each other, or such that together the detectable example, intercalating agents. Such as ethidium bromide and labels emit a signal that is not emitted by either detectable SYBR dyes, fluoresce more intensely when intercalated into label alone. Exemplary detectable labels include, for 45 double-stranded DNA than when bound to single-stranded instance, a fluorescent dye or fluorphore (e.g., a chemical DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos. 5,994, group that can be excited by light to emit fluorescence or 056; 6,171,785; and 6,814,934). Similarly, actinomycin D phosphorescence), “acceptor dyes' capable of quenching a fluoresces red when bound to single-stranded nucleic acids, fluorescent signal from a fluorescent donor dye, and the like. and green when bound to double-stranded nucleic acids. And Suitable detectable labels may include, for example, fluoros 50 in another example, the photoreactive psoralen 4-aminometh ceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-Carboxy yle-4-5".8-trimethylpsoralen (AMT) has been reported to fluorescein (5-FAM);5-HAT (Hydroxy Tryptamine); 6-HAT: exhibit decreased absorption at long wavelengths and fluo 6-JOE: 6-carboxyfluorescein (6-FAM); FITC); Alexafluors rescence upon intercalation into double-stranded DNA (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, (Johnston et al. Photochem. Photobiol. 33:785-791 (1981). 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluoro 55 For example, U.S. Pat. No. 4,257,774 describes the direct phores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, binding of fluorescent intercalators to DNA (e.g., ethidium 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, salts, daunomycin, mepacrine and acridine orange, 4',6-dia 650/665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, midino-O-phenylindole). Non-intercalating agents (e.g., TMR, TMR-X conjugate, TMR-X, SE, TR, TRATP TR-X minor groove binders such as Hoechst 33258, distamycin, SE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, 60 netropsin) may also be suitable for use. For example, Hoechst AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, 33258 (Searle, et al. Nucleic Acids Res. 18:3753-3762 coumarin phalloidin, hydroxycoumarin, CMFDA, methoxy (1990)) exhibits altered fluorescence with an increasing coumarin), calcein, calcein AM, calcein blue, calcium dyes amount of target. Exemplary detectable DNA binding agents (e.g., calcium crimson, calcium green, calcium orange, cal may include, for example, acridine derivatives (e.g., acridine cofluor white), Cascade Blue, Cascade Yellow; CyTM dyes 65 homodimer, acridine orange, acridine yellow, 9-amino-6- (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP cyclic AMP chloro-2-methoxyacridine (ACMA), proflavin), actinomy Fluorosensor (FiCRhR), fluorescent proteins (e.g., green cins (e.g., actinomycin D (Jain, et al. J. Mol. Biol. 68: 1-10 US 9,309,565 B2 19 20 (1972), 7-amino-actinomycin D (7-AAD)), anthramycin, may be affected by, for example, Sample quality (e.g., purity, auramine, azure B, BOBOTM-1, BOBOTM-3, BO-PROTM-1, presence of reaction inhibitors, and the like) or sequence (e.g., BO-PROTM-3, chromomycin (e.g., A3), crystal violet, cya G/C content, mismatches, and the like). As used herein, nine dyes, DAPI (Kapuscinski, et al. Nucleic Acids Res. amplification efficiency refers to any product that may be 6:3519-3534 (1979)), 4,6-diamidino-2-phenylindole quantified to determine copy number (e.g., a PCR amplicon, (DAPI), daunomycin, distamycin (e.g., distamycin D), dyes an LCR ligation product, or similar product). Variations in described in U.S. Pat. No. 7,387,887, ellipticine, ethidium amplification due to factors other than copy number may salts (e.g., ethidium bromide, ethidium homdimer-1, thereby be limited. ethidium homdimer-2, dihydroethidium (also known as For example, when using PCR, it may be optimal to hydroethidine), ethidium monoazide), fluorcoumanin, fluo 10 amplify amplicons of a similar size (e.g., within about ten rescent intercalators as described in U.S. Pat. No. 4,257,774, percent of the length of any other amplicon) and amplify with GelStar R(Cambrex Bio Science Rockland Inc., Rockland, similar amplification efficiency (e.g., within about ten percent Me...), hexidium iodide, Hoechst 33258 (Searle, et al., (su of one another). PCR-related amplification efficiencies can be pra)), Hoechst 33342, Hoechst 34580, homidium, hydrox tested as described by Livak, et al. (Supra), or as is otherwise ystilbamidine, JO-JO-1, JO-PROTM-1, LDS 751, LOLO-1, 15 known in the art. Briefly, Livak, et al. teach that, as the AC LO-PROTM-1, malachite green, mepacrine (e.g., orange), varies with template dilution, amplifications can be per mithramycin, netropsin, the Nissl Substance, 4',6-diamidino formed over a particular range (e.g., a 100-fold range) and the C-phenylindole, proflavine, POPOTM-1, POPOTM-3, AC calculated at each point. A log of dilution versus AC is PO-PROTM-1, propidium iodide, ruthenium polypyridyls, made and if the absolute value of the slope is close to zero, the Sevron dyes (e.g., Brilliant Red 2B, Brilliant Red 4G, Bril efficiencies are similar. For PCR-based assays, the resultant liant Red B, Orange, Yellow L), SYBR 101, SYBR 102, amplicons are typically about 70 to 125 nucleotides in length, SYBER 103, SYBR(R) Gold, SYBR(R) Green I (U.S. Pat. Nos. and can be less than or equal to about 110 nucleotides in 5,436,134 and 5,658,751), SYBR(R) Green II, SYTOXR Blue, length. The amplification efficiency is typically controlled by SYTOX(R) Green, SYTOX(R) Orange, SYTOR 1, SYTOR 11, amplifying amplicons of similar G/C content, length, or melt SYTOR 13, SYTOR 14, SYTO(R) 15, SYTOR 16, SYTOR) 25 ing temperature. The primers used in the test and control 17, SYTOR 18, SYTOR 20, SYTOR 21, SYTOR 22, assays are also typically of similar character (e.g., G/C con SYTOR 23, SYTOR 24, SYTOR 25, SYTOR 40, SYTOR) tent, length, melting temperature). Variations of these param 43, SYTOR 44, SYTOR. 45, SYTOR 59, SYTOR) 60, eters can also be used, as would be understood by the skilled SYTOR 61, SYTOR 62, SYTOR) 63, SYTOR 64, SYTOR) artisan. 80, SYTOR 81, SYTOR 82, SYTOR, 83, SYTOR) 84, 30 The methods described herein typically require a target SYTOR 85, thiazole orange (Aldrich Chemical Co., Milwau locus to be present on the one or more chromosomes being kee, Wis.), TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, studied (exceptions include, for example, the absence of a TOTO-2, TOTOTM-3, YO-PROR)-1, YO-PROR)-3, YOYO-1, target locus such as when the Y chromosome is assayed in a and YOYOR-3 (Molecular Probes, Inc., Eugene, Oreg.), normal (i.e., XX) female sample). Pre-existing bioinformat among others. SYBRR) Green I (see, e.g., U.S. Pat. Nos. 35 ics algorithms can be used to generate "qualified pre-char 5,436,134; 5,658,751; and/or 6.569,927), for example, has acterized genome targets and to design assays against those been used to monitor a PCR reaction by amplifying the target targets. Qualified genome targets may include, for example, sequence in the presence of the dye, exciting the biological genes, exons, introns, intergenic regions, junctions thereof, sample with light at a wavelength absorbed by the dye and evolutionarily conserved regions ("cold spots not prone to detecting the emission therefrom; and, determining a melting 40 change), or other regions within the genome. In certain profile of the amplified target sequence. The presence of embodiments, multiple target loci can be interrogated on a amplified products and, therefore, the target sequence in the chromosome. This can be used to control for unanticipated sample, can thereafter be determined by, for example, per abnormalities in regions containing target loci. It is possible forming a melting curve analysis (i.e., non-linear least that Such regions can include a duplication, deletion, or other squares regression of the Sum of multiple gaussians). It is to 45 abnormality that may result in an inaccurate karyotype deter be understood that the use of the SYBRR) Green dye is pre mination. By targeting multiple target loci of a chromosome, sented as an example and that many such dyes may be used in the user can control for Such unanticipated abnormalities. the methods described herein. Other nucleic acid binding Thus, in certain embodiments, one, more than one, at least agents can also be suitable as would be understood by one of two, or two or more loci are targeted from at least one chro skill in the art. 50 mosome or arm thereof, but optionally include more than one The nature of the test and control amplified products can arm, to be interrogated. In certain embodiments, three, four, vary depending on the type of assay system that is utilized. five, six, seven, eight, nine, 10 or more loci may be interro For example, the product of PCR amplification of DNA is gated. In one embodiment, four loci are interrogated on each typically referred to as an 'amplicon'. In contrast, the Liga chromosome. In some embodiments, it may also be important tion Chain Reaction product is instead referred to as “LCR 55 to separate the target loci by a particular approximate number product' or “ligation product’. In PCR, primers are utilized of nucleotides which can, in Some embodiments, be calcu but in other methods, such as LCR ligation probes, can be lated as is known in the art to further insure against interro utilized. It is known that both PCR and LCR functions gating a CNVR. It may also be beneficial to separate each through exponential amplification or linear amplification. It is target loci from any other target loci by approximately the important to note, however, that regardless of the kind of 60 same number of nucleotides (e.g., along the arm of a chro method used to perform amplification or detection of target mosome). Many suitable targets exist on each chromosome. loci, any target loci should exhibit similar “amplification Exemplary targets (which can serve as qualified targets) efficiency' to one another (e.g., within about 10 percent). This and their corresponding PCR primers that can be used to means that within a particular reaction, the target loci being interrogate human chromosomes are shown in Table 3. The compared should amplify to approximately the same extent 65 probes that can be used in combination with particular prim (e.g., within about 10 percent) under similar reaction condi ers or primer pairs, and the sequences amplified using par tions. It is understood in the art that amplification efficiency ticular primer pairs are shown in Table 4. One or more of such US 9,309,565 B2 21 22 target loci can be amplified from a biological sample (e.g., Tables 3 and 4 can be used in the methods disclosed herein. It gDNA). It should be understood that non-CNVR targets and is noted that chromosomes of other organisms may be simi corresponding probes or primers other than those listed in larly interrogated but that different target loci are used. TABLE 3 Exemplary Targets and PCR Primers

SEQ SEQ Chromosome Gene ID ID location Cytoband Symbol Forward Primer NO Reverse Primer NO

1: 366 699 68- 1-p34.3 C1orf1 O2 CCAGGGCTGCCTAT 1 GCTGATCCGGCAGAC 2 36670 038 TGACTT ACT

1 : 19882 4 69- 1p36. 13 TMCO4 CCCACGGCCTTCAG 3. GGGCTCTGCACACCC 4 19882569 GAT A.

