Advanced multiplex PCR method and composition

Abstract

To provide a method for simultaneously amplifying plural nucleic acid regions of an object with one reaction volume, and a method for selecting a primer library for use in the amplification method, and provide a primer library having a desired property such as minimum formation of an amplification primer dimer or other non-target amplification product.SOLUTION: The invention provide a method for amplifying a target gene locus in a nucleic acid sample, comprising: (a) a step for bringing the nucleic acid sample into contact with a test primer library which hybridizes simultaneously to at least different 1000 target gene loci, for producing a reaction mixture; and (b) a step for subjecting the reaction mixture to a primer extension reaction condition for producing an amplification product including a target amplification product.SELECTED DRAWING: Figure 29

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Utility Model Application No. 13 / 683,604 filed November 21, 2012, and U.S. Provisional Patent Application No. 61 / 675,020 filed July 24, 2012. U.S. Utility Model Application No. 13 / 683,604 is a continuation of U.S. Utility Model Application No. 13 / 300,235 filed November 18, 2011, which in turn is a continuation of U.S. Utility Model Application No. 13 / 110,685 filed May 18, 2011, and claims the benefits of U.S. Provisional Patent Application No. 61 / 675,020 filed July 24, 2012. U.S. Utility Model Application No. 13 / 110,685 claims the interests of U.S. Provisional Patent Application No. 61 / 395,850 filed on 18 May 2010; U.S. Provisional Patent Application No. 61 / 398,159 filed on 21 June 2010; U.S. Provisional Patent Application No. 61 / 462,972 filed on 9 February 2011; U.S. Provisional Patent Application No. 61 / 448,547 filed on 2 March 2011; and U.S. Provisional Patent Application No. 61 / 516,996 filed on 12 April 2011. U.S. Utility Model Application No. 13 / 300,235 claims the interests of U.S. Provisional Patent Application No. 61 / 571,248 filed on 23 June 2011. The entirety of all these patent applications is incorporated herein by reference for all their teachings.

[0002] Description of research and development funded by the federal government. This invention was made possible with the support of the National Institutes of Health (NIH) under authorization number 5R44HD60423-3. The U.S. Government may retain all patent rights arising from this application.

[0003] Field of Invention The present invention generally relates to a method and composition for simultaneously amplifying multiple target nucleic acid regions in a single reaction volume. [Background technology]

[0004] To increase assay throughput and enable more efficient use of nucleic acid samples, simultaneous amplification of multiple target nucleic acids in a sample can be performed by mixing multiple oligonucleotide primers with the sample and then subjecting the sample to polymerase chain reaction (PCR) conditions in a process known in the art as multiplex PCR. The use of multiplex PCR is a very simple experimental procedure and can reduce the time required for nucleic acid analysis and detection. However, when multiple pairs are added to the same PCR reaction, non-target amplification products, such as amplified primer dimers, may be generated. The likelihood of such product generation increases with the number of primers. These non-target amplification products significantly limit the subsequent analysis and / or use of the amplified products in assays. Therefore, improved methods are needed to reduce the formation of non-target amplification products during multiplex PCR.

[0005] Improved multiplex PCR techniques will be useful for a variety of applications, including non-invasive prenatal genetic diagnosis (NPD). Specifically, current prenatal diagnostic methods can alert physicians and parents to abnormalities in developing fetuses. Without prenatal diagnosis, 1 in 50 infants are born with significant physical or mental disabilities, and 1 in 30 have some form of congenital malformation. Unfortunately, standard methods are either inaccurate or involve invasive procedures that carry a risk of miscarriage. Methods based on maternal blood hormone levels or ultrasound measurements are non-invasive but similarly inaccurate. Methods such as amniocentesis, chorionic villus sampling, and fetal blood sampling are highly accurate but invasive and carry significant risks. In the United States, amniocentesis was performed on approximately 3% of all pregnancies, but its usage has decreased over the past 15 years.

[0006] In normal humans, all healthy diploid cells have two sets of 23 chromosomes, with one copy originating from each parent. Aneuploidy, a condition in nuclear cells with too many and / or too few chromosomes, is thought to be involved in implantation failure, miscarriage, and the majority of genetic diseases. Detecting chromosomal abnormalities can, in addition to increasing the chances of successful pregnancy, identify individuals or embryos with conditions such as Down syndrome, Klinefelter syndrome, and Turner syndrome. Testing for chromosomal abnormalities is particularly important because it is estimated that at least 40% of embryos are abnormal when the mother is between 35 and 40 years old, and more than half are abnormal when the mother is over 40.

[0007] Recently, it has been discovered that cell-free fetal DNA and intact fetal cells can enter the maternal blood circulation system. Therefore, it is thought that early NPD (Natural Pregnancy-Developing) may be possible by analyzing this genetic material. Improved methods should ideally offer increased sensitivity and specificity, and reduce the time and cost required for NPD. [Overview of the project] [Means for solving the problem]

[0008] In one embodiment, the present invention is characterized by a method for amplifying target gene loci in a nucleic acid sample. In some embodiments, the method includes (i) contacting a nucleic acid sample with a test primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target gene loci to generate a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing target amplification products. In some embodiments, the method also includes the step of determining the presence or absence of at least one target amplification product (e.g., at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of target amplification products). In some embodiments, the method also includes the step of determining the sequence of at least one target amplification product (e.g., at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target amplification product).

[0009] In various embodiments of any aspect of the present invention, at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the amplification product is the target amplification product. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification product is primer dimers. In some embodiments, the library of test primers includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 test primer pairs, each primer pair including a forward test primer and a reverse test primer that hybridize to the same target locus. In some embodiments, the library of test primers includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 individual test primers that hybridize to different target loci, and the individual primers are not part of a primer pair.

[0010] In various embodiments of any aspect of the present invention, the concentration of each test primer is less than 100, 75, 50, 25, 10, 5, 2, or 1 nM. In various embodiments, the GC content of the test primer is 30-80%, for example, 40-70% or 50-60%. In some embodiments, the range of GC content of the test primer (e.g., maximum GC content - minimum GC content, for example, 80%-60%=20%) is less than 30, 20, 10, or 5%. In some embodiments, the melting temperature (T) of the test primer is less than 100, 75, 50, 25, 10, 50%. mThe melting temperature range of the test primer is 40–80°C, for example, 50–70°C, 55–65°C, or 57–60.5°C. In some embodiments, the melting temperature range of the test primer is less than 20, 15, 10, 5, 3, or 1°C. In some embodiments, the length of the test primer is 15–100 nucleotides, for example, 15–75 nucleotides, 15–40 nucleotides, 17–35 nucleotides, 18–30 nucleotides, or 20–65 nucleotides. In some embodiments, the test primer includes a non-target-specific tag, for example, a tag that forms an internal loop structure. In some embodiments, the tag is located between two DNA-binding regions. In various embodiments, the test primer includes a 5' region specific to the target locus, an internal region that forms a loop structure not specific to the target locus, and a 3' region specific to the target locus. In various embodiments, the length of the 3' region is at least 7 nucleotides. In some embodiments, the length of the 3' region is 7 to 20 nucleotides, e.g., 7 to 15 nucleotides, or 7 to 10 nucleotides. In various embodiments, the test primer includes a 5' region that is not specific to the target locus (e.g., a tag or universal primer binding site), followed by a region specific to the target locus, an inner region that forms a loop structure that is not specific to the target locus, and a 3' region specific to the target locus. In some embodiments, the length of the test primer ranges to 50, 40, 30, 20, 10, or less than 5 nucleotides. In some embodiments, the length of the target amplification product is 50 to 100 nucleotides, e.g., 60 to 80 nucleotides, or 60 to 75 nucleotides. In some embodiments, the length of the target amplification product ranges to 50, 25, 15, 10, or less than 5 nucleotides.

[0011] In various embodiments of any aspect of the present invention, the primer extension reaction conditions are polymerase chain reaction (PCR) conditions. In various embodiments, the length of the annealing step is 3, 5, 8, 10, or more than 15 minutes. In various embodiments, the length of the extension step is 3, 5, 8, 10, or more than 15 minutes.

[0012] In various embodiments of any aspect of the present invention, test primers are used to simultaneously amplify at least 1,000 different target gene loci in a sample containing maternal DNA and fetal DNA from the mother during pregnancy of a fetus for determining the presence or absence of fetal chromosomal abnormalities. In various embodiments, the method includes the steps of ligating a universal primer binding site to a DNA molecule in the sample; amplifying the ligated DNA molecule using at least 1,000 specific primers and a universal primer to produce a first amplified product assembly; and amplifying the first amplified product assembly using at least 1,000 pairs of specific primers to produce a second amplified product assembly. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different primer pairs are used.

[0013] In various embodiments of any aspect of the present invention, test primers are used to simultaneously amplify at least 1,000 different target gene loci in a sample containing DNA from a person believed to be the father of the fetus, and further to simultaneously amplify target gene loci in a sample containing maternal DNA from the mother during pregnancy and fetal DNA, thereby determining whether the person believed to be the father is the biological father of the fetus.

[0014] In various embodiments of any aspect of the present invention, test primers are used to simultaneously amplify at least 1,000 different target gene loci in one or more cells derived from an embryo to determine the presence or absence of chromosomal abnormalities. In various embodiments, cells derived from a collection of two or more embryos are analyzed, and one embryo is selected for in vitro fertilization.

[0015] In various embodiments of any aspect of the present invention, test primers are used to simultaneously amplify at least 1,000 different target gene loci in a forensic nucleic acid sample. In various embodiments, the length of the annealing step is 3, 5, 8, 10, or more than 15 minutes.

[0016] In various embodiments of any aspect of the present invention, the method includes the steps of: simultaneously amplifying at least 1,000 different target loci in a control nucleic acid sample using test primers to generate a set of first target amplification products; simultaneously amplifying target loci in a test nucleic acid sample to generate a second set of target amplification products; and comparing the first and second sets of target amplification products to determine whether a target locus is present in one sample but not in the other, or whether the target loci are present at different levels in the control sample and the test sample. In various embodiments, the test sample is derived from an individual that has the target disease or phenotype (e.g., cancer) or is suspected of having an increased risk of the target disease or phenotype, and further includes one or more target loci that are associated with an increased risk of the target disease or phenotype, or are associated with the target disease or phenotype (e.g., polymorphisms or other mutations). In various embodiments, the method includes the steps of simultaneously amplifying 1,000 different target loci in a control sample containing RNA using test primers to generate a set of first target amplification products, and further simultaneously amplifying target loci in a test sample containing RNA to generate a set of second target amplification products, and comparing the first and second sets of target amplification products to determine whether there is a difference in RNA expression levels between the control sample and the test sample. In various embodiments, RNA is mRNA. In various embodiments, the test sample is derived from an individual suspected of having the target disease or phenotype (e.g., cancer) or suspected of having an increased risk of the target disease or phenotype (e.g., cancer), and one or more target loci include sequences (e.g., polymorphisms or other mutations) associated with an increased risk of the target disease or phenotype or associated with the target disease or phenotype. In some embodiments, the test sample is derived from an individual diagnosed with the target disease or phenotype (e.g., cancer), in which case the difference in RNA expression levels between the control sample and the test sample indicates that the target locus contains a sequence (e.g., polymorphism or other mutation) associated with an increased or decreased risk of the target disease or phenotype.

[0017] In some embodiments of any aspect of the present invention, the test primer is selected from a library of candidate primers based on one or more parameters, such as primer selection using any method of the present invention. In some embodiments, the test primer is selected from the candidate primer library based at least in part on the primer dimerization ability of the candidate primers.

[0018] In one embodiment, the present invention features a method for selecting test primers from a candidate primer library. In various embodiments, the selection includes: (i) using a computer to calculate undesirability scores for most or all possible combinations of two candidate primers from the library, wherein each undesirability score is at least partly based on the likelihood of dimerization between the two candidate primers; (ii) removing the candidate primer from the candidate primer library having the highest undesirability score; (iii) if the candidate primer removed in step (ii) is a member of a primer pair, removing the other member of the primer pair from the candidate primer library; and (iv) optionally repeating steps (ii) and (iii) to select a test primer library. In some embodiments, the selection method is carried out until the undesirability scores for all candidate primer combinations remaining in the library are below a minimum threshold. In some embodiments, the selection method is carried out until the number of candidate primers remaining in the library is reduced to a desired number. In various embodiments, the undesirability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% of the possible combinations of candidate primers in the library. In various embodiments, the candidate primers remaining in the library can simultaneously amplify at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci. In various embodiments, the method also includes the steps of (v) contacting a nucleic acid sample containing the target locus with the candidate primers remaining in the library to generate a reaction mixture; and (vi) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing the target amplification product.

[0019] In one embodiment, the present invention is characterized by a method for selecting test primers from a candidate primer library. In various embodiments, the method for selecting test primers from a candidate primer library includes: (i) using a computer to calculate undesirability scores for most or all possible combinations of two candidate primers from the library, wherein each undesirability score is at least partly based on the likelihood of dimerization between the two candidate primers; (ii) removing candidate primers from the candidate primer library that are part of the largest number of combinations of two candidate primers having undesirability scores exceeding a first minimum threshold; (iii) if the candidate primers removed in step (ii) are members of a primer pair, removing the remaining members of a primer pair from the candidate primer library; and (iv) optionally repeating steps (ii) and (iii) to select a test primer library. In some embodiments, the selection method is carried out until the undesirability scores for all combinations of candidate primers remaining in the library are below a first minimum threshold. In some embodiments, the selection method is carried out until the number of candidate primers remaining in the library is reduced to a desired number. In various embodiments, the undesirability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% of the possible candidate primer combinations in the library. In various embodiments, the candidate primers remaining in the library can simultaneously amplify at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci. In various embodiments, the method also includes the steps of (v) contacting a nucleic acid sample containing the target locus with the candidate primers remaining in the library to generate a reaction mixture; and (vi) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing the target amplification product.

[0020] In various embodiments of any aspect of the present invention, the selection method includes the step of further reducing the number of candidate primers remaining in the library by lowering the first minimum threshold used in step (ii) to a smaller second minimum threshold, and optionally repeating steps (ii) and (iii). In some embodiments, the selection method includes the step of raising the first minimum threshold used in step (ii) to a larger second minimum threshold, and optionally repeating steps (ii) and (iii). In some embodiments, the selection method is performed until the undesirability scores for all combinations of candidate primers remaining in the library are less than or equal to the second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number.

[0021] In various embodiments of any aspect of the present invention, the method includes, prior to step (i), identifying or selecting primers that hybridize to a target locus. In some embodiments, multiple primers (or primer pairs) hybridize to the same target locus, but one primer (or one primer pair) is selected for this target locus using a selection method based on one or more parameters. In various embodiments, the method includes, prior to step (ii), removing from the library any primer pairs that produce target amplification products that overlap with those produced by other primer pairs. In various embodiments, candidate primers are selected from a group of two or more candidate primers having equivalent undesirability scores based on one or more other parameters for the purpose of removal from the candidate primer library. In some embodiments, the candidate primers remaining in the library are used as a test primer library by any method of the present invention. In some embodiments, the resulting test primer library includes any of the primer libraries of the present invention.

[0022] In various embodiments of any aspect of the present invention, the undesirability score is based in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, prevalence related to the sequence (e.g., polymorphism) of the target locus, disease penetrant related to the sequence (e.g., polymorphism) of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product.

[0023] In various embodiments of any aspect of the present invention, the undesirability score is based at least in part on one or more parameters selected from the group consisting of heterozygosity of target loci, specificity of candidate primers to target loci, size of candidate primers, melting temperature of target amplification product, GC content of target amplification product, amplification efficiency of target amplification product, and size of target amplification product, and at least 1,000 different target loci in a sample containing maternal DNA and fetal DNA from the mother during pregnancy for determining the presence or absence of fetal chromosomal abnormalities, using test primers. In various embodiments, the method includes the steps of ligating a universal primer binding site to a DNA molecule in the sample, amplifying the ligated DNA molecule using at least 1,000 specific primers and a universal primer to produce a first set of amplification products, and amplifying the first set of amplification products using at least 1,000 pairs of specific primers to produce a second set of amplification products. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different primer pairs are used. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0024] In various embodiments of any aspect of the present invention, the undesirability score is based at least in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product, and the test primer is used to amplify at least 1,000 different target loci in a sample containing DNA from a person believed to be the father of the fetus, and simultaneously amplifies target loci in a sample containing maternal DNA from the mother during pregnancy and fetal DNA to determine whether the person believed to be the father is the biological father of the fetus. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0025] In various embodiments of any aspect of the present invention, the undesirability score is based at least in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product, and the presence or absence of chromosomal abnormalities is determined by simultaneously amplifying at least 1,000 different target loci in one or more cells derived from an embryo using the test primer. In various embodiments, cells derived from a collection of two or more embryos are analyzed, and one embryo is selected for in vitro fertilization. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0026] In various embodiments of any aspect of the present invention, the undesirability score is based at least in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product, and at least 1,000 different target loci in a forensic nucleic acid sample are amplified simultaneously using the test primer. In various embodiments, the length of the annealing step is 3, 5, 8, 10, or more than 15 minutes. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0027] In various embodiments of any aspect of the present invention, the undesirability score is based in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, prevalence related to the sequence (e.g., polymorphism) of the target locus, disease penetrant related to the sequence (e.g., polymorphism) of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product, and the method includes the steps of simultaneously amplifying at least 1,000 different target locus in a control nucleic acid sample using a test primer to generate a first set of target amplification products, simultaneously amplifying target locus in a test nucleic acid sample to generate a second set of target amplification products, and comparing the first and second sets of target amplification products to determine whether the target locus is present in one sample and not in the other, or whether the target locus is present at different levels in the control sample and the test sample. In various embodiments, the test sample is derived from an individual suspected of having the target disease or phenotype, or an increased risk of the target disease or phenotype, in which case one or more target loci contain sequences (e.g., polymorphisms) associated with or related to the increased risk of the target disease or phenotype at the target locus. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0028] In various embodiments of any aspect of the present invention, the undesirability score is based in part on one or more parameters selected from the group consisting of heterozygosity of the target locus, prevalence related to the sequence (e.g., polymorphism) of the target locus, disease penetrant related to the sequence (e.g., polymorphism) of the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product, and the method includes the steps of simultaneously amplifying 1,000 different target locuses in a control nucleic acid sample containing RNA using a test primer to generate a first set of target amplification products, simultaneously amplifying target locuses in a test sample containing RNA to generate a second set of target amplification products, and comparing the first and second sets of target amplification products to determine whether there is a difference in RNA expression levels between the control sample and the test sample. In various embodiments, RNA is mRNA. In various embodiments, the test sample is derived from an individual suspected of having an increased risk of the target disease or phenotype (e.g., cancer), in which case one or more target loci contain sequences (e.g., polymorphisms or other mutations) associated with an increased risk of the target disease or phenotype. In some embodiments, the test sample is derived from an individual diagnosed with the target disease or phenotype (e.g., cancer), in which case the difference in RNA expression levels between the control sample and the test sample indicates that the target loci contain sequences (e.g., polymorphisms or other mutations) associated with an increase or decrease in the target disease or phenotype. In various embodiments, at least 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci are amplified.

[0029] In one embodiment, the present invention is characterized by a primer library. In some embodiments, primers are selected from a candidate primer library using any of the methods of the present invention. In some embodiments, the library includes primers that simultaneously hybridize to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci. In some embodiments, the library includes primers that simultaneously amplify at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci. In some embodiments, the library includes primers that simultaneously amplify at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci, such that less than 60, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification product forms primer dimers. In some embodiments, the library includes primers that simultaneously amplify 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target gene loci, such that at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the amplified products are target amplified products. In some embodiments, the library includes primers that simultaneously amplify target loci such that at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of target loci are amplified from 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci.In some embodiments, the primer library includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 primer pairs, each primer pair including a forward test primer and a reverse test primer, and each test primer pair hybridizes to a target locus. In some embodiments, the primer library includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 individual primers, each hybridizing to a different target locus, and the individual primers are not part of a primer pair.

[0030] In various embodiments of any aspect of the present invention, the concentration of each primer is less than 100, 75, 50, 25, 10, 5, 2, or 1 nM. In various embodiments, the GC content of the primer is 30 to 80%, for example, 40 to 70% or 50 to 60%. In some embodiments, the GC content range of the primer is less than 30, 20, 10, or 5%. In some embodiments, the melting temperature of the primer is 40 to 80°C, for example, 50 to 70°C, 55 to 65°C, or 57 to 60.5°C. In some embodiments, the melting temperature range of the primer is less than 15, 10, 5, 3, or 1°C. In some embodiments, the length of the primer is 15 to 100 nucleotides, for example, 15 to 75 nucleotides, 15 to 40 nucleotides, 17 to 35 nucleotides, 18 to 30 nucleotides, or 20 to 65 nucleotides. In some embodiments, the primer includes a non-target-specific tag, e.g., a tag that forms an internal loop structure. In some embodiments, the tag is located between two DNA-binding regions. In various embodiments, the primer includes a 5' region specific to the target locus, an internal region that is not specific to the target locus and forms a loop structure, and a 3' region specific to the target locus. In various embodiments, the length of the 3' region is at least 7 nucleotides. In some embodiments, the length of the 3' region is 7 to 20 nucleotides, e.g., 7 to 15 nucleotides, or 7 to 10 nucleotides. In various embodiments, the primer includes a 5' region that is not specific to the target locus (e.g., another tag or universal primer-binding site), followed by a region specific to the target locus, an internal region that is not specific to the target locus and forms a loop structure, and a 3' region specific to the target locus. In some embodiments, the length of the primer ranges from 50, 40, 30, 20, 10, or less than 5 nucleotides. In some embodiments, the length of the target amplification product is 50 to 100 nucleotides, for example, 60 to 80 nucleotides or 60 to 75 nucleotides. In some embodiments, the length of the target amplification product is in the range of 50, 25, 15, 10, or less than 5 nucleotides.

[0031] In one embodiment, the present invention provides a kit comprising any of the primer libraries of the present invention for amplifying a target gene locus in a nucleic acid sample. In some embodiments, the kit includes instructions for amplifying the target gene locus using the libraries.

[0032] In one embodiment, the present invention features a method for determining the chromosomal ploidy status of a fetus during pregnancy. In some embodiments, the method includes the step of contacting a nucleic acid sample with a primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different polymorphic loci to generate a reaction mixture, wherein the nucleic acid sample comprises maternal DNA from the fetus's mother and fetal DNA from the fetus. In some embodiments, the reaction mixture is subjected to primer extension reaction conditions to produce an amplified product, sequencing data is generated from the amplified product using a high-throughput sequencer, the number of alleles at polymorphic loci is calculated by computer based on the sequencing data, multiple ploidy hypotheses are created by computer, each associated with a different possible ploidy state of the chromosome, a joint distribution model for the predicted number of alleles at the polymorphic locus of the chromosome is constructed by computer for each ploidy hypothesis, the relative probability of each ploidy hypothesis is calculated by computer using the joint distribution model and the number of alleles, and the ploidy state of the fetus is called by selecting the ploidy state corresponding to the hypothesis with the highest probability.

[0033] In one embodiment, the present invention is characterized by a method for determining the ploidy status of a fetus during pregnancy. In one embodiment, the method for determining the ploidy status of a fetus during pregnancy includes the steps of: obtaining a first DNA sample containing maternal DNA from the fetus's mother and fetal DNA from the fetus; preparing the first sample by isolating the DNA so that a prepared sample can be obtained; measuring the DNA in the prepared sample at multiple polymorphic loci on the chromosome; calculating the number of alleles at multiple polymorphic loci using a computer from the DNA measurements performed on the prepared sample; generating multiple ploidy hypotheses using a computer, each relating to a different possible ploidy status on the chromosome; constructing a joint distribution model using a computer for each ploidy hypothesis, for the predicted number of alleles at multiple polymorphic loci on the chromosome; determining the relative probability of each ploidy hypothesis using a computer with the joint distribution model and the number of alleles measured in the prepared sample; and calling the ploidy status of the fetus by selecting the ploidy status corresponding to the hypothesis with the highest probability.

[0034] In one embodiment, the present invention is characterized by a method for examining the abnormal distribution of chromosomes in a sample containing a mixture of maternal and fetal DNA. In some embodiments, the method comprises (i) contacting a sample with a primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci to generate a reaction mixture, wherein the target loci originate from multiple different chromosomes, and the multiple different chromosomes are suspected to have an abnormal distribution in the sample, and at least one second chromosome is presumed to be normally distributed in the sample; and (ii) the reaction mixture The method includes (iii) subjecting the compound to primer extension reaction conditions to generate an amplification product, (iii) sequencing the amplification product to obtain a plurality of sequence tags aligned to target loci, wherein the sequence tags are of sufficient length to be assigned to specific target loci, (iv) assigning the plurality of sequence tags to their corresponding target loci using a computer, (v) determining using a computer the number of sequence tags to be assigned to target loci on the first chromosome and the number of sequence tags to be assigned to target loci on the second chromosome, and (vi) comparing the numbers from step (v) using a computer to determine whether or not there is an abnormal distribution on the first chromosome.

[0035] In one embodiment, the present invention provides a method for detecting the presence or absence of aneuploidy in a fetus. In some embodiments, the method includes (i) contacting a sample containing a mixture of maternal and fetal DNA with a primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different non-polymorphic target loci derived from multiple different chromosomes to generate a reaction mixture; (ii) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing target amplification products; (iii) quantifying the relative frequencies of target amplification products derived from the first and second chromosomes of the subject using a computer; (iv) comparing the relative frequencies of target amplification products derived from the first and second chromosomes of the subject using a computer; and (v) determining the presence or absence of aneuploidy based on the compared relative frequencies of the first and second chromosomes of the subject. In some embodiments, the first chromosome is suspected to be euploid. In some embodiments, the second chromosome is suspected to be aneuploid.

[0036] In one embodiment, a method for determining the presence or absence of fetal aneuploidy in a maternal tissue sample containing fetal genomic DNA and maternal genomic DNA, comprising: (a) obtaining a mixture of fetal genomic DNA and maternal genomic DNA from the maternal tissue sample; (b) performing large-scale parallel DNA sequencing of randomly selected DNA fragments from the mixture of fetal genomic DNA and maternal genomic DNA obtained in step (a) to determine the sequence of the DNA fragments; (c) identifying the chromosome to which the sequence obtained in step (b) belongs; (d) using the data from step (c) to determine the amount of at least one first chromosome in the mixture of maternal genomic DNA and fetal genomic DNA, wherein the at least one first chromosome is estimated to be euploid in the fetus; and (e) using the data from step (c) to determine the amount of at least one first chromosome in the mixture of maternal genomic DNA and fetal genomic DNA A method is disclosed for determining the amount of a second chromosome, the steps being: (f) calculating the proportion of fetal DNA in a mixture of fetal DNA and maternal DNA; (g) if the second target chromosome is euploid, calculating the predicted distribution of the amount of the second target chromosome using the number from step (d); (h) if the second target chromosome is aneuploid, calculating the predicted distribution of the amount of the second target chromosome using the first number from step (d) and the proportion of fetal DNA in a mixture of fetal DNA and maternal DNA calculated in step (f); and (i) determining, using the maximum likelihood method or the maximum retrospective method, whether the amount of the second chromosome determined in step (e) is more likely to be part of the distribution calculated in step (g) or step (h), thereby indicating the presence or absence of fetal aneuploidy.

[0037] In various embodiments of any aspect of the present invention, the method also includes the step of obtaining genotype data from one or both parents of a fetus. In some embodiments, the step of obtaining genotype data from one or both parents of a fetus includes the step of preparing parental DNA, which includes preferentially enriching DNA at multiple polymorphism loci to obtain prepared parental DNA; optionally, the step of amplifying the prepared parental DNA; and the step of measuring the parental DNA in the prepared sample at multiple polymorphism loci.