1 : 176197341 - 1d25.2 SEC16B AACCCACCAGCCTG 5. TGAGCTCAGTCAGTA 6 176197427 AACTG CATCAGAGAT

1: 203386468- 1d32.1 DSTYK CACAATGCTGCCTG 7 TGCTTTGTTGGCAGC 8 2O3386.559 ACATCAT TGGATA

2 : 5106.2327- 2p16.3 NRXN1 TGACAGCTTTTGGC 9 CCTGTCACTGGGAGG 1 O 51O 62418 TCAGAAATTAGA AAATCCATA

2 : 2,742 6789- 2p23.3 GTF3 C2 CACGATGAGGGTTG 11 GATTCTTGAGCCAGC 12 27.42688 O AGGGAAAA AGCTGTA

2 : 141646.278 - 2 q22.1 LRP1B GCCAGGATGCTCCA 13 AAGGATTATTCTGAC 14 14164 63 66 TGTAGTA GGTTCATGTTGT

2 : 1667734O6- 2d24.3 SCN9A AGGTGAAAAAAGTA 15 TTGATCAAAGCTTAT 16 1667735.15 CTTATGAGGATGAT GGCTCTATCAACT GAAT

3 : 71541265- 3p14.1 FOXP1 AAACAGGAGAATGA. 17 CCATAGACAGATTAG 18 71541358 ATGAATGAATATGC CCTGCTTC TT T

3 : 51489,723 - 3p21. 2 WPRBP GCCATCCTCCTTTT 1.9 TGGGTTTGGTGCCTC 2 O 51489.813 TCTCATCCT ACA

3 : 98.908486- 3 q11.2 EPHA6 TGGCATGTGAGATG 21 AAAGGGTACCACTAG 22 989 O8579 TGTTCAAGAAA AGAGGCA

3 : 142551967- 3 q23 ZBTB38 TCCCTCAAGTTTAT 23 AGAGCAAGACGTCCA 24 142552O67 TCAGTCTCCTTATG AATTGAAGTA T

4 : 46733909 - 4p12 GABRB1 CATTTCCAAGTACA 25 CCTCAGTGGTGCAAA. 26 4 6734 O18 GTAACTCCACAGTA AACAGTT

4 : 69314 64- 4p16.1 KIAAO232 CCCAAGCACCTGTA. 27 CCTCAAACATCTGCC 28 6931545 GCATCATC TTCATGTGT

4 : 100651173 - 4q23 C4 or f17 GGAATGTTACCACG 29 CAGCTGTCTTCATCC 3 O 1 OO 651274 TTGCTAAGC AGTTTCTCA

4 : 1469 06234 - 4q31.22 ZNF827 TTTCTGGGAGCCAT 31 CAGCGAATCAAATAG 32 1469 O 6321 CTGAATCATC CCCCTCTT

5 : 26772993- 5p14.1 LOC1 OO131678 GGTATGACCTGGAC 33 AGGAGGGAGATAAAA. 34 2 6f 73 084 ACATTAAGCA ATAATGAGAAATGTA AGC

5 : 137918. Of- 5p15.2 DNAH5 GCAAGCCACAGGAA 35 TGTACAATTTGACTT 36 13791884 GAGGAA GGTGCCAGAA

5 : 1087734.00- 5q21.3 PJA2 GCGGAACAAGCCAG 37 GAGGTTCGAGCGCTG 38 108773485 ACTGAA TTCT

5 : 122258322- 5q23.2 SNX24 GTTTTAGAAGGAAG. 39 AGAATGTGCAGTCAA 4 O 122258422 AGGGCTAGGAA CACTCAGAA

6 : 1297.0453 - 6p24. 1 PHACTR1 GTATAGGCAGCGAC 41 AGATCTCCACAGGAC 42 12970.529 AGCACTT TCACCAA

6 : 3672898- 6p25.2 C6orf145 GAAAACCAAAGCAA 43 GGGAGATGGGAGTAA 44 36 72977 CAAGGTGAGT GTTCCAAAC

US 9,309,565 B2 29 30 TABLE 3 - continued Exemplary Targets and PCR Primers

SEQ SEQ Chromosome Gene ID ID location Cytoband Symbol Forward Primer NO Reverse Primer NO

Y: 2O211350 Yo11.222 CYorf15A CTTCCAGGGAAATT 189 GTTTCCCTGTTGAGA19 O 2O21145.5 CAC CTCTTCT ATGTTTCCAT

Y: 21.33 O 672 Yo11.223 RPS4Y2 GAAAGGGAAGAGCA 191GGCCT CAAAGTCCCA.192 21.33 Of 47 CACAGTCT CACAA

TABLE 4 Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence NO (amplicon) NO

1: 366 699 68 1p34.3 C1orf1 O2 1/2 TCTTCCAA 193 CCAGGGCTG 289 3 667 OO38 GACCTGCA CCTATTGAC CATCCG TTACTCGGA TGTGCAGGT CTTGGAAGA AGATGAGGA GTGTCTGCC GGATCAGC

1 : 19882 4 69 1p36.13 TMCO4 3/4 CAGGAGAC 194 CCCACGGCC 29 O 19882569 AGTGAGTG TTCAGGATG AGCACC GCATCCATC TGCTTGGCA TAGTCCAGG TGGCCGCTG ACCTGCAGG AGACAGTGA GTGAGCACC ACCGTGGGT GTGCAGAGC CC

1: 176 197341 1q25.2 SEC16B 5/6 CCCCAGCT 195 AACCCACCA 291 176197427 TCAGCAGC GCCTGAACT TTG GGACTCCAA GCTGCTGAA GCTGGGGGA TCATCCCGC TCCGGGGCA TCTCTGATG TACTGACTG AGCTCA

1: 2O3386468 1c32.1 DSTYK 7/8 CCGTGCCA 196 CACAATGCT 292 2O3386.559 AGATCACT GCCTGACAT GACTTA CATGGCCTC TGGCTTGCA GAATCCTAA GTCAGTGAT CTTGGCACG GTTC TGCTT ATCCAGCTG CCAACAAAG CA

2 : 510 62327 2p16.3 NRXN1 9/10 AACCACCA 197 TGACAGCTT 293 51062418 ACAAAGGG TTGGCTCAG ATTCT AAATTAGAA AATGAAATG ATAACCACC AACAAAGGG ATTCTGCAG CTGAGTATG GATTTCCTC. CCAGTGACA GG

US 9,309,565 B2 39 40 TABL 4- Continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence (amplicon) NO

GGCCGGGTT CCAGCCTCT GCAGAAGTG

8 : 61648,551 - 8q12. 1 RAB2A. 61/62 ACAAGGCA 223 GGGACCTGG 319 61.648656 AGACAGAG GTTTCCTGA ATGTAC TACTTCCTA TGTGTCACA GTTTTCCCT TAAATGATA ACCGTACAT CTCTGTCTT GCCTTGTCC TTGAATTGT CCACTCGAC TCTGGGT

8 : 754.39579 8q21.11. GDAP1 63/64 CTTTGACC 224 TGGAGAGAA 32O 754.396 60 TCAGTGTT CCCCAGGCT AATTTT TTATATGTA TACTTTGAC

TGCTTATGA ATCACACAC ATTGCTTTA G

9 : 269 49592 9p21.2 IFT74 65/66 CTCCAGTC 225 AGATGGGAT 321 26949 694 TCAACAGC CAAGGGTAA CATTCC ATCAGAGTA AGATTGATC TTGAATGAG AGAAGGAAT GGCTGTTGA GACTGGAGG GCAGGATGG ATGTAGAGA AGTTTATGT GCCC

9 : 4 O8149 9p24.3 DOCK8 67/68 CCAAGGAA 226 TGGCCATAG 322 408232 GACAGCAC CAGGGAATA TATTC ATTTCAATT

9 : 78.498 063 9q21.13 PRUNE2 69/70 CTGCTGAG 227 GGATGGGCA 323 78.498161. TAATTCAC TTTCCC AATCTCCAA

GGTAGGTGC TTAAGAGAT

9 : 1 OOO233 O1 71/72 CCTGCCCA 228 CCAGTGCCT 324 1 OOO23371 GGAGCTAG TGTGCAGGA TG TCTTCACTA GCTCCTGGG CAGGGAGAG GGAAGAATC TTGTGAGCT CAGGGAGG

US 9,309,565 B2 43 44 TABLE 4 - continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence (amplicon)

TTGTTTCGG TTGTTTCGG CGATTTGTC CGCTTC TCG GAGGGGGGC AGAAGCTTC CTTGCCTCC TCCTTGTAC T

11 : 1309.99952 11q25 NTM 87/88 ATCAGGCA 236 GGAATCCAG 332 131OOOO23 GCCAGGAT GAGGTGGTG TT ATGATCAGG CAGCCAGGA TTTCTGTCT CCACAGAGC ATACAGTTT AGTTGGCTG

12: 2734 6341 12p11.23 STK38L 89/90 ACCTCTTC 237 AAGAAATTA. 333 273.46426 ATCTGCTA GAAGTGGCC ATCCTTC ATGGAAGAA GAAGGATTA. GCAGATGAA GAGGTAATG TAATTACCT ATAATTACT GTACAAAAG CACCC

12: 16 O36914 - 12p12. 3 DERA 91/92 CAGCCCAG 238 CAGGTCAAT 334 160370.09 CAAATGCA CTACTGCTA CACAT AGGGATTTC AGCCCAGCA AATGCACAC ATTAAGAAT AATGCCAGA ATGTAGAAA AGTGGCTTT ATCTTTCAG TAGCGG

12: 426 O5583 12C12 TMEM117 93/94 CTGCGGGC 239 AGGCTGGTG 335 426 O5676 TTTAGGAC GAGGCTAGT TCCA GCTCCGCCA CAGCTGCGG GCTTTAGGA CTCCACCTC GTCAGTCAT CCATGCCAA TGGTATGGT TTCCCAGGG TTTC

12: 92044805 - 12q22 LOC643339 95/96 ACTTCCTA 24 O CTTGGGATG 336 92O4 4882 TGACAGCC TTTTATAAG AATCAC TGTCTGTCT GTACTTCCT ATGACAGCC AATCACATC CAACCTATC CTCAGGTCC CTTCCC

1.3 : 1966 O225 13 q12. 11 GJB2 97/98 AAGCCATC 241 TTGACATGA 337 1966 O311 ACTAGGAA GGCCATTTG CTTCT CTATCATAA GCCATCACT AGGAACTTC TAGTCTGTC TCACTCGAT

US 9,309,565 B2 47 48 TABL 4- Continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence NO (amplicon) NO

CCCTACAAG CCCCACGTC CTGGGGGGC TGACTCCAA CTGGGGGTG CTGCTTCCA AGCCTCATG CC

15 : 33605950 15q14 ATPBD4 113/114 ACGTGGCT 249 GAGCACCCA 345 3.3606033 CAGCACTG CTGTGTACG TATAC AGTACACAA AGTGACCAC GTGGCTCAG CACTGTATA CAAATAAGT ATGGCATAA TTCTGCTGG TGT

15 : 43 667323 - 15q21. 1 PLDN 11.5/116 ACGTCACC 25 O GGGATGGAT 346 43 667413 TCTCTGAA TTACCAGAA TTAT AGATGATCA. GCTTATAAT TCAGAGAGG TGACGTATC CTATAATAT TGACCACTC ATGAAATGC TTGGTCAGC

15 : 775.33137 15q25.1 KIAA1024 11.7/118 CTGGGCCT 251 ACTCAGGCT 347 7753,3242 TGGTTTTC TACCATATT CA TGTTTGTAC TTCTTTTAT TCACTTCAG GAGACACTG GGCCTTGGT TTTCCAAAT

TTTCCTTTG CCATGCA

15: 869 71227 15q26. 1 AEN 119/12 O CTGTGGGC 252 AGGTGATAA 348 869.71330 TTTACAAA ATAGCTTAC TTTTA ATTTTAGAG TTTGCT, TTC TGT AGT CAT AAAATTTGT

ACGTTCAGG

16 : 3124 O261 16p11.2 ITGAM 121/122 ACTGAGAG 253 TGATGGTTT 349 3124 O341 TCAAGGCA ATCAT

TACTGAGAG TCAAGGCAA TCATGGAGT TCAATCCCA GGGAAGTGG

16 : 4814 659 16p13.3 N-PAC 123/124 ACCATGTC 254 GCCCAAGAG 350 4814.737 CTCC TCTG CCTGAGTTC TCCGTC TCCTGAGAC GGACACAGG AGGACATGG

US 9,309,565 B2 51 52 TABLE 4 - continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence NO (amplicon) NO 18: 13.731517 - 18p11.21 RNMT 137/138 ATGACAGA 261 GATTGACAA 357 1373.1609 CAAACTGA ATTTCGTGA CAACTGC CCCACAAAT GTGTTTTGA CATCTGCAG TTGTCAGTT TGTCTGTCA TTAC. TCATT TGAGTCTTA TGAGCAGGC TGA

18: 2013 48- 18p11.32 USP14 139/14 O CAAATGAG 262 TCCAGAGCT 358 2O1426 GTGAAACA TTAGAGGAA TAAACCC GACACATAG GTGGGTTTA TGTTTCACC TCATTTGGA ACAAAAGAG GACAGAAGC AGACCAC

18: 57674840 - 18q21.33 RNF152 141/142 AAGATTGC 263 TGCTGTCCT 359 57674938 TCGACCAC TACT CATTC CCCTCC CACATCCTT ACTAGAGGT GAGGGGTTG GGGGAGGGG TGGTCGAGC AATCTTTTG TACTTTTGA GGGTCTGCA AACTTAGGC

18: 74.964920 - 18q23 ATP9B 143/144 TAGTGGTG 264 GTGGTCCAT 360 74.964997 AGAACACC ATGGTCCCT CATCTTC TCTTAAAGA AGATGGGTG TTCT CACCA CTATTTACA

CCCTTT

19: 17313717 – 19p13. 11 GTPBP3 145/146 CCAACCCG 265 CCGTTCGAC 361 1731.3790 GATGCCCC CCTTGATGC TGGGGCATC CGGGTTGGG ATGGAGATA GGAGGATCT CAGTATATT TGTTTCGTA GT

19: 1450 7730 – 19p13. 12 GPSN2 147/148 CAGGACAG 266 GGTGACTCA 362 14507818 AAGGGACT CTGGGCAGA CCACC TTCTCCTGG TGGAGTCCC TTCTGTCCT GGCTGTAGC TTTGTACTT AGGCCATTT CTTTGCTGT AGGCACTC

19: 47551898 - 19q13. 2 MEGF8 14.9/15 O CACTGCCG 267 GCTCTGCCG 363 47ss 1977 CATGGCTC ATGTCCTCA T GGGCTGGGC TGGCCCACA CTGCCGCAT GGCTCTGTG TCCTGAGAA

US 9,309,565 B2 57 58 TABLE 4 - continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence NO (amplicon) NO

TGGAACGCT GCAAGCAGA AGGACCTAC TGGAGCAGA TGATGGCCG AGATGATTG GCGAGTTCC CAGAC

X: 50512491 Xp11.22 SHROOM4 177/178 ACAGGCCC 281 GGAAATTGT 377 5 OS12 567 CATTAATT CAGGTCAGC TATG TCAGTGCCT ACAGGCCCC ATTAATTTA TGTCTCCTT CTTCTGAGA GGACATGAA ATGGG

X: 24.1416 O6 Xp22.11 ZFX 179/18 O CCAAATGC 282 TGCTTTAGG 378 24141705 CAGTCAAA. CAGGTGTGA GTCA ACTCCAGCC CAAATGCCA GTCAAAGTC AAGGCATGG GTTTTCCTA GCCTATCTT ANAGGAAAT TCCTGTACC TTCTTGGCC C

X: 1095,81619 - Xq22.3 RGAG1 181/182 CACTGGCG 283 CCCCATGGT 379 1095,81695 GATTAGAC CAACACAAA. ATCATT ATGTAGACT CTGAAATGA TGTCTAATC CGCCAGTGA GAGCAACAG CCTCTGGGG TGATG

X: 123351536 ODZ.1 183/184 ACCTTCCT 284 GGTCAGAAA 123351615 CACCCAGA GTTACCAGG ATAA ACTTGTCTT GATACCTTA TTCTGGGTG AGGAAGGTC TTATTTTTG TCCACTGCA GACACTGG

Y: 727 1634 Yp11.2 PRKY 185/186 ACTCTCCT 285 GTGATCTGA 381 7271743 CCCTTTGC ATGATGTTG TTCTC AACAAGCAT TATCAAAGA ATTCCACGA TGAGAAGCA AAGGGAGGA

CATTGTCTG GCTATCCGT GT

Y: 5423177 Yp11.2 PCDH11Y 187/188 CAGGGCCA 286 TCCTGCAAA 382 5423278 AGTAGTAA ATGACTTGA AGACCT GTTGGTAAA GTAGAAAGT TTTATGACT ACAAATTTC AGGGCCAAG US 9,309,565 B2 59 60 TABLE 4- Continued Exemplary Amplified Sequences

Primer Pair Probe Amplified Amplicon Chromosome Gene SEQ ID Probe SEQ ID Sequence SEQ ID location Cytoband Symbol NO) Sequence NO (amplicon) NO

TAGTAAAGA

Y: 2O211350- Yo11.222 CYorf15A 189/19 O AACTATAC 287 383 2O21145.5 GATTTGAA AAATTCACC ACAAAATT TCTTCTATA C GAAGAGTTT GTTTTGAAC TATACGATT TGAAACAAA. ATTCTTTTT TTGGAGACT ATGGAAACA TTCTCAACA GGGAAAC

Y: 21.33 O 672- Yo11.223 RPS4Y2 191/192 CCGATATT 288 GAAAGGGAA 384 21.33 Of 47 GCTGGACC GAGCACACA ACCTCT GTCTGTGTT AAGAGGTGG TCCAGCAAT ATCGGCAGG GCTTGTGTG GGACTTTGA GGCC