[0038] In various embodiments of any aspect of the present invention, the step of constructing a joint distribution model for the predicted probability of allele numbers at multiple polymorphic loci on a chromosome is performed using genetic data obtained from one or both parents. In some embodiments, the step of isolating a sample (e.g., a first sample) from maternal plasma and obtaining genotype data from the mother is performed by estimating the maternal genotype data from DNA measurements performed on the prepared sample.

[0039] In one embodiment, a diagnostic box is disclosed that is useful for determining the ploidy status of chromosomes in a fetus during pregnancy, and which can perform any of the preparation and measurement steps of the method of the present invention.

[0040] In various embodiments of any aspect of the present invention, the number of alleles is probabilistic rather than binary. In some embodiments, measurements of DNA in a prepared sample at multiple polymorphism loci are also used to determine whether the fetus has one or more disease-linked haplotypes.

[0041] In various embodiments of any aspect of the present invention, the step of constructing a joint distribution model for the probability of allele numbers is carried out by modeling the dependencies between polymorphic alleles on a chromosome using data on the probability of chromosomal crossovers at different locations within the chromosome. In some embodiments, the steps of constructing a joint distribution model for allele numbers and determining the relative probability of each hypothesis are carried out using a method that does not require the use of a reference chromosome.

[0042] In various embodiments of any aspect of the present invention, the estimated fraction of fetal DNA in the prepared sample is used in the step of determining the relative probability of each hypothesis. In some embodiments, the DNA measurements from the prepared sample used in the step of calculating the probability of allele numbers and the step of determining the relative probability of each hypothesis include primary gene data. In some embodiments, the step of selecting the ploidy state corresponding to the hypothesis with the highest probability is performed using maximum likelihood estimation or maximum aposterior estimation.

[0043] In various embodiments of any aspect of the present invention, the step of calling the ploidy status of the fetus also includes the step of combining the relative probabilities of each ploidy hypothesis, determined using a joint distribution model and the probabilities of allele numbers, with the relative probabilities of each ploidy hypothesis, calculated using statistical techniques selected from a group consisting of read count analysis, heterozygosity comparison, statistics available only when parental genetic information is used, probabilities of genotype signals normalized for a particular parental situation, statistics calculated using estimated fetal fractions of a sample (e.g., a first sample) or a prepared sample, and combinations thereof.

[0044] In various embodiments of any aspect of the present invention, confidence estimates are calculated for the called ploidy state. In some embodiments, the method also includes taking a clinical action, selected from either aborting or maintaining the pregnancy, based on the called ploidy state of the fetus.

[0045] In various embodiments of any aspect of the present invention, the method can be performed on a fetus during the following periods: between weeks 4 and 5 of pregnancy; between weeks 5 and 6 of pregnancy; between weeks 6 and 7 of pregnancy; between weeks 7 and 8 of pregnancy; between weeks 8 and 9 of pregnancy; between weeks 9 and 10 of pregnancy; between weeks 10 and 12 of pregnancy; between weeks 12 and 14 of pregnancy; between weeks 14 and 20 of pregnancy; between weeks 20 and 40 of pregnancy; in the first trimester; in the second trimester; in the third trimester; or in any combination thereof.

[0046] In various embodiments of any aspect of the present invention, the method is used to produce a report indicating the determined chromosomal ploidy status in a fetus during pregnancy. In some embodiments, a kit is disclosed for determining the ploidy status of a target chromosome in a fetus during pregnancy, designed for use in any aspect of the present invention, comprising: a plurality of inner forward primers and optionally a plurality of inner reverse primers, each of which is designed to hybridize with a region of DNA immediately upstream and / or downstream of one of the polymorphic sites on the target chromosome; and optionally another chromosome, wherein the hybridizing region is separated from the polymorphic site by a small number of bases, the small number being selected from the group consisting of 1, 2, 3, 4, 5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-60, and combinations thereof.

[0047] In one embodiment, the present invention is characterized by a method for determining whether a person who is believed to be the father is the biological father of a fetus being conceived by a pregnant mother. In some embodiments, the method includes (i) simultaneously amplifying a plurality of polymorphic loci, including at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 polymorphic loci on different genetic material derived from a person suspected to be the father, to generate a first set of amplification products; (ii) simultaneously amplifying a plurality of corresponding polymorphic loci of a mixed DNA sample, including fetal DNA and maternal DNA derived from a blood sample of a pregnant mother, to generate a second set of amplification products; (iii) determining, using a computer, the probability that the person suspected to be the father is the biological father of the fetus using genotype measurements based on the sets of first and second amplification products; and (iv) determining whether or not the person suspected to be the biological father of the fetus is using the determined probability that he is the biological father of the fetus. In various embodiments, the method further includes the step of simultaneously amplifying a plurality of corresponding polymorphic loci on maternal genetic material to produce a third set of amplification products, in which case the probability that a person identified as the father is the biological father of the fetus is determined using genotype measurements based on the sets of first, second, and third amplification products.

[0048] In one embodiment, the present invention provides a method for estimating the relative likelihood of developing each embryo from an embryo aggregate as desired. In some embodiments, the method comprises the steps of generating a reaction mixture for each embryo by contacting a sample from each embryo with a primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci, wherein the sample is obtained from cells from one or more embryos, respectively. In some embodiments, each reaction mixture is subjected to primer extension reaction conditions to produce an amplification product. In some embodiments, the method comprises the steps of using a computer to determine one or more properties of at least one cell from each embryo based on the amplification product, and using a computer to estimate the relative likelihood of developing each embryo as desired based on one or more properties of at least one cell from each embryo.

[0049] In one embodiment, the present invention is characterized by a method for measuring the amount of two or more target gene loci in a nucleic acid sample. In some embodiments, the method is (i) to amplify a nucleic acid sample containing a first reference gene locus, a second reference gene locus, a first target gene locus, and a second target gene locus using PCR to form an amplification product, wherein the first reference gene locus and the first target gene locus have the same number of nucleotides but have different sequences at the position of one or more nucleotides, and the second reference gene locus and the second target gene locus have the same number of nucleotides but have different sequences at the position of one or more nucleotides; and (ii) to sequence the amplification product. The method comprises (iii) determining a reference ratio for comparing the relative amount of amplified first reference locus to a second reference locus, wherein the reference ratio indicates a difference in PCR efficiency for amplification of the first and second reference locus; (iii) determining a target ratio for comparing the relative amount of amplified first target locus to amplified second target locus; and (iv) determining the relative amounts of the first and second target locus in the sample by adjusting the target ratio from step (iii) based on the reference ratio from step (ii). In various embodiments, the method includes determining the absolute amounts of the first and second target locus in the sample. In various embodiments, the method further includes the step of determining the presence or absence of target loci in the sample (e.g., at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci). In various embodiments, the method includes the step of using one of the primer libraries of the present invention. In various embodiments, the method includes the step of simultaneously amplifying 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci.

[0050] In one embodiment, the present invention features a method for quantitatively measuring multiple genetic targets in an analytical sample. In some embodiments, the method includes (i) mixing genetic material derived from the analytical sample with a plurality of target-specific amplification reagents and a plurality of reference sequences corresponding to the targets of the target-specific amplification reagents; (ii) amplifying target regions and reference sequences of the genetic material to produce target amplification products and reference sequence amplification products; and (iii) measuring the amounts of the produced target amplification products and reference sequence amplification products. In some embodiments, the genetic material is present in a gene library. In some embodiments, the genetic targets are polymorphic loci (such as SNPs). In some embodiments, the step of measuring the amount is achieved by counting sequences. In some embodiments, the method further includes measuring the estimated copy number of at least one chromosome in the sample from which the gene library is derived, and the measurement includes comparing the number of sequence reads of the target amplification product with the number of sequence reads of the reference amplification product. In some embodiments, the reference sequences and gene library include universal priming sites that can be primed with the same primers. In some embodiments, the mixing step includes at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 different target-specific amplification reagents and at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 reference sequences. In various embodiments, the method includes a step of using one of the primer libraries of the present invention. In various embodiments, the method includes the step of simultaneously amplifying 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target regions. In some embodiments, the relative amounts of each reference sequence are known.In some embodiments, the relative amounts of each sequence are calibrated against a reference genome. In some embodiments, the analytical sample comprises a mixture of fetal and maternal genomes. In some embodiments, the analytical sample is derived from the blood or plasma of a pregnant woman. In some embodiments, the reference genome has at least one aneuploidy, such as aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

[0051] In one embodiment, the present invention is characterized by a mixture comprising a plurality of reference sequences, the relative amount of each reference sequence in the mixture is determined by calibration against a reference genome. In various embodiments, the mixture comprises at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 reference sequences. In various embodiments, the reference sequence comprises a first universal priming site, a second universal priming site, a first target-specific priming site, a second target-specific priming site, and a marker sequence located between the first and second target-specific priming sites, wherein the first and second target-specific priming sites are located between the first and second universal priming sites. In various embodiments, calibration includes the step of using one of the primer libraries of the present invention. In various embodiments, calibration includes the step of simultaneously amplifying 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target regions. In some embodiments, the reference genome has at least one aneuploidy, such as aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

[0052] In one embodiment, the present invention is characterized by a method for generating a set of calibrated reference sequences. In some embodiments, the method includes (i) forming an amplification reaction mixture comprising a gene library prepared from a reference genome, a set of multiple target-specific amplification primer reagents, and multiple reference sequences corresponding to the set of target-specific amplification reagents; (ii) amplifying the gene library and reference sequences to produce amplification products derived from target sequences and amplification products derived from reference sequences; (iii) measuring the amounts of amplification products derived from target sequences and amplification products derived from reference sequences; and (iv) determining the relative amounts of each reference sequence to each other, thereby calibrating the multiple reference sequences. In various embodiments, at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 reference sequences are used. In various embodiments, the method includes the step of using one of the primer libraries of the present invention. In various embodiments, the present invention includes the step of simultaneously amplifying 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different sequences. In some embodiments, the reference genome has at least one aneuploidy, such as aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

[0053] In one aspect, the present invention provides a set of genetic reference sequences that have been calibrated by any of the methods of the present invention. In one aspect, the present invention provides a set of genetic reference sequences that can be calibrated before, during, and after the methods described above are performed.

[0054] In one embodiment, the present invention is characterized by a method for measuring the copy number of a target gene containing at least one allele having a deletion. In some embodiments, the method includes (i) mixing genetic material from an analytical sample with an amplification reagent specific to the target gene and not significantly amplifying the deletion containing the allele of the target gene, a reference sequence corresponding to the target gene, an amplification reagent specific to a reference sequence, and a reference sequence corresponding to the reference sequence; (ii) amplifying the target gene sequence, the reference sequence corresponding to the target gene, the reference sequence, and the reference sequence corresponding to the reference sequence to produce a target gene amplification product, a reference sequence amplification product, and a reference sequence amplification product; and (iii) measuring the amounts of the produced target amplification product and reference sequence amplification product. In some embodiments, the amount measurement is achieved by counting sequence reads. In some embodiments, the method further includes a step of calculating the estimated copy number of chromosomes in a sample from which at least one gene library is derived, the calculation step including a step of comparing the number of sequences in the target amplification product with the number of sequences in the reference amplification product. In some embodiments, the reference sequence and the gene library contain universal priming sites that can be primed with the same primers. In some embodiments, the relative amounts of each sequence are calibrated against a reference genome. In various embodiments, at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 gene reference sequences are used. In various embodiments, the method includes the step of using one of the primer libraries of the present invention. In various embodiments, the method includes the step of simultaneously amplifying 1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 different target regions. In some embodiments, the reference genome is diploid. In some embodiments, the sample for analysis is derived from blood.

[0055] In various embodiments of any aspect of the present invention, the step of preferentially enriching DNA in a sample (e.g., a first sample) at a target locus (e.g., multiple polymorphic loci) is performed using a plurality of pre-circular probes, each probe targeting one of the loci (e.g., polymorphic loci), preferably designed so that the 3' and 5' ends of the probe hybridize to a region of DNA separated from the polymorphic region of the locus by a few bases, where the few bases are 1, 2, 3, 4, The method includes the steps of obtaining a probe which is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the pre-circular probe with DNA derived from a sample (e.g., a first sample); filling the gaps between the hybridized probe ends using DNA polymerase; circularizing the pre-circular probe; and amplifying the circularized probe.

[0056] In various embodiments of any aspect of the present invention, the step of preferentially enriching DNA at a target locus (e.g., multiple polymorphic loci) is performed using multiple ligation-mediated PCR probes, each PCR probe targeting one of the target loci (e.g., multiple polymorphic loci), and the upstream and downstream PCR probes are designed to hybridize with a region of DNA on one strand of DNA separated from the polymorphic region of the locus by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 The method includes the steps of obtaining a PCR probe which is 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof; hybridizing the ligation-mediated PCR probe with DNA derived from a sample (e.g., a first sample); filling the gaps between the ends of the ligation-mediated PCR probe using DNA polymerase; ligating the ligation-mediated PCR probe; and amplifying the ligated ligation-mediated PCR probe.

[0057] In some embodiments of various aspects of the present invention, the step of preferentially enriching DNA at a target locus (e.g., multiple polymorphic loci) includes the steps of obtaining multiple hybrid capture probes targeting the locus (e.g., multiple polymorphic loci), hybridizing the hybrid capture probes with DNA in a sample (e.g., a first sample), and physically removing some or all of the DNA that has not been hybridized from the DNA sample (e.g., a first sample).

[0058] In some embodiments of any aspect of the present invention, the hybrid capture probe is designed to hybridize with a region adjacent to but not overlapping with a polymorphic site. In some embodiments, the hybrid capture probe is designed to hybridize with a region adjacent to but not overlapping with a polymorphic site, and the length of the adjacent capture probe can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybrid capture probe is designed to hybridize with a region overlapping with a polymorphic site, and the multiple hybrid capture probes include at least two hybrid capture probes for each polymorphic locus, and each hybrid capture probe is designed to be complementary to another allele at one polymorphic locus.

[0059] In some embodiments of any aspect of the present invention, the step of preferentially enriching DNA from multiple polymorphic loci is to obtain a plurality of inner forward primers, each primer targeting one of the polymorphic loci, the 3' end of which is designed to hybridize with a region of DNA upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, and the small number of primers is selected from the group consisting of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6-10 base pairs, 11-15 base pairs, 16-20 base pairs, 21-25 base pairs, 26-30 base pairs, or 31-60 base pairs, and optionally, a plurality The method comprises the steps of obtaining an inner reverse primer, each primer targeting one of the polymorphic loci, where the 3' end of the inner reverse primer is designed to hybridize with a region of DNA located upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, and a small number of primers selected from the group consisting of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6-10 base pairs, 11-15 base pairs, 16-20 base pairs, 21-25 base pairs, 26-30 base pairs, or 31-60 base pairs; hybridizing the inner primer with DNA; and amplifying the DNA using a polymerase chain reaction to form an amplification product.

[0060] In some embodiments of any aspect of the present invention, the method further comprises the steps of: obtaining a plurality of outer forward primers, each of which is designed to target one of a target locus (e.g., polymorphism locus) and hybridize with a region of DNA upstream of an inner forward primer; optionally, obtaining a plurality of outer reverse primers, each of which is designed to target one of a target locus (e.g., polymorphism locus) and hybridize with a region of DNA immediately downstream of an inner reverse primer; hybridizing the first primer with DNA; and amplifying the DNA using a polymerase chain reaction.

[0061] In some embodiments of any aspect of the present invention, the method further comprises the steps of: obtaining a plurality of outer reverse primers, each of which is designed to target one of a target locus (e.g., polymorphism locus) and to hybridize with a region of DNA immediately downstream of an inner reverse primer; optionally, obtaining a plurality of outer forward primers, each of which is designed to target one of a polymorphism locus and to hybridize with a region of DNA upstream of an inner forward primer; hybridizing the first primer with DNA; and amplifying the DNA using a polymerase chain reaction.

[0062] In some embodiments of any aspect of the present invention, the sample preparation step (e.g., first sample) further includes the step of adding a universal adapter to the DNA in the sample (e.g., first sample) and the step of amplifying the DNA in the sample (e.g., first sample) using a polymerase chain reaction. In some embodiments, at least a portion of the amplified amplification product is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp, where the portion is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.

[0063] In some embodiments of any aspect of the present invention, the step of amplifying DNA is carried out in one or more individual reaction volumes, each of which contains more than 100 pairs of different forward primers and reverse primers, more than 200 pairs of different forward primers and reverse primers, more than 500 pairs of different forward primers and reverse primers, more than 1,000 pairs of different forward primers and reverse primers, more than 2,000 pairs of different forward primers and reverse primers, more than 5,000 pairs of different forward primers and reverse primers, more than 10,000 pairs of different forward primers and reverse primers, more than 20,000 pairs of different forward primers and reverse primers, more than 50,000 pairs of different forward primers and reverse primers, or more than 100,000 pairs of different forward primers and reverse primers.

[0064] In some embodiments of any aspect of the present invention, the step of preparing a sample (e.g., a first sample) further includes dividing the sample (e.g., a first sample) into a plurality of parts, wherein DNA in each part is preferentially enriched in a subset of target loci (e.g., a plurality of polymorphic loci). In some embodiments, inner primers are selected by identifying primer pairs that may form undesirable primer doubles, and removing at least one of the primer pairs identified as potentially forming undesirable primer doubles from the plurality of primers. In some embodiments, the inner primers contain a region designed to hybridize either upstream or downstream of a target locus (e.g., a polymorphic locus) and optionally contain a universal priming sequence designed to enable PCR amplification. In some embodiments, at least a portion of the primers further contain different random regions for each individual primer molecule. In some embodiments, at least a portion of the primers further contain a molecular barcode.

[0065] In some embodiments of any aspect of the present invention, preferential enrichment results in an average allele bias between the prepared sample and the sample (e.g., the first sample) of a degree selected from the group consisting of 2x or less, 1.5x or less, 1.2x or less, 1.1x or less, 1.05x or less, 1.02x or less, 1.01x or less, 1.005x or less, 1.002x or less, 1.001x or less, and 1.0001x or less. In some embodiments, the multiple polymorphic loci are SNPs. In some embodiments, the step of measuring the DNA in the prepared sample is performed by sequencing.

[0066] In some embodiments of any aspect of the present invention, the target locus is located on the same nucleic acid as the control (e.g., the same chromosome or the same region of a chromosome). In some embodiments, at least some target loci are located on different nucleic acids of the subject (e.g., different chromosomes). In some embodiments, the nucleic acid sample comprises fragmented or digested nucleic acid. In some embodiments, the nucleic acid sample comprises genomic DNA, cDNA, or mRNA. In some embodiments, the nucleic acid sample comprises DNA derived from a single cell. In some embodiments, the nucleic acid sample is a substantially cell-free blood or plasma sample. In some embodiments, the nucleic acid sample comprises or is derived from blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretion, dental plaque, gastrointestinal tissue, feces, urine, hair, bone, body fluids, tears, tissue, skin, nails, blastomeres, embryos, amniotic fluid, chorionic villi samples, bile, lymph, cervical mucus, or forensic samples. In some embodiments, the target locus is a segment of human nucleic acid. In some embodiments, the target locus contains or is composed of a single nucleotide polymorphism (SNP). In some embodiments, the primer is a DNA molecule.

[0067] In some embodiments of any aspect of the present invention, the DNA in the sample (e.g., the first sample) is derived from maternal plasma. In some embodiments, the step of preparing the sample (e.g., the first sample) further includes the step of amplifying the DNA. In some embodiments, the step of preparing the sample (e.g., the first sample) further includes the step of preferentially enriching the DNA in the sample (e.g., the first sample) at a target locus (e.g., a plurality of polymorphic loci).

[0068] In various embodiments, the primer extension reaction or polymerase chain reaction involves the addition of one or more nucleotides by polymerase. In various embodiments, the primer extension reaction or polymerase chain reaction does not involve ligation-mediated PCR. In various embodiments, the primer extension reaction or polymerase chain reaction does not involve the ligation of two primers by ligase. In various embodiments, the primers do not involve a ligated reverse probe (LIP). This probe is also called a pre-circularized probe, pre-circularizing probe, circulating probe, Padlock probe, or molecular inversion probe (MIP).

[0069] All aspects and embodiments of the present invention described herein will be understood to include the terms "comprising," "consisting," and "consisting essentially of."

[0070] definition A single nucleotide polymorphism (SNP) refers to a single nucleotide that may differ between the genomes of two members of the same species. The use of this term should not imply any limitation on the frequency of each variant occurring.

[0071] A sequence can refer to a DNA sequence or gene sequence. A sequence can also refer to the primary physical structure of an individual's DNA molecule or strand. A sequence can refer to the sequence of nucleotides found in a DNA molecule or its complementary strand. A sequence can refer to the information contained within a DNA molecule, as expressed in silico.

[0072] A locus refers to a specific region of an individual's DNA, and can be a site of a possible insertion or deletion, or a site of several other related genetic variations; it can also refer to a SNP. A disease-linked SNP may refer to a disease-linked locus.

[0073] Polymorphic alleles, and similarly, "polymorphic loci," refer to alleles or loci whose genotypes vary among individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications, and inversions.

[0074] Polymorphic regions refer to specific nucleotides found in polymorphic regions that vary between individuals.

[0075] An allele refers to a gene that occupies a specific gene locus.

[0076] Genetic data, and similarly "genotype data," refers to data describing the characteristics of the genome of one or more individuals. This can refer to a single locus or set of loci, a partial or complete sequence, a portion or entire chromosome, or the entire genome. It may also refer to the identity of one or more nucleotides, which may refer to a sequential set of nucleotides, nucleotides originating from different locations within the genome, or combinations thereof. Genotype data is generally in silico, but it is also possible to consider physical nucleotides within a sequence as chemically encoded genetic data. Genotype data can be described as "on," "of," "at," "from," or "relating to" an individual(s). When these measurements are performed on genetic material, genotype data may refer to the output measurements from a genotyping platform.

[0077] Genetic material, and similarly, "genetic sample," refers to physical material such as tissue or blood derived from one or more individuals that contains DNA or RNA.

[0078] Noisy genetic data refers to genetic data that contains any of the following: allele dropout, uncertain base pair measurements, inaccurate base pair measurements, missing base pair measurements, uncertain measurements of insertions or deletions, uncertain measurements of chromosome segment copy numbers, false signals, missing measurements, other errors, or a combination thereof.

[0079] Confidence refers to the statistical likelihood that the number of called SNPs, alleles, sets of alleles, ploidy calls, or determined chromosomal segment copies accurately reflects the actual genetic state of an individual.

[0080] Ploidy calling, similarly known as "chromosome copy number calling" or "copy number calling" (CNC), can refer to the act of determining the quantity and / or identity of one or more chromosomes present in a cell.

[0081] Aneuploidy refers to the presence of an incorrect number of chromosomes in a cell (e.g., an incorrect number of complete chromosomes or an incorrect number of chromosomal segments, e.g., the presence of deletions or copies of chromosomal segments). In the case of human somatic cells, aneuploidy may refer to a cell that does not contain 22 pairs of autosomes and 1 pair of sex chromosomes. In the case of human gametes, aneuploidy may refer to a cell that does not contain one of each of the 23 types of chromosomes. In the case of monochromosomes, aneuploidy may refer to the presence of approximately two homologous but not identical chromosome copies, or two chromosome copies originating from the same parent. In some embodiments, the deletion of a chromosomal segment is a microdeletion.

[0082] Ploidy refers to the quantity and / or chromosomal identity of one or more chromosome types in a cell.

[0083] The term "chromosome" can also refer to a single chromosome copy, which means a single molecule of DNA present in 46 copies in a normal somatic cell, an example of which is "maternally derived chromosome 18." Alternatively, the term "chromosome" can refer to a chromosome type, of which there are 23 copies in a normal human somatic cell, an example of which is "chromosome 18."

[0084] Chromosomal identity can refer to the number of chromosomes it indicates, i.e., the chromosome type. A normal human has 22 numbered autosomal chromosomes and two sex chromosomes. Chromosomal identity can also refer to chromosomes of parental origin. Chromosomal identity can also refer to specific chromosomes inherited from parents. Chromosomal identity can also refer to other identifiable features of a chromosome.

[0085] The state of genetic material, or simply "genetic state," can refer to the identity of a set of SNPs on DNA, the haplotype of phased genetic material, and the sequence of DNA including insertions, deletions, repeats, and mutations. It can also refer to the ploidy status of one or more chromosomes, a chromosomal segment, or a set of chromosomal segments.

[0086] Allele data refers to a collection of genotype data relating to one or more alleles. Allele data may refer to phase-specific haplotype data. Allele data may also refer to SNP identity, or to DNA sequence data including insertions, deletions, repeats, and mutations. Allele data may encompass each allele of parental origin.

[0087] The allele state refers to the actual state of a gene within a set of one or more alleles. The allele state can also refer to the actual state of a gene as described in allele data.

[0088] The allelic ratio, or allele ratio, refers to the ratio of the amounts of each allele present at a locus in a sample or individual. When the sample is measured by sequencing, the allele ratio may refer to the ratio of sequence reads mapped to each allele at the locus. When the sample is measured by intensity-based methods, the allele ratio may refer to the ratio of the amounts of each allele present at the locus estimated by the measurement method.

[0089] The allele number refers to the number of sequences mapped to a particular gene locus. If the locus is polymorphic, the allele number refers to the number of sequences mapped to each allele. If each allele is counted in a binary manner, the allele number will be an integer. If alleles are counted probabilistically, the allele number can be a fraction.

[0090] Allele probability refers to the number of sequences that can map to a set of alleles at a particular locus or polymorphism locus, combined with the mapping probability. Note that an allele probability where the mapping probability for each counted sequence is binary (0 or 1) is equal to the allele probability. In some embodiments, the allele probability may be binary. In some embodiments, the allele probability can be set to be equal to the DNA measurement.

[0091] Allele distribution, or "allele count distribution," refers to the relative amount of each allele present at each locus within a set of loci. Allele distribution may also refer to an individual, a sample, or a set of measurements obtained for a sample. In relation to sequencing, allele distribution refers to the number of reads or expected reads that map to a particular allele for each allele within a set of polymorphic loci. Allele measurements can be processed probabilistically, i.e., the likelihood that a given allele represents a given sequence read is a fraction between 0 and 1; or allele measurements can be processed in a binary manner, i.e., any given read is considered to be exactly 0 copies or 1 copy of a particular allele.

[0092] An allele distribution pattern refers to a set of different allele distributions for different parental conditions. A specific allele distribution pattern may indicate a particular ploidy state.

[0093] Allele bias refers to the degree to which the measured allele ratios at a heterozygous locus differ from the ratios present in the original DNA sample. The degree of allele bias at a particular locus is equal to the measured allele ratio at that locus divided by the allele ratio in the original DNA sample at that locus. Allele bias can be defined as greater than 1; therefore, if the calculation of the degree of allele bias results in a value x less than 1, the degree of allele bias can be rephrased as 1 / x. Allele bias can result from amplification bias, purification bias, or several other phenomena that affect different alleles differently.

[0094] Primers, and similarly "PCR probes," refer to a single DNA molecule (DNA oligomer) or a group of DNA molecules (DNA oligomers) that are identical or nearly identical. Primers contain a region designed to hybridize with a target locus (e.g., a target polymorphic or non-polymorphic locus) and may contain a priming sequence designed to enable PCR amplification. Primers may also contain molecular barcodes. Primers may contain different random regions for each individual molecule. The terms "test primer" and "candidate primer" are not limiting and may refer to any of the primers disclosed herein.