Any of the exemplary loci shown in Table 3, or any other NOS. 185, 186, 187, 188, 189, 190, 191, 192 (Y chromo suitable target loci that are outside of a CNVR, can be used in Some). Typically, the primers are used in pairs as set forth in the karyotyping methods described herein. Such target loci 35 Tables 3 and 4. It is understood that other primers and primer can also be combined with target loci that fall withina CNVR, pairs not listed in Tables 3 and 4 can be used to interrogate if desired. Particular locican be targeted on each chromosome chromosomes or parts of chromosomes 1-22, X and Y. The and any such target loci can be combined with any other target probes (e.g., SEQ ID NOS. 193-288) are typically used to loci to form a Subset, or screening panel. For instance, any of detect their corresponding amplified sequence (e.g., SEQID SEQID NOS. 1-192 can be used to interrogate chromosomes 40 NOS. 289-384) (Table 4). When multiple chromosomes are 1-22, XorY as shown below: SEQID NOS. 1,2,3,4,5,6,7, interrogated, any of SEQ ID NOS. 1-192 can be used to 8 (chromosome 1); SEQID NOS. 9, 10, 11, 12, 13, 14, 15, 16 interrogate their respective chromosomes. Any one or more of (chromosome 2); SEQID NOS.: 17, 18, 19, 20, 21, 22, 23, 24 SEQID NOS. 1-192 can be used with any other one or more (chromosome 3); SEQID NOS. 25, 26, 27, 28, 29, 30, 31, 32 45 of SEQID NOS. 1-192 to perform such assays. For instance, (chromosome 4), 33, 34,35, 36, 37,38, 39, 40 (chromosome any one or more of SEQID NOS. 1-8 can be used with any 5); SEQID NOS. 41, 42, 43,44, 45,46, 47, 48 (chromosome one or more of SEQ ID NOS. 9-16 to interrogate chromo 6); SEQID NOS. 49, 50, 51, 52,53,54, 55, 56 (chromosome somes 1 and 2, respectively. Other similar subsets of SEQID 7); SEQID NOS. 57,58, 59, 60, 61, 62,63, 64 (chromosome NOS. 1-192 can also be suitable foruse, as would be contem 8): SEQID NOS. 65, 66, 67,68, 69,70, 71, 72 (chromosome 50 plated by one of skill in the art. At least one additional locus 9); SEQID NOS. 73,74, 75,76, 77, 78,79, 80 (chromosome can also be interrogated as a control (e.g., “control gene'). 10); SEQID NOS. 81, 82.83, 84,85,86, 87,88 (chromosome Such additional loci can be, for example, the aforementioned 11); SEQID NOS. 89,90,91, 92,93,94, 95, 96 (chromosome RNase P (chromosome 14, cytoband 14q11.2) or TERT 12); SEQ ID NOS. 97, 98, 99, 100, 101, 102, 103, 104 (chromosome 5, cytoband 5p15.33). (chromosome 13): SEQ ID NOS. 105, 106, 107, 108, 109, 55 In certain embodiments, the karyotyping methods 110, 111, 112 (chromosome 14); SEQ ID NOS. 113, 114, described herein are used to simultaneously interrogate mul 115, 116, 117, 118, 119, 120 (chromosome 15); SEQ ID tiple chromosomes. For example, using a human g|NA NOS. 121, 122, 123, 124, 125, 126, 127, 128 (chromosome sample, more than one chromosome (i.e., chromosomes 1-22, 16); SEQ ID NOS. 129, 130, 131, 132, 133, 134, 135, 136 X and Y) can be simultaneously interrogated in the same or (chromosome 17); SEQ ID NOS. 137, 138, 139, 140, 141, 60 separate assays. Furthermore, a Subset of the total comple 142, 143, 144 (chromosome 18); SEQ ID NOS. 145, 146, ment of chromosomes can be interrogated in the same or 147, 148, 149, 150, 151, 152 (chromosome 19); SEQ ID separate assays. Individual chromosomes can be targeted by NOS. 153, 154, 155, 156, 157, 158, 159, 160 (chromosome amplifying loci specific to Such chromosomes that are outside 20); SEQID NOS. 161, 162, 163, 164, 165, 166, 167, 168 of CNVRs. For example, chromosome 1 can be interrogated (chromosome 21); SEQ ID NOS. 169, 170, 171, 172, 173, 65 along with any one or more of chromosomes 2-22, X or Y: 174, 175, 176 (chromosome 22); SEQ ID NOS. 177, 178, chromosome 2 can be interrogated along with any one or 179, 180, 181, 182, 183, 184 (X chromosome); or SEQ ID more of chromosomes 1 and 3-22, XorY chromosome 3 can US 9,309,565 B2 61 62 be interrogated along with any one or more of chromosomes polymorphisms, and the like. The methods disclosed herein 1-2, 3-22, X or Y chromosome 4 can be interrogated along can be used to compare chromosome copy number between, with any one or more of chromosomes 1-3, 5-22, X or Y: for example, cell types (e.g., lymphocyte, epithelial cell), chromosome 5 can be interrogated along with any one or tissue types (e.g., neural tissue, skeletal muscle tissue), dis more of chromosomes 1-4, 6-22, X or Y chromosome 6 can 5 ease states (e.g., cancerous, non-cancerous), or types of be interrogated along with any one or more of chromosomes organisms (e.g. human, mouse, plant, fruitfly). Other uses for 1-5, 7-22, X or Y chromosome 7 can be interrogated along the methods described herein will be apparent to one of skill with any one or more of chromosomes 1-6, 8-22, X or Y: in the art. chromosome 8 can be interrogated along with any one or The methods described herein can be used to detect chro more of chromosomes 1-7, 9-22, X or Y chromosome 9 can 10 mosomal abnormalities. Exemplary abnormalities include, be interrogated along with any one or more of chromosomes but are not limited to, deletion, duplication, translocation, 1-8, 10-22, XorY: chromosome 10 can be interrogated along mosaicism, aneuploidy (e.g., nullisomy, disomy, trisomy and with any one or more of chromosomes 1-9, 11-22, X or Y: tetrasomy), inversion, ring formation, isochromosome for chromosome 11 can be interrogated along with any one or mation, or chromosomal instability syndromes. Pathologies more of chromosomes 1-10, 12-22, X or Y chromosome 12 15 have been associated with abnormalities in particular human can be interrogated along with any one or more of chromo chromosomes including, for example, chromosome 1 (e.g., somes 1-11, 13-22, X or Y chromosome 13 can be interro acute lymphoblastic leukemia, acute megakaryoblastic leu gated along with any one or more of chromosomes 1-12, kemia, alveolar rhabdomyosarcoma), chromosome 2 (e.g., 14-22, XorY chromosome 14 can be interrogated along with anaplastic large cell lymphoma, alveolar rhabdomyosar any one or more of chromosomes 1-13, 15-22, X or Y; chro coma), chromosome 3, chromosome 4 (e.g., Wolf-Hir mosome 15 can be interrogated along with any one or more of schhorn syndrome, acute lymphoblastic leukemia), chromo chromosomes 1-14, 16-22, X or Y chromosome 16 can be Some 5 (e.g., Cri du chat, also known as Chromosome 5q. interrogated along with any one or more of chromosomes deletion syndrome, anaplastic large cell lymphoma), chromo 1-15, 17-22. X or Y; chromosome 17 can be interrogated Some 6, chromosome 7 (e.g., Williams syndrome), chromo along with any one or more of chromosomes 1-16, 18-22, X 25 Some 8 (e.g., Burkitt's lymphoma, acute lymphoblastic leu orY chromosome 18 can be interrogated along with any one kemia, acute myeloblastic leukemia with maturation), or more of chromosomes 1-17, 19-22, XorY chromosome 19 chromosome 9 (e.g., Trisomy 9, Warkany syndrome 2, acute can be interrogated along with any one or more of chromo lymphoblastic leukemia, Philadelphia chromosome), chro somes 1-18, 20-22, X or Y chromosome 20 can be interro mosome 10, chromosome 11 (e.g., Jacobsen syndrome, gated along with any one or more of chromosomes 1-19, 21. 30 mantle cell lymphoma, multiple myeloma, acute lympho 22, X or Y chromosome 21 can be interrogated along with blastic leukemia, Ewings sarcoma, desmoplastic Small any one or more of chromosomes 1-20, 22, X or Y chromo round cell tumor), chromosome 12 (e.g., acute lymphoblastic Some 22 can be interrogated along with any one or more of leukemia, myxoid liposarcoma), chromosome 13 (e.g., Patau chromosomes 1-21, X or Y; X chromosome can be interro syndrome, alveolar rhabdomyosarcoma, breast and ovarian gated along with any one or more of chromosomes 1-22 orY. 35 cancers, deafness, Wilson's disease), chromosome 14 (e.g., Y chromosome can be interrogated along with any one or Burkitt's lymphoma, follicular lymphoma, acute lympho more of chromosomes 1-22 or X. Other subsets of chromo blastic leukemia), chromosome 15 (e.g., Angelman syn Somes can also be interrogated as would be understood by one drome, Prader-Willi syndrome, acute promyelocytic leuke ofskill in the art. For PCR-based assays, any of the primer sets mia, Marfan syndrome, Tay-Sachs disease), chromosome 16 shown in Tables 3 or 4 can be used as may any other suitable 40 (e.g., Trisomy 16, myxoid liposarcoma, polycystic kidney primer set. disease, C.-thalassemia), chromosome 17 (e.g., Miller-Dieker Described herein are karyotyping methods for determining syndrome, Smith-Magenis syndrome, acute promyelocytic the copy number of one or more chromosomes in a test leukemia, dermatofibrosarcoma protuberans, Charcot biological sample (Such as a test g|NA). In certain embodi Marie-Tooth disease), chromosome 18 (e.g., Edwards Syn ments, the methods can be used to determine the karyotype of 45 drome, Burkitt's lymphoma, follicular lymphoma, mantle a test genome. The karyotype information typically relates to cell lymphoma, multiple myeloma, Synovial sarcoma, Nie one or more chromosomes of a genome. man-Pick disease, pancreatic cancer), chromosome 19 (e.g., Such methods described herein can be used in prenatal acute lymphoblastic leukemia), chromosome 20, chromo diagnostic assays to Screen for and detect chromosomal Some 21 (e.g., Down syndrome, acute lymphoblastic leuke abnormalities in a fetus or embryo, such as Down's Syn 50 mia, acute myeloblastic leukemia with maturation), chromo drome (Trisomy 21), Edwards Syndrome (Trisomy 18) and Some 22 (e.g., Di George's syndrome, Trisomy 22, acute Patau Syndrome (Trisomy 13). These assays can be per lymphoblastic leukemia, Philadelphia chromosome, acute formed on biological samples such as fetal cells or cell-free megakaryoblastic leukemia, Ewing's sarcoma, dermatofibro fetal DNA in maternal blood, amniotic fluid, trophoblast sarcoma protuberans, desmoplastic Small round cell tumor), cells, chorionic Villus samples and percutaneous umbilical 55 X chromosome (e.g., Fragile X syndrome, Turner syndrome, cord blood. Furthermore, the methods disclosed herein can Triple X syndrome, Klinefelter's syndrome, synovial sar also be performed on embryos used in in vitro fertilization coma, mixed gonadal dysgenesis, XX gonadal dysgenesis, (IVF) wherein blastocyst cells or cells biopsied from the uniparental disomy, Duchenne muscular dystrophy, X-linked trophectoderm are analyzed from embryos that are 3 to 6 days diseases, hemophilia, adrenoleukodystrophy, Hunter's dis post-fertilization, in order to determine if they should be 60 ease), or Y chromosome (e.g., Klinefelter's syndrome, uni implanted in utero. parental disomy, acute myeloid leukemia). Any of these dis The methods described herein can be useful in, for orders, or any other disorders that may be detected by example, stem cell analysis, quality control assays of cell karyotyping as would be known by the skilled artisan, can be cultures, analysis of samples of limited quantity (e.g. single studied using the methods described herein. cell, formalin-fixed paraffin-embedded (FFPE)), compari 65 Kits for determining the copy number of multiple chromo Sons between cell or tissue types, comparisons between dis somes are also provided. For use with a PCR-based assay, the eased or non-diseased tissues, detection of chromosomal kit can include at least a set of primers for amplifying at least US 9,309,565 B2 63 64 one test locus on a chromosome (e.g., any one or more of SEQ are further described in the following examples. These ID NOS. 1-192) or corresponding probes (e.g., any one or embodiments are provided as examples only and are not more of SEQ ID NOS. 193-288). The kit can also include intended to limit the scope of the claims in any way. samples of pre-determined amplified sequences such as those listed in Table 4 (e.g., any one or more of SEQ ID NOS. EXAMPLES 289-384), which can optionally be affixed to a solid support A. Karyotyping of Chromosomes 2, 3, 4, 5, 6, 20 and (e.g. a membrane, glass slide, multi-well plate) for use as a 22 control reaction. The kit can also optionally include Stock Solutions, buffers, enzymes, detectable labels or reagents The primers and probes used to interrogate the targets of required for detection, tubes, membranes, and the like that 10 interest described below were designed using masked can be used to complete the amplification reaction. In some genomic DNA sequences (the human genome assembly B36) embodiments, multiple primer sets are included. The kits can by an Applied Biosytems proprietary TaqMan(R) Copy Num also comprise the reagents required to use a reference (e.g., ber Assay Design Pipeline. The existing pre-designed Taq primers and probes for amplifying or detecting a reference Man(R) copy number assay protocols were utilized. TaqMan(R) sequence such as RNase Por TERT) or calibrator sample (i.e., 15 copy number assays targeting chromosomal regions located a control genome of known karyotype, a chromosome outside of copy number variable regions (CNVR) on each of sample, or the like). In some embodiments, the kits can con the 24 chromosomes were designed (the data shown below, tain multiple sets of primers for amplifying multiple target however, relates only to chromosomes 2,3,4, 5, 6, 20 and 22.) loci on a single chromosome or among two or more chromo As described above, to cover all 24 chromosomes on a 384 Somes (e.g., any combination of primers sets and probes well plate or TaqMan(R) array card (TLDA), four assays can be shown in Tables 3 and 4, or any primer/probe sets that target selected for each chromosome, using two assays on each arm loci outside of CNVRs). In one embodiment, a multi-well of each chromosome whenever possible. The assays were run plate contains within its wells an array of replicate wells for as duplex TaqMan(R) real-time PCR reactions. AFAMTM dye use in carrying out assays. For example, a 384-well array based assay was used to detect test target loci and the VIC(R) containing four replicate wells for each of chromosomes 25 dye-based assay was used for the reference locus RNase P 1-22, X and Y comprising primer/probe sets targeting loci (PN 431 6844 from Applied Biosystems). Each assay utilized outside of CNVRs for each of the chromosomes, is provided, 10 ng g|DNA, 1x TaqMan(R) probe?primer mix (900 nM of optionally including reagents such as Stock Solutions, buffers, each primer, 250 nM of each probe) in 1x TaqMan(R) Geno enzymes such as polymerases, and detectable labels. Other typing Master Mix in a 10 Jul reaction (run in quadruplicate). embodiments of particular systems and kits are also contem 30 PCR reactions were incubated in an Applied Biosystems plated which would be understood by one of skill in the art. In 7900HT SDS instrument for 10 minutes at 95°C., followed addition, the arrays can be customized depending on the by 40 cycles of 15 seconds at 95°C. and 60 seconds at 60° C. target samples being analyzed, for example, arrays compris Real-time data was collected by the SDS 2.3 software. The ing one, two, three or more chromosomes comprising one, relative quantification analysis was performed to calculate two, or more than two loci to be analyzed per chromosome. 35 estimated copy number of each sample for the gene of interest Furthermore, the number of replicate wells or samples per by CopyCallerTM software. chromosome per array can vary (e.g., one, two, three, four, or These methods were used to interrogate loci on chromo more than four). somes 2, 3, 4, 5, 6, 20 and 22 of human g|DNA samples (a set All references cited within this disclosure are hereby incor of 92 African American and Caucasian gldNA samples from porated by reference in their entirety. Certain embodiments Coriell Institute) using the following primer sets and probes: TABLE 5

SEQ ID NO. SEQ ID (primer NO. Amplified sequence SEO ID NO. Chromosome pairs) Probe (probe) (amplicon) (amplicon) 2 9/10 AACCACCA 197 TGACAGCTTTTGGCTCAGAAAT 293 ACAAAGGG TAGAAAATGAAATGATAACCAC ATTCT CAACAAAGGGATTCTGCAGCTG AGTATGGATTTCCTCCCAGTGA CAGG

11/12 ACCTTCCC 198 CACGATGAGGGTTGAGGGAAAA 294 AGTTTGAT ACAGTTGGTAGGAACAAGTGCT AAGCAC TATCAAACTGGGAAGGTGCTCT TATTTACAGCTGCTGGCTCAAG AATC

13/14 CTGGCCTG 199 GCCAGGATGCTCCATGTAGTAT 295 TGTGTACT TGCATTATAACATGGTCTCAGC TCT TGGCCTGTGTGTACTTCTACAA CATGAACCGTCAGAATAATCCT

15/16 ACCCATGC 2OO AGGTGAAAAAAGTACTTATGAG 296 CTCT, TTCT GATGATGAATAGTTGGAAAAAC AATAAC TAGTAAAATAGGGTTGGTTATT AGAAAGAGGCATGGGTAGTTGA TAGAGCCATAAGCTTTGATCAA

US 9,309,565 B2 67 68 TABLE 5- continued

SEO ID NO. SEO ID (primer NO. Amplified sequence SEQ ID NO. Chromosome pairs) Probe (probe) (amplicon) (amplicon)

43/44 CCTCAAGC 214 GAAAACCAAAGCAACAAGGTGA 31 O TCCGACCC GTCCTCAGGAGGGGTCGGAGCT CTCCT TGAGGTTTTGGAGTTTGGAACT TAC TCCCATCTCCC

45/46 CATGTATA 215 GGGTTGCAGGGATGGTGTACAA 311 GCTGCATA CAGGTCCTAGCATGTATAGCTG GATTTC CATAGATTTCTTCACCTGATCT TTGTGTGGAAGATCAGAATGAA TGCA

47/48 CAGTCTGA 216 GGGATCTGTAACTTGACCAAGG 312 TGGTCCCA TCAAAGAGCTTGAAATTTCAAC AGTTGA TTGGGACCATCAGACTGAAAAC CTGCAATCTGAAACACTTTGCT GTGCTGC

2O 153/154 CAGTGAAG 269 CCACTTCCCT CAATCATGTGAC 365 CCACC TTT ACCACTTCAGTGAAGCCACCTT AAATCA. TAAATCATCTGTTTTTGAATTT GTCTGGAATCCAGAAAAAGTTG GCAAAACCC

155/156 AATGGGCA 27 O GTGCGAAGGTTAGTTTCTGAGG 366 TCATCATC AAGGAAAAAAGTTGATGATGAT AACTTT GCCCATTGTCAGGTCTGTAACT AACTTGCTGGGTTCCTCTC

157/158 CCCACCAC 271 CCCATGGGAGATGCTCTTGAGA 367 CAGAATAG AAGACTATTCTGGTGGTGGGTG TCTTT CGGGATCCTGTCAGGGGGAAGA GATGGAACCAGGAGACC

159/16O CTGTGTGC 272 GCCCTGTCCCTGCTTCTGGAAA 368 AGATGTCG AAGAATTTTCGACATCTGCACA AAAAT CAGACAGTTGTGAAAAAGGAGG AGAAGCAGCTACTGGCTAAGGG GCACCT

22 169/17O CCCAGACC 277 CCTTACCCATCCCAACTAGCCT 373 AGAAGAGT TACCCATCCTCGCCTCTCTCCT TCCAC CAGCCCAGACCAGAAGAGTTCC ACCAGCGATCCAACGTCACACT CACTCTAC

171/1.72 CTGTGCAT 278 CCTCCTGATCATACTCTGGTAC 374 CGGCCTCC CTGGCCTGTGCATCGGCCTCCT TGC GCTTCATGTCAACCTCCTACTC CTGCCAGGGAATGTG

173/174 CCAGCAGC 279 CACATGAGGCTTTCGGAGGAAT 375 CTCGATTT AAATTGGTCTGAAATCGAGGCT CAGAC GCTGGCTTTTGTGTTAATAANT GTGTAAAAGCTATCCAGGGCAT CAAGG

175/176 CTGCTTGC 28O CCATCCCTCTCCACAGCAAGGA 376 AGCGTTCC TGACGTGGAACGCTGCAAGCAG ACGTC AAGGACCTACTGGAGCAGATGA TGGCCGAGATGATTGGCGAGTT CCCAGAC

The resultant data is shown in FIG.1 (chromosome 2), FIG. B. Detection of Disease Pathology by Karyotype 2 (chromosome 3), FIG.3 (chromosome 4), FIG. 4 (chromo 60 Analysis some 5), FIG. 5 (chromosome 6), FIG. 6 (chromosome 20), FIG. 7 (chromosome 20), and FIG. 8 (chromosome 22). As Many forms of cancer have been associated with various chromosomal abnormalities (see discussion hereinabove). shown therein, the methods described herein (e.g., interrogat Detection of diseases or susceptibility to diseases is critical ing target loci located outside CNVRs) has been used to 65 for proper diagnosis or treatment. For example, certain breast accurately karyotype various chromosomes found within the and ovarian cancers are associated with abnormalities in human genome. chromosome 13. Primers and probes useful for interrogation US 9,309,565 B2 69 70 of non-CNVR loci of chromosome 13 can be used to deter 1x TaqMan(R) Genotyping Master Mix in a 10ul reaction (run mine whether a patient has or is susceptible to having a breast in quadruplicate). PCR reactions are incubated in an Applied or ovarian cancer. Biosystems 7900HT SDS instrument for 10 minutes at 95° The primers and probes used to interrogate the chromo C., followed by 40 cycles of 15 seconds at 95° C. and 60 seconds at 60° C. Real-time data is collected by the SDS 2.3 Some 13 target loci are designed using masked genomic DNA Software. The relative quantification analysis is performed as sequences (the human genome assembly B36) by an Applied described herein above to calculate estimated copy number of Biosystems proprietary TaqMan(R) Copy Number Assay each sample for the gene of interest by CopyCallerTM soft Design Pipeline and interrogated using copy number assays Wa. of format 1 and using virtual calibrator assay no. 1. Test 10 Identification of abnormal chromosomal copy number and samples are obtained from normal and tumor cell and tissue. karyotype of chromosome 13 indicates the presence or Sus As described above, four assays are selected for chromosome ceptibility to the breast or ovarian cancer associated with 13, using two assays on each arm of the chromosome. The chromosome 13. assays are run as duplex TaqMan(R) real-time PCR reactions. While certain embodiments have been described in terms A FAMTM dye-based assay is used to detect test target loci and 15 of the preferred embodiments, it is understood that variations the VICR) dye-based assay is used for the reference locus and modifications will occur to those skilled in the art. There RNase P (PN 431 6844 from Applied Biosystems) in assay fore, it is intended that the appended claims cover all such format 1. Each assay uses 10 ng gldNA, 1x TaqMan(R) probe/ equivalent variations that come within the scope of the fol primer mix (900 nM of each primer, 250 nM of each probe) in lowing claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 384

<21 Oc SEO ID NO 1 <211 LENGTH: 2O <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <22 Os FEATURE; OTHER INFORMATION: Forward Primer sequence <4 OOs SEQUENCE: 1 Ccagggctgc ct attgactt

SEO ID NO 2 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Reverse Primer sequence <4 OOs SEQUENCE: 2

gctgatc.cgg cagacact 18

SEO ID NO 3 LENGTH: 17 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Forward Primer sequence <4 OOs SEQUENCE: 3

Cccacggcct tcaggat 17

SEO ID NO 4 LENGTH: 16 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Reverse Primer sequence <4 OOs SEQUENCE: 4

gggct Ctgca caccca 16

<21 Os SEQ ID NO 5 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,309,565 B2 71 72 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 5 aacccaccag cct gaactg 19

<210s, SEQ ID NO 6 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 6 tgagct cagt cagtacat ca gagat 25

<210s, SEQ ID NO 7 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 7 cacaatgctg cct gacatca t 21

<210s, SEQ ID NO 8 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 8 tgctttgttg gcagotggat a 21

<210s, SEQ ID NO 9 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 9 tgacagcttt tectic agaa attaga 26

<210s, SEQ ID NO 10 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 10

Cctgtcactg ggaggaaatc cata 24

<210s, SEQ ID NO 11 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 11 cacgatgagg gttgagggaa aa 22 US 9,309,565 B2 73 74 - Continued

<210s, SEQ ID NO 12 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 12 gatt Cttgag cca.gcagotg ta 22

<210s, SEQ ID NO 13 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 13 gccaggatgc ticcatgtagt a 21

<210s, SEQ ID NO 14 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 14 aaggattatt Ctgacggttc atgttgt 27

<210s, SEQ ID NO 15 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 15 aggtgaaaaa agtact tatg aggatgatga at 32

<210s, SEQ ID NO 16 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 16 ttgatcaaag cittatggctic tat caact 28

<210s, SEQ ID NO 17 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 17 aaac aggaga atgaatgaat gaatatgct 29

<210s, SEQ ID NO 18 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence US 9,309,565 B2 75 76 - Continued

<4 OOs, SEQUENCE: 18 c catagacag attagcctgc titctt 25

<210s, SEQ ID NO 19 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 19 c catagacag attagcctgc titctt 25

<210s, SEQ ID NO 2 O &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 2O tgggtttggit gcct caca 18

<210s, SEQ ID NO 21 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 21 tggcatgtga gatgtgttca agaaa 25

<210s, SEQ ID NO 22 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 22 aaaggg tacc act agagagg Ca 22

<210s, SEQ ID NO 23 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 23 t ccct caagt ttatt cagtic ticcittatgt 29

<210s, SEQ ID NO 24 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 24 agagcaagac gtc.calaattg aagta 25

<210s, SEQ ID NO 25 US 9,309,565 B2 77 - Continued

&211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 25 catttic caag tacagtaact c cacagta 28

<210s, SEQ ID NO 26 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 26

Ccticagtggit gcaaaaacag tt 22

<210s, SEQ ID NO 27 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 27 cc caag cacc tdtag.cat catc 22

<210s, SEQ ID NO 28 <211 LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 28 cctcaaacat citgcct tcat gtgt 24

<210s, SEQ ID NO 29 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 29 ggaatgttac Cacgttgcta agc 23

<210s, SEQ ID NO 3 O &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 30 cagotgtc.tt catccagttt citca 24

<210s, SEQ ID NO 31 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 31 US 9,309,565 B2 79 80 - Continued tittctgggag ccatctgaat catc 24

<210s, SEQ ID NO 32 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 32 cagogaatca aatago cc ct citt 23

<210s, SEQ ID NO 33 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 33 gg tatgacct ggacac atta agca 24

<210s, SEQ ID NO 34 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 34 aggagggaga taaaaataat gagaaatgta agc 33

<210s, SEQ ID NO 35 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 35 gcaa.gc.caca ggaagaggaa

<210s, SEQ ID NO 36 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 36 tgtaca attt gaCttggtgc Cagaa 25

<210s, SEQ ID NO 37 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OO > SEQUENCE: 37 gcggaacaag C cagactgaa

<210s, SEQ ID NO 38 &211s LENGTH: 19 &212s. TYPE: DNA US 9,309,565 B2 81 82 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 38 gaggttcgag cqctgttct 19

<210s, SEQ ID NO 39 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 39 gttittagaag galagagggct aggaa 25

<210s, SEQ ID NO 4 O &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 4 O agaatgtgca gtcaac actic agaa 24

<210s, SEQ ID NO 41 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 41 gtataggcag cacagcact t 21

<210s, SEQ ID NO 42 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 42 agat ct coac aggact cacc aa 22

<210s, SEQ ID NO 43 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 43 gaaalaccalaa goaacaaggit gagt 24

<210s, SEQ ID NO 44 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 44 gggagatggg agtaagttcc aaac 24 US 9,309,565 B2 83 84 - Continued

<210s, SEQ ID NO 45 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 45 gggttgcagg gatggtgta 19

<210s, SEQ ID NO 46 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 46 tgcatt catt ctdatctitcc acaca 25

<210s, SEQ ID NO 47 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 47 gggatctgta acttgaccala ggit 23

<210s, SEQ ID NO 48 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 48 gcagcacagc aaagttgtttic ag 22

<210s, SEQ ID NO 49 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 49 gatggitat ct cocacaagtic actac 25

<210s, SEQ ID NO 50 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 50

Caatttgcag cacaaggagc ta 22

<210s, SEQ ID NO 51 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 85 86 - Continued <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 51

Ctgtctgatt gttalagaggg Cttgt 25

<210s, SEQ ID NO 52 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 52 gtcaca actt gttcttggga gtttg 25

<210s, SEQ ID NO 53 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 53 gatagatggc ctggcagtaa gaac 24

<210s, SEQ ID NO 54 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 54 gcct tc tagg gactgactitc aa 22

<210s, SEQ ID NO 55 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 55 tgaacctittg acacct tccc aaa 23

<210s, SEQ ID NO 56 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 56 tgacagcaaa tattaccgaa ggtgat 26

<210s, SEQ ID NO 57 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OO > SEQUENCE: 57 gatcto ct ct tctgtgccta catc 24 US 9,309,565 B2 87 88 - Continued

<210s, SEQ ID NO 58 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 58 tgtgatgaca aaaacatalaa tag actgag t 31

<210s, SEQ ID NO 59 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 59 gttct citctg gcc.gtcaata t citt 24

<210s, SEQ ID NO 60 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 60 Cact tctgca gaggctggaa

<210 SEQ ID NO 61 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 61 gggacctggg ttt Cotgata ct 22

<210s, SEQ ID NO 62 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 62 acccagagtic gagtggacaa t 21

<210s, SEQ ID NO 63 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 63 tggagagaac CCC aggctitt at 22

<210s, SEQ ID NO 64 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence US 9,309,565 B2 89 90 - Continued

<4 OOs, SEQUENCE: 64 ctaaagcaat gtgttgttgatt cataa.gca 28

<210s, SEQ ID NO 65 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 65 agatgggat.c aagggit aaat cagagt 26

<210s, SEQ ID NO 66 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 66 gggcacataa acttct ctac atcca 25

<210s, SEQ ID NO 67 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence < 4 OO SEQUENCE: 67 tggc catagc agggaataat ttca 24

<210s, SEQ ID NO 68 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 68 accagacaca totgcaccaa

<210s, SEQ ID NO 69 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 69 ggatgggcaa ttt taggitaa tict coaa 27

<210s, SEQ ID NO 70 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OO > SEQUENCE: 7 O atct cittaag cacct accct ggit 23

<210s, SEQ ID NO 71 &211s LENGTH: 19 US 9,309,565 B2 91 92 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 71

Ccagtgcctt gtgcaggat 19

<210s, SEQ ID NO 72 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 72 cctic cctdag ct cacaagat t 21

<210s, SEQ ID NO 73 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 73 aaatagaata tdagacatga gtaaatatgc ccttitt 36

<210s, SEQ ID NO 74 &211s LENGTH: 24 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 74 t caagttgta gtcagtgcca caaa 24

<210s, SEQ ID NO 75 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 75 caaaagctac aagc.ca.gaga tacga 25

<210s, SEQ ID NO 76 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OO > SEQUENCE: 76 acacatatgt acagagagac Caggaa 26

<210s, SEQ ID NO 77 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OO > SEQUENCE: 77 US 9,309,565 B2 93 94 - Continued alagacaatgt gcagdaaaag at agc 25

<210s, SEQ ID NO 78 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OO > SEQUENCE: 78 ggcct ctitco td tatttgca gttt 24

<210s, SEQ ID NO 79 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 79 caaattittac ccacacagcc tdaaa 25

<210s, SEQ ID NO 8O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 80 gCttaagttg tatgtgtgca gagtt 25

<210s, SEQ ID NO 81 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 81 aaaacaagta gggcactgga agaa 24

<210s, SEQ ID NO 82 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 82 tgcc ctitt ca actgctgatt ctaat 25

<210s, SEQ ID NO 83 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 83 cattgcagaa citcCagggaa ct 22

<210s, SEQ ID NO 84 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,309,565 B2 95 96 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 84