[0095] A primer library refers to a collection of two or more primers. In various embodiments, the library contains at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different primers. In various embodiments, the library contains at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different primer pairs, each primer pair containing a forward test primer and a reverse test primer, and each test primer pair hybridizes to a target gene locus. In some embodiments, the primer library includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 distinct individual primers, each hybridizing to a different target locus, where the individual primers are not part of a primer pair. In some embodiments, the library includes both (i) primer pairs and (ii) individual primers that are not part of a primer pair (e.g., universal primers).

[0096] A hybrid capture probe refers to any nucleic acid sequence, sometimes modified, that is generated by various methods such as PCR or direct synthesis, and is intended to be complementary to one strand of a specific target DNA sequence in a sample. An exogenous hybrid capture probe can be added to a prepared sample and hybridized through a denaturation-re-annealing process to form an exogenous-endogenous fragment double helix. These double helixes can then be physically separated from the sample by various means.

[0097] A sequence read refers to data that shows the sequence of nucleotide bases measured using clonal sequencing. Clonal sequencing can produce sequence data that represents a single original DNA molecule, a clone of a single original DNA molecule, or a cluster of a single original DNA molecule. Sequence reads may also have an associated quality score that indicates the probability that a nucleotide is accurately called at each base position in the sequence.

[0098] Sequence read mapping is the process of determining the origin of a sequence read within the genome sequence of a specific organism. The origin of a sequence read is based on the similarity of the nucleotide sequences between the read and the genome sequence.

[0099] Congruent copy error, similarly known as "constant chromosomal aneuploidy" (MCA), refers to a state of aneuploidy in which a single cell contains two identical or nearly identical chromosomes. This type of aneuploidy can occur during gamete formation in meiosis and can be called meiotic nondisjunction error. This type of error can occur during mitosis. Congruent trisomy may refer to a case where three copies of a given chromosome exist in an individual, and two of the copies are identical.

[0100] Mismatched copy errors, and similarly, "unique chromosome aneuploidy" (UCA), refer to a state of aneuploidy in which a single cell contains two chromosomes that are of the same parent and may be homologous but not identical. This type of aneuploidy can occur during meiosis and can be called a meiotic error. Mismatched trisomy can refer to a case where three copies of a given chromosome exist in an individual, two of which are of the same parent and homologous but not identical. Note that mismatched trisomy can also refer to a case where two homologous chromosomes exist from one parent, and some segments of the chromosome are identical, while other segments are simply homologous.

[0101] Homologous chromosomes refer to chromosomal copies that contain the same set of genes that typically pair up during meiosis.

[0102] An identical chromosome refers to a chromosomal copy containing the same set of genes. For each gene, an identical chromosome has the same set of alleles that are identical or nearly identical.

[0103] Allele dropout (ADO) refers to a situation in which at least one of the base pairs within a set of homologous chromosome-derived base pairs in a given allele is not detected.

[0104] Locus dropout (LDO) refers to a situation in which neither of the base pairs within a set of base pairs originating from homologous chromosomes in a given allele is detected.

[0105] Homozygosity refers to having the same allele as the corresponding chromosomal locus.

[0106] Heterozygosity refers to having non-uniform alleles at the corresponding chromosomal locus.

[0107] Heterozygosity refers to the percentage of individuals in a population who possess heterozygous alleles at a given locus. Heterozygosity may also refer to the predicted or measured ratio of alleles at a given locus in an individual or DNA sample.

[0108] A single nucleotide polymorphism (HISNP) with high informational value refers to an SNP in which a fetus possesses an allele that is not present in the mother's genotype.

[0109] A chromosomal region refers to a segment of a chromosome or an entire chromosome.

[0110] A chromosomal segment refers to a section of a chromosome that can range in size from a single base pair to an entire chromosome.

[0111] A chromosome refers to either a complete chromosome or a segment or section of a chromosome.

[0112] A copy refers to the number of copies of a chromosomal segment. This can refer to identical or non-identical homologous copies of a chromosomal segment. Different copies of a chromosomal segment contain substantially similar sets of loci, differing in one or more alleles. Note that in some cases of aneuploidy, such as M2 copy errors, a given chromosomal segment may have some identical copies and some non-identical copies.

[0113] A haplotype generally refers to a combination of alleles at multiple loci on the same chromosome that are inherited together. Depending on the number of recombination events that have occurred between a given set of loci, a haplotype can refer to as few as two loci or even an entire chromosome. A haplotype can also refer to a set of single nucleotide polymorphisms (SNPs) on a statistically related single chromatid.

[0114] Haplotype data, similarly referred to as “phase-specific data” or “ordered genetic data,” refers to data from a single chromosome of a diploid or polyploid genome, i.e., data from either a separated maternal or paternal copy of a chromosome in a diploid genome.

[0115] Phase identification refers to the process of determining an individual's haplotype genetic data by considering unordered, diploid (or polyploid) genetic data. Phase identification can also refer to the process of determining which of the two alleles found on a set of alleles on a single chromosome is associated with each of the individual's two homologous chromosomes.

[0116] Data with identified phases refers to genetic data in which one or more haplotypes have been determined.

[0117] A hypothesis refers to a set of possible ploidy states in a given set of chromosomes or a set of possible allele states in a given set of loci. The set of possibilities may contain one or more elements.

[0118] The copy number hypothesis, and similarly the "ploidy status hypothesis," refers to a hypothesis about the number of copies of an individual's chromosomes. This can also refer to a hypothesis about the identity of each chromosome, including which parent each chromosome originates from and which of the two parental chromosomes is present in the individual. This can also refer to a hypothesis about which, if any, of the relevant individual-derived chromosomes or chromosomal segments are genetically corresponding to a given individual-derived chromosome.

[0119] A target individual refers to an individual whose genetic state is determined. In some embodiments, only a limited amount of DNA is available from the target individual. In some embodiments, the target individual is a fetus. In some embodiments, there may be two or more target individuals. In some embodiments, fetuses born from a pair of parents can each be considered a target individual. In some embodiments, the genetic data to be determined is a call for one allele or a set of allele calls. In some embodiments, the genetic data to be determined is a ploidy call.

[0120] Related individuals refer to any individual that is genetically related to the target individual and therefore shares a haplotype block with the target individual. In some situations, related individuals may be the target individual's genetic parents, or any genetic material derived from parents, such as sperm, polar bodies, embryos, fetuses, or offspring. Related individuals may also refer to siblings, parents, or grandparents.

[0121] A sibling refers to any individual whose genetic parents are the same as the individual in question. In some embodiments, a sibling may refer to a newborn, embryo, or fetus, or one or more cells derived from a newborn, embryo, or fetus. A sibling may also refer to a haploid individual originating from one parent, such as a sperm, polar body, or a collection of genetic material of any other haplotype. An individual can be considered a sibling in itself.

[0122] "Fetal" refers to "of the fetus" or "of the region of the placenta that is genetically similar to the fetus." In pregnant women, a portion of the placenta is genetically similar to the fetus, and floating fetal DNA found in maternal blood may originate from a portion of the placenta with a genotype matching that of the fetus. It should be noted that half of the genetic information of the fetus's chromosomes is inherited from the fetus's mother. In some embodiments, the DNA from these maternally inherited chromosomes originating from fetal cells is considered to be of "fetal origin" rather than "of maternal origin."

[0123] Fetal DNA refers to DNA that was originally part of a cell whose genotype was basically the same as that of the fetus.

[0124] Maternal DNA refers to DNA that was originally part of a cell whose genotype was basically the same as that of the mother.

[0125] The term "offspring" may refer to an embryo, blastomer, or fetus. It should be noted that in the embodiments disclosed herein, the concepts described apply equally well to an individual which is a newborn offspring, fetus, embryo, or collection of cells derived therefrom. The use of the term "offspring" implies that an individual simply referred to as an offspring is a genetic descendant of the parents.

[0126] Parents refer to the genetic mother or father of an individual. Generally, an individual has two parents, a mother and a father; however, this is not always the case, for example, in the chimeric phenomenon of genes or chromosomes. Parents can be considered individuals.

[0127] Parental status refers to the genetic state of a given SNP in each of the two related chromosomes for one or both of the two target parents.

[0128] To develop as desired, and similarly to “to develop normally,” means to implant a viable embryo in the uterus, resulting in pregnancy, and / or to continue the pregnancy and result in birth, and / or to ensure that the child born is free from chromosomal abnormalities, and / or to ensure that the child born is free from other undesirable genetic conditions, such as disease-linked genes. The term “to develop as desired” means to encompass anything that may be desired by the parents or healthcare assistants. In some cases, “to develop as desired” may refer to an undevelopable or viable embryo that is useful for medical research or other purposes.

[0129] Uterine insertion refers to the process of transferring an embryo into the uterine cavity in relation to in vitro fertilization.

[0130] Maternal plasma refers to the plasma portion of blood derived from a pregnant woman.

[0131] A clinical decision refers to any decision to take or not take action that has an outcome affecting the health or survival of an individual. In the context of prenatal diagnosis, a clinical decision may refer to a decision to miscarry or not miscarry a fetus. A clinical decision may also refer to a decision to perform further tests, take measures to reduce an undesirable phenotype, or take measures to prepare for the birth of a child with abnormalities.

[0132] A diagnostic box refers to a machine or combination of machines designed to carry out one or more embodiments of the methods disclosed herein. In some embodiments, the diagnostic box may be placed in a patient care setting. In some embodiments, the diagnostic box may carry out targeted amplification, followed by sequencing. In some embodiments, the diagnostic box may function alone or with the assistance of a technician.

[0133] An informatics-based method refers to a method that relies heavily on statistics to interpret large amounts of data. In the context of prenatal diagnosis, an informatics-based method refers to a method designed to determine the ploidy status of one or more chromosomes or the allele status of one or more alleles, not by directly physically measuring the status, but by statistically inferring the most likely status by considering large amounts of genetic data, for example, from molecular arrays or sequencing. In certain embodiments of this disclosure, the informatics-based technique may be one disclosed in this patent. In certain embodiments of this disclosure, the informatics-based technique may be PARENTAL SUPPORT®.

[0134] Primary gene data refers to the analog intensity signals output from a genotyping platform. In relation to SNP arrays, primary gene data refers to the intensity signals before any genotype calls are made. In relation to sequencing, primary gene data refers to chromatogram-like analog measurements produced by a sequencer before any base pair identity is determined and before the sequence is mapped to the genome.

[0135] Secondary gene data refers to processed gene data output from a genotyping platform. In relation to SNP arrays, secondary gene data refers to allele calls performed by software accompanying the SNP array reader, which determine whether a given allele is present or absent in the sample. In relation to sequencing, secondary gene data refers to the determination of base pair identity of a sequence, and in some cases, the mapping of the sequence to the genome.

[0136] Non-invasive prenatal diagnosis (NPD), or similarly, non-invasive prenatal screening (NPS), refers to a method in which a mother determines the genetic status of her fetus during pregnancy using genetic material found in her blood, which is obtained by drawing venous blood from the mother.

[0137] Preferential enrichment of DNA corresponding to a locus, or preferential enrichment of DNA at a locus, refers to any method that makes the percentage of DNA molecules in the enriched DNA mixture corresponding to that locus higher than the percentage of DNA molecules in the unenriched DNA mixture corresponding to that locus. The method may include selective amplification of DNA molecules corresponding to a locus. The method may include the step of removing DNA molecules that do not correspond to a locus. The method may include a combination of methods. The degree of enrichment is defined as the percentage of DNA molecules in the enriched mixture corresponding to that locus divided by the percentage of DNA molecules in the unenriched mixture corresponding to that locus. Preferential enrichment can be performed at multiple loci. In some embodiments of this disclosure, the degree of enrichment is greater than 20. In some embodiments of this disclosure, the degree of enrichment is greater than 200. In some embodiments of this disclosure, the degree of enrichment is greater than 2,000. When preferential enrichment is performed at multiple loci, the degree of enrichment may refer to the average degree of enrichment of all loci within the set of loci.

[0138] Amplification refers to a method of increasing the number of copies of a DNA molecule.

[0139] Selective amplification can refer to a method of increasing the number of copies of a specific DNA molecule or a specific region of DNA. It may also refer to a method of increasing the number of copies of a specific target DNA molecule or region of DNA more than increasing the number of copies of unlabeled DNA molecules or regions. Selective amplification may be a method of preferential enrichment.

[0140] A universal priming sequence refers to a DNA sequence that can be added to a population of target DNA molecules, for example, by ligation, PCR, or ligation-mediated PCR. After addition to the population of target molecules, the target population can be amplified using a single pair of amplification primers with primers specific to the universal priming sequence. Universal priming sequences are generally independent of target sequences.

[0141] Universal adapter, or "Ligation adapter," or "Library tag" This is a DNA molecule containing a universal priming sequence that can be covalently ligated to the 5' and 3' ends of a population of target double-stranded DNA molecules. By adding the adapter, the universal priming sequence is provided to the 5' and 3' ends of a target population from which PCR amplification can be performed, and all molecules from the target population are amplified using a single pair of amplification primers.

[0142] Targeting refers to a method used to selectively amplify, or otherwise preferentially enrich, DNA molecules corresponding to a set of loci in a mixture of DNA.

[0143] A joint distribution model is a model that defines the probability of a predefined event with respect to multiple random variables, considering multiple predefined random variables in the same probability space, where the probabilities of the variables are related. In some embodiments, degenerate cases in which the probabilities of the variables are not related can be used.

[0144] The embodiments disclosed herein are further described with reference to the accompanying drawings, and similar structures are referred to by similar figures throughout several overviews. The figures shown are not necessarily to a constant scale and are generally intended to illustrate the principles of the embodiments disclosed herein. [Brief explanation of the drawing]

[0145] [Figure 1] This is a schematic diagram of the direct multiplex mini-PCR method. [Figure 2] This is an explanatory diagram of the seminested mini-PCR method. [Figure 3] This is an explanatory diagram of the fully nested mini-PCR method. [Figure 4] This is an explanatory diagram of the heminested mini-PCR method. [Figure 5] This is an explanatory diagram of the triple heminested mini-PCR method. [Figure 6] This is an explanatory diagram of the one-sided nested mini-PCR method. [Figure 7] This is an explanatory diagram of the one-sided mini-PCR method. [Figure 8] This is an explanatory diagram of the reverse seminested mini-PCR method. [Figure 9] This is a diagram illustrating several possible workflows for the semi-nested method. [Figure 10] This is a diagram illustrating the loop ligation adapter. [Figure 11] This is an explanatory diagram of a primer with internal tags. [Figure 12] Here are some examples of primers with internal tags. [Figure 13] This is an explanatory diagram of a method using a primer having a ligation adapter binding region. [Figure 14] This graph shows the accuracy of simulated polyploidy calls for counting methods using two different analytical techniques. [Figure 15]This figure shows the ratio of two alleles for multiple SNPs in the cell line of Experiment 4. [Figure 16] This figure shows the ratio of two alleles for multiple SNPs in the cell lines of Experiment 4, separated by chromosome. [Figure 17A] Figures 17A-D show the ratio of two alleles for multiple SNPs in plasma samples from four pregnant women, separated by chromosome. [Figure 17B] Figures 17A-D show the ratio of two alleles for multiple SNPs in plasma samples from four pregnant women, separated by chromosome. [Figure 17C] Figures 17A-D show the ratio of two alleles for multiple SNPs in plasma samples from four pregnant women, separated by chromosome. [Figure 17D] Figures 17A-D show the ratio of two alleles for multiple SNPs in plasma samples from four pregnant women, separated by chromosome. [Figure 18] This graph shows the proportion of data that can be explained by the binomial variance before and after data correction. [Figure 19] This graph shows the relative enrichment of fetal DNA in the sample after a short library preparation protocol. [Figure 20] This graph compares direct PCR and semi-nested PCR in terms of read depth. [Figure 21] This graph shows a comparison of the read depths of direct PCR for three types of genome samples. [Figure 22] This graph shows a comparison of read depths from semi-nested mini-PCR of three different samples. [Figure 23] This graph shows a comparison of read depths for 1,200 plex reactions and 9,600 plex reactions. [Figure 24] This graph shows the read number ratios across three chromosomes for six different cell types. [Figure 25]This figure shows the allele ratios for two 3-cell reactions in three types of chromosomes, and for a third reaction performed on 1 ng of genomic DNA. [Figure 26] This figure shows the allele ratios for two single-cell reactions in three types of chromosomes. [Figure 27] This figure shows the number of loci with specific minor allele frequencies targeted by each of the two primer libraries. [Figure 28-1] Figure 28A: Graph of the electrophoresis of PCR products. [Figure 28-2] Figures 28B to 28M are electrophoretic diagrams of lanes 1 to 12 in Figure 28A. [Figure 28-3] Figures 28B to 28M are electrophoretic diagrams of lanes 1 to 12 in Figure 28A. [Figure 28-4] Figures 28B to 28M are electrophoretic diagrams of lanes 1 to 12 in Figure 28A. [Figure 29] Figures 29A-29E: These are illustrative representations of the present invention's method for determining fetal aneuploidy (Figure 29A). Multiple independent hypotheses for each possible fetal ploidy state are generated in silico using maternal and paternal genotype data (from blood or cheek swab) and crossover frequency data from a hapmap database (Figure 29B). Each of these hypotheses is extended to include secondary hypotheses that take into account different possible crossover points. The data model predicts the likely sequencing data to be generated for each hypothetical fetal genotype and the proportion of different fetal cfDNAs (predicted allele distribution), and this is compared to the actual sequencing data (Figure 29C). The likelihood for each hypothesis is determined using Bayesian statistics. In this example, the hypothesis with the highest likelihood (euploidy) is determined (Figure 29D). The individual likelihoods for each copy number hypothesis family (monosomy, disomy, or triploidy) in Figure 29C are summed up. The maximum likelihood hypothesis is called the ploidy state, reveals the fetal fraction, and demonstrates sample-specific computational accuracy (Figure 29E). [Figure 30-1]Figures 30A–30H: Representative graphical representations of euploidy (Figures 30A–30C), monosomy (Figure 30D), and trisomy (Figures 30E–30H). In all plots, the x-axis represents the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis represents the number of A allele reads as a percentage of the total (A+B) allele reads. Maternal and fetal genotypes, as well as the position of the y-axis center of the bands, are shown on the right side of the plot. If necessary for clarity, the plots may be color-coded according to the maternal genotype, with red representing AA, blue representing BB, and green representing AB. If necessary, the contribution of the maternal allele may be indicated by color in the "Fetal Genotype" column. The allele contribution is expressed in the form of maternal|fetal, such as AA|AB for alleles where the mother is AA and the fetus is AB. Figure 30A is a plot generated when two chromosomes are present and the fetal cfDNA fraction is 0%. This plot is from a non-pregnant woman and therefore represents the pattern when the genotype is entirely maternal. Thus, the allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). Figure 30B shows a plot generated when two chromosomes are present and the fetal fraction is 12%. Due to the contribution of fetal alleles to the proportion of A allele reads, the position of some allele spots shifts up and down along the y-axis. As a result, the bands are centered around 1 (AA|AA allele), 0.94 (AA|AB allele), 0.56 (AB|AA allele), 0.50 (AB|AB allele), 0.44 (AB|BB allele), 0.06 (BB|AB allele), and 0 (BB|BB allele). Figure 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern including two red and two blue peripheral bands and a triplet of green bands in the center is easily visible (colors are not shown in the figure).The bands are centered at 1 (AA|AA allele), 0.87 (AA|AB allele), 0.63 (AB|AA allele), 0.50 (AB|AB allele), 0.37 (AB|BB allele), 0.13 (BB|AB allele), and 0 (BB|BB allele). Figure 30D shows the plot generated when one chromosome is present and the fetal fraction is 26%. The Hallmark pattern of one outer red and one outer blue peripheral band, as well as two central green bands, indicates maternal monosomy (colors are not shown in the figure). Since the fetus contributes only one allele (A or B) to each allele lead, the inner peripheral red and blue bands are absent, and the central triple band is compressed into two bands (colors are not shown in the figure). The bands are centered at 1 (AA|A allele), 0.57 (AB|A allele), 0.43 (AB|B allele), and 0 (BB|B allele). Figure 30E is a plot generated when three chromosomes are present and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.88 (AA|AAB allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.12 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30F is a plot generated when three chromosomes are present and the fetal fraction is 14%. This pattern of three red and three blue peripheral bands, as well as two central green bands, indicates paternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.93 (AA|AAB allele), 0.87 (AA|ABB allele), 0.60 (AB|AAA allele), 0.53 (AB|AAB allele), 0.47 (AB|ABB allele), 0.40 (AB|BBB allele), 0.13 (BB|AAB allele), 0.07 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30G is a plot generated when three chromosomes are present and the fetal fraction is 35%.This pattern of two red and two blue peripheral bands and four green bands indicates maternal mitotic trisomy (colors not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.85 (AA|AAB allele), 0.72 (AB|AAA allele), 0.57 (AB|AAB allele), 0.43 (AB|ABB allele), 0.28 (AB|BBB allele), 0.15 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30H is a plot generated when three chromosomes are present and the fetal fraction is 25%. This pattern of two red and two blue peripheral bands, as well as four central green bands, indicates paternal mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternal mitotic trisomy (like Figure 30G) by the position of the inner peripheral bands. Specifically, the main bands are 1 (AA|AAA allele), 0.78 (AA|ABB allele), 0.67 (AB|AAA allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.33 (AB|BBB allele), 0.22 (BB|AAB allele), and 0 (BB|BBB allele). [Figure 30-2]Figures 30A–30H: Representative graphical representations of euploidy (Figures 30A–30C), monosomy (Figure 30D), and trisomy (Figures 30E–30H). In all plots, the x-axis represents the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis represents the number of A allele reads as a percentage of the total (A+B) allele reads. Maternal and fetal genotypes, as well as the position of the y-axis center of the bands, are shown on the right side of the plot. If necessary for clarity, the plots may be color-coded according to the maternal genotype, with red representing AA, blue representing BB, and green representing AB. If necessary, the contribution of the maternal allele may be indicated by color in the "Fetal Genotype" column. The allele contribution is expressed in the form of maternal|fetal, such as AA|AB for alleles where the mother is AA and the fetus is AB. Figure 30A is a plot generated when two chromosomes are present and the fetal cfDNA fraction is 0%. This plot is from a non-pregnant woman and therefore represents the pattern when the genotype is entirely maternal. Thus, the allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). Figure 30B shows a plot generated when two chromosomes are present and the fetal fraction is 12%. Due to the contribution of fetal alleles to the proportion of A allele reads, the position of some allele spots shifts up and down along the y-axis. As a result, the bands are centered around 1 (AA|AA allele), 0.94 (AA|AB allele), 0.56 (AB|AA allele), 0.50 (AB|AB allele), 0.44 (AB|BB allele), 0.06 (BB|AB allele), and 0 (BB|BB allele). Figure 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern including two red and two blue peripheral bands and a triplet of green bands in the center is easily visible (colors are not shown in the figure).The bands are centered at 1 (AA|AA allele), 0.87 (AA|AB allele), 0.63 (AB|AA allele), 0.50 (AB|AB allele), 0.37 (AB|BB allele), 0.13 (BB|AB allele), and 0 (BB|BB allele). Figure 30D shows the plot generated when one chromosome is present and the fetal fraction is 26%. The Hallmark pattern of one outer red and one outer blue peripheral band, as well as two central green bands, indicates maternal monosomy (colors are not shown in the figure). Since the fetus contributes only one allele (A or B) to each allele lead, the inner peripheral red and blue bands are absent, and the central triple band is compressed into two bands (colors are not shown in the figure). The bands are centered at 1 (AA|A allele), 0.57 (AB|A allele), 0.43 (AB|B allele), and 0 (BB|B allele). Figure 30E is a plot generated when three chromosomes are present and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.88 (AA|AAB allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.12 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30F is a plot generated when three chromosomes are present and the fetal fraction is 14%. This pattern of three red and three blue peripheral bands, as well as two central green bands, indicates paternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.93 (AA|AAB allele), 0.87 (AA|ABB allele), 0.60 (AB|AAA allele), 0.53 (AB|AAB allele), 0.47 (AB|ABB allele), 0.40 (AB|BBB allele), 0.13 (BB|AAB allele), 0.07 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30G is a plot generated when three chromosomes are present and the fetal fraction is 35%.This pattern of two red and two blue peripheral bands and four green bands indicates maternal mitotic trisomy (colors not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.85 (AA|AAB allele), 0.72 (AB|AAA allele), 0.57 (AB|AAB allele), 0.43 (AB|ABB allele), 0.28 (AB|BBB allele), 0.15 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30H is a plot generated when three chromosomes are present and the fetal fraction is 25%. This pattern of two red and two blue peripheral bands, as well as four central green bands, indicates paternal mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternal mitotic trisomy (like Figure 30G) by the position of the inner peripheral bands. Specifically, the main bands are 1 (AA|AAA allele), 0.78 (AA|ABB allele), 0.67 (AB|AAA allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.33 (AB|BBB allele), 0.22 (BB|AAB allele), and 0 (BB|BBB allele). [Figure 30-3]Figures 30A–30H: Representative graphical representations of euploidy (Figures 30A–30C), monosomy (Figure 30D), and trisomy (Figures 30E–30H). In all plots, the x-axis represents the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis represents the number of A allele reads as a percentage of the total (A+B) allele reads. Maternal and fetal genotypes, as well as the position of the y-axis center of the bands, are shown on the right side of the plot. If necessary for clarity, the plots may be color-coded according to the maternal genotype, with red representing AA, blue representing BB, and green representing AB. If necessary, the contribution of the maternal allele may be indicated by color in the "Fetal Genotype" column. The allele contribution is expressed in the form of maternal|fetal, such as AA|AB for alleles where the mother is AA and the fetus is AB. Figure 30A is a plot generated when two chromosomes are present and the fetal cfDNA fraction is 0%. This plot is from a non-pregnant woman and therefore represents the pattern when the genotype is entirely maternal. Thus, the allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). Figure 30B shows a plot generated when two chromosomes are present and the fetal fraction is 12%. Due to the contribution of fetal alleles to the proportion of A allele reads, the position of some allele spots shifts up and down along the y-axis. As a result, the bands are centered around 1 (AA|AA allele), 0.94 (AA|AB allele), 0.56 (AB|AA allele), 0.50 (AB|AB allele), 0.44 (AB|BB allele), 0.06 (BB|AB allele), and 0 (BB|BB allele). Figure 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern including two red and two blue peripheral bands and a triplet of green bands in the center is easily visible (colors are not shown in the figure).The bands are centered at 1 (AA|AA allele), 0.87 (AA|AB allele), 0.63 (AB|AA allele), 0.50 (AB|AB allele), 0.37 (AB|BB allele), 0.13 (BB|AB allele), and 0 (BB|BB allele). Figure 30D shows the plot generated when one chromosome is present and the fetal fraction is 26%. The Hallmark pattern of one outer red and one outer blue peripheral band, as well as two central green bands, indicates maternal monosomy (colors are not shown in the figure). Since the fetus contributes only one allele (A or B) to each allele lead, the inner peripheral red and blue bands are absent, and the central triple band is compressed into two bands (colors are not shown in the figure). The bands are centered at 1 (AA|A allele), 0.57 (AB|A allele), 0.43 (AB|B allele), and 0 (BB|B allele). Figure 30E is a plot generated when three chromosomes are present and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.88 (AA|AAB allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.12 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30F is a plot generated when three chromosomes are present and the fetal fraction is 14%. This pattern of three red and three blue peripheral bands, as well as two central green bands, indicates paternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.93 (AA|AAB allele), 0.87 (AA|ABB allele), 0.60 (AB|AAA allele), 0.53 (AB|AAB allele), 0.47 (AB|ABB allele), 0.40 (AB|BBB allele), 0.13 (BB|AAB allele), 0.07 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30G is a plot generated when three chromosomes are present and the fetal fraction is 35%.This pattern of two red and two blue peripheral bands and four green bands indicates maternal mitotic trisomy (colors not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.85 (AA|AAB allele), 0.72 (AB|AAA allele), 0.57 (AB|AAB allele), 0.43 (AB|ABB allele), 0.28 (AB|BBB allele), 0.15 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30H is a plot generated when three chromosomes are present and the fetal fraction is 25%. This pattern of two red and two blue peripheral bands, as well as four central green bands, indicates paternal mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternal mitotic trisomy (like Figure 30G) by the position of the inner peripheral bands. Specifically, the main bands are 1 (AA|AAA allele), 0.78 (AA|ABB allele), 0.67 (AB|AAA allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.33 (AB|BBB allele), 0.22 (BB|AAB allele), and 0 (BB|BBB allele). [Figure 30-4]Figures 30A–30H: Representative graphical representations of euploidy (Figures 30A–30C), monosomy (Figure 30D), and trisomy (Figures 30E–30H). In all plots, the x-axis represents the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis represents the number of A allele reads as a percentage of the total (A+B) allele reads. Maternal and fetal genotypes, as well as the position of the y-axis center of the bands, are shown on the right side of the plot. If necessary for clarity, the plots may be color-coded according to the maternal genotype, with red representing AA, blue representing BB, and green representing AB. If necessary, the contribution of the maternal allele may be indicated by color in the "Fetal Genotype" column. The allele contribution is expressed in the form of maternal|fetal, such as AA|AB for alleles where the mother is AA and the fetus is AB. Figure 30A is a plot generated when two chromosomes are present and the fetal cfDNA fraction is 0%. This plot is from a non-pregnant woman and therefore represents the pattern when the genotype is entirely maternal. Thus, the allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). Figure 30B shows a plot generated when two chromosomes are present and the fetal fraction is 12%. Due to the contribution of fetal alleles to the proportion of A allele reads, the position of some allele spots shifts up and down along the y-axis. As a result, the bands are centered around 1 (AA|AA allele), 0.94 (AA|AB allele), 0.56 (AB|AA allele), 0.50 (AB|AB allele), 0.44 (AB|BB allele), 0.06 (BB|AB allele), and 0 (BB|BB allele). Figure 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern including two red and two blue peripheral bands and a triplet of green bands in the center is easily visible (colors are not shown in the figure).The bands are centered at 1 (AA|AA allele), 0.87 (AA|AB allele), 0.63 (AB|AA allele), 0.50 (AB|AB allele), 0.37 (AB|BB allele), 0.13 (BB|AB allele), and 0 (BB|BB allele). Figure 30D shows the plot generated when one chromosome is present and the fetal fraction is 26%. The Hallmark pattern of one outer red and one outer blue peripheral band, as well as two central green bands, indicates maternal monosomy (colors are not shown in the figure). Since the fetus contributes only one allele (A or B) to each allele lead, the inner peripheral red and blue bands are absent, and the central triple band is compressed into two bands (colors are not shown in the figure). The bands are centered at 1 (AA|A allele), 0.57 (AB|A allele), 0.43 (AB|B allele), and 0 (BB|B allele). Figure 30E is a plot generated when three chromosomes are present and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.88 (AA|AAB allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.12 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30F is a plot generated when three chromosomes are present and the fetal fraction is 14%. This pattern of three red and three blue peripheral bands, as well as two central green bands, indicates paternal meiotic trisomy (colors are not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.93 (AA|AAB allele), 0.87 (AA|ABB allele), 0.60 (AB|AAA allele), 0.53 (AB|AAB allele), 0.47 (AB|ABB allele), 0.40 (AB|BBB allele), 0.13 (BB|AAB allele), 0.07 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30G is a plot generated when three chromosomes are present and the fetal fraction is 35%.This pattern of two red and two blue peripheral bands and four green bands indicates maternal mitotic trisomy (colors not shown in the figure). The bands are centered at 1 (AA|AAA allele), 0.85 (AA|AAB allele), 0.72 (AB|AAA allele), 0.57 (AB|AAB allele), 0.43 (AB|ABB allele), 0.28 (AB|BBB allele), 0.15 (BB|ABB allele), and 0 (BB|BBB allele). Figure 30H is a plot generated when three chromosomes are present and the fetal fraction is 25%. This pattern of two red and two blue peripheral bands, as well as four central green bands, indicates paternal mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternal mitotic trisomy (like Figure 30G) by the position of the inner peripheral bands. Specifically, the main bands are 1 (AA|AAA allele), 0.78 (AA|ABB allele), 0.67 (AB|AAA allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.33 (AB|BBB allele), 0.22 (BB|AAB allele), and 0 (BB|BBB allele). [Figure 31-1] Figure 31: Graph representation of euploid (Figure 31A), T13 (Figure 31B), T18 (Figure 31C), T21 (Figure 31D), 45, X (Figure 31E), and 47, XXY (Figure 31F) test samples. Each chromosome is shown at the top of the plot, and fetal and maternal genotypes are shown to the right of the plot. The x-axis represents the linear position of SNPs along each chromosome, and the y-axis represents the number of A allele reads as a percentage of the total reads. Note that the cluster positions are changed based on the fetal fraction, as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown to the right of the plot, and chromosome identification information is shown at the top of the plot. [Figure 31-2]Figure 31: Graph representation of euploid (Figure 31A), T13 (Figure 31B), T18 (Figure 31C), T21 (Figure 31D), 45, X (Figure 31E), and 47, XXY (Figure 31F) test samples. Each chromosome is shown at the top of the plot, and fetal and maternal genotypes are shown to the right of the plot. The x-axis represents the linear position of SNPs along each chromosome, and the y-axis represents the number of A allele reads as a percentage of the total reads. Note that the cluster positions are changed based on the fetal fraction, as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown to the right of the plot, and chromosome identification information is shown at the top of the plot. [Figure 31-3] Figure 31: Graph representation of euploid (Figure 31A), T13 (Figure 31B), T18 (Figure 31C), T21 (Figure 31D), 45, X (Figure 31E), and 47, XXY (Figure 31F) test samples. Each chromosome is shown at the top of the plot, and fetal and maternal genotypes are shown to the right of the plot. The x-axis represents the linear position of SNPs along each chromosome, and the y-axis represents the number of A allele reads as a percentage of the total reads. Note that the cluster positions are changed based on the fetal fraction, as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown to the right of the plot, and chromosome identification information is shown at the top of the plot. [Figure 31-4] Figure 31: Graph representation of euploid (Figure 31A), T13 (Figure 31B), T18 (Figure 31C), T21 (Figure 31D), 45, X (Figure 31E), and 47, XXY (Figure 31F) test samples. Each chromosome is shown at the top of the plot, and fetal and maternal genotypes are shown to the right of the plot. The x-axis represents the linear position of SNPs along each chromosome, and the y-axis represents the number of A allele reads as a percentage of the total reads. Note that the cluster positions are changed based on the fetal fraction, as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown to the right of the plot, and chromosome identification information is shown at the top of the plot. [Figure 32]This graph shows that the prevalence of composite births due to sex chromosome aneuploidy is higher than that due to autosomal aneuploidy. [Modes for carrying out the invention]