Ctgtctgcga C9ggalaga 18

<210s, SEQ ID NO 85 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 85 cc.ccggctitc ggttct 16

<210s, SEQ ID NO 86 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 86 agtacaagga ggaggcaagg aa 22

<210s, SEQ ID NO 87 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 87 ggaatcCagg aggtggtgat g 21

<210s, SEQ ID NO 88 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 88 cago caacta aactgtatgc tictdt 25

<210s, SEQ ID NO 89 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 89 aagaaattag aagtggc.cat ggalaga 26

<210s, SEQ ID NO 90 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 90 gggtgcttitt gtacagtaat tataggit 27 US 9,309,565 B2 97 98 - Continued

<210s, SEQ ID NO 91 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 91

Cagg to aatc tactgctaag ggatt 25

<210s, SEQ ID NO 92 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 92 cc.gctactgaaagataaag.c cactt 25

<210s, SEQ ID NO 93 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 93 aggctggtgg aggctagt 18

<210s, SEQ ID NO 94 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 94 gaaaccctgg gaalaccatac cat 23

<210s, SEQ ID NO 95 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 95 cittgggatgt tittataagtg totgtctgt 29

<210s, SEQ ID NO 96 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 96 gggaagggac Ctgaggatag g 21

<210s, SEQ ID NO 97 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence US 9,309,565 B2 99 100 - Continued

<4 OO > SEQUENCE: 97 ttgacatgag gccatttgct atca 24

<210s, SEQ ID NO 98 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 98 gcaccitaa.ca acattgtagc ct caa 25

<210s, SEQ ID NO 99 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 99 agatctggga caaggit ctgt git 22

<210s, SEQ ID NO 100 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 1.OO gctaatgtca acgatgtc.ca toaag 25

<210s, SEQ ID NO 101 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 101 acttct coag titgaaagggit atcca 25

<210s, SEQ ID NO 102 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 102 ggtaatggag act catgaat gacact 26

<210s, SEQ ID NO 103 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 103 gcct agaata t cctgcagtg gtaga 25

<210s, SEQ ID NO 104 US 9,309,565 B2 101 102 - Continued

&211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 104 tgct tccagt cittggcatct titt 23

<210s, SEQ ID NO 105 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 105 ggagga aggc Cagcagaag 19

<210s, SEQ ID NO 106 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 106 acaa.ca.gcag ctic caacca 19

<210s, SEQ ID NO 107 & 211 LENGTH; 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 107 cagocttitt.c accaac ctitc aaa 23

<210s, SEQ ID NO 108 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 108 accagacacic accitatgatt gga 23

<210s, SEQ ID NO 109 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 109 gataggcgca C caggaatga

<210s, SEQ ID NO 110 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 110 US 9,309,565 B2 103 104 - Continued

Cagtggccaa acacga atta aagt 24

<210s, SEQ ID NO 111 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 111 catccactica ccactgtatic ca 22

<210s, SEQ ID NO 112 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 112 ggcatgaggc titggaa.gca 19

<210s, SEQ ID NO 113 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 113 gagg acccac ttgtacga 19

<210s, SEQ ID NO 114 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 114 acaccagoag aattatgcca tactt 25

<210s, SEQ ID NO 115 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 115 gggatggatt taccagaaag atgat cag 28

<210s, SEQ ID NO 116 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 116 agct gaccaa gcattt catg agt 23

<210s, SEQ ID NO 117 &211s LENGTH: 29 &212s. TYPE: DNA US 9,309,565 B2 105 106 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 117 acticaggctt accatatttgtttgtactt 29

<210s, SEQ ID NO 118 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 118 tgcatggcaa aggaaatcCC a 21

<210s, SEQ ID NO 119 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 119 aggtgataaa tagcttacat tittagagttt gct 33

<210s, SEQ ID NO 120 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 120 c catacct ga acgtgatgcc a 21

<210s, SEQ ID NO 121 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 121 tgatggittitt ctdgtgtc.cc tittag 25

<210s, SEQ ID NO 122 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 122 c cact tcc ct gggattgaac t 21

<210s, SEQ ID NO 123 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 123 gcc.calaga.gc ctgagttct 19 US 9,309,565 B2 107 108 - Continued

<210s, SEQ ID NO 124 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 124 ccgggc catc gatgaagag 19

<210s, SEQ ID NO 125 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 125 c catcgtaag gcttggaatc gaa 23

<210s, SEQ ID NO 126 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 126 ggct acttico Cagcaaggta at 22

<210s, SEQ ID NO 127 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 127 ggtc.ca.gc.cc ttct caacac

<210s, SEQ ID NO 128 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 128 accago actic accogctaaa 19

<210s, SEQ ID NO 129 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 129 t cctgg tagg gatacagctic att 23

<210s, SEQ ID NO 130 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 109 110 - Continued <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 130 gtctggitttg Caggga cact

<210s, SEQ ID NO 131 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 131 ggcago.cgat ggt cagta 18

<210s, SEQ ID NO 132 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 132 ggccttgggc agaacagtaa ata 23

<210s, SEQ ID NO 133 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 133 c cct ctdctg gcacctittaa g 21

<210s, SEQ ID NO 134 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 134 gccCaggtgg gactgaga 18

<210s, SEQ ID NO 135 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 135 gcttagagcc ticcatttctt toct 24

<210s, SEQ ID NO 136 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 136 agactitt.cca to cagtgaga t cca 24 US 9,309,565 B2 111 112 - Continued

<210s, SEQ ID NO 137 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 137 gattgacaaa titt cqtgacc cacaa 25

<210s, SEQ ID NO 138 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 138 t cagcc tigct cataag actic aaatg 25

<210s, SEQ ID NO 139 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 139 tccagagctt tagaggaaga Cacat 25

<210 SEQ ID NO 140 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 140 gtggtctgct tctgtc.ct ct titt 23

<210s, SEQ ID NO 141 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 141 tgctgtcc tit act cattcca catc 24

<210s, SEQ ID NO 142 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 142 gcctaagttt gcagaccctic aa 22

<210s, SEQ ID NO 143 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence US 9,309,565 B2 113 114 - Continued

<4 OOs, SEQUENCE: 143 gtggtc. cata tdgtocct to ttaaa 25

<210s, SEQ ID NO 144 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 144 aaagggactic titc.cggttct td 22

<210s, SEQ ID NO 145 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 145 cc.gttcgacc cittgatgct 19

<210s, SEQ ID NO 146 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence < 4 OO SEQUENCE: 146 actacgaaac aaatatactg agatcctic ct 3 O

<210s, SEQ ID NO 147 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 147 ggtgacticac togcagatt C 21

<210s, SEQ ID NO 148 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 148 gagtgcctac agcaaagaaa tig 23

<210s, SEQ ID NO 149 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 149 gctctg.ccga tigt cctica 18

<210s, SEQ ID NO 150 &211s LENGTH: 2O US 9,309,565 B2 115 116 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 150 Cagtgtgggc attgcagttc

<210s, SEQ ID NO 151 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 151

Cagtactggit gagggaaatc titt 24

<210s, SEQ ID NO 152 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 152 tccaggctta taacgtagga cacta 25

<210s, SEQ ID NO 153 &211s LENGTH: 23 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 153 c cact tcc ct caat catgtg aca 23

<210s, SEQ ID NO 154 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 154 gggttittgcc aacttitttct ggatt 25

<210s, SEQ ID NO 155 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OO > SEQUENCE: 155 gtgc galaggt tagtttctgaggaa 24

<210s, SEQ ID NO 156 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 156 US 9,309,565 B2 117 118 - Continued gagaggalacc cagcaagtta gtt 23

<210s, SEQ ID NO 157 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 157 cc catgggag atgcticttga

<210s, SEQ ID NO 158 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 158 ggtc.t.cct gg titc catct ct tc 22

<210s, SEQ ID NO 159 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 159 gCCCtgtcCC ticttctg 18

<210s, SEQ ID NO 160 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 160 aggtgc.ccct tagc.cagta 19

<210s, SEQ ID NO 161 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 161 Caggcagctt Cttggit caga

<210s, SEQ ID NO 162 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 162 gtgtc.ccggc atggatgt 18

<210s, SEQ ID NO 163 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,309,565 B2 119 120 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 163 agcatccitat gcc tittgaca aaga 24

<210s, SEQ ID NO 164 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 164

Cagtaa.gcaa gcaggctagg t 21

<210s, SEQ ID NO 165 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 165 gttctgattt c tactic ct col acaca 25

<210s, SEQ ID NO 166 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 166 ggaatcagtic tittgcatttg catct 25

<210s, SEQ ID NO 167 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 167

Ctgatgtgtc. ttggCagg to at 22

<210s, SEQ ID NO 168 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 168 c catggat.ca ggacgt.cgaa ag 22

<210s, SEQ ID NO 169 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 169 ccttacccat cocaactago citta 24 US 9,309,565 B2 121 122 - Continued

<210s, SEQ ID NO 170 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 170 gtagagtgag ttgacgttg gat 23

<210s, SEQ ID NO 171 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 171 cctic ctdatc at actctggit acct 24

<210s, SEQ ID NO 172 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 172

Cacatt coct ggcaggagta g 21

<210s, SEQ ID NO 173 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 173

Cacatgaggc titt cqgagga ataa 24

<210s, SEQ ID NO 174 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 174 ccttgatgcc ctdgatagct tttac 25

<210s, SEQ ID NO 175 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OO > SEQUENCE: 175 c catcc ct ct c cacagdaa 19

<210s, SEQ ID NO 176 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence US 9,309,565 B2 123 124 - Continued

<4 OOs, SEQUENCE: 176 gtctgggaac togccalatca t 21

<210s, SEQ ID NO 177 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 177 ggaaattgtc. aggtoagctic agt 23

<210s, SEQ ID NO 178 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 178 cc cattt cat gtcct ct cag aagaa 25

<210s, SEQ ID NO 179 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OO > SEQUENCE: 179 tgctittaggc aggtgtgaac t 21

<210s, SEQ ID NO 18O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 18O gggccaagaa ggtacaggaa titt 23

<210s, SEQ ID NO 181 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 181 c cc catggtc. aacacaaaat gtag 24

<210s, SEQ ID NO 182 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 182 catcaccc.ca gaggctgtt 19

<210s, SEQ ID NO 183 US 9,309,565 B2 125 126 - Continued

&211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 183 ggtcagaaag ttaccaggac ttgt 24

<210s, SEQ ID NO 184 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 184

Ccagtgtctg. Cagtggacaa aa 22

<210s, SEQ ID NO 185 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 185 gtgatctgaa tatgttgaa caa.gca 26

<210s, SEQ ID NO 186 & 211 LENGTH 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 186 acacggatag ccagacaatgaaatac 26

<210s, SEQ ID NO 187 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 187 t cctgcaaaa tacttgagt tta 25

<210s, SEQ ID NO 188 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence

<4 OOs, SEQUENCE: 188 acatgtcagg cctttittatt ttgtagc 27

<210s, SEQ ID NO 189 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence

<4 OOs, SEQUENCE: 189 US 9,309,565 B2 127 128 - Continued ctitccaggga aatticacctic ttct 24

<210s, SEQ ID NO 190 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 190 gttt coctdt tdagaatgtt to cat 25

<210s, SEQ ID NO 191 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer sequence <4 OOs, SEQUENCE: 191 gaaagggaag agcacacagt ct 22

<210s, SEQ ID NO 192 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer sequence <4 OOs, SEQUENCE: 192 ggcct caaag toccacacaa

<210s, SEQ ID NO 193 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 193 tott coaaga cct gcacatc cq 22

<210s, SEQ ID NO 194 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 194

Caggagacag tagtgagca cc 22

<210s, SEQ ID NO 195 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 195 c cccagctitc agcagottg 19

<210s, SEQ ID NO 196 &211s LENGTH: 22 &212s. TYPE: DNA US 9,309,565 B2 129 130 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 196 cc.gtgccaag at cactgact ta 22

<210s, SEQ ID NO 197 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OO > SEQUENCE: 197 aaccaccaac aaagggattic t 21

<210s, SEQ ID NO 198 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 198 acct tcc.cag tittgataagc ac 22

<210s, SEQ ID NO 199 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 199 ctggcc tigtg td tact tct 19

<210s, SEQ ID NO 2 OO &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 2OO acccatgcct ctittctaata ac 22

<210s, SEQ ID NO 2 O1 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 2O1 cctgcc tacc aaagaggata ca 22

<210s, SEQ ID NO 202 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 2O2 tcc.ca.gc.cca citgitaaatg 19 US 9,309,565 B2 131 132 - Continued

<210s, SEQ ID NO 203 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 2O3 aatgccacca gtgacct tca g 21

<210s, SEQ ID NO 204 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 204 tottctittgc ticcitgaga cc tic 22

<210s, SEQ ID NO 205 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 205 at aggtgtcc ctgtaaag.ca ac 22

<210s, SEQ ID NO 206 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 2O6 t cct gctgcc cactgacctg

<210s, SEQ ID NO 2 O7 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 2O7 ccacac aggc tcc ttggagt aa 22

<210s, SEQ ID NO 208 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 208 ttgctgttgg ctgaat cact

<210s, SEQ ID NO 209 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 133 134 - Continued

<223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 209 aatggagaag ggaaaattac

<210s, SEQ ID NO 210 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 210

Cagggaga.ca aaacagaaat at 22

<210s, SEQ ID NO 211 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 211 ccggagcc.gc tigCacat 17

<210s, SEQ ID NO 212 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 212 c tagg tactg cqc cacttitt

<210s, SEQ ID NO 213 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 213 atgcaactica togctgaattt a 21

<210s, SEQ ID NO 214 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 214 cct caa.gctic cqaccc ct cot 21

<210s, SEQ ID NO 215 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 215 catgtatago togcatagatt to 22 US 9,309,565 B2 135 136 - Continued

<210s, SEQ ID NO 216 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 216

Cagt ctgatg gtc.ccaagtt ga 22

<210s, SEQ ID NO 217 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 217 ccaccott to tacatt to to c 21

<210s, SEQ ID NO 218 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 218 t ccttgccaa citagaaacta td 22

<210 SEQ ID NO 219 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 219 aaagaatcag gCaggtaaag ct 22

<210s, SEQ ID NO 220 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 220 acgctgggct attt catcat ct 22

<210s, SEQ ID NO 221 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 221 citt.cccc.cag caaagttagt td 22

<210s, SEQ ID NO 222 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence US 9,309,565 B2 137 138 - Continued

< 4 OOs SEQUENCE: 222 ccacggtc.ca citctgtccac git 22

SEQ ID NO 223 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 223 acaaggcaag acagagatgt ac 22

SEQ ID NO 224 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 224 ctittgacctic agtgttaatt tt 22

SEQ ID NO 225 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 225 citccagt citc aacago catt co 22

SEQ ID NO 226 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 226 cCaaggaaga cagcactatt C 21

SEO ID NO 227 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 227 ctgctgagta attcactitt c cc 22

SEQ ID NO 228 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 228

Cctgcc Cagg agctagtg 18

SEQ ID NO 229 LENGTH: 2O US 9,309,565 B2 139 140 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 229 acaaccagcc acatggattic

<210s, SEQ ID NO 230 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 23 O ctgagcaa.gc atttgatcct gc 22

<210s, SEQ ID NO 231 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 231

Cttcacagac Cagataaac g 21

<210s, SEQ ID NO 232 &211s LENGTH: 19 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 232 tcqgtc.ttct gcatct tcc 19

<210s, SEQ ID NO 233 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 233 acccaacaca acago attaa git 22

<210s, SEQ ID NO 234 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 234

Caagggctgg cactcc.ca 18

<210s, SEQ ID NO 235 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 235 US 9,309,565 B2 141 142 - Continued titt.cggttgt titcggcgatt to 22

<210s, SEQ ID NO 236 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 236 at Cagg cagc Caggattt 18

<210s, SEQ ID NO 237 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 237 acct citt cat citgctaatcc titc 23

<210s, SEQ ID NO 238 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 238

CagcCC agca aatgcacaca t 21

<210s, SEQ ID NO 239 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 239 Ctgcgggctt taggacticca

<210s, SEQ ID NO 24 O &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 240 actitcc tatg acagocaatc ac 22

<210s, SEQ ID NO 241 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 241 aagc catcac taggaact to t 21

<210s, SEQ ID NO 242 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,309,565 B2 143 144 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 242 ttgagc.ca.gc agaaatgtt 19

<210s, SEQ ID NO 243 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 243 cagat cagtg cagtgtttct ca 22

<210s, SEQ ID NO 244 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 244 ttgctic catt coagaagata gc 22

<210s, SEQ ID NO 245 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 245 ctgagaagat gcaaccaatic ct 22

<210s, SEQ ID NO 246 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 246 aag.cgatgat caattacgaa aact 24

<210s, SEQ ID NO 247 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 247 cc.gc.catata citt coctaaa got 23

<210s, SEQ ID NO 248 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 248 totcct coct ttgtttitccc US 9,309,565 B2 145 146 - Continued

SEQ ID NO 249 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 249 acgtggctica gcactgtata C 21

SEO ID NO 250 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 250 acgt cacctic tictdaattat

SEQ ID NO 251 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 251

Ctgggccttg gttitt.cca 18

SEQ ID NO 252 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 252 ctgtgggctt tacaaattitt a 21

SEO ID NO 253 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence SEQUENCE: 253 actgagagtic aaggcaatca t 21

SEQ ID NO 254 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence

SEQUENCE: 254 accatgtc.ct c ct gtgtc.cg to 22

SEO ID NO 255 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe sequence US 9,309,565 B2 147 148 - Continued

<4 OO > SEQUENCE: 255 caactgct cattggittattt to 22

<210s, SEQ ID NO 256 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 256 aag.ccc.ttga gccatctitt 19

<210s, SEQ ID NO 257 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OO > SEQUENCE: 257 titcggcaatig accatc ctitt g 21

<210s, SEQ ID NO 258 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 258 cct cittgggc atgtctitt cot 21

<210s, SEQ ID NO 259 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 259

Cagataggag ccttgaagaa aca 23

<210s, SEQ ID NO 260 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 260 ttgcgaaacg cgattgcc.ca

<210s, SEQ ID NO 261 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 261 atgacagaca aactgacaac togc 23

<210s, SEQ ID NO 262 US 9,309,565 B2 149 150 - Continued

&211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 262 caaatgaggit gaalacatalaa CCC 23

<210s, SEQ ID NO 263 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 263 aagattgctic gaccaccc ct c c 22

<210s, SEQ ID NO 264 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 264 tagtggtgag aac acc catc titc 23

<210s, SEQ ID NO 265 & 211 LENGTH; 16 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 265 c caa.cccgga tigc.ccc 16

<210s, SEQ ID NO 266 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 266

Caggacagaa gggacticcac C 21

<210s, SEQ ID NO 267 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 267 cactg.ccgca tdgctict 17

<210s, SEQ ID NO 268 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 268 US 9,309,565 B2 151 152 - Continued caagtic ctac agtgcattca td 22

<210s, SEQ ID NO 269 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 269 cagtgaagcc acctittaaat ca 22

<210s, SEQ ID NO 270 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 270 aatgggcatc atcatcaact tt 22

<210s, SEQ ID NO 271 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 271 cccaccacca gaatagt citt t 21

<210s, SEQ ID NO 272 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 272

Ctgtgtgcag atgtcgaaaa t 21

<210s, SEQ ID NO 273 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 273 Catctggc cc tigcgtacca

<210s, SEQ ID NO 274 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 274 cCaggtgttg Ctgagttt at

<210s, SEQ ID NO 275 &211s LENGTH: 19 &212s. TYPE: DNA US 9,309,565 B2 153 154 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OO > SEQUENCE: 275

Cactatggcg taattgttg 19

<210s, SEQ ID NO 276 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 276 c cattcacca gccaaagtt 19

<210s, SEQ ID NO 277 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OO > SEQUENCE: 277 cc.ca.gaccag aagagttcca C 21

<210s, SEQ ID NO 278 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OO > SEQUENCE: 278

Ctgtgcatcg gccticctic 19

<210s, SEQ ID NO 279 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OO > SEQUENCE: 279 c cagcago: ct cqattt caga c 21

<210s, SEQ ID NO 280 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 280 ctgcttgcag cqttccacgt c 21

<210s, SEQ ID NO 281 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 281 acaggc.ccca ttaattitatg US 9,309,565 B2 155 156 - Continued

<210s, SEQ ID NO 282 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 282 c caaatgcca gtcaaagtica

<210s, SEQ ID NO 283 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 283

Cactggcgga ttagacat catt 22

<210s, SEQ ID NO 284 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 284 acct tcctica cccagaataa

<210s, SEQ ID NO 285 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 285 actictoctoc ctittgcttct c 21

<210s, SEQ ID NO 286 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OOs, SEQUENCE: 286

Cagggccaag tagtaaagac ct 22

<210s, SEQ ID NO 287 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe sequence

<4 OO > SEQUENCE: 287 aactatacga tittgaaacaa aattic 25

<210s, SEQ ID NO 288 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 157 158 - Continued

<223> OTHER INFORMATION: Probe sequence <4 OOs, SEQUENCE: 288 ccgatattgc tiggaccacct ct 22

<210s, SEQ ID NO 289 &211s LENGTH: 71 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 289 cCagggctgc ctattgactt act cqgatgt gcaggtottg gaagaagatg aggagtgtct 6 O gccggat.cag C 71.

<210s, SEQ ID NO 290 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 290

Cccacggcct t caggatggc atcCatctgc titggCatagt ccaggtggcc gctgacctgc 6 O aggaga cagt gagtgagcac caccgtgggt gtgcagagcc C

<210s, SEQ ID NO 291 & 211 LENGTH; 87 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 291 alacccaccag cctgaactgg act coaa.gct gctgaagctgggggatcatC cc.gct Coggg 6 O gcatct ctda td tactgact gagctica 87

<210s, SEQ ID NO 292 &211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 292

Cacaatgctg. cctgacat catcCtctgg Cttgcagaat cctaagt cag tdatcttggc 6 O acggttctgc titat coagct gccaacaaag ca 92

<210s, SEQ ID NO 293 &211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 293 tgacagcttt tectic agaa attagaaaat gaaatgataa ccaccaacaa agggattctg 6 O

Cagctgagta tigatttic ct cccagtgaca gg 92

<210s, SEQ ID NO 294 &211s LENGTH: 92 US 9,309,565 B2 159 160 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 294 cacgatgagg gttgagggaa aaa.cagttgg taggaacaag tictitat caa actgggalagg 6 O tgct cittatt tacagotgct ggct caagaa to 92

<210s, SEQ ID NO 295 &211s LENGTH: 89 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 295 gccaggatgc ticcatgtagt attgcatt at alacatggtct cagctggcct gtgtgtactt 6 O ctacaa catg aacct caga ataatcctt 89

<210s, SEQ ID NO 296 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 296 aggtgaaaaa agtact tatg aggatgatga at agttggala aaact agtaa aatagggttg 6 O gttatt agaa agaggcatgg gtagttgata gagccataag Ctttgat Caa 11 O

<210s, SEQ ID NO 297 &211s LENGTH: 94 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OO > SEQUENCE: 297 aaac aggaga atgaatgaat gaatatgcta atacaac cac citctgitatic c tictittgg tag 6 O gCaggaggca agaagcaggc taatctgtct atgg 94

<210s, SEQ ID NO 298 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 298 gccatcct cotttittct cat cct gtggggg tact tatgat gtgatgc.cat ttacagtggg 6 O

Ctgggattac aggtgtgagg caccaaac cc a 91

<210s, SEQ ID NO 299 &211s LENGTH: 94 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 299 tggcatgtga gatgtgttca agaaacticaa gacta aggga atgaatgaat gaatgccacc 6 O US 9,309,565 B2 161 162 - Continued agtgacct tc agtgcct ct c tagtggtacc ctitt 94

<210s, SEQ ID NO 3 OO &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 3 OO t ccct caagt ttatt cagtic ticcittatgta at cagtaatt citatcaaatc ct cittctittg 6 O citcc tdagac ct caccitact t caatttgga cqt cittgctic t

<210s, SEQ ID NO 3 O1 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 301 catttic caag tacagtaact c cacagtact atcctgttgc titt acaggga caccitatgcc 6 O tttitttctitc agaataaaga acattgcaaa citgtttittgc accactgagg 11 O