[0146] The above figures illustrate the embodiments disclosed herein, but other embodiments are also intended, as mentioned in the discussion. This disclosure provides examples of embodiments, not limitations. Those skilled in the art can devise numerous other modifications and embodiments that fall within the scope of the principles and spirit of the embodiments disclosed herein.

[0147] This invention is partly based on the unexpected discovery that a relatively small number of primers in a primer library are often responsible for the formation of a significant amount of amplified primer dimers during multiplex PCR reactions. A method has been developed to select the most undesirable primers for removal from a candidate primer library. By reducing the amount of primer dimers to a negligible level (about 0.1% of the PCR product), these methods enable the simultaneous amplification of multiple target loci in just one multiplex PCR reaction using the generated primer library. The number of different target loci that can be amplified increases because the primers hybridize to the target loci and amplify them without hybridizing to other primers and forming amplified primer dimers. It has also been found that using lower-than-usual primer concentrations and considerably longer annealing times increases the likelihood that primers hybridize to target loci rather than hybridizing to each other and forming primer dimers.

[0148] During PCR amplification and sequencing of 19,488 target loci in genomic samples, 99.4–99.7% of these sequencing reads mapped to the genome, and 99.99% mapped to target loci. For plasma samples with 10 million sequencing reads, typically at least 19,350 (99.3%) of the 19,488 target loci were amplified and sequenced. The ability to simultaneously amplify such a large number of target loci significantly reduces the time and amount of DNA required to analyze thousands of target loci. For example, single-cell DNA is sufficient to simultaneously analyze thousands of target loci, which are important in DNA-limited applications such as single-cell genetic testing of embryos before in vitro fertilization or genetic testing of forensic samples containing small amounts of DNA. Furthermore, the ability to analyze target loci in a single reaction volume (e.g., one container or well) without splitting the sample into multiple separate reactions reduces potential variability during the reaction. Furthermore, the method described above is designed to use a reference standard to correct for amplification biases that may occur between different target loci. For example, differences in amplification efficiency between target loci due to factors such as GC content may result in different amounts of PCR product being produced for target loci, even though the amounts should actually be the same. By using a reference standard similar to the target locus, such amplification biases can be detected and corrected during the quantification of the target locus.

[0149] During sequencing of PCR products, artifacts such as primer dimers are detected, hindering the detection of the target amplification product. Due to this limitation, microarrays equipped with hybridization probes are often used for detection because of their low sensitivity to primer dimer interference. High levels of multiplexing, including the minimum currently achievable non-target amplification product, make PCR and subsequent sequencing usable as an alternative to microarrays.

[0150] The multiplex PCR method of the present invention can be used for a variety of applications. For example, it can be used for genotype analysis, detection of chromosomal abnormalities (e.g., fetal chromosomal aneuploidy), analysis of gene mutations and polymorphisms (e.g., single nucleotide polymorphisms, SNPs), gene deletion analysis, paternity testing, analysis of genetic differences within a population, forensic analysis, predisposition to disease, quantitative analysis of mRNA, and detection and identification of infectious pathogens (e.g., bacteria, parasites, and viruses). Furthermore, the multiplex PCR method can also be used for non-invasive prenatal genetic testing, such as paternity testing or detection of fetal chromosomal abnormalities.

[0151] Typical primer design methods High-level multiplexing PCR can often produce a very high proportion of product DNA resulting from unproductive side reactions, such as primer dimerization. In one embodiment, specific primers most likely to cause unproductive side reactions can be removed from the primer library to obtain a primer library that yields a high proportion of amplified DNA that maps to the genome. The step of removing problematic primers, i.e., those that may stabilize dimers, unexpectedly enabled very high levels of PCR multiplexing for subsequent sequencing analysis. In systems such as sequencing where performance is significantly degraded by primer dimers and / or other adverse products, multiplexing levels of more than 10, 50, and 100 times higher than those described elsewhere were achieved. Note that this is in contrast to probe-based detection methods, e.g., microarrays, TAQMAN, and PCR, where excess primer dimers do not significantly affect the results to a sense. Note also that, in common practice in the art, multiplexed PCR for sequencing is limited to approximately 100 assays per well. Fluidigm and Rain Dance provide a platform for performing 48 or 1000 PCR assays in parallel reactions on a single sample.

[0152] Several methods exist for selecting primers for libraries that minimize the amount of non-mapping primer dimers or other adverse primer products. Empirical data have shown that a small number of "bad" primers are involved in a large number of non-mapping primer dimer side effects. By removing these "bad" primers, the percentage of sequence reads that locate at the target gene locus can be increased. One method for identifying "bad" primers is to examine the sequencing data of DNA amplified by targeted amplification, and by removing the primer dimers found most frequently, a primer library with a significantly lower probability of producing non-mapping byproduct DNA can be created. There are also publicly available programs that can calculate the binding energies of various primer combinations, and by removing the primer combinations with the highest binding energies, a primer library with a significantly lower probability of producing non-mapping byproduct DNA can similarly be created.

[0153] In some embodiments for primer selection, an initial candidate primer library is constructed by designing one or more primers or primer pairs for candidate target loci. The set of candidate target loci (e.g., SNPs) can be selected based on publicly available information regarding desired parameters of the target loci, such as the frequency of SNPs in the target population or the heterozygosity of SNPs. In one embodiment, PCR primers can be designed using the Primer3 program (primer3.sourceforge.net worldwide web; libprimer3 release 2.2.3, (which is incorporated herein in its entirety by reference)). If necessary, primers can be designed to anneal within a specific annealing temperature range, to have a specific range of GC content, to fall within a specific size range, to produce a target amplification product within a specific size range, and / or to have other parameter characteristics. Starting with multiple primers or primer pairs per candidate target locus increases the likelihood that primers or primer pairs for most or all target loci will remain in the library. In one embodiment, the selection criterion may be that at least one primer pair per target locus must remain in the library. In this way, using the final primer library will amplify almost all or all target gene loci. This is desirable for applications such as deletion screening or screening for replication at multiple sites in the genome or for screening for multiple sequences (e.g., polymorphisms or other mutations) associated with disease or increased risk of disease. If a primer pair from the library produces a target amplification product that overlaps with the target amplification product produced by another primer pair, one of the primer pairs can be removed from the library to prevent interference.

[0154] In some embodiments, an "undesirability score" (higher scores indicate less desirability) is calculated (e.g., by computer) for almost all or all possible combinations of two primers from the candidate primer library. In various embodiments, the undesirability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% of possible combinations of candidate primers in the library. Each undesirability score is at least partly based on the likelihood of dimerization between the two candidate primers. Where necessary, the undesirability score may also be based on one or more other parameters selected from the group consisting of heterozygosity of the target locus, prevalence associated with the sequence (e.g., polymorphism) at the target locus, disease penetrant associated with the sequence (e.g., polymorphism) at the target locus, specificity of the candidate primer to the target locus, size of the candidate primer, melting temperature of the target amplification product, GC content of the target amplification product, amplification efficiency of the target amplification product, and size of the target amplification product. When considering multiple factors, the undesirability score may be calculated based on a weighted average of various parameters. The parameters can be assigned different weights based on their importance to the specific application in which the primers are used. In some embodiments, the primer with the highest undesirability score is removed from the library. If the removed primer is a member of a primer pair that hybridizes to one target locus, the other member of the primer pair can also be removed from the library. The primer removal process can be repeated as needed. In some embodiments, the selection method is carried out until the undesirability scores of all candidate primer combinations remaining in the library are below a minimum threshold. In some embodiments, the selection method is carried out until the number of candidate primers remaining in the library is reduced to a desired number.

[0155] In various embodiments, after the undesirability score is calculated, candidate primers that are part of the largest number of candidate primer combinations having an undesirability score above a first minimum threshold are removed from the library. This step ignores interactions below the first minimum threshold because these interactions are of low importance. If the removed primer is a member of a primer pair that hybridizes to one target locus, the other member of the primer pair can be removed from the library. The primer removal process can be repeated as needed. In some embodiments, the selection method is carried out until the undesirability scores of all candidate primer combinations remaining in the library are below the first minimum threshold. If the number of candidate primers remaining in the library is greater than the required number, the number of primers can be reduced by lowering the first minimum threshold to a second minimum threshold and repeating the primer removal process. If the number of candidate primers remaining in the library is less than the required number, the method can be continued by increasing the first minimum threshold to a larger second minimum threshold and repeating the primer removal process using the original candidate primer library, thereby leaving more candidate primers in the library. In some embodiments, the selection method is performed until the undesirability scores of all remaining complementary primer combinations in the library fall below a second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number.

[0156] If necessary, primer pairs that produce target amplification products that overlap with those produced by other primer pairs can be separated into separate amplification reactions. Multiplex PCR amplification reactions may be preferable for applications where it is desirable to analyze all candidate target loci (rather than excluding candidate target loci from analysis due to overlapping target amplification products).

[0157] These selection methods minimize the number of candidate primers that need to be removed from the library to achieve the desired reduction in primer dimers. By removing fewer candidate primers from the library, more (or all) target loci can be amplified using the resulting primer library.

[0158] Multiplexing a large number of primers imposes considerable constraints on the assays that can be included. Assays that unintentionally interact can result in false amplification products. The size constraints of miniPCRs can impose further constraints. In one embodiment, it is possible to start with a very large number of potential SNP targets (between approximately 500 and over 1 million) and attempt to design primers to amplify each SNP. If primers can be designed, it is possible to attempt to identify primer pairs that are likely to form false products by evaluating the likelihood of false primer double-strand formation between all possible primer pairs using published thermodynamic parameters for DNA double-strand formation. Primer interactions can be ranked by a score function related to the interaction, and the primers with the worst interaction scores can be eliminated until the desired number of primers is met. If potentially heterozygous SNPs are most useful, it is possible to similarly rank the list of assays and select the assay that best fits heterozygosity. Experiments have verified that primers with high interaction scores are most likely to form primer dimers. In advanced multiplexing, it is impossible to eliminate all spurious interactions, but primers or primer pairs with the highest interaction scores in silico must be removed, as they dominate the overall reaction and significantly limit amplification from the intended target. This procedure was used to create multiplexed primer sets reaching, and in some cases exceeding, 10,000 primers. The improvement from this procedure is substantial, enabling amplification of over 80%, 90%, 95%, 98%, and even over 99% of the target product compared to 10% from reactions where the worst primers were not removed, as determined by sequencing of all PCR products. When combined with previously described partially semi-nested techniques, amplification of over 90% and even over 95% of the products can be mapped to the target sequence.

[0159] It should be noted that other methods exist for determining which PCR probes are likely to form dimers. In some embodiments, analysis of a pool of DNA amplified using an unoptimized set of primers may be sufficient to identify problematic primers. For example, the analysis can be performed using sequencing, and the dimer present in the largest number can be determined as the most likely to form dimers and removed.

[0160] This method has several potential applications, such as SNP genotyping, heterozygosity determination, copy number measurement, and other targeted sequencing. In some embodiments, the primer design method can be used in combination with the mini-PCR method described elsewhere in this document. In some embodiments, the primer design method can be used as part of a large-scale multiplex PCR method.

[0161] Using tags on primers can reduce the amplification and sequencing of primer dimer products. In some embodiments, the primer includes an internal region that forms a loop structure with a tag. In certain embodiments, the primer includes a 5' region specific to the target locus, an internal region that forms a loop structure not specific to the target locus, and a 3' region specific to the target locus. In some embodiments, the loop region may be located between two binding regions designed to bind to adjacent or adjacent regions of the template DNA. In various embodiments, the length of the 3' region is at least 7 nucleotides. In some embodiments, the length of the 3' region is 7 to 20 nucleotides, for example, 7 to 15 nucleotides, or 7 to 10 nucleotides. In various embodiments, the primer includes a 5' region that is not specific to the target locus (e.g., a tag or universal primer binding site), followed by a region specific to the target locus, an internal region that forms a loop structure not specific to the target locus, and a 3' region specific to the target locus. Using tag-primers, the required target-specific sequences can be shortened to less than 20 base pairs, less than 15 base pairs, less than 12 base pairs, and even less than 10 base pairs. This can be discovered incidentally during standard primer design when the target sequence is fragmented within the primer binding site, or it can be planned into primer design. The advantages of this method include an increased number of assays that can be designed for a given maximum amplification product length, and shortened sequencing of "non-informational" primer sequences. This method can also be used in combination with internal tagging (see other parts of this document).

[0162] In some embodiments, the relative amount of unproductive products in multi-target PCR amplification can be reduced by increasing the annealing temperature. When amplifying a library having the same tag as the target-specific primers, the annealing temperature can be increased relative to that of genomic DNA because the tag contributes to primer binding. In some embodiments, considerably lower primer concentrations than previously reported are used, along with longer annealing times than those reported elsewhere. In some embodiments, the annealing time may be greater than 3 minutes, greater than 5 minutes, greater than 8 minutes, greater than 10 minutes, greater than 15 minutes, greater than 20 minutes, greater than 30 minutes, greater than 60 minutes, greater than 120 minutes, greater than 240 minutes, greater than 480 minutes, and even greater than 960 minutes. In some embodiments, longer annealing times than previously reported are used, which allows for lower primer concentrations. In various embodiments, longer extension times than usual are used, for example, greater than 3, 5, 8, 10, or 15 minutes. In some embodiments, primer concentrations are as low as 50 nM, 20 nM, 10 nM, 5 nM, 1 nM, and less than 1 μM. Surprisingly, this results in robust performance for highly multiplexed reactions, such as 1,000 plex reactions, 2,000 plex reactions, 5,000 plex reactions, 10,000 plex reactions, 20,000 plex reactions, 50,000 plex reactions, and even 100,000 plex reactions. In one embodiment, amplification is performed using one, two, three, four, or five cycles with long annealing times, followed by more PCR cycles than usual using tagged primers.

[0163] To select a target location, we can start with a pool of candidate primer-pair designs, create a thermodynamic model of potentially harmful interactions between primer-pairs, and then use this model to eliminate designs that do not fit with the other designs in the pool.

[0164] After the selection process, the primers remaining in the library can be used in any of the methods of the present invention.

[0165] Representative primer library In one embodiment, the present invention features a library of primers, for example, primers selected from a candidate primer library using any of the methods of the present invention. In some embodiments, the library includes primers that simultaneously hybridize (or are capable of simultaneously hybridizing) or simultaneously amplify (or are capable of simultaneously amplifying) at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci in one reaction volume. In various embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) 1,000-2,000; 2,000-5,000; 5,000-7,500; 7,500-10,000; 10,000-20,000; 20,000-25,000; 25,000-30,000; 30,000-40,000; 40,000-50,000; 50,000-75,000; and 75,000-100,000 different target gene loci in one reaction volume. In various embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) 1,000 to 100,000 different target loci in one reaction volume, for example, 1,000 to 50,000; 1,000 to 30,000; 1,000 to 20,000; 1,000 to 10,000; 2,000 to 30,000; 2,000 to 20,000; 2,000 to 10,000 different target loci. In some embodiments, the library includes primers that simultaneously amplify (or are capable of simultaneously amplifying) the target gene locus in one reaction volume, thereby resulting in primer dimers accounting for less than 60, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.5% of the amplified product. In various embodiments, the amount of primer dimer amplified product is between 0.5 and 60%, for example, 0.1-40%, 0.1-20%, 0.25-20%, 0.25-10%, 0.5-20%, 0.5-10%, 1-20%, or 1-10%.In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) the target locus in one reaction volume, thereby amplifying at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the amplified product which is the target amplified product. In various embodiments, the amount of the amplified product which is the target amplified product is 50-99.5%, for example, 60-99%, 70-98%, 80-98%, 90-99.5%, or 95-99.5%. In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) the target locus in one reaction volume, thereby amplifying at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target locus. In various embodiments, the amount of amplified target locus is 50–99.5%, for example, 60–99%, 70–98%, 80–99%, 90–99.5%, 95–99.9%, or 98–99.99%. In some embodiments, the primer library contains at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 primer pairs, where each pair of primers contains a forward test primer and a reverse test primer, and each test primer pair hybridizes to the target locus. In some embodiments, the primer library includes at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 individual primers, each hybridizing to a different target locus, wherein the individual primers are not part of a primer pair.

[0166] In various embodiments, the concentration of each primer is less than 100, 75, 50, 25, 20, 10, 5, 2, or 1 nM, or less than 500, 100, 10, or 1 uM. In various embodiments, the concentration of each primer is between 1 uM and 100 nM, for example, 1 uM to 1 nM, 1 to 75 nM, 2 to 50 nM, or 5 to 50 nM. In various embodiments, the GC content of the primer is between 30 and 80%, for example, 40 to 70%, or 50 to 60%. In some embodiments, the range of the GC content of the primer is less than 30, 20, 10, or 5%. In some embodiments, the range of the GC content of the primer is between 5 and 30%, for example, 5 to 20% or 5 to 10%. In some embodiments, the melting temperature (T) of the test primer is specified. m The temperature is 40-80°C, for example, 50-70°C, 55-65°C, or 57-60.5°C. In some embodiments, T mThis is calculated by the primer3 program (libprimer3 release 2.2.3) using the built-in SantaLucia parameter (worldwide web at primer3.sourceforge.net). In some embodiments, the primer melting temperature range is less than 15, 10, 5, 3, or 1°C. In some embodiments, the primer melting temperature range is 1 to 15°C, for example, 1 to 10°C, 1 to 5°C, or 1 to 3°C. In some embodiments, the primer length is 15 to 100 nucleotides, for example, 15 to 75 nucleotides, 15 to 40 nucleotides, 17 to 35 nucleotides, 18 to 30 nucleotides, or 20 to 65 nucleotides. In some embodiments, the primer length range is less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the primer length range is 5 to 50 nucleotides, for example, 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides. In some embodiments, the length of the target amplification product is 50 to 100 nucleotides, for example, 60 to 80 nucleotides or 60 to 75 nucleotides. In some embodiments, the length range of the target amplification product is less than 50, 25, 15, 10, or 5 nucleotides. In some embodiments, the length range of the target amplification product is 5 to 50 nucleotides, for example, 5 to 25 nucleotides, 5 to 15 nucleotides, or 5 to 10 nucleotides.

[0167] These primer libraries can be used in any of the methods of the present invention.

[0168] Typical primer kits In one embodiment, the present invention features a kit comprising any of the primer libraries of the present invention (for example, a kit for amplifying a target gene locus in a nucleic acid sample). In some embodiments, a kit can be formulated comprising multiple primers designed to accomplish the methods described herein. The primers may be outer forward and reverse primers, inner forward and reverse primers, as disclosed herein, and may be primers designed to have low binding affinity to other primers in the kit, as disclosed in the section on primer design, and may be hybrid capture probes or pre-circularization probes or some combination thereof, as described in the relevant section. In some embodiments, a kit can be constructed for determining the ploidy status of a target chromosome in a pregnant fetus, designed for use in the methods disclosed herein, comprising multiple inner forward primers, and optionally multiple inner reverse primers, and optionally outer forward and outer reverse primers, each of which is designed to hybridize with a region of DNA immediately upstream and / or downstream of one of the target sites (e.g., polymorphic sites) on the target chromosome and optionally another target site on another chromosome. In some embodiments, the primer kit can be used in combination with a diagnostic box as described elsewhere in this document. In some embodiments, the kit includes instructions for amplifying target gene loci using a library.

[0169] Typical multiplex PCR method In one embodiment, the present invention is characterized by a method for amplifying target gene loci in a nucleic acid sample, the method comprising (i) contacting the nucleic acid sample with a primer library that simultaneously hybridizes to at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target gene loci to generate a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (e.g., PCR conditions) to generate an amplification product containing target amplification products. In some embodiments, the method also comprises the step of determining the presence or absence of at least one target amplification product (e.g., at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of target amplification products). In some embodiments, the method also includes the step of determining the sequence of at least one target amplification product (e.g., at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target amplification product). In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of the target locus is amplified. In various embodiments, amplification products of less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% are primer dimers.

[0170] In one embodiment, the method disclosed herein uses highly efficient advanced multiple-target PCR to amplify DNA and then perform high-throughput sequencing to determine the allele frequencies at each target locus. It is novel and non-trivial that approximately 50 or more PCR primers can be multiplexed in a single reaction volume so that the majority of the resulting sequence reads map to the target loci. One technique that enables advanced multiple-target PCR to be carried out in a highly efficient manner involves designing primers that are less likely to hybridize with each other. PCR probes, commonly referred to as primers, are selected by creating thermodynamic models of potentially harmful interactions between at least 500, at least 1,000, at least 2,000, at least 5,000, at least 7,500, at least 10,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 potential primer pairs, or unintended interactions between primers and sample DNA, and then using these models to eliminate designs that do not fit with other designs in the pool. Another technique that enables highly multi-target PCR to be performed in a highly efficient manner is to perform targeted PCR using partial or full nesting techniques. Using one or a combination of these methods, it becomes possible to multiplex at least 300, at least 800, at least 1,200, at least 4,000, or at least 10,000 primers in a single pool, and the resulting amplified DNA, upon sequencing, contains the majority of DNA molecules that map to the target locus. Using one or a combination of these methods, it becomes possible to multiplex a large number of primers in a single pool, and the resulting amplified DNA contains more than 50%, more than 60%, more than 67%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.5% of DNA molecules that map to the target locus.

[0171] In some embodiments, detection of target genetic material can be performed in a multiplexing manner. The number of target sequences of genes that can be performed in parallel can range from 1 to 10, 10 to 100, 100 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1,000,000, or 1,000,000 to 10,000,000. Previous attempts to multiplex more than 100 primers per pool have resulted in significant problems, including undesirable side reactions such as primer dimerization.

[0172] Targeted PCR In some embodiments, PCR can be used to target specific locations in the genome. In plasma samples, the original DNA is highly fragmented (typically less than 500 bp, with an average length of less than 200 bp). In PCR, both the forward and reverse primers anneal to the same fragment to enable amplification. Therefore, if the fragment is short, the PCR assay must amplify a similarly relatively short region. If the location of the polymorphism is too close to the polymerase binding site, as in MIPS, it can lead to a bias in amplification from different alleles. Currently, PCR primers targeting polymorphic regions, such as those containing SNPs, are typically designed so that the 3' end of the primer hybridizes to a base immediately adjacent to one or more polymorphic bases. In some embodiments of this disclosure, the 3' ends of both the forward and reverse PCR primers are designed to hybridize to a base that is only one or a few positions away from the location of the mutation (polymorphic region) in the target allele. The number of bases between the polymorphic site (SNP or other type) and the base designed to hybridize the 3' end of the primer may be 1, 2, 3, 4, 5, 6, 7–10, 11–15, or 16–20 bases. Forward and reverse primers can be designed to hybridize with different numbers of bases away from the polymorphic site.

[0173] While numerous PCR assays can be generated, interactions between different PCR assays make it difficult to multiplex them beyond approximately 100 assays. Various complex molecular techniques can increase the level of multiplexing, but even then, it may still be limited to fewer than 100, perhaps 200, or even 500 assays per reaction. Samples with large amounts of DNA can be split into multiple secondary reactions and then recombined before sequencing. For samples where either the entire DNA molecule or a subset of subpopulations is limited, splitting the sample introduces statistical noise. In some embodiments, small or limited amounts of DNA may refer to amounts less than 10 pg, between 10 pg and 100 pg, between 100 pg and 1 ng, between 1 ng and 10 ng, or between 10 ng and 100 ng. While this method is particularly useful for small amounts of DNA where other methods involving the step of splitting into multiple pools can introduce significant problems associated with probabilistic noise, it should be noted that this method offers the benefit of minimizing bias regardless of the amount of DNA sample. In these situations, a universal pre-amplification step can be used to increase the overall sample volume. Ideally, this pre-amplification step should not alter the allele distribution to a degree that would be perceptible.

[0174] In some embodiments, the method of this disclosure can generate PCR products specific to a large number of target loci, specifically 1,000 to 5,000 loci, 5,000 to 10,000 loci, or more than 10,000 loci, from a limited sample such as a single cell or DNA derived from bodily fluids, for genotyping by sequencing or several other genotyping methods. Currently, performing multiplex PCR reactions of more than 5 to 10 targets presents significant challenges, often due to interference from primer byproducts, such as primer dimers, and other artifacts. When detecting target sequences using microarrays with hybridization probes, primer dimers and other artifacts are not detected and can therefore be ignored. However, when sequencing is used as the detection method, the majority of sequencing reads sequence such artifacts, and the desired target sequences in the sample are not sequenced. Methods using prior art that involve multiplexing more than 50 or 100 reactions in a single reaction volume and then sequencing them generally result in more than 20%, often more than 50%, often more than 80%, and in some cases more than 90% off-target sequence reads.

[0175] Generally, to perform targeted sequencing on a large number of (n) targets (over 50, over 100, over 500, or over 1,000) in a sample, the sample can be split into several parallel reactants to amplify a single individual target. This is done in multi-well PCR plates or on commercial platforms, such as FLUIDIGM ACCESS ARRAY (48 reactions per sample in a microfluidic chip) or DROPLET PCR from RAIN DANCE TECHNOLOGY (100 to several thousand targets). Unfortunately, these split-and-pool methods are problematic for samples with limited DNA quantities because the number of copies of the genome present is often insufficient to ensure that there is one copy of each region of the genome in each well. This is a particularly serious problem when targeting polymorphic loci, as the probabilistic noise introduced by splitting and pooling leads to a very poorly accurate measurement of the proportion of alleles present in the original DNA sample, while the relative proportion of alleles at the polymorphic locus is required. A method for effectively and efficiently amplifying many PCR reaction products is described herein, applicable when only a limited amount of DNA is available. In some embodiments, the method can be applied to analyze single cells, body fluids, DNA mixtures, for example, floating DNA found in maternal plasma, biopsy materials, environmental samples, and / or forensic samples.

[0176] In one embodiment, target sequencing may include one, more, or all of the following steps: a) Generate and amplify a library with adapter sequences at both ends of a DNA fragment. b) After library amplification, split into multiple reactions. c) Generate and amplify a library with adapter sequences at both ends of a DNA fragment, optionally. d) Perform 1,000 to 10,000 plex amplification of the selected target using one target-specific "forward" primer and one tag-specific primer per target. e) Perform a second amplification from this product using a "reverse" target-specific primer and one (or more) universal tag-specific primers introduced as part of the target-specific forward primer in the first round. f) Perform 1,000 plex pre-amplification of the selected target for a limited number of cycles. g) Divide the product into multiple fractions and amplify the target subpool in individual reactions (e.g., 50 to 500 plexes (this method can be used for any plex up to a single plex)). h) Pool the products of the parallel subpool reactions. i) During these amplifications, the primers can retain sequencing-compatible tags (partial length or full length), thereby enabling sequencing of the product.

[0177] Advanced multiplex PCR This specification discloses a method for the targeted amplification of over 100 to tens of thousands of target sequences (e.g., SNP loci) derived from nucleic acid samples such as genomic DNA obtained from plasma. The amplified samples contain relatively few primer dimer products and exhibit less allelic bias at the target loci. If a sequencing compatibility adapter is added to the product during or after amplification, these products can be analyzed by sequencing.

[0178] When performing highly multiplicative PCR amplification using methods known in the art, the desired amplification product is often produced in excess, along with primer dimer products unsuitable for sequencing. These can be empirically reduced by eliminating the primers that form these products or by performing in silico selection of primers. However, this problem becomes more difficult as the number of assays increases.

[0179] One solution is to split the 5,000-plex reaction into several lower-plex amplifications, e.g., 100 50-plex reactions or 50 100-plex reactions, or to use microfluidics, or even to split the sample into individual PCR reactions. However, when sample DNA is limited, such as in non-invasive prenatal diagnosis from pregnancy plasma, splitting the sample between numerous reactions should be avoided, as this will lead to bottlenecking.

[0180] This specification describes a method for first amplifying plasma DNA from a sample overall, and then splitting the sample into multiple target enrichment reactions with a moderate number of target sequences per reaction. In some embodiments, the method of the Disclosure may be used to preferentially enrich a DNA mixture at multiple loci, and the method comprises one or more of the following steps: generating and amplifying a library from a mixture of DNA, wherein the molecules in the library have adapter sequences ligated to both ends of DNA fragments; splitting the amplified library into multiple reactions; and carrying out a first round of multiple amplification of selected targets using one target-specific "forward" primer per target and one or more adapter-specific universal "reverse" primers. In some embodiments, the method of the Disclosure further comprises carrying out a second amplification using "reverse" target-specific primers and one or more primers specific to the universal tag introduced as part of the target-specific forward primer in the first round. In some embodiments, the method may involve fully nested PCR, heminested PCR, semi-nested PCR, one-sided fully nested PCR, one-sided heminested PCR, or one-sided semi-nested PCR. In some embodiments, the method of the disclosure is used to preferentially enrich a DNA mixture at multiple loci, and the method includes the steps of performing pre-multiplexing amplification of selected targets for a limited number of cycles, dividing the product into several fixed amounts, amplifying subpools of targets in individual reactions, and pooling the products of parallel subpool reactions. It should be noted that this method can be used to perform targeted amplification for 50 to 500 loci, 500 to 5,000 loci, 5,000 to 50,000 loci, or even 50,000 to 500,000 loci, such that allelic bias is low. In some embodiments, the primers carry partial-length or full-length sequencing-compatible tags.

[0181] The workflow may include the steps of (1) extracting DNA such as plasma DNA, (2) preparing a fragment library having universal adapters at both ends of the fragments, (3) amplifying the library using universal primers specific to the adapters, (4) dividing the amplified sample "library" into several fixed volumes, (5) performing multiplex amplification on the fixed volumes (e.g., about 100 plexes, 1,000, or 10,000 plexes using one target-specific primer and one tag-specific primer per target), (6) pooling fixed volumes of one sample, (7) barcoding the samples, (8) mixing the samples and adjusting the concentration, and (9) sequencing the samples. The workflow may include several substeps containing one of the listed steps (for example, step (2) preparing the library may include three enzymatic steps (blunt-end termination, dA tailing, and adapter ligation) and three purification steps). The workflow steps can be combined, separated, or performed in different orders (e.g., barcoding and pooling of samples).

[0182] It is important to note that library amplification can be performed in a way that biases it to more efficiently amplify shorter fragments. Thus, shorter sequences, such as mononucleosome DNA fragments, can be preferentially amplified as cell-free fetal DNA (of placental origin) found in the circulation of pregnant women. Note that PCR assays may have tags, such as sequencing tags (usually in the form of 15-25 base pairs). After multiplexing, the PCR multiplexed products of the sample are pooled, and then tagging is completed by tag-specific PCR (which can also be done by ligation) (including barcoding). Alternatively, a complete sequencing tag can be added to the same reaction as multiplexing. In the first cycle, the target can be amplified using target-specific primers, after which tag-specific primers become dominant to complete the complete SQ adapter sequence. PCR primers do not need to carry the tag. Sequencing tags can be added to the amplified product by ligation.

[0183] In one embodiment, the material amplified using advanced multiplex PCR followed by clonal sequencing can be evaluated for various applications, such as detecting fetal aneuploidy. While conventional multiplex PCR evaluates up to 50 loci simultaneously, the method described herein can enable the simultaneous evaluation of more than 50 loci, more than 100 loci, more than 500 loci, more than 1,000 loci, more than 5,000 loci, more than 10,000 loci, more than 50,000 loci, and more than 100,000 loci. Experiments have shown that up to 10,000, including 10,000, and more than 10,000 distinct loci can be simultaneously evaluated in a single reaction with sufficiently good efficiency and specificity, enabling non-invasive prenatal aneuploidy diagnosis and / or copy number calling with high accuracy. The assay can combine the entire sample, such as a cfDNA sample isolated from maternal plasma, a fraction thereof, or a further processed derivative of the cfDNA sample in a single reaction. The sample (e.g., cfDNA or derivative) can also be split into multiple parallel reactions. The optimal sample splitting and multiplexing is determined by trade-offs of various performance specifications. Because the amount of material is limited, splitting the sample into multiple fractions can lead to increased sampling noise, handling time, and the potential for errors. Conversely, high multiplexing can result in increased false amplification and amplification inequality, both of which degrade the assay performance.

[0184] Two extremely important related considerations in the application of the methods described herein are the limited amount of the original sample (e.g., plasma) and the number of original molecules in the material from which allele frequencies or other measurements are obtained. If the number of original molecules falls below a certain level, random sampling noise becomes significant and can affect the accuracy of the test. Generally, when performing measurements on a sample containing the equivalent of 500 to 1000 original molecules per target locus, data of sufficient quality can be obtained for non-invasive prenatal aneuploidy diagnosis. There are several ways to increase the number of separate measurements, for example, increasing the volume of the sample. Each operation applied to the sample also potentially results in loss of material. To avoid losses that can degrade the performance of the test, it is essential to characterize the losses incurred by various operations and to avoid specific operations or improve their yields as necessary.

[0185] In certain embodiments, it is possible to reduce potential losses in subsequent steps by amplifying all or a portion of the original sample (e.g., cfDNA sample). Various methods are available to amplify all of the genetic material in the sample and thereby increase the amount available for downstream procedures. In certain embodiments, ligation-mediated PCR (LM-PCR) DNA fragments are amplified by PCR after ligation of either one separate adapter, two separate adapters, or many separate adapters. In certain embodiments, multiple displacement amplification (MDA) is used with phi-29 polymerase to isothermally amplify all of the DNA. In DOP-PCR and its variants, random priming is used to amplify the original material DNA. Each method has specific characteristics, such as the uniformity of amplification across all regions of a representative genome, the efficiency of capture and amplification of the original DNA, and amplification performance according to fragment length.

[0186] In certain embodiments, LM-PCR can be used with a single heteroduplex adapter having 3' tyrosine. The heteroduplex adapter allows for the use of a single adapter molecule that can convert, during the first round of PCR, into two distinct sequences on the 5' and 3' ends of the original DNA fragment. In certain embodiments, it is possible to fractionate the amplified library by size separation, or products such as AMPURE, TASS, or other similar methods. Prior to ligation, the sample DNA is made blunt-ended and then a single adenosine base is added to the 3' end. Prior to ligation, the DNA can be cleaved using a restriction enzyme or some other cleavage method. During ligation, the ligation efficiency can be enhanced by the 3' adenosine of the sample fragment and the complementary 3' tyrosine overhang of the adapter. The extension step of PCR amplification can be limited from a time perspective to reduce amplification from fragments longer than about 200 bp, about 300 bp, about 400 bp, about 500 bp or about 1,000 bp. Since the longer DNA found in maternal plasma is almost exclusively maternal, this can result in an enrichment of 10-50% of fetal DNA and an improvement in test performance. Several reactions were carried out using the conditions specified by a commercial kit, resulting in a successful ligation of less than 10% of the sample DNA molecules. Through a series of optimizations of the reaction conditions for this, the ligation was improved to approximately 70%.

[0187] Mini-PCR The Mini-PCR method described below is suitable for samples containing short nucleic acids such as cfDNA, digested nucleic acids, or fragmented nucleic acids. Conventional PCR assay designs result in significant loss of characteristic fetal molecules, but this loss can be significantly reduced by designing very short PCR assays, known as mini-PCR assays. Fetal cfDNA in maternal serum is highly fragmented, with fragment sizes distributed in a roughly Gaussian manner, with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of approximately 100 bp, and a maximum size of approximately 220 bp. The distribution of start and end positions of fragments with respect to target polymorphisms is not necessarily random, but varies widely among individual targets and collectively among all targets, and a polymorphic site at one particular target locus can occupy any position from start to end among the various fragments originating from that locus. Note that the term mini-PCR can equally and equally refer to conventional PCR without further restriction or limitation.

[0188] During PCR, amplification occurs only from template DNA fragments containing both forward and reverse primer sites. Since fetal cfDNA fragments are short, the likelihood of both primer sites being present, and the likelihood of a fetal fragment of length L containing both forward and reverse primer sites, is the ratio of the length of the amplified product to the length of the fragment. Under ideal conditions, assays with amplification products of 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, or 70 bp successfully amplify 72%, 69%, 66%, 63%, 59%, or 56% of the available template fragment molecules, respectively. The length of the amplification product is the distance between the 5' ends of the forward and reverse priming sites. Amplification products shorter than those commonly used by those skilled in the art may result in more efficient measurement of desired polymorphic loci using only the required short sequence reads. In one embodiment, the substantial fraction of the amplification product should be less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0189] It should be noted that short assays, such as those described herein, are not necessary in methods known from the prior art and are generally avoided because they impose considerable constraints on primer design due to limitations on primer length, annealing properties, and the distance between forward and reverse primers.

[0190] It should also be noted that if the 3' end of any primer is within approximately 1–6 bases of the polymorphic region, amplification bias is potentially present. This single-base difference at the site where polymerase first binds can lead to preferential amplification of one allele, which can alter the observed allele frequency and reduce performance. All of these constraints make it very difficult to identify primers that successfully amplify a particular locus, and in addition, to design a large set of primers that fit the same multiplexing reaction. In some embodiments, the 3' ends of the inner forward and reverse primers are designed to hybridize to a region of DNA upstream of the polymorphic region, separated from it by a small number of bases. Ideally, the number of bases may be between 6 and 10, but equally good results can be achieved with 4 to 15, 3 to 20, 2 to 30, or 1 to 60 bases.

[0191] Multiplex PCR may involve a single round of PCR in which all targets are amplified, or it may involve a single round of PCR followed by one or more rounds of nested PCR or some variation of nested PCR. Nested PCR consists of one or more rounds of PCR amplification using one or more new primers that bind at least one base pair more internally than the primers used in the previous round. Nested PCR reduces the number of false amplification targets by amplifying only the amplification product from the previous reaction, which has the correct internal sequence, in the subsequent reaction. The reduction in false amplification targets improves the number of useful measurements that can be obtained, particularly in sequencing. Nested PCR generally involves designing primers that are completely internal to the previous primer binding site, which inevitably increases the minimum DNA segment size required for amplification. For samples such as maternal plasma cfDNA, where the DNA is highly fragmented, the larger assay size reduces the number of distinct cfDNA molecules from which measurements can be obtained. In one embodiment, to counteract this effect, a partial nesting technique can be used in which one or both of the second-round primers overlap the first binding site, which extends several bases internally, thereby achieving an additional specificity while minimizing the expansion of the overall assay size.

[0192] In one embodiment, a multipool of PCR assays is designed to amplify one or more potentially heterozygous SNPs or other polymorphic or non-polymorphic loci on a chromosome, and these assays are used in a single reaction to amplify the DNA. The number of PCR assays may be between 50 and 200, between 200 and 1,000, between 1,000 and 5,000, or between 5,000 and 20,000 (50-200 plexes, 200-1,000 plexes, 1,000-5,000 plexes, 5,000-20,000 plexes, and over 20,000 plexes, respectively). In one embodiment, a multi-pool of approximately 10,000 PCR assays (10,000 plexes) is designed to amplify potentially heterozygous SNP loci on the X, Y, 13, 18, and 21 chromosomes, and on 1 or 2 chromosomes. These assays are used in a single reaction to amplify cfDNA obtained from material plasma samples, chorionic villi samples, amniocentesis samples, single or a small number of cells, other bodily fluids or tissues, cancer or other genetic material. The SNP frequency of each locus can be determined by clonal sequencing of the amplified product or by several other methods. Statistical analysis of the allele frequency distribution or ratios of all assays can be used to determine whether a sample contains one or more trisomies of the chromosomes included in the test. In another embodiment, the original cfDNA sample is split into two samples, and parallel 5,000 plex assays are performed. In another embodiment, the original cfDNA sample is divided into n samples, and parallel (approximately 10,000 / n) plex assays are performed, where n is between 2 and 12, or between 12 and 24, or between 24 and 48, or between 48 and 96. The data are collected and analyzed as already described. It should be noted that this method is equally good applicable to detecting translocations, deletions, duplications, and other chromosomal abnormalities.

[0193] In some embodiments, tails that do not have homology to the target genome may be added to either the 3' or 5' end of the primer. These tails facilitate subsequent manipulation, procedures, or measurements. In some embodiments, the tail sequence may be the same for the target-specific forward primer and target-specific reverse primer. In some embodiments, different tails may be used for the target-specific forward primer and target-specific reverse primer. In some embodiments, multiple different tails may be used for different loci or sets of loci. Certain tails may be shared among all loci or among subsets of loci. For example, direct sequencing after amplification may be possible using forward and reverse tails corresponding to the forward and reverse sequences required for any of the current sequencing platforms. In some embodiments, the tails may be used as general priming sites that can be used to add other useful sequences among all amplified targets. In some embodiments, the inner primer may contain a region designed to hybridize either upstream or downstream of the target locus (e.g., a polymorphic locus). In some embodiments, the primer may contain a molecular barcode. In some embodiments, the primers may contain a universal priming sequence designed to enable PCR amplification.

[0194] In one embodiment, a 10,000-plex PCR assay pool is prepared such that the forward and reverse primers have tails corresponding to the required forward and reverse sequences needed for a high-throughput sequencing instrument, such as HISEQ, GAIIX, or MYSEQ, available from ILLUMINA. Furthermore, the 5' end of the sequencing tail contains yet another sequence that can be used as a priming site for adding nucleotide barcode sequences to the amplified product in subsequent PCR, thereby enabling multiple samples to be multiplexed and sequenced on a single lane of the high-throughput sequencing instrument.

[0195] In one embodiment, a 10,000-plex PCR assay pool is prepared such that the reverse primers have tails corresponding to the required reverse sequences needed for a high-throughput sequencing instrument. After amplification in the first 10,000-plex assay, subsequent PCR amplification can be performed using another 10,000-plex pool having partially nested forward primers (e.g., 6-base nested) for all targets and reverse primers corresponding to the reverse sequencing tails included in the first round. This subsequent round of partially nested amplification using only one target-specific primer and a universal primer limits the size required for the assay, reduces sampling noise, and significantly reduces the number of false amplification products. The sequencing tag can be attached to the attached ligation adapter and / or as part of the PCR probe, so that the tag becomes part of the final amplification product.

[0196] The fetal fraction affects the performance of the test. Several methods exist to enrich the fetal fraction of DNA found in maternal plasma. The fetal fraction can be increased by the LM-PCR method already discussed above, as well as by targeted removal of long maternal fragments. In one embodiment, an additional multiplex PCR reaction can be performed before the multiplex PCR amplification of the target locus to selectively remove long, large maternal fragments corresponding to the locus targeted in the subsequent multiplex PCR. Additional primers are designed to anneal to sites further from the polymorphism than would be expected to be present among cell-free fetal DNA fragments. These primers can be used in a single cycle of multiplex PCR before the multiplex PCR of the target polymorphism locus. These distal primers are tagged with molecules or portions that can enable selective recognition of tagged DNA fragments. In one embodiment, these DNA molecules can be covalently modified with biotin molecules that allow for the removal of newly formed double-stranded DNA containing these primers after one cycle of PCR. The double-stranded DNA formed during that first round appears to be of maternal origin. Removal of hybrid materials can be achieved by using magnetic streptavidin beads. Other tagging methods exist that can function equally well. In one embodiment, a size selection method can be used to enrich the sample for shorter DNA, such as DNA less than approximately 800 bp, less than approximately 500 bp, or less than approximately 300 bp. Amplification of the short fragments is then carried out as usual.

[0197] The mini-PCR method described herein enables highly multiplexed amplification and analysis of hundreds, thousands, or even millions of loci from a single sample in a single reaction. Simultaneously, the detection of the amplified DNA can be multiplexed, and by using barcoding PCR, tens to hundreds of samples can be multiplexed in a single sequencing lane. This multiplexed detection has been successfully tested up to 49 plexes, and much higher levels of multiplexing are possible. In effect, this makes it possible to determine genotypes at thousands of SNPs in hundreds of samples in a single sequencing run. For these samples, the method allows for the determination of genotype and heterozygosity, and simultaneously, copy number, both of which can be used to detect aneuploidy. This method is particularly useful in detecting aneuploidy in a pregnant fetus from floating DNA found in maternal plasma. This method can be used as part of a method for fetal sex determination and / or prediction of fetal paternity. This method can be used as part of a method for mutational load determination. This method can be used on any amount of DNA or RNA, and the target region may be an SNP, another polymorphic region, a non-polymorphic region, or a combination thereof.

[0198] In some embodiments, ligation-mediated universal PCR amplification of fragmented DNA can be used. Using ligation-mediated universal PCR amplification, plasma DNA can be amplified and then split into multiple parallel reactions. Ligation-mediated universal PCR amplification can also be used to preferentially amplify shorter fragments, thereby enriching the fetal fraction. In some embodiments, tagging fragments by ligation may allow for the detection of shorter fragments, the use of shorter target sequence-specific portions of primers, and / or annealing at higher temperatures to reduce nonspecific reactions.

[0199] The methods described herein can be used for several purposes when a set of target DNA is present mixed with a certain amount of contaminating DNA. In some embodiments, the target DNA and contaminating DNA may originate from genetically related individuals. For example, a genetic abnormality in a fetus (target) can be detected from maternal plasma containing fetal (target) DNA and similarly maternal (contaminating) DNA, and abnormalities include whole-chromosome abnormalities (e.g., aneuploidy), partial chromosomal abnormalities (e.g., deletions, duplications, inversions, translocations), polynucleotide polymorphisms (e.g., STR), single nucleotide polymorphisms, and / or other genetic abnormalities or differences. In some embodiments, the target DNA and contaminating DNA may originate from the same individual, but for example, in the case of cancer, the target DNA and contaminating DNA may differ by one or more mutations (see, for example, H. Mamon et al. Preferred Amplification of Apoptotic DNA from Plasma: Potential for Enhancing Detection of Minor DNA Alterations in Circulating DNA. Clinical Chemistry 54:9 (2008)). In some embodiments, DNA can be found in the (apoptotic) supernatant of a cell culture. In some embodiments, apoptosis can be induced in a biological sample (e.g., blood) for subsequent library preparation, amplification, and / or sequencing. Several possible workflows and protocols for achieving this objective are shown elsewhere in this disclosure.

[0200] In some embodiments, the target DNA may be derived from a single cell, a DNA sample consisting of less than one copy of the target genome, a small amount of DNA, DNA from mixed origins (e.g., pregnancy plasma: placenta and maternal DNA; cancer patient plasma and tumor: mixture of healthy and cancer DNA, grafts, etc.), other bodily fluids, cell cultures, culture supernatants, forensic DNA samples, ancient DNA samples (e.g., insects captured in amber), other DNA samples, and combinations thereof.

[0201] In some embodiments, short amplification product sizes can be used. Short amplification product sizes are particularly suitable for fragmented DNA (see, for example, A. Sikora et al., Detection of increased amounts of cell-free fetal DNA with short PCR amplicons. Clin Chem. January 2010; Vol. 56 (No. 1): pp. 136-138).

[0202] Using a short amplification product size can yield several important benefits. A shorter amplification product size can lead to optimized amplification efficiency. A shorter amplification product size generally results in shorter products, and therefore, a lower likelihood of nonspecific priming. Shorter products can cluster more densely on the sequencing flow cell, as the cluster size decreases. It should be noted that the method described herein may work equally well with longer PCR amplification products. The length of the amplification product can be increased if necessary, for example, when sequencing a larger range of sequences. Experiments using 146-plex targeted amplification with an assay of 100-200 bp length as the first step of a nested PCR protocol were performed on single cells and genomic DNA, yielding positive results.

[0203] In some embodiments, the methods described herein can be used to amplify and / or detect SNPs, copy numbers, nucleotide methylation, mRNA levels, expression levels of other types of RNA, and the morphology and / or epigenetic forms of other genes. The mini-PCR methods described herein can be used in conjunction with next-generation sequencing, and the methods can be used in conjunction with other downstream methods, such as microarrays, counting by digital PCR, real-time PCR, and mass spectrometry.

[0204] In some embodiments, the mini-PCR amplification methods described herein can be used as part of a method for accurately quantifying small populations. The method can be used for absolute quantification using a spike calibrator. The method can be used for quantification of variant / trace alleles by ultradeep sequencing and can be performed in a highly multiplexed manner. The method can be used for standard paternity and identity testing of relatives or ancestors in humans, animals, plants, or other living organisms. The method can be used for forensic testing. The method can be used for rapid genotyping and copy number analysis (CN) of any type of material, e.g., amniotic fluid and CVS, sperm, and product of conception (POC). The method can be used for single-cell analysis, such as genotyping of biopsy samples from embryos. The method can be used for rapid embryo analysis (less than 1 day, 1 day, or within 2 days of biopsy) by targeted sequencing using min-PCR.

[0205] In some embodiments, the mini-PCR amplification method can be used for tumor analysis: tumor biopsies are often a mixture of healthy and tumor cells. Targeted PCR allows for deep sequencing of SNPs and loci that are not nearby background sequences. The method can be used for the analysis of copy number and heterozygosity loss in tumor DNA. The tumor DNA may be present in many different bodily fluids or tissues of tumor patients. The method can be used for the detection of tumor recurrence and / or tumor screening. The method can be used for seed quality control testing. The method can be used for breeding or fisheries. It should be noted that any of these methods can be used equally well for targeting non-polymorphic loci for polyploidy calls.

[0206] Some of the documents that describe some of the basic methods underlying the present disclosure include: (1) Wang HY, Luo M, Tereshchenko IV, Frikker DM, Cui X, Li JY, Hu G, Chu Y, Azaro MA, Lin Y, Shen L, Yang Q, Kambouris ME, Gao R, Shih W, Li H. Genome Res. 2005 Feb;15(2):276-83. Department of Molecular Genetics, Microbiology and Immunology / The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903, USA. (2) High-throughput genotyping of single nucleotide polymorphisms with high sensitivity. Li H, Wang HY, Cui X, Luo M, Hu G, Greenawalt DM, Tereshchenko IV, Li JY, Chu Y, Gao R. Methods Mol Biol. 2007;396-PubMed PMID:18025699. (3) Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Varley KE, Mitra RD. Genome Res. 2008 Nov;18(11):1844-50. Epub 2008 Oct 10 (this document describes a method that includes multiplexing of an average of 9 assays for sequencing). It should be noted that the methods disclosed herein enable multiplexing on a much larger scale than those in the above references.

[0207] Variant of target PCR - nesting Many workflows are possible when performing PCR, and several workflows typical of the methods disclosed herein are described. The steps outlined herein are not intended to exclude other possible steps, nor do they imply that any of the steps described herein are necessary for the method to function properly. Numerous parameter variations or other modifications are known in the literature and can be made without affecting the core of the invention. One particular general workflow is shown below, followed by several possible variants. Variants generally refer to possible secondary PCR reactions, e.g., different types of nesting that can be performed (step 3). It is important to note that variants can be performed at different times or in different orders than those explicitly described herein. Examples using polymorphic loci for illustrative purposes can be readily adapted to amplification of non-polymorphic loci as needed. 1. DNA in a sample can often be tagged with a ligation adapter, often referred to as a library tag or ligation adapter tag (LT), which contains a universal priming sequence followed by universal amplification. In some embodiments, this can be done using a standard protocol designed to prepare a sequencing library after fragmentation. In some embodiments, the DNA sample can be blunt-ended and then an A can be added to the 3' end. A Y-adapter with a T-overhang can be added and ligated. In some embodiments, other sticky ends besides A or T overhangs can be used. In some embodiments, other adapters, such as loop ligation adapters, can be added. In some embodiments, the adapter may have a tag designed for PCR amplification. 2. Specific Target Amplification (STA): Hundreds, thousands, tens of thousands, or even hundreds of thousands of targets can be multiplexed in pre-amplification within a single reaction volume. STA is generally performed for 10–30 cycles, but can be performed for 5–40 cycles, 2–50 cycles, or even 1–100 cycles. For example, tails can be attached to primers for a simpler workflow or to avoid sequencing of most dimers. Note that, generally, dimers of both primers possessing the same tag will not be efficiently amplified or sequenced. In some embodiments, PCR can be performed for 1 to 10 cycles, in some embodiments, for 10 to 20 cycles, in some embodiments, for 20 to 30 cycles, in some embodiments, for 30 to 40 cycles, and in some embodiments, for more than 40 cycles. Amplification may be linear amplification. The number of PCR cycles can be optimized to yield an optimal read depth (DOR) profile. Different DOR profiles may be desirable for different purposes. In some embodiments, a more uniform distribution of reads across all assays is desirable; however, if the DOR is very small for some assays, the probabilistic noise may be too high for the data to be very useful, while if the read depth is very deep, the marginal usefulness of each additional read is relatively small. Primer tails can improve the detection of fragmented DNA from universally tagged libraries. Hybridization can be improved if the library tag and primer tail contain homologous sequences (e.g., melting temperature (T)). M(To lower the base pair), the primer can only be extended if a portion of the primer target sequence is present in the DNA fragment of the sample. In some embodiments, 13 or more target-specific base pairs can be used. In some embodiments, 10 to 12 target-specific base pairs can be used. In some embodiments, 8 to 9 target-specific base pairs can be used. In some embodiments, 6 to 7 target-specific base pairs can be used. In some embodiments, STA can be performed on pre-amplified DNA, e.g., MDA, RCA, other whole-genome amplification, or adapter-mediated universal PCR. In some embodiments, STA can be performed on samples where specific sequences and populations have been enriched or depleted, for example, by size selection, target capture, or directed degradation. 3. In some embodiments, secondary multiplex PCR or primer extension reactions can be performed to increase specificity and reduce undesirable products. For example, full nesting, semi-nesting, heminesting, and / or subdivision into parallel reactions of smaller assay pools are all techniques that can be used to increase specificity. Experiments have shown that dividing a sample into three 400-plex reactions yielded product DNA with higher specificity than a single 1,200-plex reaction using exactly the same primers. Similarly, experiments have shown that dividing a sample into four 2,400-plex reactions yielded product DNA with higher specificity than a single 9,600-plex reaction using exactly the same primers. In some embodiments, it is possible to use target-specific and tag-specific primers with the same and opposite orientations. 4. In some embodiments, the DNA sample produced by the STA reaction (diluted, purified, or otherwise) can be amplified using tag-specific primers and "universal amplification," i.e., many or all of the pre-amplified and tagged targets can be amplified. The primers may contain additional functional sequences required for sequencing on a high-throughput sequencing platform, such as barcodes or complete adapter sequences.

[0208] These methods can be used to analyze any DNA sample and are particularly useful when the DNA sample is very small, or when the DNA sample originates from two or more individuals, for example, in the case of maternal plasma. These methods can be used for DNA samples such as single or small numbers of cells, genomic DNA, plasma DNA, amplified plasma libraries, amplified apoptotic supernatant libraries, or other mixed DNA samples. In some embodiments, these methods can be used when cells with different genetic makeup may be present in a single individual, for example, in cancer or grafts.

[0209] Modifications of the protocol (modifications and / or additions to the above workflow) Direct multiplex mini-PCR: Specific target amplification (STA) of multiple target sequences using tagged primers is shown in Figure 1. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with PCR primers. 104 shows the final PCR product. In some embodiments, STA can be performed on targets greater than 100, greater than 200, greater than 500, greater than 1,000, greater than 2,000, greater than 5,000, greater than 10,000, greater than 20,000, greater than 50,000, greater than 100,000, or greater than 200,000. In subsequent reactions, all target sequences are amplified by tag-specific primers, and a tag containing all sequences necessary for sequencing, including the sampling index, is extended. In some embodiments, the primers do not need to be tagged, or only specific primers may be tagged. The sequencing adapter can be added by conventional adapter ligation. In some embodiments, the first primer may carry the tag.

[0210] In one embodiment, the primer is designed so that the length of the DNA being amplified is unexpectedly short. Prior art has demonstrated that those skilled in the art can generally design amplification products of 100+ bp. In one embodiment, the amplification product can be designed to be less than 80 bp. In one embodiment, the amplification product can be designed to be less than 70 bp. In one embodiment, the amplification product can be designed to be less than 60 bp. In one embodiment, the amplification product can be designed to be less than 50 bp. In one embodiment, the amplification product can be designed to be less than 45 bp. In one embodiment, the amplification product can be designed to be less than 40 bp. In one embodiment, the amplification product can be designed to be less than 35 bp. In one embodiment, the amplification product can be designed to be between 40 bp and 65 bp.

[0211] The experiment was performed using this protocol with 1200 plex amplification. Both genomic DNA and pregnancy plasma were used; approximately 70% of the sequenced reads mapped to the target sequence. Further details are provided elsewhere in this document. Sequence determination of 1042 plexes without assay design and selection resulted in >99% of the sequences becoming primer dimer products.

[0212] Following sequential PCR:STA1, multiple fixed amounts of the product can be amplified in parallel using a reduced-complexity pool with the same primers. The first amplification may yield sufficient material for splitting. This method is particularly excellent for small samples, e.g., approximately 6–100 pg, 100 pg–1 ng, 1 ng–10 ng, or 10 ng–100 ng. A protocol was performed in which 1200plex was used for three 400plex steps. Sequencing read mapping increased from approximately 60–70% with 1200plex alone to over 95%.

[0213] Semi-nested mini-PCR: (See Figure 2) After STA1, a second STA is performed consisting of multiple sets of inner nested forward primers (103B, 105b) and one (or a few) tag-specific reverse primers (103A). 101 shows double-stranded DNA with the target polymorphic locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with forward primer B and reverse primer A. 104 shows the PCR product from 103. 105 shows the product from 104 with the hybridized nested forward primer b and reverse tag A, which is part of the molecule from the PCR already generated between 103 and 104. 106 shows the final PCR product. Using this workflow, typically more than 95% of the sequence is mapped to the intended target. Nested primers may overlap with the outer forward primer sequence, but introduce additional 3' terminal bases. In some embodiments, between 1 and 20 extra 3' bases can be used. Experiments have shown that using 9 or more extra 3' bases works well in 1200plex designs.

[0214] Fully nested mini-PCR: (See Figure 3) After STA step 1, a second multiplex PCR (or a less complex parallel mpPCR) can be performed using two nested primers carrying tags (A, a, B, b). 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with forward primer B and reverse primer A. 104 shows the PCR product from 103. 105 shows the product from 104 hybridized with nested forward primer b and nested reverse primer a. 106 shows the final PCR product. In some embodiments, a complete set of two primers can be used. Using a fully nested mini-PCR protocol, 146-plex amplification was performed on single cells and groups of three cells without step 102, after adding a universal ligation adapter.

[0215] Heminested mini-PCR: (See Figure 4) It is possible to use target DNA with adapters at the ends of the fragment. Perform an STA consisting of multiple sets of forward primers (B) and one (or a few) tag-specific reverse primers (A). A second STA can be performed using a universal tag-specific forward primer and a target-specific reverse primer. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with reverse primer A. 104 shows the PCR product from 103 amplified using reverse primer A and ligation adapter tag primer LT. 105 shows the product from 104 hybridized with forward primer B. 106 shows the final PCR product. This workflow uses target-specific forward and reverse primers in separate reactions, thereby reducing reaction complexity and preventing dimerization of the forward and reverse primers. Note that in this example, primers A and B can be considered the first primers, and primers "a" and "b" can be considered the inner primers. This method is as good as direct PCR but offers a significant improvement over direct PCR by avoiding primer dimerization. After the first round of the heminested protocol, approximately 99% of non-target DNA is generally observed, but this generally improves significantly after the second round.

[0216] Triple-heminested mini-PCR: (See Figure 5) It is possible to use target DNA with adapters at the ends of the fragment. Perform an STA consisting of multiple sets of forward primers (B) and one (or a few) tag-specific reverse primers (A) and (a). A second STA can be performed using a universal tag-specific forward primer and a target-specific reverse primer. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with reverse primer A. 104 shows the PCR product from 103 amplified using reverse primer A and ligation adapter tag primer LT. 105 shows the product from 104 hybridized with forward primer B. 106 shows the PCR product from 105 amplified using reverse primer A and forward primer B. 107 shows the product from 106 hybridized with reverse primer "a". 108 shows the final PCR product. Note that in this example, primers "a" and B can be considered as inner primers, and A can be considered as the first primer. If necessary, both A and B can be considered as the first primer, and "a" can be considered as the inner primer. The names of the reverse and forward primers can be switched. In this workflow, target-specific forward and reverse primers are used in separate reactions, thereby reducing the complexity of the reaction and preventing dimerization of the forward and reverse primers. This method is as good as direct PCR but is a significant improvement over direct PCR because it avoids primer dimerization. After the first round of the heminested protocol, approximately 99% of non-target DNA is generally observed, but this generally improves significantly after the second round.

[0217] One-sided nested mini-PCR: (See Figure 6) It is possible to use target DNA with adapters at the ends of the fragment. STA can also be performed using multiple sets of nested forward primers and a ligation adapter tag as the reverse primer. A second STA can then be performed using a set of nested forward primers and a universal reverse primer. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows universally amplified single-stranded DNA hybridized with forward primer A. 104 shows the PCR product from 103 amplified using forward primer A and ligation adapter tag reverse primer LT. 105 shows the product from 104 hybridized with a nested forward primer. 106 shows the final PCR product. This method allows for the detection of shorter target sequences than standard PCR by using primers that overlap in the first and second STAs. The method described above is generally performed by subtracting the DNA sample that has already undergone the STA Step 1 - universal tag addition and amplification described above, with two nested primers on only one side and a library tag on the other side. The method was performed on libraries of apoptotic supernatant and pregnancy plasma. Using this workflow, approximately 60% of the sequences were mapped to the intended target. It should be noted that reads containing reverse adapter sequences were not mapped, and therefore this number is expected to be higher when reads containing reverse adapter sequences are mapped.

[0218] Unilateral mini-PCR: It is possible to use target DNA with adapters at the ends of the fragment (see Figure 7). STA can be performed using multiple sets of forward primers and one (or a few) tag-specific reverse primers. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows single-stranded DNA hybridized with forward primer A. 104 shows the PCR product from 103 amplified using forward primer A and ligation adapter tag reverse primer LT, which is the final PCR product. This method can detect shorter target sequences than standard PCR. However, because only one target-specific primer is used, it may be relatively nonspecific. The effectiveness of this protocol is half that of unilateral nested mini-PCR.

[0219] Reverse seminested mini-PCR: This method allows the use of target DNA with adapters at the ends of the fragment (see Figure 8). STA can be performed using multiple sets of forward primers and one (or a few) tag-specific reverse primers. 101 shows double-stranded DNA with the target polymorphism locus at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. 103 shows single-stranded DNA hybridized with reverse primer B. 104 shows the PCR product from 103 amplified using reverse primer B and ligation adapter tag forward primer LT. 105 shows PCR product 104 hybridized with forward primer A and the inner reverse primer "b". 106 shows the PCR product amplified from 105 using forward primer A and reverse primer "b", which is the final PCR product. This method allows for the detection of shorter target sequences than standard PCR.

[0220] Further variations of the above methods, which are simply repetitions or combinations, may also exist, such as double-nested PCR using three sets of primers. Another variation is one-sided semi-nested mini-PCR, in which STA can also be performed using multiple sets of nested forward primers and one (or a few) tag-specific reverse primers.

[0221] It should be noted that in all of these variants, the identity of the forward and reverse primers can be interchanged. It should also be noted that in some embodiments, the nested variant can be performed equally well without the addition of adapter tags and the preparation of the initial library, which includes a universal amplification step. In some embodiments, an additional round of PCR may be included with additional forward and / or reverse primers and amplification steps, and it should be noted that these additional steps may be particularly useful when it is desirable to further increase the percentage of DNA molecules corresponding to the target locus.

[0222] Nesting Workflow Many methods exist for performing amplification with varying degrees of nesting and multiplexing. Figure 9 shows flowcharts along with some possible workflows. Note that the use of 10,000 plex PCR is just an example, and these flowcharts work equally well for other degrees of multiplexing.

[0223] Loop ligation adapter For example, when attaching a universally tagged adapter to create a library for sequencing, there are several methods for ligating the adapter. One method is to blunt-end the sample DNA, perform A-tailing, and ligate it with an adapter having a T-overhang. There are several other methods for ligating the adapter. There are also several adapters that can be ligated. For example, a Y-adapter can be used, consisting of two strands of DNA, one strand having a double-stranded region and a region designated by a forward primer region, and the other strand being designated by a double-stranded region complementary to the double-stranded region on the first strand and a region with a reverse primer. When annealing, the double-stranded region may contain a T-overhang in order to ligate with double-stranded DNA having an A-overhang.

[0224] In one embodiment, the adapter may be a loop of DNA having complementary terminal regions and containing a forward-tagged region (LFT), a reverse-tagged region (LRT), and a cleavage site between the two (see Figure 10). 101 refers to double-stranded blunt-ended target DNA. 102 refers to target DNA with an A tail. 103 refers to a loop ligation adapter having a T overhang "T" and a cleavage site "Z". 104 refers to target DNA to which the loop ligation adapter has been attached. 105 refers to target DNA to which the ligation adapter has been attached, cleaved at the cleavage site. LFT refers to the ligation adapter forward tag, and LRT refers to the ligation adapter reverse tag. The complementary region may end in a T overhang or other form that can be used to ligate with the target DNA. The cleavage site may be a series of uracils for cleavage along the UNG, or a sequence that can be recognized and cleaved by restriction enzymes or other cleavage methods or simply basic amplification. These adapters can be used, for example, to prepare any library for sequencing. These adapters can be used in combination with any of the other methods described herein, for example, the mini-PCR amplification method.

[0225] Primer with internal tags When sequencing is used to determine the alleles present at a given polymorphism locus, sequence reads are generally started upstream of the primer binding site (a), followed by the polymorphism site (X). The tags are generally positioned as shown on the left side of Figure 11. 101 refers to a single-stranded target DNA having the target polymorphism locus "X" and primer "a" with tag "b" attached. To avoid nonspecific hybridization, the primer binding site (the region of target DNA complementary to "a") is generally 18–30 bp long. The sequence tag "b" is generally about 20 bp, and theoretically, these can be any length longer than about 15 bp, although many people use primer sequences sold by sequencing platform companies. The distance "d" between "a" and "X" may be at least 2 bp to avoid allelic bias. When performing W multiplex PCR amplification using the methods disclosed herein or other methods, careful primer design is necessary to avoid excessive primer-primer interactions. The acceptable window for the distance "d" between "a" and "X" can vary considerably: 2 bp to 10 bp, 2 bp to 20 bp, 2 bp to 30 bp, or even greater than 2 bp to 30 bp. Therefore, when using the primer configuration shown on the left side of Figure 11, the sequenced reads must be at least 40 bp to obtain reads long enough to measure polymorphic loci, and depending on the lengths of "a" and "d", sequenced reads may need to be up to 60 bp or 75 bp. Generally, the longer the sequenced read, the greater the cost and time required to sequence a given number of reads; therefore, minimizing the required read length can save both time and money. Furthermore, on average, reads with earlier bases are more accurate than reads with later bases, so reducing the required sequenced read length can also improve the accuracy of measuring polymorphic regions.

[0226] In one embodiment, as shown in Figure 11, 103, a primer-binding site (a), referred to as an internally tagged primer, is divided into multiple segments (a', a'', a''''....), and a sequence tag (b) is placed on the DNA segment in the middle of the two primer-binding sites. This arrangement allows the sequencer to produce shorter sequence reads. In one embodiment, a'+a'' should be at least about 18 bp and may be 30 bp, 40 bp, 50 bp, 60 bp, 80 bp, 100 bp, or more than 100 bp. In one embodiment, a'' should be at least about 6 bp and in one embodiment, between about 8 bp and 16 bp. All other factors are equivalent, and by using internally tagged primers, the required sequence read length can be shortened to at least 6 bp, 8 bp, 10 bp, 12 bp, 15 bp, or even 20 bp or 30 bp. This can result in significant savings in money, time, and accuracy. An example of a primer with an internal tag is shown in Figure 12.

[0227] Primer having a ligation adapter binding region One problem with fragmented DNA is that, due to its shorter length, polymorphisms are more likely to be located near the ends of the DNA strand than in longer strands (e.g., 101, Figure 10). Since PCR capture of polymorphisms requires primer binding sites of appropriate length on both sides of the polymorphism, a significant number of DNA strands containing the target polymorphism are not captured due to insufficient overlap between the primer and the target binding site. In some embodiments, a ligation adapter 102 can be added to the target DNA 101, and the target primer 103 may have a region (cr) complementary to a ligation adapter tag (lt) added upstream of the designed binding region (a) (see Figure 13); therefore, if the binding region (the region of 101 complementary to a) is shorter than the 18 bp generally required for hybridization, the region (cr) of the primer complementary to the library tag can increase the binding energy to a point where PCR can proceed. It should be noted that any specificity lost due to a shorter binding region can be compensated for by other PCR primers having a sufficiently long target binding region. It should be noted that this embodiment can be used in combination with direct PCR or any of the other methods described herein, such as nested PCR, semi-nested PCR, heminested PCR, one-sided nested or semi-nested or heminested PCR, or other PCR protocols.

[0228] When determining ploidy using sequencing data in combination with analytical methods that involve comparing observed allele data with predicted allele distributions for various hypotheses, each additional read from an allele with a low read depth provides more information than reads from an allele with a high read depth. Therefore, ideally, it is desirable to observe a uniform read depth (DOR) where each locus has a similar number of representative sequence reads. Thus, it is desirable to minimize the variance of the DOR. In some embodiments, it is possible to reduce the coefficient of variation of the DOR (which can be defined as the standard deviation of the DOR / mean DOR) by increasing the annealing time. In some embodiments, the annealing temperature may be greater than 2 minutes, greater than 4 minutes, greater than 10 minutes, greater than 30 minutes, greater than 1 hour or even longer. Since annealing is an equilibrium process, there is no limit to the improvement in the variance of the DOR with increasing annealing time. In some embodiments, the variance of the DOR is reduced by increasing the primer concentration.

[0229] Representative whole-genome amplification methods In some embodiments, DNA amplification, such as whole-genome applications, may be included to amplify the nucleic acid sample before amplifying only the target gene locus. DNA amplification is the process of converting a small amount of genetic material into a larger amount of genetic material containing a collection of similar genetic data, and can be carried out by a wide variety of methods, including, but not limited to, polymerase chain reaction (PCR). One method of amplifying DNA is whole-genome amplification (WGA). Several methods are available for WGA: ligation-mediated PCR (LM-PCR), degenerate oligonucleotide-primer PCR (DOP-PCR), and multiple substitution amplification (MDA). In LM-PCR, short DNA sequences called adapters are ligated to the blunt ends of the DNA. These adapters contain universal amplification sequences, which are used to amplify the DNA by PCR. In DOP-PCR, random primers containing similar universal amplification sequences are used in the first round of annealing and PCR. Then, a second round of PCR is used to further amplify the sequence using the universal primer sequences. MDA utilizes phi-29 polymerase, a highly processable and nonspecific enzyme for DNA replication, which is used for single-cell analysis. Major limitations to amplification of single-cell derived material are (1) the need to use extremely diluted DNA concentrations or very small volumes of reaction mixtures, and (2) the difficulty in reliably dissociating DNA from proteins across the entire genome. Nevertheless, single-cell whole-genome amplification has been successfully employed for various applications over the years. Other methods exist for amplifying DNA from DNA samples. DNA amplification converts the initial DNA sample into a much larger quantity of DNA with a similar sequence set. In some cases, amplification may not be necessary.

[0230] In some embodiments, DNA can be amplified using universal amplification, such as WGA or MDA. In some embodiments, DNA can be amplified by targeted amplification, such as targeted PCR or using a circularized probe. In some embodiments, DNA can be preferentially enriched using a targeted amplification method or a method that results in complete or partial separation of desired and undesirable DNA, such as capture by a hybridization technique. In some embodiments, DNA can be amplified by using a combination of a universal amplification method and a preferential enrichment method. A more detailed description of some of these methods can be found elsewhere in this document.

[0231] Representative enrichment and sequencing methods In some embodiments, the methods disclosed herein employ selective enrichment techniques that preserve the relative allele frequencies present at each target locus (e.g., each polymorphic locus) from a set of target loci (e.g., each polymorphic locus) within a sample of the original DNA. While enrichment is particularly useful as an analytical method for polymorphic loci, these enrichment methods can be readily adapted to non-polymorphic loci as needed. In some embodiments, amplification and / or selective enrichment techniques may involve PCR, such as ligation-mediated PCR, fragment capture by hybridization, molecular inversion probes, or other cyclized probes. In some embodiments, the method for amplification or selective enrichment may involve the use of probes such that, when precisely hybridized with the target sequence, the 3' or 5' end of the nucleotide probe is separated from the polymorphic site of the allele by a small number of nucleotides. This separation reduces the preferential amplification of one allele, known as allele bias. This is an improvement over methods involving the use of probes in which the precisely hybridized probe's 3' or 5' end is directly adjacent to, or very close to, the polymorphic site of the allele. In some embodiments, probes whose hybridized region may contain, or certainly contains, a polymorphic site are excluded. The presence of a polymorphic site at the hybridization site may lead to uneven hybridization in some alleles, or the hybridization may be inhibited overall, resulting in preferential amplification of a particular allele. These embodiments are an improvement over other methods involving targeted amplification and / or selective enrichment in that they better preserve the original allele frequencies at each polymorphic locus in the sample, whether the sample is a pure genomic sample from a single individual or a mixture of individuals.

[0232] Several unexpected advantages can be provided by using techniques to enrich a sample of DNA in a set of target loci and then sequence it as part of a method for non-invasive prenatal allele calling or ploidy calling. In some embodiments of this disclosure, the method includes a step of measuring genetic data for use in an informatics-based method, e.g., PARENTAL SUPPORT® (PS). In some embodiments, the final outcome is readily usable genetic data of an embryo or fetus. As part of the embodied methods, there are many methods that can be used to measure genetic data of an individual and / or related individual. In some embodiments, a method for enriching the concentration of a target allele set is disclosed herein, the method comprising one or more of the following steps: targeting and amplifying genetic material; adding locus-specific oligonucleotide probes; ligating a specific DNA strand; isolating a desired DNA set; removing undesirable components of the reaction; detecting the sequence of a specific DNA by hybridization; and detecting the sequence of one or more DNA strands by a DNA sequencing method. In some cases, the DNA strand may refer to the target genetic material; in some cases, the DNA strand may refer to the primer; and in some cases, the DNA strand may refer to the synthesized sequence or a combination thereof. These steps can be carried out in several different orders.

[0233] For example, a universal amplification step of DNA prior to targeted amplification may offer several advantages, such as eliminating the risk of bottlenecks and reducing allelic bias. DNA can be mixed with oligonucleotide probes that can hybridize to two adjacent regions on either side of a target sequence. After hybridization, the ends of the probes can be joined by adding polymerase, a means of ligation, and any necessary reagents to enable the circularization of the probes. After circularization, the uncircularized genetic material can be digested by adding exonuclease, and then the circularized probes can be detected. DNA can also be mixed with PCR primers that can hybridize to two adjacent regions on either side of a target sequence. After hybridization, the ends of the probes can be joined by adding polymerase, a means of ligation, and any necessary reagents to complete the PCR amplification. The amplified or unamplified DNA may also be targets for hybrid capture probes targeting a set of loci, and after hybridization, the probes can be localized and separated from the mixture to yield a mixture of DNA enriched with the target sequence.

[0234] Several unexpected advantages can be handed down by using methods that target specific gene loci as part of allele calling or ploidy calling, followed by sequencing. Some methods that can target or preferentially enrich DNA include capture methods by hybridization such as circularized probes, linked inverted probes (LIP, MIP), and SURESELECT, and the use of targeted PCR or ligation-mediated PCR amplification strategies.

[0235] In some embodiments, the methods of this disclosure include a step of measuring genetic data for use in an informatics-based method, for example, PARENTAL SUPPORT® (PS), which is further described herein. PARENTAL SUPPORT® is an informatics-based method for manipulating genetic data, and its embodiments are described herein. The final outcome of some embodiments is readily available genetic data of an embryo or fetus, and subsequent clinical decisions based on readily available data. The algorithms behind the PS method can increase the accuracy of determining the genetic status of the target individual by obtaining measured genetic data from a target individual, often an embryo or fetus, and measured genetic data from related individuals. In some embodiments, the measured genetic data is used in a situation where ploidy determination is made during prenatal genetic diagnosis. In some embodiments, the measured genetic data is used in a situation where ploidy determination or allele calling is made to an embryo during in vitro fertilization. There are many methods that can be used to measure the genetic data of an individual and / or related individuals in the above-described situations. Different methods involve several steps, which often include steps of amplifying genetic material, adding oligonucleotide probes, ligating specific DNA strands, isolating the desired DNA aggregate, removing undesirable components of the reaction, detecting specific DNA sequences by hybridization, and detecting the sequences of one or more DNA strands by a DNA sequencing method. In some cases, a DNA strand refers to the target genetic material; in other cases, a DNA strand refers to a primer; and in other cases, a DNA strand refers to a synthesized sequence or a combination thereof. These steps can be performed in several different orders.

[0236] It should be noted that, theoretically, it is possible to target any number of loci in the genome, from one locus to more than one million. When a DNA sample is targeted and then sequenced, the proportion of alleles read by the sequencer is enriched compared to their naturally occurring amounts in the sample. The degree of enrichment can range from 1 percent (or even lower) to 10 times, 100 times, 1,000 times, or even more, up to one million times. The human genome contains approximately 3 billion base pairs and nucleotides, including approximately 75 million polymorphism loci. The more loci targeted, the less enrichment is possible. The fewer loci targeted, the greater the degree of enrichment is possible, and at those loci, a greater read depth can be achieved for a given number of sequence reads.

[0237] In some embodiments of this disclosure, targeting or preference can be entirely focused on SNPs. In some embodiments, targeting or preference can be focused on any polymorphic site. Numerous commercial targeting products are available for exon enrichment. Surprisingly, exclusively targeting SNPs or exclusively polymorphic loci is particularly advantageous when using methods for NPD that rely on allele distribution. There are also published methods for NPD using sequencing, for example, U.S. Patent No. 7,888,017, which involves read count analysis where read counts are focused on counting the number of reads that map to a given chromosome, and the analyzed sequenced reads do not focus on polymorphic genomic regions. These types of methodologies that do not focus on polymorphic alleles are not as useful as targeting or preferentially enriching a set of alleles.

[0238] In some embodiments of this disclosure, a targeting method can be used to enrich a gene sample in polymorphic regions of the genome by focusing on SNPs. In some embodiments, it is possible to focus on a small number of SNPs, e.g., between 1 and 100 SNPs, or a larger number, e.g., between 100 and 1,000, between 1,000 and 10,000, between 10,000 and 100,000, or more than 100,000 SNPs. In some embodiments, it is possible to focus on one or a few chromosomes that correlate with birth in a surviving trisomy, e.g., chromosome 13, chromosome 18, chromosome 21, the X chromosome and the Y chromosome, or a combination of some of these. In some embodiments, it is possible to enrich the target SNPs by a small factor, e.g., between 1.01 and 100, or by a larger factor, e.g., between 100 and 1,000,000, or more than 1,000,000. In some embodiments of this disclosure, a targeted method can be used to produce a sample of DNA preferentially enriched in polymorphic regions of a genome. In some embodiments, this method can be used to produce a mixture of DNA having any of these properties, wherein the DNA mixture also includes maternal DNA and floating fetal DNA. In some embodiments, this method can be used to produce a mixture of DNA having any combination of these coefficients. For example, using the method described herein, a mixture of DNA preferentially enriched in DNA corresponding to 200 SNPs, all of which are located on either chromosome 18 or chromosome 21, and are enriched 1,000 times on average, can be produced. In another example, the method can be used to produce a mixture of DNA preferentially enriched in 10,000 SNPs, all or most of which are located on chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y, and where the average enrichment per locus exceeds 500 times. A mixture of DNA preferentially enriched at a specific gene locus can be prepared using any of the targeting methods described herein.

[0239] In some embodiments, the method of the present disclosure includes the step of measuring DNA in a mixed fraction using a high-throughput DNA sequencer, wherein the DNA in the mixed fraction contains a disproportionate number of sequences derived from one or more chromosomes, and the one or more chromosomes are selected from the group including chromosome 13, chromosome 18, chromosome 21, X chromosome, Y chromosome, and combinations thereof.

[0240] This specification describes three methods—multiplex PCR, targeted capture by hybridization, and ligated reverse probe (LIP)—which can be used to obtain and analyze measurements from a sufficient number of polymorphic loci derived from maternal plasma samples to detect fetal aneuploidy. This does not preclude other methods that selectively enrich target loci. Other methods can be used equally well without altering the core of the aforementioned methods. In each case, the polymorphisms assayed may include single nucleotide polymorphisms (SNPs), small insertions / deletions, or STRs. The preferred method involves the use of SNPs. Each method yields allele frequency data, and fetal ploidy can be determined by analyzing the allele frequency data for each target locus and / or the co-allele frequency distribution from these loci. Each method has its own considerations due to the limited source material and the fact that maternal plasma consists of a mixture of maternal and fetal DNA. These methods can be combined with other methods to yield more accurate determinations. In some embodiments, this method can be combined with sequence counting techniques, such as those described in U.S. Patent No. 7,888,017. The described methods can also be used to non-invasively detect fetal paternity from maternal plasma samples. Furthermore, each method can be applied to mixtures of other DNA or pure DNA samples to detect the presence or absence of aneuploid chromosomes, to genotype a large number of SNPs derived from a degraded DNA sample, to detect segmented copy number variations (CNVs), to detect the genotype status of other subjects or some combination thereof.

[0241] Accurate measurement of allele distribution in a sample Current sequencing methods can be used to estimate the distribution of alleles in a sample. One such method, called shotgun sequencing, involves a step of randomly sampling sequences from a pool of DNA. The proportion of a particular allele in sequencing data is generally very low and can be determined by simple statistics. The human genome contains approximately 3 billion base pairs. Therefore, if the sequencing method used generates 100 bp reads, a particular allele is measured approximately once every 30 million sequence reads.

[0242] In one embodiment, the method of this disclosure is used to determine the presence or absence of two or more different haplotypes containing the same set of loci in a DNA sample, based on the measured allele distribution of the loci from which the chromosome originates. Different haplotypes may represent two different homologous chromosomes from one individual, three different homologous chromosomes from an individual with trisomy, three different homologous haplotypes from the mother and fetus, one of which is shared between the mother and fetus, three or four haplotypes from the mother and fetus, one or two of which are shared between the mother and fetus, or other combinations. Alleles that are polymorphic between haplotypes tend to be more informative, but any allele for which neither the mother nor the father is homozygous for the same allele can provide useful information beyond what is available from simple read number analysis through the measured allele distribution.

[0243] However, shotgun sequencing of such samples is highly inefficient because it yields many sequences of non-polymorphic regions or non-target chromosomes among different haplotypes in the sample, and therefore does not provide information about the proportion of the target haplotype. This specification describes methods for increasing the yield of allelic information obtained by sequencing by specifically targeting and / or preferentially enriching segments of DNA in a sample that are more likely to be polymorphic within the genome. It should be noted that, for allelic distributions measured in enriched samples that truly represent the actual amount present in the target individual, it is significant if the preferential enrichment of one allele is slight or absent compared to other alleles at a given locus within the target segment. Current methods known in the art for targeting polymorphic alleles are designed to ensure that at least a portion of any alleles present are detected. However, these methods were not designed to measure the unbiased allelic distribution of polymorphic alleles present in the original mixture. It is not obvious that any particular method of targeted enrichment can produce an enriched sample, and that the measured allele distribution will more accurately reflect the allele distribution present in the original, unamplified sample than any other method. While theoretically many enrichment methods can be predicted to achieve such an objective, those skilled in the art are well aware that current amplification, targeting, and other preferential enrichment methods have a considerable degree of probabilistic or deterministic bias. One embodiment of the method herein makes it possible to amplify or preferentially enrich multiple alleles found in a mixture of DNA corresponding to a given locus in the genome such that the degree of enrichment of each allele is approximately the same. In other words, the method makes it possible to increase the overall relative amount of alleles present in the mixture while the ratios between alleles corresponding to each locus remain essentially the same as the ratios in the original mixture of DNA. Some reported methods can result in allele biases of more than 1%, more than 2%, more than 5%, and even more than 10%.This preferential enrichment may be due to capture bias when using capture by hybridization techniques, or amplification bias that may be small for each cycle but can become large when compounded over 20, 30, or 40 cycles. For the purposes of this disclosure, "the ratio remains essentially the same" means that the ratio of alleles in the original mixture divided by the ratio of alleles in the resulting mixture is between 0.95 and 1.05, between 0.98 and 1.02, between 0.99 and 1.01, between 0.995 and 1.005, between 0.998 and 1.002, between 0.999 and 1.001, or between 0.9999 and 1.0001. It should be noted that the calculation of allele ratios presented herein cannot be used to determine the ploidy status of a target individual and may only be used as a metric to measure allele bias.

[0244] In one embodiment, once the mixture is preferentially enriched in a set of target loci, sequencing can be performed using any one of the previous, current, or next-generation sequencing instruments to sequence clonal samples (samples generated from single molecules; examples include ILLUMINA GAIIx, ILLUMINA HiSeq, Life Technologies SOLiD, and 5500XL). The ratio can be evaluated by sequencing through specific alleles within the target region. These sequencing reads can be analyzed and counted according to the allele type and, therefore, the assignment of different alleles. For variations ranging from one to several bases in length, allele detection is performed by sequencing, and it is essential that the sequencing reads span the allele in question to assess the allele composition of the captured molecule. The total number of captured molecules to assay for genotype can be increased by increasing the length of the sequencing reads. Complete sequencing of all molecules ensures the collection of the maximum amount of data available in the enriched pool. However, sequencing is currently costly, and methods that can measure allele distribution using a small number of sequencing reads are highly valuable. Furthermore, as read length increases, technical and accuracy limitations arise regarding the maximum possible read length. The alleles with the greatest utility are those with a length of 1 to a few base pairs, but theoretically, any allele shorter than the length of the sequencing read can be used. Allele variation occurs in all types, but the examples provided herein focus on SNPs or variants contained within just a few adjacent base pairs. Larger variants, e.g., variants of copy numbers divided into segments, can often be detected by summing these smaller variations, as the overall population of SNPs within the segments often overlaps. Variants larger than a few base pairs, e.g., STRs, require special consideration and some targeted technique studies, but not others.

[0245] Several targeting techniques exist that can be used to specifically isolate and enrich the locations of one or more variants within a genome. Generally, these rely on utilizing non-mutated sequences adjacent to the variant sequence. Other researchers have reported on targeting in sequencing when the substrate is maternal plasma (see, e.g., Liao et al., Clin. Chem. 2011; 57(1): pp. 92-101). However, these techniques use targeting probes that target exons and do not focus on targeting polymorphic regions of the genome. In some embodiments, the method of the present disclosure includes the step of using a targeting probe that exclusively or nearly exclusively focuses on polymorphic regions. In some embodiments, the method of the present disclosure includes the step of using a targeting probe that exclusively or nearly exclusively focuses on SNPs. In some embodiments of the present disclosure, the target polymorphic site comprises at least 10% SNPs, at least 20% SNPs, at least 30% SNPs, at least 40% SNPs, at least 50% SNPs, at least 60% SNPs, at least 70% SNPs, at least 80% SNPs, at least 90% SNPs, at least 95% SNPs, at least 98% SNPs, at least 99% SNPs, at least 99.9% SNPs, or exclusively SNPs.

[0246] In some embodiments, the methods of the disclosure can be used to determine genotypes (the base composition of DNA at a particular locus) and the relative proportions of these genotypes from a mixture of DNA molecules, which may originate from one or more genetically distinct individuals. In some embodiments, the methods of the disclosure can be used to determine genotypes in a set of polymorphic loci and the relative ratios of the amounts of different alleles present at these loci. In some embodiments, the polymorphic loci may consist entirely of SNPs. In some embodiments, the polymorphic loci may include SNPs, single tandem repeats, and other polymorphisms. In some embodiments, the methods of the disclosure can be used to determine the relative distribution of alleles in a set of polymorphic loci in a mixture of DNA, which may include DNA originating from the mother and DNA originating from the fetus. In some embodiments, the co-allele distribution can be determined for a mixture of DNA isolated from the blood of a pregnant woman. In some embodiments, the allele distribution in a set of loci can be used to determine the ploidy status of one or more chromosomes in a pregnant fetus.

[0247] In some embodiments, the mixture of DNA molecules may originate from DNA extracted from multiple cells of a single individual. In some embodiments, if the individual is mosaic (germline or somatic), the original population of cells from which the DNA originates may include a mixture of diploid or haploid cells of the same or different genotypes. In some embodiments, the mixture of DNA molecules may originate from DNA extracted from a single cell. In some embodiments, the mixture of DNA molecules may originate from DNA extracted from two or more cells of the same individual or a mixture of two or more cells of different individuals. In some embodiments, the mixture of DNA molecules may originate from DNA isolated from a biological material that has already been freed from cells, such as plasma, which is known to contain cell-free DNA. In some embodiments, this biological material may be a mixture of DNA from one or more individuals, as has been shown in the case of pregnancy where fetal DNA is present in the mixture. In some embodiments, the biological material may originate from a mixture of cells found in maternal blood, some of which originate from the fetus. In some embodiments, the biological material may be cells derived from pregnant blood enriched with fetal cells.

[0248] Circularized probe Some embodiments of this disclosure involve amplifying a target locus before or after amplification using non-LIP primers in the multiplex PCR method of the present invention, using a “LIP” “Lipulated Reverse Probe” (LIP) as previously described in the literature. LIP is a general term encompassing techniques involving the construction of a circular DNA molecule, the probe being designed to hybridize with regions of target DNA on both sides of a target allele, and thus, with the addition of a suitable polymerase and / or ligase, and appropriate conditions, buffers, and other reagents, a circular loop of DNA is constructed that captures information found in the target allele, completing complementary reverse regions of DNA across the target allele. LIPs are also referred to as pre-circularized probes, pre-circularizing probes, or circularized probes. LIP probes may be linear DNA molecules between 50 and 500 nucleotides in length, and in some embodiments, between 70 and 100 nucleotides in length, and in some embodiments, may be longer or shorter than those described herein. Other embodiments of this disclosure involve different embodiment of LIP technology, such as Padlock probes and molecular reverse probes (MIPs).

[0249] One method for targeting specific locations for sequencing involves synthesizing a probe such that its 3' and 5' ends are adjacent to the target DNA and annealed in a reverse manner at locations on both sides of the target region. Therefore, by adding DNA polymerase and DNA ligase, extension from the 3' end occurs, a base is added to a single-stranded probe complementary to the target molecule (gap filling), and then the new 3' end ligates with the 5' end of the original probe, resulting in a circular DNA molecule that can later be isolated from background DNA. The probe ends are designed to be adjacent to the target region of interest. One aspect of this technique is commonly referred to as MIP and is used in conjunction with array techniques to determine the nature of the sequence being filled. One drawback of using MIP in situations where allele ratios are being measured is that the hybridization, circularization, and amplification steps do not occur at equivalent rates for different alleles at the same locus. As a result, allele ratios are measured that do not represent the actual allele ratios present in the original mixture.

[0250] In one embodiment, the cyclic probe is constructed such that a region of the probe designed to hybridize upstream of a target polymorphic locus and a region of the probe designed to hybridize downstream of the target polymorphic locus are covalently linked through a non-nucleic acid backbone. This backbone may be any biocompatible molecule or combination of biocompatible molecules. Some examples of possible biocompatible molecules include poly(ethylene glycol), polycarbonate, polyurethane, polyethylene, polypropylene, sulfone polymers, silicone, cellulose, fluoropolymers, acrylic compounds, styrene block copolymers, and other block copolymers.

[0251] In some embodiments of this disclosure, the method is modified to be readily sequenced as a means of examining fillings within a sequence. To preserve the proportion of the original alleles in the original sample, at least one important consideration must be taken into account. The location of variability between different alleles within the gap-filling region should not be too close to the probe binding site, as there may be initiation bias by DNA polymerase resulting in mutant differentiation. Another consideration is that there may be further variability at the probe binding site correlated with the mutant within the gap-filling region, which may result in uneven amplification from different alleles. In some embodiments of this disclosure, the 3' and 5' ends of the pre-circularized probe are designed to hybridize with bases that are one or a few positions away from the location of the mutation (polymorphic site) of the target allele. The number of bases between the polymorphic site (SNP or other types) and the bases designed to hybridize the 3' and / or 5' ends of the pre-circularization probe may be 1, 2, 3, 4, 5, 6, 7–10, 11–15, or 16–20, 20–30, or 30–60 bases. Forward and reverse primers can be designed to hybridize with different numbers of bases away from the polymorphic site. Numerous circularized probes can be generated using current DNA synthesis techniques, allowing for the generation and potential pooling of a very large number of probes to simultaneously investigate many loci. Working with over 300,000 probes has been reported. Two papers that discuss methods involving circularized probes that can be used to measure genomic data of target individuals are Porreca et al., Nature Methods, 2007, Vol. 4 (No. 11), pp. 931-936; and Turner et al., Nature Methods, 2009, Vol. 6 (No. 5), pp. 315-316. The methods described in these papers can be used in combination with other methods described herein.Certain steps of the methods from these two papers can be used in combination with other steps from other methods described herein.

[0252] In some embodiments of the methods disclosed herein, the genetic material of a target individual is amplified as needed, then hybridized with a pre-circularized probe, gap-filling is performed to fill the bases between the two ends of the hybridized probe, the two ends are ligated to form a circularized probe, and the circularized probe is amplified, for example, using rolling circle amplification. Once the genetic information of the desired target allele is captured in a well-designed circularized oligonucleotide probe, for example, in a LIP system, the genetic sequence of the circularized probe can be measured to obtain the desired sequence data. In some embodiments, a well-designed oligonucleotide probe can be directly circularized in the unamplified genetic material of the target individual and then amplified. It should be noted that the original genetic material can be amplified or the LIP can be circularized using several amplification procedures, including rolling circle amplification, MDA, or other amplification protocols. Genetic information on the target genome can be measured using different methods, for example, high-throughput sequencing, Sanger sequencing, other sequencing methods, capture by hybridization, capture by circularization, multiplex PCR, other hybridization methods, and combinations thereof.

[0253] By measuring an individual's genetic material using one or a combination of the above methods, or an informatics-based method, such as the PARENTAL SUPPORT® method, in conjunction with appropriate genetic measurements, it is then possible to determine the ploidy status of one or more chromosomes in the individual, and / or the genetic status of one or more alleles or sets of alleles (specifically, alleles correlated with the disease or genetic condition of interest). It should be noted that the use of LIP for multiplexed capture of gene sequences and subsequent genotyping using sequencing has been reported. However, the use of sequencing data resulting from LIP-based strategies to amplify genetic material found in single cells, a small number of cells, or extracellular DNA has not been used to determine the ploidy status of a target individual.

[0254] The application of informatics-based methods for determining the ploidy status of an individual from genetic data measured by hybridization arrays, such as the ILLUMINA INFINIUM array or AFFYMETRIX gene chip, is described in the references elsewhere in this document. However, the methods described herein represent improvements over methods previously described in the literature. For example, LIP-based methods, followed by high-throughput sequencing, unexpectedly yield better genotyping data because they have superior multiplexing capabilities, superior capture specificity, superior uniformity, and less allele bias. Greater multiplexing allows for targeting more alleles, resulting in more accurate results. Superior uniformity allows for the measurement of more target alleles, resulting in more accurate results. A lower rate of allele bias reduces the rate of false calls, resulting in more accurate results. More accurate results lead to improved clinical outcomes and better medical care.

[0255] It is important to note that LIP can be used as a method to target specific gene loci in a DNA sample for genotyping by methods other than sequencing. For example, LIP can be used to target DNA for genotyping using SNP arrays or other DNA or RNA-based microarrays.

[0256] Ligation-mediated PCR Ligation-mediated PCR can be used to amplify target loci before or after PCR amplification using unligated primers. Ligation-mediated PCR is a PCR method used to preferentially enrich a DNA sample by amplifying one or more loci in a mixture of DNA, the method comprising the step of obtaining a set of primer pairs, each primer in the pair containing a target-specific sequence and a non-target sequence, preferably the target-specific sequence is designed to anneal to a target region, one upstream of a polymorphic site and the other downstream of a polymorphic site, and the target-specific sequence is located 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 2 from the polymorphic site. The reaction comprises steps that may be separated by 1-30, 31-40, 41-50, 51-100, or more than 100 units; a step of polymerizing DNA from the 3' end of an upstream primer to fill the single-stranded region between it and the 5' end of a downstream primer having nucleotides complementary to the target molecule; a step of ligating the last polymerized base of the upstream primer with the 5' base of the adjacent downstream primer; and a step of amplifying only the polymerized and ligated molecules using a non-target sequence containing the 5' end of the upstream primer and the 3' end of the downstream primer. Primer pairs for different targets can be mixed in the same reaction. The non-target sequence functions as a universal sequence, and therefore all successfully polymerized and ligated primer pairs can be amplified using a single pair of amplification primers.

[0257] Capture by hybridization In some embodiments, the methods of this disclosure may include, in addition to amplifying a target locus using multiplex PCR, a step of using capture by one of the following hybridization methods. Preferential enrichment of a specific set of sequences in a target genome can be achieved in several ways. While other parts of this document describe how LIP can be used to target a specific set of sequences, in all of these applications, other targeting and / or preferential enrichment methods can be used equally well for the same purpose. One example of another targeting method is capture by a hybridization technique. Some examples of capture by commercial hybridization technologies include AGILENT's SURE SELECT and ILLUMINA's TruSeq. In hybridization capture, a set of oligonucleotides complementary or nearly complementary to the desired target sequence is hybridized with a mixture of DNA, and then physically separated from the mixture. Once the desired sequence has hybridized with the targeted oligonucleotide, the target sequence is also extracted by physically removing the targeted oligonucleotide. Once the hybridized oligonucleotides are isolated, they can be amplified by heating them above their melting point. Several methods for physically isolating targeted oligonucleotides involve covalently bonding the targeted oligonucleotides to a solid support, such as magnetic beads or chips. Another method for physically isolating targeted oligonucleotides involves covalently bonding them to a molecular moiety that has a strong affinity for another molecular moiety. An example of such a molecular pair is biotin and streptavidin, used, for example, in SURE SELECT. Thus, the target sequence can be covalently attached to a biotin molecule, and after hybridization, the biotinylated oligonucleotide with the target sequence hybridized can be pulled down using a solid support to which streptavidin has been added.

[0258] Hybrid capture involves hybridizing a target molecule with a probe complementary to the target. Hybrid capture probes were originally developed to target and enrich a large portion of the genome that exhibits relative homogeneity among targets. In its application, it was important that all amplified targets had sufficient homogeneity so that all regions could be detected by sequencing, but care was not taken to preserve the allele proportions in the original sample. After capture, the alleles present in the sample can be determined by direct sequencing of the captured molecules. These sequencing reads can then be analyzed and counted according to allele type. However, with current techniques, the allele distribution of the measured captured sequences generally does not represent the original allele distribution.

[0259] In some embodiments, allele detection is performed by sequencing. To capture allele identity at polymorphic sites and to evaluate the allele composition of the captured molecule, sequencing reads must span the allele in question. Since captured molecules often vary in length, sequencing must be performed on the entire molecule to guarantee overlapping mutation sites. However, due to cost considerations regarding the maximum possible length and sequencing read accuracy, as well as technical limitations, sequencing of the entire molecule is not feasible. In some embodiments, increasing read lengths from approximately 30 to 50 or even 70 base pairs significantly increases the number of reads that overlap with mutation sites within the target sequence.

[0260] Another method for increasing the number of reads used to locate the target is to reduce the probe length, provided that this does not result in a bias in the underlying enriched alleles. The length of the synthesized probe should be sufficient for two probes, designed to hybridize with two different alleles found at a single locus, to hybridize with approximately equal affinity to the various alleles in the original sample. Methods currently known in the art generally describe probes longer than 120 bases. In current embodiments, when the allele is one or a few bases, the capture probe may be less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than 30 bases, and less than about 25 bases, and this amount is sufficient to ensure equal enrichment from all alleles. When the DNA mixture enriched using hybrid capture technology is a mixture containing floating DNA isolated from blood, such as maternal blood, the average length of the DNA is fairly short, generally less than 200 bases. Using shorter probes increases the likelihood that the hybrid capture probe will capture the desired DNA fragment. Larger variations may require longer probes. In some embodiments, the variations in question are 1 (SNP) to a few bases long. In some embodiments, a target region within the genome can be preferentially enriched using a hybrid capture probe, where the length of the hybrid capture probe is less than 90 bases, and may be less than 80, 70, 60, 50, 40, 30, or 25 bases. In one embodiment, to increase the likelihood of sequencing the desired allele, the length of the probe, which is designed to hybridize with a region adjacent to the location of the polymorphic allele, can be reduced from over 90 bases to about 80 bases, or about 70 bases, or about 60 bases, or about 50 bases, or about 40 bases, or about 30 bases, or about 25 bases.

[0261] To enable capture, there must be a minimum overlap between the synthesized probe and the target molecule. While this synthesized probe can be as short as possible, it is still larger than this minimum required overlap. The benefit of using shorter probe lengths to target polymorphic regions is that more molecules overlap with the target allele region. The fragmentation state of the original DNA molecule also affects the number of reads that overlap with the target allele. Some DNA samples, such as plasma samples, are already fragmented due to biological processes occurring in vivo. However, for samples with longer fragments, fragmentation before sequencing library preparation and enrichment is beneficial. When both the probe and fragments are short (approximately 60–80 bp), maximum specificity can be achieved with relatively few sequence reads that do not overlap with the critical region of target.

[0262] In some embodiments, hybridization conditions can be adjusted to maximize uniformity in capturing different alleles present in the original sample. In some embodiments, the hybridization temperature is reduced to minimize differences in hybridization bias between alleles. Methods known in the art avoid using lower temperatures for hybridization because reducing the temperature has the effect of increasing hybridization between the probe and unintended targets. However, if the objective is to preserve the allele ratio with maximum fidelity, a method using lower hybridization temperatures yields an optimally accurate allele ratio, despite the fact that teachings in the current art avoid this approach. The hybridization temperature can also be increased to require a greater overlap between the target and the synthesized probe so that only targets with substantial overlap of the target region are captured. In some embodiments of the present disclosure, the hybridization temperature is reduced from the normal hybridization temperature to about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C.

[0263] In some embodiments, a hybrid capture probe can be designed such that the region of the capture probe having DNA complementary to the DNA found in a region adjacent to the polymorphic allele is not immediately adjacent to the polymorphic site. Instead, the capture probe can be designed such that the region of the capture probe designed to hybridize with DNA adjacent to the target polymorphic site is separated from the portion of the capture probe that is in van der Waals contact with the polymorphic site by a small distance equal to one or a few bases. In some embodiments, a hybrid capture probe is designed to hybridize with a region adjacent to the polymorphic allele but not crossing it; this is referred to as an adjacent capture probe. The length of the adjacent capture probe may be less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, or less than about 25 bases. The genomic region targeted by the adjacent capture probe may be separated from the polymorphism locus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, or more than 20 base pairs.

[0264] This document describes disease screening tests using targeted sequence capture. It covers custom-ordered targeted sequence capture, such as those currently offered by AGILENT (SURE SELECT), ROCHE-NIMBLEGEN, or ILLUMINA. Capture probes can be custom-designed to ensure capture of various types of mutations. For point mutations, one or more probes overlapping the point mutation should be sufficient to capture and sequence the mutation.

[0265] For small insertions or deletions, one or more probes overlapping the mutation may be sufficient to capture and sequence the fragment containing the mutation. Hybridization may generally be less efficient than probe-limited capture efficiencies designed to reference genome sequences. To ensure capture of the fragment containing the mutation, two probes can be designed: one matching the normal allele and another matching the mutated allele. Longer probes can enhance hybridization. Multiple overlapping probes can enhance capture. Finally, by placing probes immediately adjacent but not overlapping, the mutation may allow for relatively similar capture efficiencies for both the normal and mutated alleles.

[0266] For simple tandem repeats (STRs), probes overlapping these highly variable sites are less likely to successfully capture the fragments. To enhance capture, the probe can be placed adjacent to the variable sites but without overlap. The fragments can then be sequenced as usual to determine the length and composition of the STRs.

[0267] For large deletions, a series of overlapping probes, a common technique currently used in exon capture systems, can function. However, this technique can make it difficult to determine whether an individual is heterozygous. By targeting and evaluating SNPs within the captured region, it is possible to potentially indicate a loss of heterozygosity across that region, indicating that the individual is a carrier. In some embodiments, non-overlapping probes or singleton probes can be placed across the potentially deleted region, and the number of captured fragments can be used as a measure of heterozygosity. If an individual has a large deletion, it is expected that half the number of fragments will be available for capture compared to a non-deleted (diploid) reference locus. Therefore, the number of reads obtained from the deleted region should be approximately half the number of reads obtained from a normal diploid locus. By summing and averaging the sequencing read depths from multiple singleton probes across the potentially deleted region, the signal can be augmented and the reliability of the diagnosis can be improved. The two approaches—targeting SNPs to identify loss of heterozygosity and using multiple singleton probes to obtain a quantitative measure of the amount of underlying fragments from that locus—can also be combined. Either or both of these strategies, combined with other strategies, can yield better results.

[0268] During the test, the detection of cfDNA in male fetuses, indicated by the presence of a Y chromosome fragment captured and sequenced in the same test, and the presence of either an X-linked dominant mutation unaffected by both mother and father, or a maternal dominant mutation unaffected by mother, indicates an increased risk to the fetus. The detection of two mutated recessive alleles within the same gene in an unaffected mother means that the fetus inherited the mutated allele from the father and potentially the second mutated allele from the mother. In all cases, follow-up examinations by amniocentesis or chorionic villus sampling may also be indicated.

[0269] Targeted capture-based disease screening tests can be combined with non-invasive prenatal diagnostic tests based on targeted capture for aneuploidy. Several methods exist to reduce read depth (DOR) variability: for example, primer concentrations can be increased, longer targeted amplification probes can be used, or more STA cycles can be performed (e.g., over 25, over 30, over 35, or even over 40).

[0270] Representative method for determining the number of DNA molecules in a sample A method for determining the number of DNA molecules in a sample is described herein by generating a uniquely identified molecule for each of the original DNA molecules in the sample during the first round of DNA amplification. Procedures that achieve the above objective, followed by single-molecule sequencing or clonal sequencing, are described herein.

[0271] The method includes the steps of targeting one or more specific gene loci, and generating copies of the original molecules tagged with a unique tag for each target locus, so that these barcodes can be distinguished from each other when sequenced using clonal or single-molecule sequencing. Each unique sequenced barcode indicates a unique molecule in the original sample. Simultaneously, the sequencing data is used to identify the locus from which the molecule originates. This information can be used to determine the number of unique molecules in the original sample for each locus.

[0272] This method can be used for any application requiring a quantitative assessment of the number of molecules in the original sample. Furthermore, the number of unique molecules of one or more targets can be correlated with the number of unique molecules of one or more other targets to determine relative copy numbers, allele distributions, or allele ratios. Alternatively, the number of copies detected from various targets can be modeled by distribution to identify the most likely number of copies of the original target. Applications include, but are not limited to, the detection of insertions and deletions, e.g., those found in carriers of Duchenne muscular dystrophy; the quantification of deletions or duplications of chromosomal segments, e.g., those observed in copy number variants; the chromosome copy number of samples from living individuals; and the chromosome copy number of samples from unborn individuals, e.g., embryos or fetuses.

[0273] The method described above can be combined with the evaluation of simultaneous variations contained in sequence targeting. This can be used to determine the number of molecules representing each allele in the original sample. This copy number method can be combined with the evaluation of SNPs or other sequence variations to determine the chromosomal copy numbers of born and unborn individuals; to identify and quantify copies from loci that have short sequence variations but can be amplified from multiple target regions by PCR, for example, for the purpose of detecting carriers of spinalis atrophy; to determine the copy numbers of various sources of molecules from a sample consisting of a mixture of different individuals, for example, for the purpose of detecting fetal aneuploidy from floating DNA obtained from maternal plasma.

[0274] In one embodiment, a method relating to a single target locus may include one or more of the following steps: (1) designing a standard pair of oligomers for PCR amplification of a particular locus; (2) during synthesis, adding a specific sequence of bases to the 5' end of one of the target-specific oligomers that has no complementarity to, or minimal complementarity to, the target locus or genome. This sequence, referred to as the tail, is a known sequence used for subsequent amplification, followed by a sequence of random nucleotides. These random nucleotides contain random regions. The random regions contain randomly generated nucleic acid sequences that are probabilistically different between each probe molecule. Thus, after synthesis, the tailed oligomer pool consists of a population of oligomers that begin with a known sequence, followed by an unknown sequence that is different between molecules, and then followed by the target-specific sequence; (3) performing one round of amplification (denaturation, annealing, extension) using only the tailed oligomers. (4) Adding exonuclease to the reaction to effectively stop the PCR reaction, incubating the reaction to an appropriate temperature to remove the template and the forward single-stranded oligo that did not anneal, and extending it to form a double-stranded product. (5) Incubating the reaction to a high temperature to denature the exonuclease and eliminate its activity. (6) Adding a new oligonucleotide complementary to the tail of the oligomer used in the first reaction to the reaction along with other target-specific oligomers to enable PCR amplification of the product produced in the first round of PCR. (7) Continuing the amplification to produce a sufficient product for downstream clonal sequencing. (8) Measuring the amplified PCR product, which has a sufficient number of bases linked to the sequence, by a number of methods, such as clonal sequencing.

[0275] In some embodiments, the method of the present disclosure includes the step of targeting multiple loci in parallel or in other ways. Primers for different target loci can be generated independently and mixed to create a multiplex PCR pool. In some embodiments, the original sample can be divided into subpools, and each subpool can be recombined and sequenced after targeting different loci. In some embodiments, amplification can be performed by tagging the pool before subdividing it and carrying out several amplification cycles to ensure efficient targeting of all targets, then splitting and refining, and then continuing amplification using smaller sets of primers in the subdivided pools.

[0276] One application where this technique is particularly useful is non-invasive prenatal aneuploidy diagnosis, where the ratio of alleles at a given locus or the distribution of alleles at several loci can be used to determine the number of chromosome copies present in the fetus. In this situation, it is desirable to amplify the DNA present in the initial sample while maintaining the relative amounts of various alleles. In some cases, especially when the amount of DNA present is very small, for example, fewer than 5,000 genome copies, fewer than 1,000 genome copies, fewer than 500 genome copies, and fewer than 100 genome copies, a phenomenon called bottlenecking can occur. This means that a small number of copies of any given allele are present in the initial sample, and as a result of amplification bias, the ratio of those alleles in the amplified DNA pool is significantly different from the ratio of those alleles in the initial DNA mixture. By applying a unique or nearly unique set of barcodes to each DNA strand prior to standard PCR amplification, it is possible to eliminate n-1 copies of DNA from a set of n identical molecules of sequenced DNA originating from the same original molecule.

[0277] For example, consider a mixture of DNA from an individual, where each allele is represented by a single nucleotide polymorphism (SNP) within the individual's genome, and the original DNA sample containing 10 molecules of each allele. After amplification, there may be 100,000 DNA molecules corresponding to that locus. Due to the probabilistic process, the DNA ratio can be anywhere from 1:2 to 2:1, but because each of the original molecules is tagged with a unique tag, it is possible to determine that the DNA in the amplified pool originates precisely from 10 DNA molecules from each allele. Therefore, this method provides a more accurate relative measure of the amount of each allele than methods that do not use this technique. This method provides accurate data for methods where minimizing the relative bias of alleles is desirable.

[0278] The association between sequenced fragments and target loci can be achieved in several ways. In one embodiment, a molecular barcode corresponding to the target sequence, as well as a sufficiently long sequence obtained by tying a sufficient number of unique bases to the target fragment, allows for the explicit identification of the target locus. In another embodiment, a molecular barcoding primer containing a randomly generated molecular barcode may also contain a locus-specific barcode (locus barcode) that identifies the target to which it is associated. This locus barcode is identical among all molecular barcoding primers for each individual target, and therefore among all the resulting amplification products, but different from all other targets. In one embodiment, the tagging method described herein can be combined with a one-sided nesting protocol.

[0279] In one embodiment, the design and generation of molecular barcoding primers can be carried out as follows: The molecular barcoding primer may consist of a sequence not complementary to the target sequence, followed by a random molecular barcode region, followed by a target-specific sequence. The 5' sequence of the molecular barcode can be used for partial sequence PCR amplification and may contain sequences useful in converting the amplified product into a library for sequencing. Random molecular barcode sequences can be generated in numerous ways. A preferred method is to synthesize molecularly tagged primers to contain all four bases for the reaction during the synthesis of the barcode region. All bases or various combinations of bases can be specified using IUPACDNA ambiguous codes. Thus, the population of synthesized molecules contains a random mixture of sequences within the molecular barcode region. The length of the barcode region determines how many primers contain unique barcodes. The number of unique sequences is N L The barcode region is associated with the length of the barcode, where N is the number of bases, generally 4, and L is the length of the barcode. A 5-base barcode can result in up to 1024 unique sequences, and an 8-base barcode can result in 65536 unique barcodes. In one embodiment, DNA can be measured by a sequencing method, and the sequence data represents the sequence of a single molecule. This may include a method of directly sequencing a single molecule, or a method referred to herein as clonal sequencing, in which a single molecule is amplified to form a clone detectable by a sequencing instrument, but still representing a single molecule.

[0280] Typical Methods and Reagents for Quantifying Amplification Products Quantification of specific nucleic acid sequences of interest is typically performed using quantitative real-time PCR techniques such as TAQMAN (LIFE TECHNOLOGIES) and INVADER probes (THIRD WAVE TECHNOLOGIES). Such techniques have many drawbacks, including limited ability to perform parallel simultaneous analysis (multiplexing) of multiple sequences and the ability to generate accurate quantitative data only within a narrow range of possible amplification cycles (e.g., the logarithmic ratio of PCR amplification output to the number of cycles is within a linear range). DNA sequencing technologies, particularly high-throughput next-generation sequencing technologies (often called high-volume parallel sequencing technologies), such as those employed by MYSEQ (ILLUMINA), HISEQ (ILLUMINA), ION TORRENT (LIFE TECHNOLOGIES), GENOME ANALYZERILX (ILLUMINA), and GSFLEX+ (ROCHE454), can be used to quantitatively measure the copy number of target sequences present in a sample, thereby obtaining quantitative information about the starting material, such as copy number or transcription level. High-throughput gene sequencers use barcoding (i.e., tagging samples using characteristic nucleic acid sequences) to identify specific samples from individual populations, thereby enabling simultaneous analysis of multiple samples in a single operation of the DNA sequencer. The number of times a particular region of the genome in a library preparation (or other target nucleic acid preparation) is sequenced (read count) will likely be proportional to the copy number of that sequence in the target genome (or, in the case of a cDNA-containing preparation, the expression level). However, gene library preparation and sequencing (and preparations from similar genomes) can introduce many biases that interfere with obtaining accurate quantitative reads of the target nucleic acid sequence. For example, different nucleic acid sequences may result in different amplification efficiencies during the nucleic acid amplification step that occurs during gene library preparation or sample preparation.

[0281] Problems related to different amplification efficiencies can be mitigated by using specific embodiments of the present invention. The present invention includes various methods and compositions relating to the use of content criteria in the amplification process that can be used to improve the accuracy of quantification. As described herein, and in particular in U.S. Patent No. 8,008,018; U.S. Patent No. 7,332,277; International Publication No. WO2012 / 078792A2; International Publication No. WO2011 / 146632A1, the present invention is particularly useful in the area of ​​detecting fetal aneuploidy by analyzing floating fetal DNA in maternal blood. These patents are incorporated herein by reference in their entirety. Embodiments of the present invention are also useful for detecting aneuploidy in in vitro developing embryos. Commercially important detectable aneuploidy include aneuploidy of human chromosomes 13, 18, 21, X, and Y.

[0282] Embodiments of the present invention can be used for human or non-human nucleic acids, and are applicable to both animal and plant-derived nucleic acids. Furthermore, embodiments of the present invention can be used to detect and / or quantify alleles associated with other genetic disorders characterized by deletions or insertions. Deletion-containing alleles may be detected in individuals suspected of being carriers of the target allele.

[0283] One embodiment of the present invention involves a reference sequence that exists in a known quantity (relative or absolute). For example, consider a gene library constructed from a source in which chromosome 8 (containing locus A) is diploid and chromosome 21 (containing locus B) is triploid. The gene library can be generated from a sample containing an amount of sequence that is a function of the number of chromosomes present in the sample, e.g., 200 copies of locus A and 300 copies of locus B. However, if locus A is amplified with much higher efficiency than locus B, after PCR there may be 60,000 copies of A amplified product and 30,000 copies of B amplified product, thus obscuring the number of chromosome copies in the initial true genome sample for analysis by high-throughput DNA sequencing (or other quantitative nucleic acid detection techniques). To mitigate this problem, a reference sequence for locus A is employed, in which case the reference sequence is amplified with substantially the same efficiency as locus A. Similarly, a reference sequence for locus B is formed, in which case the reference sequence is amplified with substantially the same efficiency as locus B. Prior to PCR (or other amplification techniques), reference sequences for locus A and locus B are added to the mixture. These reference sequences are present in known amounts, either relative or absolute. Therefore, under the same set of conditions, if a 1:1 mixture of reference sequences A and B is added to the mixture in the previous example (before amplification), 3000 copies of the reference A amplified product and 1000 copies of the reference B amplified product are produced, demonstrating that locus A is amplified three times more efficiently than locus B.

[0284] Specific amplification and subsequent sequencing are possible for one or more selected regions of a genome containing a target SNP (or other polymorphism). This target-specific amplification can be performed during the formation of a gene library for sequencing. The library can contain many target amplification regions. In some embodiments, it may include at least 10;100;500;1,000;2,000;5,000;7,500;10,000;20,000;25,000;30,000;40,000;50,000;75,000; or 100,000 target regions. Examples of such libraries are described herein and can also be found in U.S. Patent Publication No. 2012 / 0270212, filed November 18, 2011, which is incorporated herein by reference in its entirety.

[0285] Many high-throughput DNA sequencing techniques require modification of the starting genetic material, such as ligation of universal priming sites and / or barcodes, for library formation, thereby facilitating clonal amplification of small nucleic acid fragments before subsequent sequencing reactions. In some embodiments, one or more reference sequences are added during gene library formation or to the precursor components of the gene library before library amplification. The reference sequences can be selected to mimic (but still distinguishable based on nucleotide sequence) the target genomic fragment prepared for sequencing by the high-throughput gene sequencing technique. In one embodiment, the reference sequence may be identical to the target genomic fragment except for 1, 2, 3, 4-10, or 11-20 nucleotides. In some embodiments, if the target gene sequence contains an SNP, the reference sequence may be identical to the SNP other than the polymorphic base, and can be selected to be one of four nucleotides not observed in the native site. Reference sequences can be used for highly multiplex analysis of multiple target loci (e.g., polymorphic loci). Reference sequences can be added in known amounts (relative or absolute) during the library formation (pre-amplification) process to provide a reference metric for greater accuracy in determining the amount of target sequences in the analyte. The amplification characteristics of each reference sequence with respect to its corresponding target sequence can be calibrated using a combination of information on known amounts of reference sequences used, along with information on previously characterized ploidy levels, e.g., ploidy level libraries for sequencing formed from genomes known to be entirely diploid. Variations between batch mixtures containing multiple reference sequences can also be taken into account. Given that it is often necessary to analyze a large number of loci simultaneously, generating mixtures containing many sets of reference sequences is useful. Embodiments of the present invention include mixtures containing multiple reference sequences. Ideally, the amount of each reference sequence in the mixture should be known with high precision. However, achieving this ideal is extremely difficult.The reason is that, in practice, there is considerable variation in the amount of each reference sequence in a mixture, particularly in the amount of reference sequences in a mixture containing many different synthetic oligonucleotides. This variation can have many sources, such as batch-to-batch variations in in vitro oligonucleotide synthesis efficiency, inaccuracies in volume measurement, and variations in pipetting. Furthermore, this variation can occur even between batches containing theoretically exactly the same amount of exactly the same set of reference sequences. Therefore, it is meaningful to calibrate each reference sequence batch independently. A reference sequence batch can be calibrated against a reference genome of known chromosomal composition. A reference sequence batch can be calibrated by sequencing the reference sequence batch with minimal or no amplification steps included in the sequencing protocol. Embodiments of the present invention include mixtures of various calibrated reference sequences. Other embodiments of the present invention include methods for calibrating mixtures of different reference sequences and mixtures of different calibrated reference sequences prepared by this method.

[0286] Various embodiments of the reference sequence mixtures and their uses may include at least 10; 100; 500; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 or more reference sequences, as well as various intermediate numbers. The number of reference sequences may be the same as the number of target sequences selected for analysis during the generation of the target library for DNA sequencing. However, in some embodiments, it may be advantageous to use fewer reference sequences than the number of target regions in the library being constructed. Using fewer reference sequences may be advantageous in avoiding reaching the sequencing capacity limits of the high-throughput DNA sequencer employed. The number of reference sequences may be 50% or less of the number of target regions, 40% or less of the number of target regions, 30% or less of the number of target regions, 20% or less of the number of target regions, 10% or less of the number of target regions, 5% or less of the number of target regions, 1% or less of the number of target regions, and various intermediate numbers. For example, if a gene library is constructed using 15,000 pairs of primers targeting specific SNP-containing loci, a suitable mixture containing 1,500 reference sequences corresponding to 1,500 of the 15,000 target loci can be added before the amplification step of library construction.

[0287] The amount of reference sequence added during library construction may vary considerably between individual embodiments. In some embodiments, the amount of each reference sequence may be approximately the same as the predicted amount of target sequence present in the genomic material sample used for library preparation. In other embodiments, the amount of each reference sequence may be greater or less than the predicted amount of target sequence present in the genomic material sample used for library preparation. While the initial relative amounts of target and reference sequences are not critical to the purposes of the present invention, they are preferably in the range of 100 times more to 100 times less than the amount of target sequence present in the genomic material sample used for library preparation. Excessive amounts of reference sequences may overuse much of the sequencing capacity of the DNA sequencer within a given number of operations of the instrument. Using too few reference sequences may result in insufficient data to help analyze variations in amplification efficiency.

[0288] The reference sequence can be selected to have a nucleotide base sequence that is very similar to the target region to be amplified, and preferably, the reference sequence has exactly the same primer-binding sites as the genomic region being analyzed, i.e., the “target sequence”. The reference sequence must be distinguishable from the corresponding target sequence at a given locus. For convenience, this distinguishable region of the reference sequence is called the “marker sequence”. In some embodiments, the marker sequence region of the target sequence may include a polymorphic region, e.g., an SNP, with primer-binding sites located on both sides. The reference sequence can be selected to closely match the GC content of the corresponding target sequence. In some embodiments, the primer-binding sites of the reference sequence are flanked by universal priming sites. These universal priming sites are selected to match the universal priming sites used in the genomic library for analysis. In other embodiments, the reference sequence has no universal priming sites, which are added during library formation. The reference sequence is usually provided in single-stranded form. The reference sequence is defined relative to the corresponding target sequence, and the target sequence is amplified using a sequence-specific reagent. In some embodiments, the target sequence includes a target polymorphism present in the nucleic acid sample for analysis, e.g., a SNP, deletion, or insertion. The reference sequence is a synthetic polynucleotide whose nucleotide base sequence is similar to that of the target sequence, but is still distinguishable from the target sequence by a difference of at least one nucleotide base, thereby providing a mechanism to distinguish the amplified product sequence derived from the reference sequence from the amplified product sequence derived from the target sequence. The reference sequence is selected to have substantially the same amplification characteristics as the corresponding target sequence when amplified with the same set of amplification reagents, e.g., PCR primers. In some embodiments, the reference sequence may have the same primer sequence binding sites as the corresponding target sequence. In other embodiments, the reference sequence may have different primer sequence binding sites than the corresponding target sequence. In some embodiments, the reference sequence may be selected to produce an amplified product of the same length as the amplified product derived from the corresponding target sequence.In other embodiments, the reference sequences can be selected to generate amplification products that are slightly different in length from the amplification products derived from the corresponding target sequences.

[0289] After the amplification reaction is complete, the library is sequenced on a high-throughput DNA sequencer, where individual molecules are clonally amplified and sequenced. The sequence read counts for each allele of the target sequence are counted, and similarly, the sequence read counts for the reference sequences corresponding to the target sequences are counted. This process is also performed similarly for at least one other pair of target and corresponding reference sequences. For example, with respect to locus A, X A1 reads are generated for allele 1 of locus A, X A2 reads are generated for allele 2 of locus A, and X AC reads are generated for reference sequence A. For each target locus, the ratio of (X AC plus X A1 plus X A2 ) to X is determined. As previously discussed, this process can be performed on a reference genome, for example, a genome where the entire chromosome is known to be diploid. This process can be repeated multiple times to obtain a large number of read values and measure the average read count and the standard deviation of the read counts. This process is performed on a mixture containing a large number of different reference sequences corresponding to different loci. Assuming that (1) X A1 plus X A2 corresponds to 2 for a known number of chromosomes, for example, the normal human female genome, and (2) the reference sequences have amplification (and detection) characteristics similar to their corresponding native loci, the relative amounts of different reference sequences in the multiplex reference mixture can be determined. Subsequently, the calibrated multiplex reference sequence mixture can be used to adjust the variation in amplification efficiency between different loci in the multiplex amplification reaction.

[0290] Other embodiments of the present invention include methods and compositions for measuring the copy number of a target-specific gene, such as a mutant gene characterized by a large deletion that may interfere with replication and sequencing-based quantification. Sequencing can be difficult in detecting alleles with such deletions. This problem can be mitigated by using an amplification process that includes a reference sequence.

[0291] In one embodiment of the present invention, the target sequence for analysis is a gene characterized by a wild-type (i.e., functional) and a deletion-type mutant. A typical such gene is SMN1, a deletion-type allele that causes the genetic disorder spinal muscular atrophy (SMA). Detecting individuals carrying the mutant gene using high-throughput gene sequencing techniques is meaningful. However, the application of such techniques to the detection of deletion mutations can be problematic, particularly because sequence absences are observed during sequencing (in contrast to the detection of simple point mutations or SNPs). In such embodiments, the following are used: (1) a pair of amplification primers specific to the target gene that amplify the target gene (or a portion thereof) without significantly amplifying the mutant allele; (2) a reference sequence corresponding to the wild-type allele of the target gene (i.e., the target sequence), but differing by at least one detectable nucleotide base; (3) a pair of amplification primers specific to a second target sequence that functions as a reference sequence; and (4) a reference sequence corresponding to the reference sequence.

[0292] One embodiment of the present invention provides a method for measuring the copy number of a target gene, the target gene having one intentional allele containing a deletion. The method can employ an amplification reagent specific to the target gene, such as a PCR primer, in that it amplifies at least a portion of the target gene, the entire target gene, or a region adjacent to the target gene, without amplifying the deletion-containing allele of the target gene. Furthermore, the method employs a reference sequence corresponding to the target gene, in which case the reference sequence differs from the target gene by at least one nucleotide base (so that the sequence of the reference sequence is easily distinguishable from the target natural gene). Typically, the reference sequence contains the same primer binding site as the target gene, thereby minimizing all amplification differences between the target gene and the reference sequence corresponding to the target gene. The reaction also includes an amplification reagent specific to the reference sequence. The reference sequence is a known (or at least assumed to be known) copy number sequence in the genome being analyzed. The reaction further includes a reference sequence corresponding to the reference sequence. Typically, the reference sequence corresponding to a reference sequence contains the same primer binding sites as the reference sequence, thereby minimizing all amplification differences between the reference sequence and the reference sequence corresponding to the reference sequence.

[0293] Representative nucleic acid samples In some embodiments, gene samples can be prepared and / or purified. There are several standard procedures known in the art for achieving such objectives. In some embodiments, the sample can be separated into...

Claims

1. A nested amplification method, wherein the method is (a) A step of isolating cell-free DNA (cfDNA) fragments from the sample, (b) A step of tagging at least some of the cfDNA fragments with an adapter containing a universal priming site to obtain a nucleic acid sample containing multiple target gene loci, (c) A step of performing a first amplification of the plurality of target gene loci of the nucleic acid sample using a first primer library comprising a plurality of target-specific primers and a universal primer that binds to the universal priming site, (d) The process includes a step of generating an amplification product by performing a second nested amplification on the target gene locus using a second primer library containing a plurality of internal target-specific primers and the universal primer, Herein, in step (c) or step (d), or both, the length of the annealing step between amplifications is more than 3 minutes, in the method.

2. (e) The method according to claim 1, comprising sequencing the amplification product by a high-throughput sequencing method.

3. The method according to claim 1, wherein the melting temperature is determined by the Primer3 program.

4. The method according to claim 1, wherein the melting temperature range of the primer is less than 5°C.

5. The method according to claim 1, wherein at least 10 different target gene loci are simultaneously amplified in step (c) or step (d), or both.

6. The method according to claim 1, wherein at least 50 different target gene loci are simultaneously amplified in step (c) or step (d), or both.

7. The method according to claim 1, wherein at least 100 different target gene loci are simultaneously amplified in step (c) or step (d), or both.

8. The method according to claim 1, wherein at least 10 to 10,000 different target gene loci are simultaneously amplified in step (c) or step (d), or both.

9. The method according to claim 1, wherein at least 90% of the amplification product is a target amplification product.

10. The method according to claim 1, wherein at least 90% of the target gene locus is amplified.

11. The method according to claim 1, wherein less than 20% of the amplification product is a primer dimer.

12. The method according to claim 1, wherein the concentration of each primer in the library is less than 20 nM.

13. The method according to claim 1, wherein the length of the annealing step under the reaction conditions is 5 to 60 minutes.

14. The method according to claim 1, wherein the target gene locus is an SNP gene locus.

15. The method according to claim 1, wherein the cell-free DNA includes tumor DNA.

16. The method according to claim 1, wherein the sample is a plasma sample.

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