<210s, SEQ ID NO 3 O2 &211s LENGTH: 82 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 302 cc caag cacc tdtag.cat catcgtccacgt cct gctg.ccc act gacctgt ccggctic cac 6 O acatgaaggc agatgtttga gg 82

<210s, SEQ ID NO 3 O3 &211s LENGTH: 102 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 303 ggaatgttac Cacgttgcta agctatgtaa Cat at Cttaa Calaccaggga gcc acacagg 6 O CtcCttggag taagagtgtg agaaactgga tigaagacagc tig

<210s, SEQ ID NO 3 O4 &211s LENGTH: 88 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 304 tittctgggag ccatctgaat Catctittgct gttggctgaa t cact cagag Ctgagaggga 6 O ggatgaagag gggctatttg attcgctg 88

<210s, SEQ ID NO 3 O5 &211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon US 9,309,565 B2 163 164 - Continued

<4 OOs, SEQUENCE: 305 gg tatgacct ggacacatta agcatt.cgta attitt coctt ct c cattact agatacagtg 6 O citta catt to t catta t t t t t at ct coctic ct 92

<210s, SEQ ID NO 3 O6 &211s LENGTH: 78 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 306 gcaa.gc.caca ggaagaggala gcc caaaggc agggaga caa alacagaaata tottctggc 6 O accalagt caa attgtaca 78

<210s, SEQ ID NO 3 O7 &211s LENGTH: 86 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OO > SEQUENCE: 3 O7 gcggaacaag C cagactgaa aaaaaaaaaa aaaaaccctic accgaaatgt gcagoggctic 6 O cggagcgaga acagogct cq aacct c 86

<210 SEQ ID NO 3 O8 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 308 gttittagaag galagagggct aggaagaaaa gtggcgcagt acctagtagg taagtataat 6 O ctggatgctic ccagtaattic tdagtgttga citgcacattct

<210s, SEQ ID NO 3 O9 211 LENGTH: 77 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 309 gtataggcag cacagcact ttaaattica gcatgagttg catggttggc caatgttggit 6 O gagt ccttg gagatct 77

<210s, SEQ ID NO 310 &211s LENGTH: 8O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 310 gaaalaccalaa goaacaaggit gag to ct cag gaggggtcgg agcttgaggt tttggagttt 6 O ggaact tact cocatc tocc

<210s, SEQ ID NO 311 US 9,309,565 B2 165 166 - Continued

&211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 311 gggttgcagg gatggtgtac aac aggtoct agcatgtata gctgcataga titt Cttcacc 6 O tgat Ctttgt gtggaagat.c agaatgaatg Ca 92

<210s, SEQ ID NO 312 &211s LENGTH: 95 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 312 gggatctgta acttgaccala ggtcaaagag Cttgaaattt Caacttggga c catcagact 6 O gaaaacct gc aatctgaaac actittgctgt gctgc 95

<210s, SEQ ID NO 313 &211s LENGTH: 109 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 313 gatggit at Ct CCCaCaagtic act actt CCt gtgtttittgc gaaaagctic C C C9tgagggit 60 gggtgccacc ctittctacat ttctic cctag ctic cttgtgc tigcaaattg 109

<210s, SEQ ID NO 314 &211s LENGTH: 87 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 314

Ctgtctgatt gttalagaggg Cttgt attct Cttgaaaatc at agtttct a gttggcaagg 6 O agcaaact co Caagaacaag ttgttgac 87

<210s, SEQ ID NO 315 &211s LENGTH: 89 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 315 gatagatggc ctggcagtaa gaacaagaca C9gaaagctt tacct gcct g attctitt CCt 6 O t cct tc.tttg aagttcagt co citagaaggc 89

<210s, SEQ ID NO 316 &211s LENGTH: 83 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 316 tgaacctittg acacct tccc aaaacgctgg gctattt cat catcttctac agt citt catc 6 O US 9,309,565 B2 167 168 - Continued acct tcggta at atttgctg. tca 83

<210s, SEQ ID NO 317 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 317 gatcto ct ct tctgtgccta catcaactitc ccc.ca.gcaaa gttagttgta t citttgtcta 6 O cticagt ct catttatgttitt tdt catcaca 9 O

<210s, SEQ ID NO 318 &211s LENGTH: 99 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 318 gttct citctg gcc.gtcaata t cittaatgaaagtgacattc cqttggccac gigt coactict 6 O gtcCacgtgg agggc.cgggit to cagcct ct gcagaagtg 99

<210s, SEQ ID NO 319 &211s LENGTH: 106 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 319 gggacctggg titt cotgata citt cotatgt gtcacagttt toccittaaat gataaccota 6 O catctotgtc. ttgccttgtc. cittgaattgt ccact cqact citgggit 106

<210s, SEQ ID NO 320 &211s LENGTH: 82 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 32O tggagagaac cccaggctitt atatgtatac tittgacctica gtgttaattt taaatgctta 6 O tgaat cacac acattgctitt ag 82

<210s, SEQ ID NO 321 &211s LENGTH: 103 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 321 agatgggat.c aagggit aaat cagagtaaga ttgat Cttga atgagagaag gaatggctgt 6 O tgagactgga gggcaggatg gatgtagaga agtttatgtg CCC 103

<210s, SEQ ID NO 322 &211s LENGTH: 84 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 169 170 - Continued <223> OTHER INFORMATION: Amplicon 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (62) . . (62) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 322 tggc catagc agggaataat ttcaatttga aaacaagtgg aatagtgctg. tct tccttgg 6 O tntgttggtg Cacatgtgtc. tt 84

<210s, SEQ ID NO 323 &211s LENGTH: 99 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 323 ggatgggcaa ttt taggitaa tict coaattg accta actict aatggaatgg gaaagtgaat 6 O tact cagcag atgaccacca ggg taggtgc tita agagat 99

<210s, SEQ ID NO 324 &211s LENGTH: 71 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 324

C CagtgcCtt gtgcaggat.c tt Cactagct CCtgggcagg gagagggaag alat Cttgttga 60 gctic agggag g 71.

<210s, SEQ ID NO 325 &211s LENGTH: 82 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 325 aaatagaata tdagacatga gtaaatatgc ccttittatac aaccago cac atggatt citt 6 O tgtggcactg act acaactt ga 82

<210s, SEQ ID NO 326 &211s LENGTH: 95 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 326 caaaagctac aagc.ca.gaga tacgatacaa Caaggacatt gct ctgcagg atcaaatgct 6 O tgct cagatt toctdgtctic tictd tacata tdtgt 95

<210s, SEQ ID NO 327 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 327 aaga caatgt gcagdaaaag atagotc.cat cataaccacg tttitt tatga ttgtc.tt cac 6 O US 9,309,565 B2 171 172 - Continued agaccgagat aaacgaaaaa citgcaaatac aggaagaggc C

<210s, SEQ ID NO 328 &211s LENGTH: 104 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 328 caaattittac ccacacagcc tdaaaaatac Cttgaaag.ca aacct cqgtc ttctgcatct 6 O tccaattgat tcc titt acaa actctgcaca catacaactt aagc 104

<210s, SEQ ID NO 329 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 329 aaaacaagta gggcactgga agaaaaac cc aacacaa.cag cattaagttt caaacct gca 6 O titcCaattag aat cagcagt taaagggca 9 O

<210s, SEQ ID NO 330 &211s LENGTH: 74 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 330 cattgcagaa citcCagggaa ct catgaaga gtgcaagggc tiggcact coc agc.cagt ctt 6 O cc.cgt.cgcag acag 74

<210s, SEQ ID NO 331 &211s LENGTH: 1.OO &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 331 cc.ccggct tc ggttctgc.cg gttacgcttgttt cqgttgt titcggcgatt tdt CCCtt C 6 O tcggaggggg gCagaa.gctt ccttgcct Co. tcc ttgtact

<210s, SEQ ID NO 332 &211s LENGTH: 72 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 332 ggaatcCagg aggtggtgat gat Caggcag C caggatttic tetct coaca gag catacag 6 O tittagttggc tig 72

<210s, SEQ ID NO 333 &211s LENGTH: 86 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,309,565 B2 173 174 - Continued <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 333 aagaaattag aagtggc.cat ggaagaagaa ggattagcag atgaagaggit aatgtaatta 6 O cctataatta citgtacaaaa goaccc 86

<210s, SEQ ID NO 334 &211s LENGTH: 96 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 334 cagg to aatc tactgctaag ggattitcago cca.gcaaatg cacacattaa gaataatgcc 6 O agaatgtaga aaagtggctt tat ctitt Cag tagcgg 96

<210s, SEQ ID NO 335 &211s LENGTH: 94 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 335 aggctggtgg aggctagtgc ticcgc.cacag ctg.cgggctt taggacticca cct citcagt 6 O catc catgcc aatgg tatgg titt cocaggg titt c 94

<210s, SEQ ID NO 336 &211s LENGTH: 78 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 336 cittgggatgt tittataagtg totgtctgta citt cotatga cagccaatca catccaacct 6 O atcc to aggt ccctt.ccc 78

<210s, SEQ ID NO 337 &211s LENGTH: 87 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OO > SEQUENCE: 337 ttgacatgag gccatttgct at cataagcc at cactagga acttic tagt c tdt ct cactic 6 O gattgaggct acaatgttgt taggtgc 87

<210s, SEQ ID NO 338 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 338 agatctggga caaggit ctgt gtc.cctaaga catttct cag agtggtttga gcc agcagaa 6 O atgttgcttg atggacat cq ttgacattag C 91 US 9,309,565 B2 175 176 - Continued

<210s, SEQ ID NO 339 &211s LENGTH: 81 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon"

<4 OOs, SEQUENCE: 339 acttct coag titgaaagggit atc catttga gaalacactgc act gatctgg aatatagtgt 6 O catt catgag tot coattac c 81

<210s, SEQ ID NO 340 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 340 gcct agaata t cctgcagtg gtagagtttg Ctic catt coa galagatagcc aaaaagaagc 6 O tgagaaaaaa agatgccaag actggaagca 9 O

<210s, SEQ ID NO 341 &211s LENGTH: 72 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 341 ggagga aggc cagcagaa.gc aggattggitt gcatcttct c aggaaggct g c cctggttgg 6 O agctgctgtt gt 72

<210s, SEQ ID NO 342 &211s LENGTH: 75 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 342 cagocttitt.c accaac ctitc aaaaagttitt cqtaattgat catcgct tcc tict coaatca 6 O taggtggtgt Ctggit

<210s, SEQ ID NO 343 &211s LENGTH: 98 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 343 gataggcgca ccaggaatga cc.gc.catata citt coctaaa gct caac cca cccaccagtt 6 O cagttaagaa ttatactitta attcgtgttt ggccactg 98

<210s, SEQ ID NO 344 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 344 US 9,309,565 B2 177 178 - Continued catccactica ccactgtatic catccacctic ticctic cc titt gttitt coct a caa.gc.cccac 6 O gtcCtggggg gctgacticca actgggggtg Ctgct tccaa goctoatgcc 11 O

<210s, SEQ ID NO 345 &211s LENGTH: 84 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 345 gagg acccac ttgtacgag tacacaaagt gaccacgtgg Ctcagcactg. tatacaaata 6 O agtatggcat a attctgctg gtgt 84

<210s, SEQ ID NO 346 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 346 gggatggatt taccagaaag atgat cagct tataatt cag agaggtgacg tat cotataa 6 O tattgaccac toatgaaatig cittggtcago t 91

<210s, SEQ ID NO 347 &211s LENGTH: 106 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon <4 OOs, SEQUENCE: 347 acticaggctt accatatttgtttgtactitc titt tatt cac titcaggagac actgggc citt 6 O ggttitt coaa at agggitttt tdacctggga titt cotttgc catgca 106

<210s, SEQ ID NO 348 &211s LENGTH: 104 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 348 aggtgataaa tagcttacat tittagagttt gctttctgtt ataaaagttg tacgcattga 6 O tataaaattt gtaaag.ccca cagtggcatc acgttcaggt atgg 104

<210s, SEQ ID NO 349 &211s LENGTH: 81 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amplicon

<4 OOs, SEQUENCE: 349 tgatggittitt Ctggtgtc.cc tittaggtocc agc.cagtact gagagt caag gcaat catgg 6 O agttcaatcc Cagggaagtgg 81

<210s, SEQ ID NO 350 &211s LENGTH: 79 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence