JP6997815B2 - Highly multiplex PCR method and composition - Google Patents

Description

Mutual reference to related applications This application is in US Utility Model Application Nos. 13 / 683,604 filed November 21, 2012, and US Patent Provisional Application Nos. 61 / 675,020 filed July 24, 2012. Claim interests and priorities. US Utility Model Application No. 13 / 683,604 is a partial continuation of US Utility Model Application No. 13 / 110,685 filed May 18, 2011, and is a US utility model application filed on November 18, 2011. It is a partial continuation of Nos. 13 / 300,235 and claims the interests of US Patent Provisional Application Nos. 61 / 675,020 filed on July 24, 2012. US Practical New Application No. 13 / 110,685 is US Patent Provisional Application No. 61 / 395,850 filed May 18, 2010; US Patent Provisional Application No. 61/398 filed June 21, 2010, 159; US Patent Provisional Application No. 61 / 462,972 filed February 9, 2011; US Patent Provisional Application Nos. 61 / 448,547 filed March 2, 2011; and April 12, 2011. Claims the benefit of US Patent Provisional Application No. 61 / 516,996 of the application. US Utility Model Application Nos. 13 / 300,235 claim the benefit of US Patent Provisional Application Nos. 61 / 571,248 filed June 23, 2011. All of these patent applications are incorporated herein by reference for all their teachings.

Federally Funded Description of Research and Development The invention was made with the support of the National Institutes of Health under Authorization No. 5R44HD60423-3. The US Government may hold rights to all patents derived from this application.

Fields of Invention The present invention generally relates to methods and compositions for simultaneously amplifying a plurality of nucleic acid regions of interest in one reaction volume.

To increase assay throughput and allow for more efficient use of nucleic acid samples, a number of oligonucleotide primers are mixed with the sample and then polymerase chain reaction (PCR) conditions are performed in a process known in the art as multiplex PCR. By providing the sample to the sample, simultaneous amplification of many target nucleic acids in the target sample can be performed. The use of multiplex PCR is a very simple experimental procedure that 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 produced. As the number of primers increases, the likelihood of such product formation increases. These non-target amplification products severely limit the use of amplification products in subsequent analysis and / or assays. Therefore, there is a need for improved methods to reduce the formation of non-target amplification products during multiplex PCR.

The improved multiplex PCR method will be useful in a variety of applications such as non-invasive prenatal genetic diagnosis (NPD). Specifically, current methods of prenatal diagnosis can alert physicians and parents to abnormalities in a growing fetus. Without prenatal testing, 1 in 50 babies are born with significant physical or mental handicaps, and 1 in 30 have some form of congenital malformation. Unfortunately, standard methods involve inaccurate or invasive procedures with 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 biopsy and fetal blood sampling are accurate but invasive and carry significant risks. Amniocentesis has been performed in approximately 3% of all pregnancies in the United States, but its frequency of use has declined over the last 15 years.

A normal human has two sets of 23 chromosomes in all healthy diploid cells, one copy from each parent. Aneuploidy, a condition in nuclear cells with too many and / or too few chromosomes, is thought to contribute to the majority of implantation failures, miscarriage, and genetic diseases. Detection of chromosomal abnormalities can identify individuals or embryos with conditions such as Down's syndrome, Klinefelter's syndrome, and Turner's syndrome, among other things, in addition to increasing the chances of successful pregnancy. Testing for chromosomal abnormalities estimates that at least 40% of embryos are abnormal between the ages of 35 and 40 and more than half of the embryos are abnormal beyond the age of 40. So it is especially important.

Recently, it has been discovered that cell-free fetal DNA and intact fetal cells can enter the maternal blood circulation system. Therefore, analysis of this genetic material may enable early NPD. It is desirable that the improved method increases sensitivity and specificity and reduces the time and cost required for NPD.

In one aspect, the invention features a method of amplifying a target locus in a nucleic acid sample. In some embodiments, the method comprises (i) nucleic acid samples 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 a step of contacting with a test primer library that simultaneously hybridizes to 100,000 different target loci to generate a reaction mixture, and (ii) the reaction mixture. It comprises a step of producing an amplification product containing a target amplification product by subjecting it to primer extension reaction conditions. In some embodiments, the method comprises targeting at least one target amplification product (eg, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5%). It also includes a step of determining the presence or absence of an amplification product). In some embodiments, the method comprises targeting at least one target amplification product (eg, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5%). It also includes the step of determining the sequence of the amplification product).

In various embodiments of any aspect of the 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% amplification products are target amplification products. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of target loci are amplified. In various embodiments, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplification products. Less than is a primer dimer. In some embodiments, the library of test primers is 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 containing a forward test primer and a reverse test primer that hybridize to the same target locus. In some embodiments, the library of test primers hybridizes to different target loci 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, and the individual primers are not part of the primer pair.

In various embodiments of any aspect of the 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 primers (eg, maximum GC content-minimum GC content, eg, 80% -60% = 20% range) is less than 30, 20, 10, or 5%. Is. In some embodiments, the melting temperature ( _Tm ) 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 in the range of 20, 15, 10, 5, 3, or less than 1 ° C. In some embodiments, the length of the test primer is 15-100 nucleotides, eg, 15-75 nucleotides, 15-40 nucleotides, 17-35 nucleotides, 18-30 nucleotides, 20-65 nucleotides. In some embodiments, the test primer comprises a non-target specific tag, eg, a tag that forms an internal loop structure. In some embodiments, the tag resides between two DNA binding regions. In various embodiments, the test primer comprises a 5'region specific for the target locus, an internal region that forms a loop structure that is not specific for the target locus, and a 3'region specific for 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 7-20 nucleotides, eg, 7-15 nucleotides, or 7-10 nucleotides. In various embodiments, the test primer is a 5'region that is not specific to the target locus (eg, a tag or universal primer binding site), followed by a region specific to the target locus, specific to the target locus. It contains an internal region that forms an untargeted loop structure and a 3'region that is specific to the target locus. In some embodiments, the range of test primer lengths is less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the length of the target amplification product is 50-100 nucleotides, for example 60-80 nucleotides, or 60-75 nucleotides. In some embodiments, the length range of the target amplification product is less than 50, 25, 15, 10, or 5 nucleotides.

In various embodiments of any aspect of the invention, the primer extension reaction condition is a polymerase chain reaction condition (PCR). 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.

In various embodiments of any aspect of the invention, at least 1,000 pieces in a sample containing maternal and fetal DNA from the pregnant mother of the fetus to determine the presence or absence of fetal chromosomal abnormalities. Test primers are used to simultaneously amplify different target loci. In various embodiments, the method comprises ligating a universal primer binding site to a DNA molecule in a sample and amplifying the ligated DNA molecule with at least 1,000 specific and universal primers. It comprises the step of producing a set of 1 amplification product and the step of amplifying the set of the first amplification product with at least 1,000 pairs of specific primers to generate the set of the second amplification product. 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 of any aspect of the invention, test primers are used to simultaneously amplify at least 1,000 different target loci in a sample containing DNA from a person allegedly the father of a fetus. Further, the target loci in the sample containing the maternal DNA derived from the pregnant mother of the fetus and the fetal DNA are simultaneously amplified to determine whether the person to be the father is the biological father of the fetus.

In various embodiments of any aspect of the invention, test primers are used to simultaneously amplify at least 1,000 different target loci in one embryo-derived cell or cells to chromosomally. The presence or absence of an abnormality is determined. In various embodiments, cells from a collection of two or more embryos are analyzed and one embryo is selected for in vitro fertilization.

In various embodiments of any aspect of the invention, test primers are used to simultaneously amplify at least 1,000 different target 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.

In various embodiments of any aspect of the invention, the method uses test primers to simultaneously amplify at least 1,000 different target loci in a control nucleic acid sample to produce a first target amplification product. And the steps of simultaneously amplifying the target loci in the test nucleic acid sample to generate the second target amplification product set and the first and second target amplification product sets are compared and targeted. Includes a step to determine if the locus is present in one sample but not in the other sample, or if the target locus is present at different levels in the control sample and the test sample. .. In various embodiments, the test sample is from an individual who is of interest or phenotype (eg, cancer) or who is suspected of having an increased risk of being of interest or phenotype, and one or more. Target loci include sequences (eg, polymorphisms or other mutations) that are associated with an increased risk of the disease or phenotype of interest, or are associated with the disease or phenotype of interest. In various embodiments, the method uses test primers to simultaneously amplify 1,000 different target loci in a control sample containing RNA to produce a set of first target amplification products, further. The step of simultaneously amplifying the target locus in the test sample containing RNA to generate the second target amplification product set is compared with the first and second target amplification product sets, and the control sample and the test sample are compared. Includes a step of determining the presence or absence of differences in RNA expression levels between. In various embodiments, the RNA is mRNA. In various embodiments, the test sample is from an individual suspected of having a target disease or phenotype (eg, cancer) or an increased risk of the target disease or phenotype (eg, cancer). Yes, one or more target loci are associated with an increased risk of the disease or phenotype, or include sequences associated with the disease or phenotype (eg, polymorphisms or other variants). .. In some embodiments, the test sample is from an individual diagnosed with the disease or phenotype (eg, cancer), in which case the difference in RNA expression levels between the control sample and the test sample is targeted. It is shown that the locus contains a sequence (eg, a polymorphism or other mutation) associated with an increased or decreased risk of the disease or phenotype of interest.

In some embodiments of any aspect of the invention, the test primer is selected from a library of candidate primers based on one or more parameters, such as primer selection using any of the methods of the invention. Will be done. In some embodiments, test primers are selected from the candidate primer library based at least in part on the primer dimer forming ability of the candidate primer.

In one aspect, the invention features a method of selecting test primers from a candidate primer library. In various embodiments, the selection is (i) a step of using a computer to calculate an undesignability score for most or all possible combinations of the two candidate primers from the library. , Each undesignability score is at least partially based on the likelihood of dimer formation between the two candidate primers, and (ii) candidate primers from the candidate primer library with the highest undesignability score. And (iii) if the candidate primer removed in step (iii) is a member of the primer pair, the step of removing the other member of the primer pair from the candidate primer library, and (iv). Optionally, it comprises repeating steps (ii) and (iii) to select a test primer library. In some embodiments, the selection method is performed until all undesignability scores for the combination of candidate primers remaining in the library are below the minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to the desired number. In various embodiments, the undesignability 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 are 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 can be amplified simultaneously. In various embodiments, the method comprises (v) contacting a nucleic acid sample containing a target locus with a candidate primer remaining in the library to generate a reaction mixture; and (vi) primer extension of the reaction mixture. It also includes a step of producing an amplification product containing a target amplification product by subjecting it to reaction conditions.

In one aspect, the invention features a method of selecting test primers from a candidate primer library. In various embodiments, the method of selecting test primers from the candidate primer library is to (i) use a computer to obtain an undesignability score for most or all possible combinations of the two candidate primers from the library. The steps to calculate, where each undesignability score is at least partially based on the likelihood of dimer formation between the two candidate primers, and (ii) the first minimum threshold from the candidate primer library. A step of removing a candidate primer that is part of the largest number of combinations of two candidate primers having an undesignability score exceeding, and a step of removing the candidate primer in (iii) step (ii) are primer pairs. If it is a member, a step of removing the remaining members of the primer pair from the candidate primer library and (iv) optionally repeating steps (ii) and (iii) to select a test primer library. And include. In some embodiments, the selection method is performed until the undesignability score for the combination of candidate primers remaining in the library is all below the first minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to the desired number. In various embodiments, the undesignability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% of possible candidate primer combinations in the library. In various embodiments, the candidate primers remaining in the library are 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 can be amplified simultaneously. In various embodiments, the method comprises (v) contacting a nucleic acid sample containing a target locus with a candidate primer remaining in the library to generate a reaction mixture; and (vi) primer extension of the reaction mixture. It also includes the step of producing an amplification product containing the target amplification product according to the reaction conditions.

In various embodiments of any aspect of the invention, the selection method is a candidate remaining in the library by lowering the first minimum threshold used in step (ii) to a smaller second minimum threshold. It includes a step of further reducing the number of primers and an optional step of repeating (ii) and (iii). In some embodiments, the selection method increases the first minimum threshold used in step (ii) to a larger second minimum threshold, and optionally repeats (ii) and (iii). including. In some embodiments, the selection method is until the undesignability score for the combination of candidate primers remaining in the library is all below the second minimum threshold, or the candidate primers remaining in the library. It is carried out until the number is reduced to the desired number.

In various embodiments of any aspect of the invention, the method comprises the step of identifying or selecting a primer that hybridizes to the target locus prior to step (i). In some embodiments, multiple primers (or primer pairs) hybridize to the same target locus, but one for this target locus using a selection method based on one or more parameters. A primer (or a pair of primers) is selected. In various embodiments, the method comprises removing from the library a primer pair that produces a target amplification product that overlaps with a target amplification product produced by another primer pair prior to step (ii). In various embodiments, candidate primers from a group of two or more candidate primers with comparable undesignability scores based on one or more other parameters for removal from the candidate primer library. Is selected. In some embodiments, the candidate primers remaining in the library are used as the test primer library in any of the methods of the invention. In some embodiments, the resulting test primer library comprises any of the primer libraries of the invention.

In various embodiments of any aspect of the invention, the undesignability score is the heterozygotes of the target locus, the prevalence associated with the sequence of the target locus (eg, polymorphism), the prevalence of the target locus. Disease penetration associated with the sequence (eg, polymorphism), specificity of the candidate primer for 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 at least in part based on one or more parameters selected from the group consisting of the size of the target amplification product.

In various embodiments of any aspect of the invention, the undesignability score determines the heterozygous rate of the target locus, the specificity of the candidate primer for the target locus, the size of the candidate primer, the melting temperature of the target amplification product, and the melting temperature of the target amplification product. Fetal chromosomes based on at least one or more parameters selected from the group consisting of the GC content of the target amplification product, the amplification efficiency of the target amplification product, and the size of the target amplification product, and using test primers. At least 1,000 different target loci in a sample containing maternal and fetal DNA from the pregnant mother of the fetal to determine the presence or absence of anomalies are simultaneously amplified. In various embodiments, the method involves ligating a universal primer binding site to a DNA molecule in a sample and amplifying and first amplifying the ligated DNA molecule with at least 1,000 specific and universal primers. It comprises the step of producing a set of products and the step of amplifying the set of first amplification products with at least 1,000 pairs of specific primers to produce a set of second 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.

In various embodiments of any aspect of the invention, the undesignability score determines the heterozygous rate of the target locus, the specificity of the candidate primer for the target locus, the size of the candidate primer, the melting temperature of the target amplification product, and the melting temperature of the target amplification product. It is based at least in part on one or more parameters selected from the group consisting of the GC content of the target amplification product, the amplification efficiency of the target amplification product, and the size of the target amplification product, and also using test primers. At least 1,000 different target loci are amplified in samples containing DNA from the alleged father of the fetal, and targets in samples containing maternal and fetal DNA from the pregnant mother of the fetal. The loci are simultaneously amplified to determine if the alleged father is the father of a biological fetal. 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.

In various embodiments of any aspect of the invention, the undesignability score determines the heterozygous rate of the target locus, the specificity of the candidate primer for the target locus, the size of the candidate primer, the melting temperature of the target amplification product, and the melting temperature of the target amplification product. It is based at least in part on one or more parameters selected from the group consisting of the GC content of the target amplification product, the amplification efficiency of the target amplification product, and the size of the target amplification product, and also using test primers. At least 1,000 different target loci in one embryo-derived cell or cells are simultaneously amplified to determine the presence or absence of chromosomal abnormalities. In various embodiments, cells 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.

In various embodiments of any aspect of the invention, the undesignability score determines the heterozygous rate of the target locus, the specificity of the candidate primer for the target locus, the size of the candidate primer, the melting temperature of the target amplification product, and the melting temperature of the target amplification product. It is based at least in part on one or more parameters selected from the group consisting of the GC content of the target amplification product, the amplification efficiency of the target amplification product, and the size of the target amplification product, and also using test primers. At least 1,000 different target loci in forensic nucleic acid samples are simultaneously amplified. 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.

In various embodiments of any aspect of the invention, the undesignability score is the heterozygous rate of the target locus, the prevalence associated with the sequence of the target locus (eg, polymorphism), the prevalence of the target locus. Disease permeability associated with the sequence (eg, polymorphism), specificity of the candidate primer for 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 at least in part based on one or more parameters selected from the group consisting of the size of the target amplification product, the method also uses test primers to at least 1,000 in a control nucleic acid sample. A step of simultaneously amplifying different target loci to generate a first target amplification product set, and simultaneously amplifying the target loci in the test nucleic acid sample to generate a second target amplification product set, and a first step. And the second target amplification product set, whether the target locus is present in one sample and not in the other, or at different levels of the target locus in the control and test samples Includes a step to determine if it exists in. In various embodiments, the test sample is of the subject disease or phenotype, or of an individual suspected of having an increased risk of the subject disease or phenotype, in which case one or more target loci are targeted. A locus contains a sequence (eg, a polymorphism) associated with an increased risk of a subject disease or phenotype or associated with a subject 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.

In various embodiments of any aspect of the invention, the undesignability score is the heterozygous rate of the target locus, the prevalence associated with the sequence of the target locus (eg, polymorphism), the prevalence of the target loci. Disease permeability associated with the sequence (eg, polymorphism), specificity of the candidate primer for 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 at least in part based on one or more parameters selected from the group consisting of the size of the target amplification product, and the method is 1,000 in a control nucleic acid sample containing RNA using test primers. A step of simultaneously amplifying different target loci to generate a first set of target amplification products and simultaneously amplifying the target loci in a test sample containing RNA to generate a second set of target amplification products. And the step of comparing the set of the first and second target amplification products to determine the presence or absence of a difference in RNA expression level between the control sample and the test sample. In various embodiments, the RNA is mRNA. In various embodiments, the test sample is from an individual suspected of having an increased risk of the subject disease or phenotype (eg, cancer) or the subject disease or phenotype (eg, cancer), in this case one or more. Multiple target loci include sequences (eg, polymorphisms or other mutations) that are associated with an increased risk of the target disease or phenotype or are associated with the target disease or phenotype. In some embodiments, the test sample is from an individual diagnosed with the disease or phenotype (eg, cancer), in which case the difference in RNA expression levels between the control sample and the test sample is , Indicates that the target locus contains a sequence (eg, a polymorphism or other mutation) associated with an increase or decrease in the disease or phenotype of interest. 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.

In one aspect, the invention features a primer library. In some embodiments, primers are selected from a candidate primer library using any of the methods of the invention. In some embodiments, the library is 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 contains primers that simultaneously hybridize to 100,000 different target loci. In some embodiments, the library is 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 contains primers that simultaneously amplify 100,000 different target loci. In some embodiments, the library is less than 60, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05%. At least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50 so that the amplification product is a primer dimer. 000; 75,000; or contains primers that simultaneously amplify 100,000 different target loci. In some embodiments, the library has 1 such that at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% amplification product is the target amplification product. 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 Contains a primer that simultaneously amplifies the target locus. In some embodiments, the library is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% target loci from 100,000 different target loci are amplified. As such, it contains a primer that simultaneously amplifies the target locus. In some embodiments, the primer library is 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 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 libraries hybridize to different target loci, respectively, 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, the individual primers are not part of a primer pair.

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

In one aspect, the invention provides a kit comprising any of the primer libraries of the invention for amplifying a target locus in a nucleic acid sample. In some embodiments, the kit comprises instructions for amplifying a target locus using a library.

In one aspect, the invention features a method of determining the ploidy state of a fetal chromosome during pregnancy. In some embodiments, the method comprises at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000 of nucleic acid samples. The nucleic acid sample comprises the steps of contacting a primer library that simultaneously hybridizes to 50,000; 75,000; or 100,000 different polymorphic loci to generate a reaction mixture, and the nucleic acid sample is from the fetal mother. Includes maternal DNA and fetal DNA from the fetus. In some embodiments, the reaction mixture is subjected to primer extension reaction conditions to produce an amplification product, from which the amplification product is sequenced using a high throughput sequencer and computerized on the basis of the sequencing data. The number of alleles at the morphic loci is calculated, and multiple multiple hypotheses associated with possible multiple states of each different chromosome are computer-generated and predicted at the chromosomal polymorphic locus for each multiple hypothesis. A co-distribution model for the number of alleles is computer-constructed, the relative probability of each multiple hypothesis is computer-calculated using the co-distribution model and the number of alleles, and corresponds to the hypothesis with the highest probability. Choosing a chromosomal state calls the fetal chromosomal state.

In one aspect, the invention features a method of determining the ploidy state of a fetal chromosome during pregnancy. In one embodiment, the method for determining the chromosomal polymorphism in a pregnant fetus is the step of obtaining a sample of a first DNA containing maternal DNA from the mother of the fetus and fetal DNA from the fetus, and preparation. Preparation of the first sample by isolating the DNA so that the obtained sample can be obtained, and the step of measuring the DNA in the prepared sample at multiple polymorphic loci on the chromosome. Multiple multiple hypotheses regarding the steps to computerize the number of allogens at multiple polymorphic loci from DNA measurements made on the sample and each of the possible different multiple states on the chromosome. And a computer-built co-distribution model for the expected number of allogens at multiple polymorphic loci on the chromosome for each multiple hypothesis, a co-distribution model and prepared. Fetal multiples by computer-determining the relative probability of each of the multiple hypotheses using the number of allogens measured in the sample and by selecting the polyploid state corresponding to the hypothesis with the highest probability. Includes a step to call the state.

In one aspect, the invention features a method of examining chromosomal aberrations in a sample containing a mixture of maternal and fetal DNA. In some embodiments, the method is (i) 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 a step of contacting a sample with a primer library that simultaneously hybridizes to 100,000 different target chromosomal loci to generate a reaction mixture with multiple target chromosomal loci. At least one first chromosome that is derived from a different chromosome and is suspected of having an abnormal distribution in the sample, and at least one presumed to be normally distributed in the sample. A step containing 2 chromosomes, (ii) a step of subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product, and (iii) a plurality of sequence tags in which the amplification product was sequenced and aligned with the target loci. The steps to obtain, one in which the sequence tag is long enough to be assigned to a particular target locus, and (iv) a step in which a computer assigns multiple sequence tags to their corresponding target loci, (. v) From step (v) on a computer to determine the number of sequence tags to be assigned to the target locus of the first chromosome and the number of sequence tags to be assigned to the target locus of the second chromosome. It includes a step of comparing numbers to determine the presence or absence of an abnormal distribution of the first chromosome.

In one aspect, the invention provides a method of detecting the presence or absence of fetal aneuploidy. In some embodiments, the method comprises (i) a sample containing a mixture of maternal and fetal DNA at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; With a primer library that simultaneously hybridizes to non-polymorphic target loci from 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different, different chromosomes. A step of contacting to generate a reaction mixture, (ii) subjecting the reaction mixture to primer extension reaction conditions to produce an amplification product containing a target amplification product, and (iii) a first and second subject on a computer. A step of quantifying the relative frequency of the target amplification product derived from a chromosome, (iv) a step of comparing the relative frequency of the target amplification product derived from the first and second chromosomes of the subject with a computer, and (v) the first step of the subject. It comprises the step of identifying the presence or absence of aneuploidy based on the relative frequencies of the first and second chromosomes compared. In some embodiments, the first chromosome is a chromosome suspected to be a positive polyploid. In some embodiments, the second chromosome is a chromosome suspected to be aneuploidy.

In one aspect, it is a method for determining the presence or absence of fetal heterogeneity in a maternal tissue sample containing fetal genomic DNA and maternal genomic DNA, wherein (a) the maternal tissue sample is used to determine the presence or absence of a fetal chromosomal. Large-scale parallel DNA sequencing of DNA fragments randomly selected from the steps to obtain a mixture of chromosomal DNA and maternal chromosomal DNA and (b) a mixture of fetal and maternal chromosomal DNA in step (a). The step of determining the sequence of the DNA fragment, the step of (c) identifying the chromosome to which the sequence obtained in step (b) belongs, and (d) the data of step (c) are used. A step in determining the amount of at least one first chromosome in a mixture of maternal and fetal genomic DNA, wherein the at least one first chromosome is presumed to be positive multiple in the fetal. And (e) a step of determining the amount of the second chromosome in the mixture of the maternal genomic DNA and the fetal genomic DNA using the data of the second chromosome. However, the step suspected to be an aneuploid in the fetal, (f) the step of calculating the ratio of fetal DNA in the mixture of fetal DNA and maternal DNA, and (g) the second target chromosome is positive multiple. If, the step (d) is used to calculate the predicted distribution of the amount of the second target chromosome, and (h) if the second target chromosome is heterogeneous, the step (d). ) And the step of calculating the predicted distribution of the amount of the second target chromosome using the ratio of the fetal DNA in the mixture of the fetal DNA and the maternal DNA calculated in the first number and the step (f). (I) One of the distributions calculated in step (g) or the distribution calculated in step (h) for the amount of the second chromosome determined in step (e) using the most probable method or the maximum posterior method. Methods are disclosed that include a step that determines if it is more likely to be a part, thereby indicating the presence or absence of chromosomal chromosomality in the fetal.

In various embodiments of any aspect of the invention, the method also includes the step of obtaining genotype data from one parent or parent of the fetus. In some embodiments, the step of obtaining genotype data from one parent or parent of the fetus is the step of preparing DNA from the parent, which preferentially enriches the DNA at multiple polymorphic loci. Steps involving obtaining the prepared parental DNA, optionally amplifying the prepared parental DNA, and measuring the parental DNA in the prepared sample at multiple polymorphic loci. Including steps to do.

In various embodiments of any aspect of the invention, one parent or parent obtains a step of constructing a joint distribution model for the probability of a predicted allele number at multiple polymorphic loci on a chromosome. It is performed using the obtained genetic data. In some embodiments, a sample (eg, a first sample) is isolated from the maternal plasma and a step of obtaining genotype data from the mother is performed on the prepared sample from DNA measurements to the genotype of the maternal line. This is done by estimating the data.

In one aspect, a diagnostic box is disclosed that aids in the determination of chromosomal ploidy status in a pregnant fetus and is capable of performing the preparation and measurement steps of any of the methods of the invention. Has been done.

In various embodiments of any aspect of the invention, the number of alleles is probabilistic rather than binary. In some embodiments, measurements of DNA in the prepared sample at multiple polymorphic loci are also used to determine if the fetus has one or more disease chain haplotypes.

In various embodiments of any aspect of the invention, the steps of constructing a co-distribution model for the probability of allele numbers are performed on the chromosome using data on the probability of chromosome crossover at different locations within the chromosome. This is done by modeling the dependence between polymorphic alleles. In some embodiments, the steps of constructing a joint distribution model for the number of alleles and determining the relative probabilities of each hypothesis are performed using methods that do not require the use of reference chromosomes.

In various embodiments of any aspect of the invention, an 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 samples used in the step of calculating the probability of the number of alleles 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 a posteriori estimation.

In various embodiments of any aspect of the invention, the steps to call the fetal statistic state are with the respective relative probabilities of the joint probabilities determined using the joint distribution model and the probability of the number of allogenes. , Read count analysis, Heterojunction rate comparison, Statistics available only when using parental genetic information, Probability of genotype signals normalized to a particular parental situation, Calculated using statistics calculated using the estimated fetal fraction of the sample (eg, the first sample) or the prepared sample, and statistical techniques selected from the group consisting of combinations thereof. It also includes the steps to combine with each relative probability of the multiple hypothesis.

In various embodiments of any aspect of the invention, confidence estimates are calculated for the called ploidy state. In some embodiments, the method also includes the step of taking a clinical measure of choice between abortion or maintaining pregnancy based on the ploidy status of the called fetus.

In various embodiments of any aspect of the invention, the method comprises 4-5 weeks gestation; 5-6 weeks gestation; 6-7 weeks gestation; 7-8 weeks gestation. Weeks; 8-9 weeks gestation; 9-10 weeks gestation; 10-12 weeks gestation; 12-14 weeks gestation; 14-20 weeks gestation; pregnancy It can be performed on the fetus between 20 and 40 weeks; early pregnancy; mid-pregnancy; late pregnancy; or a combination thereof.

In various embodiments of any aspect of the invention, the method is used to make a report showing the determined ploidy state of a chromosome in a pregnant fetus. In some embodiments, a kit designed for use in any of the methods of the invention, for determining the polymorphic state of a target chromosome in a pregnant fetus, with multiple inner forward primers. And, optionally, multiple inner reverse primers, each of which is designed to hybridize to regions of DNA immediately upstream and / or downstream of one of the polymorphic sites on the target chromosome. The primer and, if necessary, yet another chromosome, the hybridizing region, is separated from the polymorphic site by a small number of bases, the minority being 1, 2, 3, 4, ... Kits are disclosed that include 5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-60, and chromosomes selected from the group consisting of combinations thereof.

In one aspect, the invention comprises a method of determining whether a person allegedly a father is the biological father of a fetus in which a pregnant mother is pregnant. In some embodiments, the method is (i) at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; from the alleged father. Simultaneously amplify multiple polymorphic loci including polymorphic loci on 30,000; 40,000; 50,000; 75,000; or 100,000 different genetic material of the first amplification product. The steps to generate the assembly and (ii) simultaneously amplify the corresponding polymorphic loci of a mixed sample of fetal DNA from a pregnant mother's blood sample and DNA containing maternal DNA to produce a second amplification product. The steps to generate the set and (iii) on the computer determine the probability that the alleged father is the father of a biological fetal using genotype measurements based on the set of first and second amplification products. And (iv) the step of determining whether or not the person to be the father is the father of the biological fetal using the determined probability that the person is the father of the biological fetal. include. In various embodiments, the method further comprises the step of simultaneously amplifying a polymorphic locus on a corresponding genetic material of maternal origin to produce a third set of amplification products, in this case with the father. The probability that a person will be the father of a biological fetal is determined using genotyping based on a set of first, second, and third amplification products.

In one aspect, the invention provides a method of estimating the relative likelihood of developing each embryo from a set of embryos as desired. In some embodiments, the method comprises at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 30,000; samples from each embryo. The sample comprises the steps of contacting with a primer library that simultaneously hybridizes to 40,000; 50,000; 75,000; or 100,000 different target loci to generate a reaction mixture for each embryo. Obtained from one or more embryo-derived cells, respectively. In some embodiments, each reaction mixture is subjected to primer extension reaction conditions to produce an amplification product. In some embodiments, the method is computerized to determine the properties of one or more cells of at least one cell from each embryo based on the amplification product and computerized to at least each embryo. It comprises the step of estimating the relative likelihood of developing each embryo as desired, based on the properties of one or more cells.

In one aspect, the invention features a method of measuring the amount of two or more target loci in a nucleic acid sample. In some embodiments, the method uses (i) PCR to generate a nucleic acid comprising a first reference locus, a second reference locus, a first target loci, and a second target loci. In the step of amplifying a sample to form an amplification product, the first reference locus and the first target loci are the same number of nucleotides, but different sequences at the positions of one or more nucleotides. Steps that have the same number of nucleotides in the second reference locus and the second target loci but have different sequences at the positions of one or more nucleotides, and (ii) sequencing the amplification product. A step of determining a reference ratio for comparing the relative amount of the amplified first reference locus with respect to the amplified second reference locus, wherein the reference ratio is the first reference locus and A target ratio comparing the step showing the difference in PCR efficiency for the amplification of the second reference locus and (iii) the relative amount of the amplified first target locus compared to the amplified second target locus. And the relative of the first and second target loci in the sample by adjusting the target ratio from step (iii) based on the reference ratio from step (iv) step (iii). Includes steps to determine the amount. In various embodiments, the method comprises determining the absolute amount of a first target locus and a second target locus in a sample. In various embodiments, the method comprises a target locus in a sample (eg, at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30, It further comprises the step of determining the presence or absence of 000; 40,000; 50,000; 75,000; or 100,000 different target loci). In various embodiments, the method comprises the step of using any of the primer libraries of the invention. In various embodiments, the method is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; Includes the step of simultaneously amplifying 75,000; or 100,000 different target loci.

In one aspect, the invention features a method of quantitatively measuring a plurality of genetic targets of a sample for analysis. In some embodiments, the method comprises (i) mixing the genetic material 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) a step of amplifying a target region and a reference sequence of a genetic material to produce a target amplification product and a reference sequence amplification product, and (iii) a step of measuring the amount of the produced target amplification product and the reference sequence amplification product. including. In some embodiments, the genetic material is present in the genetic library. In some embodiments, the genetic target is a polymorphic locus (such as an SNP). In some embodiments, the step of measuring the quantity is accomplished by counting the sequences. In some embodiments, the method further comprises measuring the estimated copy number of at least one chromosome in the sample from which the gene library is derived, wherein the measurement is based on the sequence read number of the target amplification product. Includes steps to compare to the number of sequence reads in the product. In some embodiments, the reference sequence and gene library include a universal priming site that can be primed with the same primers. In some embodiments, the mixing step is 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 comprises the step of using any of the primer libraries of the invention. In various embodiments, the method is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or including the step of simultaneously amplifying 100,000 different target regions. In some embodiments, the relative amount of each of the reference sequences is known. In some embodiments, the relative amount of each of the sequences is calibrated to the reference genome. In some embodiments, the analytical sample comprises a mixture of fetal and maternal genomes. In some embodiments, the analytical sample is from the blood of a pregnant woman or from plasma. In some embodiments, the reference genome has at least one aneuploidy, such as the aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention features a mixture containing multiple gene reference sequences, the relative amount of each gene reference sequence in the mixture is determined by calibration to the reference genome. In various embodiments, the mixture is 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 contains 100,000 gene reference sequences. In various embodiments, the gene reference sequence is a first universal priming site, a second universal priming site, a first target-specific priming site, a second target-specific priming site, and first and second. It comprises a marker sequence located between the target-specific priming sites, the first target-specific site and the second target-specific priming site are located between the first and second universal priming sites. In various embodiments, the calibration comprises the step of using any of the primer libraries of the invention. In various embodiments, the calibration is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75. 000; or including the step of simultaneously amplifying 100,000 different target regions. In some embodiments, the reference genome has at least one aneuploidy, such as the aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention features a method of producing a calibrated set of genetic reference sequences. In some embodiments, the method comprises (i) a gene library prepared from a reference genome, a collection of multiple target-specific amplification primer reagents, and a plurality of genetic criteria corresponding to the collection of target-specific amplification reagents. A step of forming an amplification reaction mixture containing a sequence, (ii) a step of amplifying a gene library and a gene reference sequence to produce an amplification product derived from a target sequence and an amplification product derived from a gene reference sequence, and (iii) a target. Steps to measure the amount of sequence-derived amplification products and gene reference sequence-derived amplification products, and (iv) determine the relative amount of each gene reference sequence to each other, thereby calibrating multiple gene reference sequences. And include. 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 comprises the step of using any of the primer libraries of the invention. In various embodiments, the invention is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or including the step of simultaneously amplifying 100,000 different sequences. In some embodiments, the reference genome has at least one aneuploidy, such as the aneuploidy of chromosomes 13, 18, 21, X, or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention provides a set of genetic reference sequences calibrated by any of the methods of the invention. In one aspect, the invention provides a set of genetic reference sequences that can be calibrated before, during, and after the method is performed.

In one aspect, the invention features a method of measuring the number of copies of a gene of interest, including at least one allele with a deletion. In some embodiments, the method comprises (i) an amplification reagent, subject that is specific to the gene of interest and is unable to significantly amplify deletions containing the allelic gene of the gene of interest from the genetic material derived from the sample for analysis. The step of mixing with the reference sequence corresponding to the gene of interest, the amplification reagent specific to the reference sequence, and the reference sequence corresponding to the reference sequence, and (ii) the gene sequence of interest, the reference sequence corresponding to the gene of interest, the reference sequence. , And the step of amplifying the reference sequence corresponding to the reference sequence to generate the gene amplification product, the reference sequence amplification product, and the reference sequence amplification product of interest, and (iii) the amount of the production target amplification product and the reference sequence amplification product. Includes steps to measure. In some embodiments, the measurement of quantity is achieved by counting sequence reads. In some embodiments, the method further comprises calculating the estimated number of copies of chromosomes in a sample from which at least one gene library is derived, where the calculation step is the number of sequences of the target amplification product. Includes a step to compare with the number of sequences of the reference amplification product. In some embodiments, the reference sequence and gene library include a universal priming site that can be primed with the same primers. In some embodiments, the relative amount of each sequence is calibrated to the 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 comprises the step of using any of the primer libraries of the invention. In various embodiments, the method is 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or including the step of simultaneously amplifying 100,000 different target regions. In some embodiments, the reference genome is diploid. In some embodiments, the analytical sample is of blood origin.

In various embodiments of any aspect of the invention, the step of preferentially enriching the DNA in a sample (eg, a first sample) at a target locus (eg, a plurality of polymorphic loci) is a step. Multiple pre-cyclization probes, each of which targets one of the loci (eg, polymorphic loci), preferably the 3'and 5'ends of the probe are loci. It is designed to hybridize to a region of DNA that is separated from the polymorphic site by a small number of bases, with the minority being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ,. Steps to obtain probes 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof, and pre-cyclization probes and samples. A step of hybridizing the DNA derived from (for example, the first sample), a step of filling the gap between the hybridized probe ends with a DNA polymerase, a step of cyclizing the pre-cyclization probe, and cyclization. Includes a step of amplifying the probe.

In various embodiments of any aspect of the invention, the step of preferentially enriching DNA at a target locus (eg, a plurality of polymorphic loci) is a plurality of ligation-mediated PCR probes. Each PCR probe targets one of the target loci (eg, polymorphic loci), the upstream and downstream PCR probes of which are preferably separated from the polymorphic site of the loci with a small number of bases. It is designed to hybridize to a region of the DNA on one strand of the DNA, the minority of which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Steps to obtain a PCR probe of 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-60, or a combination thereof, and a ligation-mediated PCR probe and sample (eg,). , The step of hybridizing the DNA derived from (1st sample), the step of filling the gap between the ends of the ligation-mediated PCR probe with a DNA polymerase, and the step of ligating the ligation-mediated PCR probe. Includes a step of amplifying a ligation-mediated PCR probe.

In some embodiments of the various embodiments of the invention, the step of preferentially enriching DNA at a target locus (eg, a plurality of polymorphic loci) is a locus (eg, a polymorphic locus). A step to obtain multiple target hybrid capture probes, a step to hybridize the hybrid capture probe with DNA in a sample (eg, first sample), and a hybrid from a sample relating to DNA (eg, first sample). It comprises the steps of physically removing some or all of the non-soybean DNA.

In some embodiments of any aspect of the invention, the hybrid capture probe is designed to hybridize to regions that are adjacent to but not overlapping with the polymorphic site. In some embodiments, the hybrid capture probe is designed to hybridize to regions that are adjacent but not overlapping with the polymorphic site, and the length of the adjacent capture probe is approximately 120 bases. Less than, 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 about 25 You can choose from a group of less than bases. In some embodiments, the hybrid capture probe is designed to hybridize to a region that overlaps the polymorphic site, and the plurality of hybrid capture probes is at least two hybrid capture probes for each polymorphic locus. Each hybrid capture probe is designed to be complementary to another allele at one polymorphic locus.

In some embodiments of any aspect of the invention, the step of preferentially enriching DNA at multiple polymorphic loci is a plurality of inner forward primers, each primer being a polymorphic gene. Targeting one of the loci so that the 3'end of the inner forward primer is upstream of the polymorphic site and hybridizes to a region of DNA separated from the polymorphic site by a small number of base pairs. Designed, a few are 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. Steps to obtain primers selected from the group consisting of pairs, 26-30 base pairs or 31-60 base pairs, and optionally multiple inner reverse primers, each primer at the polymorphic locus. Designed to target one of these and hybridize with a region of DNA that has the 3'end of the inner reverse primer upstream of the polymorphic site and is separated from the polymorphic site by a small number of base pairs. A few of them are 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 to 10 base pairs, 11 to 15 base pairs, 16 to 20 base pairs, 21 to 25 base pairs, 26. A step of obtaining a primer selected from the group consisting of ~ 30 base pairs or 31 ~ 60 base pairs, a step of hybridizing the inner primer with DNA, and a step of amplifying DNA using a polymerase chain reaction to form an amplification product. Including steps to do.

In some embodiments of any aspect of the invention, the method is a plurality of outer forward primers, each primer having one of a target loci (eg, a polymorphic locus). A step to target and obtain primers designed to hybridize with regions of DNA upstream of the inner forward primer, and optionally multiple outer reverse primers, each primer being the target gene. A step to obtain a primer designed to target one of the loci (eg, a polymorphic locus) and hybridize with a region of DNA just downstream of the inner reverse primer, and a first primer. It further comprises a step of hybridizing with the DNA and a step of amplifying the DNA using a polymerase chain reaction.

In some embodiments of any aspect of the invention, the method is a plurality of outer reverse primers, each primer having one of a target loci (eg, a polymorphic locus). A step to target and obtain primers designed to hybridize with regions of DNA immediately downstream of the inner reverse primer, and optionally multiple outer forward primers, each with multiple primers. A step of targeting one of the type gene loci to obtain a primer designed to hybridize with a region of DNA upstream of the inner forward primer, and a step of hybridizing the first primer with DNA. , Further comprising the step of amplifying the DNA using a polymerase chain reaction.

In some embodiments of any aspect of the invention, the sample (eg, first sample) preparation step is the step of adding a universal adapter to the DNA in the sample (eg, first sample) and the polymerase chain. It further comprises the step of amplifying the DNA in the sample (eg, the first sample) using the 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, and a portion is 10. %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%.

In some embodiments of any aspect of the invention, the step of amplifying DNA is performed in one or more individual reaction volumes, each with more than 100 different forward primers. Reverse Primer Pairs, Over 200 Different Forward and Reverse Primer Pairs, Over 500 Different Forward and Reverse Primer Pairs, Over 1,000 Different Forward and Reverse Primer Pairs, Over 2,000 Different Forwards Primer and Reverse Primer Pairs, Over 5,000 Different Forward and Reverse Primer Pairs, Over 10,000 Different Forward and Reverse Primer Pairs, Over 20,000 Different Forward and Reverse Primer Pairs, 50 It contains more than 000 different forward and reverse primer pairs, or more than 100,000 different forward and reverse primer pairs.

In some embodiments of any aspect of the invention, the step of preparing a sample (eg, first sample) is the step of dividing the sample (eg, first sample) into multiple portions. , Further comprising the step of preferentially enriching the DNA within each moiety in a subset of target loci (eg, multiple polymorphic loci). In some embodiments, at least a step of identifying a primer pair that may form an undesired primer double chain and a pair of primers that are identified as having the potential to form an undesired primer double chain. The inner primer is selected by the step of removing one from multiple primers. In some embodiments, the inner primer contains a region designed to hybridize either upstream or downstream of the target locus (eg, a polymorphic locus) and, if desired, PCR. It contains a universal priming sequence designed to allow amplification. In some embodiments, at least some of the primers further contain different random regions for each of the individual primer molecules. In some embodiments, at least some of the primers further contain molecular barcodes.

In some embodiments of any aspect of the invention, by preferential enrichment, between the prepared sample and the sample (eg, the first sample) no more than twice, no more than 1.5 times. , 1.2 times or less, 1.1 times or less, 1.05 times or less, 1.02 times or less, 1.01 times or less, 1.005 times or less, 1.002 times or less, 1.001 times or less and 1 It results in an average allele bias to the extent of the coefficients selected from the group consisting of .0001 times or less. In some embodiments, the polymorphic locus is an SNP. In some embodiments, the step of measuring DNA in the prepared sample is performed by sequencing.

In some embodiments of any aspect of the invention, the target locus resides on the same nucleic acid of the control (eg, the same chromosome or the same region of the chromosome). In some embodiments, at least some target loci are present on different nucleic acids of interest (eg, 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 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 is blood, plasma, saliva, sperm, sperm, cell culture supernatant, mucus secretion, dentin, gastrointestinal tissue, stool, urine, hair, bone, body fluid, tears, tissue, Contains or is derived from skin, nails, blastomere, embryos, sheep's water, 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 comprises or is composed of a single nucleotide polymorphism (SNP). In some embodiments, the primer is a DNA molecule.

In some embodiments of any aspect of the invention, the DNA in the sample (eg, first sample) is derived from maternal plasma. In some embodiments, the step of preparing a sample (eg, a first sample) further comprises the step of amplifying the DNA. In some embodiments, the step of preparing a sample (eg, first sample) prioritizes the DNA in the sample (eg, first sample) at the target locus (eg, multiple polymorphic loci). Further includes steps to enrich the target.

In various embodiments, the primer extension reaction or polymerase chain reaction comprises the addition of one or more nucleotides by the polymerase. In various embodiments, the primer extension reaction or polymerase chain reaction does not include ligation-mediated PCR. In various embodiments, the primer extension reaction or polymerase chain reaction does not involve the ligation of the two primers with ligase. In various embodiments, the primer does not include a ligated reverse probe (LIP). This probe is also referred to as a pre-cyclalized probe, a pre-cyclalized probe or a cyclized probe, a cyclized probe, a padlock probe, or a molecular inversion probe (MIP).

All aspects and embodiments of the invention described herein are "comprising", embodiments "consisting", and embodiments and embodiments "comprising". It will be understood that it includes "consisting essentially of".

Definition Single nucleotide polymorphism (SNP) refers to a single nucleotide that can differ between the genomes of two members of the same species. The use of this term should not imply any limitation on the frequency with which each variant occurs.

A sequence refers to a DNA sequence or a gene sequence. A sequence can refer to the primary physical structure of an individual's DNA molecule or strand. The sequence can refer to a DNA molecule or a sequence of nucleotides found in a complementary strand of a DNA molecule. The sequence can refer to the information contained in the DNA molecule, which is displayed in silico.

A locus refers to a particular region of interest on an individual's DNA and can refer to an SNP that is the site of a possible insertion or deletion or the site of some other relevant genetic variation. .. The disease chain SNP may also refer to a disease chain locus.

A polymorphic allele, as well as a "polymorphic locus," refers to an allele or locus whose genotype varies between individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications, and inversions.

A polymorphic site refers to a specific nucleotide found in a polymorphic region that varies from individual to individual.

An allele is a gene that occupies a specific locus.

Genetic data, as well as "genotype data", refers to data that describes the aspects of the genome of one or more individuals. This can refer to a locus or set of loci, a partial or whole sequence, a part of a chromosome or an entire chromosome, or an entire genome. This may refer to the identity of one or more nucleotides, which may refer to a sequential set of nucleotides or nucleotides from different locations within the genome, or a combination thereof. The genotype data is generally in silico, but it is also possible to think of physical nucleotides in the sequence as chemically encoded genetic data. Genotype data can be referred to as an individual (s) "on", "of", "at", "from" or "on". .. Genotyping data can refer to output measurements from genotyping platforms when these measurements are made on genetic material.

A genetic material, as well as a "gene sample", refers to a physical substance such as tissue or blood from one or more individuals containing DNA or RNA.

Gene data with noise refers to genetic data with any of the following: allelic dropouts, uncertain base pair measurements, inaccurate base pair measurements, missing base pair measurements, insertions or omissions: Uncertain measurement of loss, uncertain measurement of chromosomal segment copy count, false signal, missing measurement, other error, or a combination thereof.

Confidence refers to the statistical likelihood that the number of SNPs called, alleles, sets of alleles, haploid calls or determined chromosomal segment copies accurately indicates the actual genetic state of the individual.

A polyploid call, as well as a "chromosome copy number call" or "copy number call" (CNC), is the act of determining the amount and / or identity of one or more chromosomes present in a cell. Can point to.

Aneuploidy means the presence of an incorrect number of chromosomes in a cell (eg, an incorrect number of complete chromosomes or an incorrect number of chromosomal segments, eg, the presence of deletions or duplications of chromosomal segments). .. In the case of human somatic cells, aneuploidy can refer to the case where the cell does not contain 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes. In the case of human gametes, aneuploidy can refer to the case where the cell does not contain one of each of the 23 chromosomes. In the case of a single chromosomal type, aneuploidy can refer to the presence of roughly two homologous but not identical chromosomal copies, or the presence of two chromosomal copies originating from the same parent. .. In some embodiments, the deletion of a chromosomal segment is a microdeletion.

Ploidy state refers to the amount and / or chromosomal identity of one or more chromosomal types in a cell.

Chromosome may refer to a single chromosome copy, which means a single molecule of DNA present in 46 normal somatic cells, an example of which is "maternally derived chromosome 18." The chromosome may refer to a chromosome type existing in 23 normal human somatic cells, for example, "chromosome 18".

Chromosome identity can refer to the referent number of chromosomes, i.e., the chromosomal type. Normal humans have 22 numbered autosomal types and two sex chromosomes. Chromosome identity may also refer to a chromosome of parental origin. Chromosome identity may also refer to a particular chromosome that is inherited from the parent. Chromosome identity may also refer to other identified forms of chromosomes.

The state of the genetic material or simply the "gene state" is the identity of the set of SNPs on the DNA, the haplotype of the phased genetic material, and the DNA including insertions, deletions, repetitions and mutations. Can point to an array of. This may refer to a polyploid state of one or more chromosomes, a chromosomal segment or a set of chromosomal segments.

Allele data refers to a set of genotype data relating to a set of one or more alleles. Allelic data can refer to phase-identified haplotype data. Allelic data may refer to SNP identity and allelic data may refer to DNA sequence data including insertions, deletions, repeats and mutations. The allele data may include each allele of parental origin.

Allele state refers to the actual state of a gene within a set of one or more alleles. The allelic state can refer to the actual state of the gene described in the allele data.

Allele ratio or allele ratio refers to the ratio between the amount of each allele at a locus present in a sample or individual. When the sample is measured by sequencing, the allele ratio can refer to the ratio of sequence reads mapped to each allele at the locus. When the sample is measured by an intensity-based measurement method, the allele ratio can refer to the ratio of the amount of each allele present at the locus estimated by the measurement method.

The number of alleles refers to the number of sequences mapped to a particular locus, and if the locus is polymorphic, the number of alleles refers to the number of sequences mapped to each of the alleles. If each allele is counted in binary mode, the number of alleles will be an integer. If alleles are stochastically counted, the number of alleles can be a fraction.

Allele number probability refers to the number of sequences that may be mapped to a set of alleles at a particular locus or polymorphic locus, combined with the mapping probability. Note that the probability of mapping for each of the counted sequences is binary (0 or 1) and the number of alleles is equal to the probability of the number of alleles. In some embodiments, the probability of allele number may be binary. In some embodiments, the probability of the number of alleles can be set to be equal to the DNA measurement.

Allele distribution or "allele number distribution" refers to the relative amount of each allele present at each locus within the set of loci. Allelic distribution may refer to an individual, sample or set of measurements obtained for a sample. In the context of sequencing, allele distribution refers to the number of reads or prospects mapped to a particular allele for each allele within the set of polymorphic loci. Allele measurements can be processed probabilistically, i.e. the likelihood that a given allele shows a given sequence read is a fraction between 0 and 1, or allele measurements are. Any given read that can be processed in a binary fashion is considered to be exactly 0 or 1 copy of a particular allele.

An allelic distribution pattern refers to a set of different allelic distributions for different parental situations. A particular allele distribution pattern may indicate a particular ploidy state.

Allele bias refers to the degree to which the measured allele ratio at a heterozygous locus differs from the ratio that was present in the original DNA sample. The degree of allelic bias at a particular locus is equal to measuring the allelic gene ratio observed at that locus and dividing by the ratio of allelic genes in the original DNA sample at that locus. The allele bias can be defined as greater than 1, so if the calculation of the degree of allele bias yields a value x less than 1, then the degree of allele bias can be paraphrased as 1 / x. .. Allele bias can result from amplification bias, purification bias, or some other phenomenon that affects different alleles differently.

A primer, as well as a "PCR probe", refers to a single DNA molecule (DNA oligomer) or group of DNA molecules (DNA oligomer), the DNA molecule being the same or nearly identical, and the primer being the target locus (eg, the target locus). , Target polymorphic loci or non-polymorphic loci), and may contain priming sequences designed to allow PCR amplification. The primer may also contain a molecular barcode. Primers may contain different random regions for each individual molecule. The terms "test primer" and "candidate primer" are not meant to be limiting and may mean any of the primers disclosed herein.

Primer library means a population of two or more primers. In various embodiments, the library is at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 50,000. 75,000; or 100,000 different primers. In various embodiments, the library is at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 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 locus. In some embodiments, the primer library hybridizes to different target loci at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; It contains 30,000; 40,000; 50,000; 75,000; or 100,000 different individual primers, and the individual primers are not part of a primer pair. In some embodiments, the library comprises both (i) a primer pair and (ii) an individual primer that is not part of the primer pair (eg, a universal primer).

Hybrid capture probes are generated by a variety of methods, such as PCR or direct synthesis, and are intended to be complementary to, and in some cases modified, to one strand of a specific target DNA sequence in a sample. Refers to any nucleic acid sequence. An extrinsic hybrid capture probe can be added to the prepared sample and hybridized through a denaturation-reannealing process to form a double chain of exogenous and endogenous fragments. These double chains can then be physically separated from the sample by various means.

Sequence read refers to data indicating the sequence of nucleotide bases measured by the clonal sequencing method. Clone sequencing can yield sequence data showing a single original DNA molecule or a clone of one original DNA molecule or a cluster of one original DNA molecule. Sequence reads can also have an associated quality score that indicates the probability that the nucleotide is being called correctly at the position of each base in the sequence.

Sequence read mapping is the process of determining where to start a sequence read in a sequence within the genome of a particular organism. The starting location for sequence reads is based on the similarity between the nucleotide sequences of the reads and the genomic sequences.

Match copy error, as well as "matched chromosomal aneuploidy" (MCA), refers to a aneuploidy state in which one cell contains two identical or nearly identical chromosomes. This type of aneuploidy can occur during gametogenesis in meiosis and can be referred to as meiotic inseparable error. This type of error can occur in mitosis. Trisomy can refer to the case where three copies of a given chromosome are present in an individual and two of the copies are identical.

Mismatched copy errors, as well as "unique chromosome aneuploidy" (UCA), are two chromosomes in which one cell is from the same parent and may be homologous but not identical. Refers to an aneuploidy state containing. This type of aneuploidy can occur during meiosis and can be referred to as meiosis error. Mismatch trisomy can refer to the case where three copies of a given chromosome are present in an individual, two of which are of the same parental origin, and are homologous but not identical. Note that disagreement trisomy can refer to the presence of two homologous chromosomes from one parent, some segments of the chromosome being identical, while others are merely homologous.

A homologous chromosome refers to a chromosomal copy that contains the same set of genes that normally pair during meiosis.

The same chromosome refers to a chromosome copy containing the same set of genes, and for each gene, the same chromosome has the same set of alleles that are the same or nearly identical.

Allele dropout (ADO) refers to the situation in which at least one of the base pairs in a set of base pairs from a homologous chromosome in a given allele is not detected.

Locus dropout (LDO) refers to the situation in which both base pairs within a set of base pairs from homologous chromosomes in a given allele are not detected.

Homozygosity refers to having an allele similar to the locus of the corresponding chromosome.

Heterozygosity refers to having an allele that is not similar to the locus of the corresponding chromosome.

Heterozygous rate refers to the rate at which an individual in a population has a heterozygous allele at a given locus. Heterozygous rate may also refer to the predicted allele ratio or the measured allele ratio at a given locus in an individual or DNA sample.

A single nucleotide polymorphism (HISNP) with high information value refers to an SNP in which the fetus has an allele that is not present in the genotype of the mother.

Chromosome region refers to a segment or complete chromosome of a chromosome.

A chromosomal segment is a section of a chromosome that can vary in size from one base pair to the entire chromosome.

Chromosome refers to either a complete chromosome or a segment or section of a chromosome.

Copy refers to the number of copies of a chromosomal segment. It may refer to the same or non-identical homologous copy of a chromosomal segment, where different copies of a chromosomal segment contain a collection of substantially similar loci and one or more of alleles. The pieces are different. Note that in some cases of aneuploidy, such as M2 copy errors, one may have some copies of a given chromosomal segment that are identical as well as some copies that are not identical to the same chromosomal segment. ..

A haplotype generally refers to a combination of alleles at multiple loci that are inherited together on the same chromosome. A haplotype can refer to just two loci or the entire chromosome, depending on the number of recombination events that have occurred between a given set of loci. Haplotypes may also refer to a set of single nucleotide polymorphisms (SNPs) on a single statistically relevant chromatid.

Haplotype data, as well as "phase-specific data" or "ordered genetic data," is a single chromosome in a diploid or polyploid genome, i.e., a separate maternal lineage of chromosomes in the diploid genome. Refers to data from either a copy of or a copy of the paternal lineage.

Phase identification refers to the act of determining an individual's haplotype genetic data by taking into account unordered, diploid (or polyploid) genetic data. Phase identification can refer to the act of determining which of the two allele genes is associated with each of the two homologous chromosomes of an individual for a set of alleles found on one chromosome.

Phase-specific data refers to genetic data for which one or more haplotypes have been determined.

A hypothesis refers to a set of possible polyploid states in a given set of chromosomes or possible allelic states in a given set of loci. The set of possibilities may include one or more elements.

The copy number hypothesis, as well as the "haploid state hypothesis," refers to the hypothesis regarding the number of copies of an individual's chromosomes. This may refer to a hypothesis about the identity of each chromosome, including the parent from which each chromosome originates and which of the two parental chromosomes is present in the individual. This may refer to a hypothesis as to whether a related individual-derived chromosome or chromosomal segment, if any, genetically corresponds to a given individual-derived chromosome.

A target individual is an individual whose genetic status 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 can be more than one target individual. In some embodiments, fetuses born to a pair of parents can each be considered a target individual. In some embodiments, the genetic data determined is a single allele call or a set of allele calls. In some embodiments, the genetic data determined is polyploid call.

A related individual is any individual that is genetically associated with the target individual and therefore shares a haplotype block with the target individual. In certain situations, the relevant individual may be the genetic parent of the target individual, or any genetic material derived from the parent, eg, sperm, polar body, embryo, fetus or offspring. Related individuals may also refer to siblings, parents or grandparents.

Brotherhood refers to any individual whose genetic parent is the same as the individual in question. In some embodiments, sibs can refer to a born offspring, embryo or fetus, or one or more cells derived from a born offspring, embryo or fetus. A sibling may also refer to a set of polyploid individuals originating from one of the parents, such as sperm, polar bodies or any other haplotype genetic material. An individual can consider itself a sibling.

Fetal refers to "of the fetus" or "of the region of the placenta that is genetically simillar to the fetal". .. In pregnant women, part of the placenta is genetically similar to the fetus, and the floating fetal DNA found in the blood of the maternal line can originate from the part of the placenta whose genotype matches the fetus. Note that the genetic information of half of the fetal chromosomes is inherited from the fetal mother. In some embodiments, the DNA from these maternally inherited chromosomes derived from fetal cells is not "of maternal origin" but "of fetal origin". ) ”.

Fetal-origin DNA refers to DNA that was originally part of a cell whose genotype is basically equal to that of the fetus.

Maternally-derived DNA refers to DNA that was originally part of a cell whose genotype is essentially the same as that of the mother.

A offspring can refer to an embryo, blastomere or fetus. It should be noted that in the embodiments disclosed herein, the concepts described apply equally well to individuals that are a collection of cells of birth, fetus, embryo or derived from them. The use of the term offspring simply means that the individual referred to as the offspring is the genetic offspring of the parent.

A parent is an individual's genetic mother or father. An individual generally has two parents, a mother and a father, but this is not always the case, for example, in a genetic or chromosomal chimeric phenomenon. Parents can be considered individuals.

Parental status refers to the genetic status of a given SNP on each of two related chromosomes for one or both of the two targeted parents.

To develop as desired, as well as to "generate normally", means to implant a viable embryo into the uterus and result in pregnancy, and / or to continue pregnancy and result in birth, and / Alternatively, it refers to the absence of chromosomal abnormalities in the offspring and / or the absence of other unwanted genetic states, such as disease linkage genes, in the offspring. The term "generate as desired" is meant to include anything that may be desired by a parent or health care assistant. In some cases, "developing as desired" can refer to non-growth or viable embryos that are useful for medical research or other purposes.

Insertion into the uterus refers to the process of transferring an embryo into the uterine cavity in the context of in vitro fertilization.

Maternal plasma refers to the plasma portion of blood from a pregnant woman.

A clinical decision refers to any decision to take or not take measures with outcomes that affect an individual's health or survival. In the context of prenatal diagnosis, a clinical decision can refer to the decision to miscarriage or not to have a fetus. A clinical decision may refer to the decision to take further tests, take steps to reduce unwanted phenotypes, or prepare for the birth of a child with an abnormality.

Diagnostic box refers to a machine or a combination of machines designed to perform one or more embodiments of the methods disclosed herein. In certain embodiments, the diagnostic box can be placed in a place that cares for the patient. In certain embodiments, the diagnostic box allows targeted amplification followed by sequencing. In certain embodiments, the diagnostic box may function alone or with the assistance of a technician.

Informatics-based methods refer to methods that rely heavily on statistics to unravel large amounts of data. In the context of prenatal diagnosis, informatics-based methods directly physically measure the state of a polyploid state on one or more chromosomes or the state of an allele in one or more alleles. It refers to a method designed to determine the most probable state by statistically inferring, for example, taking into account a large amount of genetic data from a molecular array or sequencing. In certain embodiments of the present disclosure, the informatics-based technique may be those disclosed in this patent. In certain embodiments of the present disclosure, the informatics-based technique may be PARENTAL SUPPORT ™.

Primary genetic data refers to the analog intensity signal output from the genotyping platform. In the context of SNP arrays, primary genetic data refers to the intensity signal before any genotype call is made. In the context of sequencing, primary genetic data refers to analog measurements similar to chromatograms that arise from the sequencer before any base pair identity is determined and before the sequence is mapped to the genome. Point to.

Secondary genetic data refers to processed genetic data output from the genotyping platform. In the context of an SNP array, secondary gene data refers to allele calls made by software associated with the SNP array reader, which may or may not be present in the sample. A call is made. In the context of sequencing, secondary genetic data refers to the determination of base pair identity of a sequence, and in some cases, similarly, to the mapping of a sequence to the genome.

Non-invasive prenatal testing (NPD) or similarly "non-invasive prenatal screening" (NPS) is the gene of a fetus in which the mother is pregnant using genetic material found in the mother's blood. Refers to a method of determining the condition of the mother, the genetic material is obtained by drawing the mother's intravenous blood.

Priority enrichment of the DNA corresponding to the locus, or preferential enrichment of the DNA at the locus, is a percentage of the DNA molecule in the enriched DNA mixture corresponding to that locus. Refers to any method of increasing the percentage of DNA molecules in the pre-enriched DNA mixture corresponding to that locus. The method may include selective amplification of the DNA molecule corresponding to the locus. The method may include the step of removing a DNA molecule that does 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 post-enrichment mixture corresponding to the locus divided by the percentage of DNA molecules in the pre-enrichment mixture corresponding to the locus. Priority enrichment can be done at multiple loci. In some embodiments of the present disclosure, the degree of enrichment is greater than 20. In some embodiments of the present disclosure, the degree of enrichment exceeds 200. In some embodiments of the present disclosure, the degree of enrichment exceeds 2,000. When preferential enrichment is performed at multiple loci, the degree of enrichment can refer to the average degree of enrichment of all loci within the set of loci.

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

Selective amplification can refer to a method of increasing the number of copies of a particular DNA molecule or DNA molecule corresponding to a particular region of DNA. Selective amplification may also refer to a method of increasing the number of copies of a particular target DNA molecule or region of the target DNA rather than increasing the unlabeled molecule or region of the DNA. Selective amplification may be a preferential enrichment method.

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 for the universal priming sequence. Universal priming sequences are generally unrelated to target sequences.

Universal adapter or "ligation adapter" or "library tag"
Is a DNA molecule containing a universal priming sequence that can be covalently linked to the 5'and 3'ends of a population of target double-stranded DNA molecules. The addition of an adapter results in a universal priming sequence at the 5'and 3'ends of the target population from which PCR amplification can be performed, and all molecules from the target population are singled with an amplification primer. Amplify using pairs.

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

A joint distribution model defines the probabilities of predefined events for multiple random variables, taking into account multiple random variables defined for the same probability space to which the probabilities of the variables are associated. Refers to the model. In some embodiments, degeneration cases in which the probabilities of variables are not associated can be used.

The embodiments disclosed herein are further described with reference to the accompanying figures, and similar structures are referred to by similar numbers throughout some overviews. The figures shown are not necessarily of constant scale and generally emphasize the illustration of the principles of the embodiments disclosed herein.

It is a schematic diagram of the direct multiplex mini-PCR method. It is explanatory drawing of the semi-nested mini-PCR method. It is explanatory drawing of the complete nested mini-PCR method. It is explanatory drawing of the heminestized mini-PCR method. It is explanatory drawing of the triple heminestized mini-PCR method. It is explanatory drawing of the one-sided nested mini-PCR method. It is explanatory drawing of the one-sided mini-PCR method. It is explanatory drawing of the reverse semi-nested mini-PCR method. It is a workflow diagram of some possibilities of the semi-nested method. It is explanatory drawing of the loop ligation adapter. It is explanatory drawing of the primer with a tag inside. Here are some examples of primers with internal tags. It is explanatory drawing of the method of using the primer which has a ligation adapter binding region. FIG. 6 is a graph showing the accuracy of simulated haploid calls for counting methods using two different analytical techniques. It is a figure which shows the ratio of two alleles for a plurality of SNPs in the cell line of Experiment 4. FIG. 5 shows the ratio of two alleles for multiple SNPs in the cell line of Experiment 4, separated by chromosome. 17A-D are diagrams showing the ratio of two alleles for multiple SNPs in plasma samples of four pregnant women, separated by chromosome. 17A-D are diagrams showing the ratio of two alleles for multiple SNPs in plasma samples of four pregnant women, separated by chromosome. 17A-D are diagrams showing the ratio of two alleles for multiple SNPs in plasma samples of four pregnant women, separated by chromosome. 17A-D are diagrams showing the ratio of two alleles for multiple SNPs in plasma samples of four pregnant women, separated by chromosome. It is a graph which shows the ratio of the data which can be explained by the binomial variance before and after the data correction. FIG. 6 is a graph showing the relative enrichment of fetal DNA in a sample after a short library preparation protocol. It is a graph comparing the direct PCR and the semi-nested method with respect to the read depth. It is a graph which shows the comparison of the read depth of the direct PCR of three kinds of genomic samples. It is a graph which shows the comparison of the read depth of the semi-nested mini-PCR of three kinds of samples. It is a graph which shows the comparison of the read depth of a 1,200-plex reaction and a 9,600-plex reaction. It is a graph which shows the read number ratio in 3 chromosomes about 6 kinds of cells. FIG. 5 shows the ratio of alleles for two 3-cell reactions on three chromosomes and for a third reaction performed on 1 ng of genomic DNA. It is a figure which shows the ratio of alleles about two single cell reactions in three kinds of chromosomes. It is a figure which shows the number of loci having a specific minor allele frequency targeted by each of two primer libraries. FIG. 28A: Graph of electrophoresis of PCR product. 28B-28M are electrophoretic diagrams of lanes 1-12 in FIG. 28A, respectively. 28B-28M are electrophoretic diagrams of lanes 1-12 in FIG. 28A, respectively. 28B-28M are electrophoretic diagrams of lanes 1-12 in FIG. 28A, respectively. 29A-29E: Image representation of the method of the invention for determining fetal aneuploidy (FIG. 29A). Maternal and paternal genotype data (from blood or buccal swabs) from the Hapmap database and crossover frequency data are used to generate multiple independent hypotheses for each possible fetal ploidy state by incilico (FIG. 29B). Each of these hypotheses is extended to include secondary hypotheses that take into account different possible crossover points. The data model predicts probable sequencing data for each hypothetical fetal genotype and different fetal cfDNA proportions (predictive allele distribution) and compares it to actual sequencing data (Figure). 29C). Bayesian statistics are used to determine the likelihood for each hypothesis. This hypothetical example determines the hypothesis with the highest likelihood (positive ploidy) (Fig. 29D). The individual likelihoods of each copy number hypothesis family (monosomy, disomy, or triploid) in FIG. 29C are summed. The maximum likelihood hypothesis, called as a ploidy state, reveals the fetal fraction and shows sample-specific computational accuracy (FIG. 29E). 30A-30H: Representative graph representations of positive ploidy (FIGS. 30A-30C), monosomy (FIGS. 30D), and trisomy (FIGS. 30E-30H). In all plots, the x-axis is the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis is the number of A allele reads as a percentage of the total (A + B) allele reads. Represents. Maternal and fetal genotypes, as well as the location of the y-axis at the center of the band, are shown on the right side of the plot. If necessary, the plots are color coded according to maternal genotype so that red indicates the maternal genotype of AA, blue indicates the maternal genotype of BB, and green indicates the maternal genotype of AB. May be good. If desired, the contribution of the maternal allele may be indicated by color in the "fetal genotype" column. Allele contributions are expressed in the form of maternal | fetus, such as AA | AB in the case of alleles where the mother is AA and the fetus is AB. FIG. 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 a pattern when the genotype is completely maternal. Therefore, allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). FIG. 30B shows a plot produced when two chromosomes are present and the fetal fraction is 12%. The contribution of fetal alleles to the proportion of A allele reads causes the position of some allele spots to move up and down along the y-axis. Therefore, the bands are 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) are central. FIG. 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern containing two red and two blue peripheral bands and a triplet of central green bands is readily visible (colors not shown in the figure). The bands are 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) are central. FIG. 30D shows a plot generated when one chromosome is present and the fetal fraction is 26%. Hallmark patterns of one outer red and one outer blue peripheral band, as well as two central green bands, showed maternal genetic monosomy (color not shown in the figure). Since the fetus contributes only to a single allele (A or B) to the allele read, there are no inner peripheral red and blue bands and the central triplet band is compressed into two bands. (Colors are not shown in the figure). The bands are centered on 1 (AA | A allele), 0.57 (AB | A allele), 0.43 (AB | B allele), and 0 (BB | B allele). FIG. 30E is a plot generated when there are 3 chromosomes and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal inherited meiotic trisomy (colors not shown in the figure). The bands are 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). FIG. 30F is a plot generated when 3 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 inherited meiotic trisomy (colors not shown in the figure). The bands are 1 (AA | AAA alligator gene), 0.93 (AA | AAB allelic gene), 0.87 (AA | ABB allelic gene), 0.60 (AB | AAA allelic gene), 0.53 (AB). | AAB allelic gene), 0.47 (AB | ABB allelic gene), 0.40 (AB | BBB allelic gene), 0.13 (BB | AAB allelic gene), 0.07 (BB | ABB allelic gene), And 0 (BB | BBB allogeneic gene) are central. FIG. 30G is a plot generated when there are 3 chromosomes and the fetal fraction is 35%. This pattern of two red and two blue peripheral bands and four green bands indicates maternally inherited mitotic trisomy (color not shown in the figure). The bands are 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) are central. FIG. 30H is a plot generated when 3 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 paternally inherited mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternally inherited mitotic trisomy (as in FIG. 30G) by the position of the medial peripheral band. Specifically, the bands are 1 (AA | AAA allele), 0.78 (AA | ABB allele), 0.67 (AB | AAA allele), 0.56 (AB | AAB allele), It is centered on 0.44 (AB | ABB allele), 0.33 (AB | BBB allele), 0.22 (BB | AAB allele), and 0 (BB | BBB allele). 30A-30H: Representative graph representations of positive ploidy (FIGS. 30A-30C), monosomy (FIGS. 30D), and trisomy (FIGS. 30E-30H). In all plots, the x-axis is the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis is the number of A allele reads as a percentage of the total (A + B) allele reads. Represents. Maternal and fetal genotypes, as well as the location of the y-axis at the center of the band, are shown on the right side of the plot. If necessary, the plots are color coded according to maternal genotype so that red indicates the maternal genotype of AA, blue indicates the maternal genotype of BB, and green indicates the maternal genotype of AB. May be good. If desired, the contribution of the maternal allele may be indicated by color in the "fetal genotype" column. Allele contributions are expressed in the form of maternal | fetus, such as AA | AB in the case of alleles where the mother is AA and the fetus is AB. FIG. 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 a pattern when the genotype is completely maternal. Therefore, allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). FIG. 30B shows a plot produced when two chromosomes are present and the fetal fraction is 12%. The contribution of fetal alleles to the proportion of A allele reads causes the position of some allele spots to move up and down along the y-axis. Therefore, the bands are 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) are central. FIG. 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern containing two red and two blue peripheral bands and a triplet of central green bands is readily visible (colors not shown in the figure). The bands are 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) are central. FIG. 30D shows a plot generated when one chromosome is present and the fetal fraction is 26%. Hallmark patterns of one outer red and one outer blue peripheral band, as well as two central green bands, showed maternal genetic monosomy (color not shown in the figure). Since the fetus contributes only to a single allele (A or B) to the allele read, there are no inner peripheral red and blue bands and the central triplet band is compressed into two bands. (Colors are not shown in the figure). The bands are centered on 1 (AA | A allele), 0.57 (AB | A allele), 0.43 (AB | B allele), and 0 (BB | B allele). FIG. 30E is a plot generated when there are 3 chromosomes and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal inherited meiotic trisomy (colors not shown in the figure). The bands are 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). FIG. 30F is a plot generated when 3 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 inherited meiotic trisomy (colors not shown in the figure). The bands are 1 (AA | AAA alligator gene), 0.93 (AA | AAB allelic gene), 0.87 (AA | ABB allelic gene), 0.60 (AB | AAA allelic gene), 0.53 (AB). | AAB allelic gene), 0.47 (AB | ABB allelic gene), 0.40 (AB | BBB allelic gene), 0.13 (BB | AAB allelic gene), 0.07 (BB | ABB allelic gene), And 0 (BB | BBB allogeneic gene) are central. FIG. 30G is a plot generated when there are 3 chromosomes and the fetal fraction is 35%. This pattern of two red and two blue peripheral bands and four green bands indicates maternally inherited mitotic trisomy (color not shown in the figure). The bands are 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) are central. FIG. 30H is a plot generated when 3 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 paternally inherited mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternally inherited mitotic trisomy (as in FIG. 30G) by the position of the medial peripheral band. Specifically, the bands are 1 (AA | AAA allele), 0.78 (AA | ABB allele), 0.67 (AB | AAA allele), 0.56 (AB | AAB allele), It is centered on 0.44 (AB | ABB allele), 0.33 (AB | BBB allele), 0.22 (BB | AAB allele), and 0 (BB | BBB allele). 30A-30H: Representative graph representations of positive ploidy (FIGS. 30A-30C), monosomy (FIGS. 30D), and trisomy (FIGS. 30E-30H). In all plots, the x-axis is the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis is the number of A allele reads as a percentage of the total (A + B) allele reads. Represents. Maternal and fetal genotypes, as well as the location of the y-axis at the center of the band, are shown on the right side of the plot. If necessary, the plots are color coded according to maternal genotype so that red indicates the maternal genotype of AA, blue indicates the maternal genotype of BB, and green indicates the maternal genotype of AB. May be good. If desired, the contribution of the maternal allele may be indicated by color in the "fetal genotype" column. Allele contributions are expressed in the form of maternal | fetus, such as AA | AB in the case of alleles where the mother is AA and the fetus is AB. FIG. 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 a pattern when the genotype is completely maternal. Therefore, allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). FIG. 30B shows a plot produced when two chromosomes are present and the fetal fraction is 12%. The contribution of fetal alleles to the proportion of A allele reads causes the position of some allele spots to move up and down along the y-axis. Therefore, the bands are 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) are central. FIG. 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern containing two red and two blue peripheral bands and a triplet of central green bands is readily visible (colors not shown in the figure). The bands are 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) are central. FIG. 30D shows a plot generated when one chromosome is present and the fetal fraction is 26%. Hallmark patterns of one outer red and one outer blue peripheral band, as well as two central green bands, showed maternal genetic monosomy (color not shown in the figure). Since the fetus contributes only to a single allele (A or B) to the allele read, there are no inner peripheral red and blue bands and the central triplet band is compressed into two bands. (Colors are not shown in the figure). The bands are centered around 1 (AA | A allele), 0.57 (AB | A allele), 0.43 (AB | B allele), and 0 (BB | B allele). FIG. 30E is a plot generated when there are 3 chromosomes and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal inherited meiotic trisomy (colors not shown in the figure). The bands are 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). FIG. 30F is a plot generated when 3 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 inherited meiotic trisomy (colors not shown in the figure). The bands are 1 (AA | AAA alligator gene), 0.93 (AA | AAB allelic gene), 0.87 (AA | ABB allelic gene), 0.60 (AB | AAA allelic gene), 0.53 (AB). | AAB allelic gene), 0.47 (AB | ABB allelic gene), 0.40 (AB | BBB allelic gene), 0.13 (BB | AAB allelic gene), 0.07 (BB | ABB allelic gene), And 0 (BB | BBB allogeneic gene) are central. FIG. 30G is a plot generated when there are 3 chromosomes and the fetal fraction is 35%. This pattern of two red and two blue peripheral bands and four green bands indicates maternally inherited mitotic trisomy (color not shown in the figure). The bands are 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) are central. FIG. 30H is a plot generated when 3 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 paternally inherited mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternally inherited mitotic trisomy (as in FIG. 30G) by the position of the medial peripheral band. Specifically, the bands are 1 (AA | AAA allele), 0.78 (AA | ABB allele), 0.67 (AB | AAA allele), 0.56 (AB | AAB allele), It is centered on 0.44 (AB | ABB allele), 0.33 (AB | BBB allele), 0.22 (BB | AAB allele), and 0 (BB | BBB allele). 30A-30H: Representative graph representations of positive ploidy (FIGS. 30A-30C), monosomy (FIGS. 30D), and trisomy (FIGS. 30E-30H). In all plots, the x-axis is the linear position of the individual polymorphic locus along each chromosome (shown at the bottom of the plot), and the y-axis is the number of A allele reads as a percentage of the total (A + B) allele reads. Represents. Maternal and fetal genotypes, as well as the location of the y-axis at the center of the band, are shown on the right side of the plot. If necessary, the plots are color coded according to maternal genotype so that red indicates the maternal genotype of AA, blue indicates the maternal genotype of BB, and green indicates the maternal genotype of AB. May be good. If desired, the contribution of the maternal allele may be indicated by color in the "fetal genotype" column. Allele contributions are expressed in the form of maternal | fetus, such as AA | AB in the case of alleles where the mother is AA and the fetus is AB. FIG. 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 a pattern when the genotype is completely maternal. Therefore, allele clusters are distributed around 1 (AA allele), 0.5 (AB allele), and 0 (BB allele). FIG. 30B shows a plot produced when two chromosomes are present and the fetal fraction is 12%. The contribution of fetal alleles to the proportion of A allele reads causes the position of some allele spots to move up and down along the y-axis. Therefore, the bands are 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) are central. FIG. 30C shows a plot generated when two chromosomes are present and the fetal fraction is 26%. A pattern containing two red and two blue peripheral bands and a triplet of central green bands is readily visible (colors not shown in the figure). The bands are 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) are central. FIG. 30D shows a plot generated when one chromosome is present and the fetal fraction is 26%. Hallmark patterns of one outer red and one outer blue peripheral band, as well as two central green bands, showed maternal genetic monosomy (color not shown in the figure). Since the fetus contributes only to a single allele (A or B) to the allele read, there are no inner peripheral red and blue bands and the central triplet band is compressed into two bands. (Colors are not shown in the figure). The bands are centered around 1 (AA | A allele), 0.57 (AB | A allele), 0.43 (AB | B allele), and 0 (BB | B allele). FIG. 30E is a plot generated when there are 3 chromosomes and the fetal fraction is 27%. This pattern of two red and two blue peripheral bands and two central green bands indicates maternal inherited meiotic trisomy (colors not shown in the figure). The bands are 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). FIG. 30F is a plot generated when 3 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 inherited meiotic trisomy (colors not shown in the figure). The bands are 1 (AA | AAA alligator gene), 0.93 (AA | AAB allelic gene), 0.87 (AA | ABB allelic gene), 0.60 (AB | AAA allelic gene), 0.53 (AB). | AAB allelic gene), 0.47 (AB | ABB allelic gene), 0.40 (AB | BBB allelic gene), 0.13 (BB | AAB allelic gene), 0.07 (BB | ABB allelic gene), And 0 (BB | BBB allogeneic gene) are central. FIG. 30G is a plot generated when there are 3 chromosomes and the fetal fraction is 35%. This pattern of two red and two blue peripheral bands and four green bands indicates maternally inherited mitotic trisomy (color not shown in the figure). The bands are 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) are central. FIG. 30H is a plot generated when 3 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 paternally inherited mitotic trisomy (colors not shown in the figure). This pattern can be distinguished from the pattern of maternally inherited mitotic trisomy (as in FIG. 30G) by the position of the medial peripheral band. Specifically, the bands are 1 (AA | AAA allele), 0.78 (AA | ABB allele), 0.67 (AB | AAA allele), 0.56 (AB | AAB allele), It is centered on 0.44 (AB | ABB allele), 0.33 (AB | BBB allele), 0.22 (BB | AAB allele), and 0 (BB | BBB allele). FIG. 31: Graph display of positive polyploid (FIG. 31A), T13 (FIG. 31B), T18 (FIG. 31C), T21 (FIG. 31D), 45, X (FIG. 31E), and 47, XXY (FIG. 31F) test samples. Is. Each chromosome is shown at the top of the plot, the fetal and maternal genotypes are shown on the right side of the plot, the x-axis represents the linear position of the SNP along each chromosome, and the y-axis represents the ratio to the total read. A Shows the number of allele reads. Note that the cluster position is changed based on the fetal fraction as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown on the right side of the plot, and chromosomal identification information is shown at the top of the plot. FIG. 31: Graph display of positive polyploid (FIG. 31A), T13 (FIG. 31B), T18 (FIG. 31C), T21 (FIG. 31D), 45, X (FIG. 31E), and 47, XXY (FIG. 31F) test samples. Is. Each chromosome is shown at the top of the plot, the fetal and maternal genotypes are shown on the right side of the plot, the x-axis represents the linear position of the SNP along each chromosome, and the y-axis represents the ratio to the total read. A Shows the number of allele reads. Note that the cluster position is changed based on the fetal fraction as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown on the right side of the plot, and chromosomal identification information is shown at the top of the plot. FIG. 31: Graph display of positive polyploid (FIG. 31A), T13 (FIG. 31B), T18 (FIG. 31C), T21 (FIG. 31D), 45, X (FIG. 31E), and 47, XXY (FIG. 31F) test samples. Is. Each chromosome is shown at the top of the plot, the fetal and maternal genotypes are shown on the right side of the plot, the x-axis represents the linear position of the SNP along each chromosome, and the y-axis represents the ratio to the total read. A Shows the number of allele reads. Note that the cluster position is changed based on the fetal fraction as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown on the right side of the plot, and chromosomal identification information is shown at the top of the plot. FIG. 31: Graph display of positive polyploid (FIG. 31A), T13 (FIG. 31B), T18 (FIG. 31C), T21 (FIG. 31D), 45, X (FIG. 31E), and 47, XXY (FIG. 31F) test samples. Is. Each chromosome is shown at the top of the plot, the fetal and maternal genotypes are shown on the right side of the plot, the x-axis represents the linear position of the SNP along each chromosome, and the y-axis represents the ratio to the total read. A Shows the number of allele reads. Note that the cluster position is changed based on the fetal fraction as described herein. Each spot represents a single SNP locus. Fetal and maternal genotypes are shown on the right side of the plot, and chromosomal identification information is shown at the top of the plot. FIG. 5 is a graph showing that the prevalence of complex births due to sex chromosome aneuploidy is higher than that due to autosomal aneuploidy.

The above figure describes the embodiments disclosed herein, but other embodiments are also intended, as mentioned in the discussion. The present disclosure exemplifies the embodiments as representatives, and is not shown as a limitation. One of ordinary skill in the art can devise a number of other modifications and embodiments that fall within the scope and intent of the embodiments disclosed herein.

The present invention is in part an unexpected finding that relatively few primers in the primer library are often responsible for the production of significant amounts of amplified primer dimers formed during multiplex PCR reactions. Is based on. Methods have been developed to select the least desirable primers for removal from the candidate primer library. By reducing the amount of primer dimer to a negligible amount (about 0.1% of the PCR product), these methods use the generated primer library to generate a large number of target genes in a single multiplex PCR reaction. Allows the loci to be amplified at the same time. Primers hybridize to target loci and amplify target loci without forming amplified primer dimers by hybridizing to other primers, thus increasing the number of different target loci that can be amplified. .. It is also clear that using lower primer concentrations and significantly longer annealing times increases the likelihood that the primers will hybridize to the target locus rather than hybridize to each other to form primer dimers. Became.

During PCR amplification and sequencing of 19,488 target loci in the genomic sample, 99.4-99.7% of these sequencing reads are mapped to the genome and 99.99% are mapped to the target loci. Was done. For plasma samples with 10 million sequencing reads, at least 19,350 (99.3%) of the 19,488 target loci were typically amplified and sequenced. The ability to simultaneously amplify such a large number of target loci at one time can significantly reduce the time and amount of DNA required to analyze thousands of target loci. Simultaneously analyze thousands of target loci that are important in low DNA applications, such as genetic testing of single cells from embryos prior to in vitro fertilization or genetic testing of forensic samples containing small amounts of DNA. Therefore, DNA derived from a single cell is sufficient. In addition, the ability to analyze the target locus in one reaction volume (eg, one vessel or well) without dividing the sample into multiple different reactions can reduce possible variability during the reaction. In addition, the method has been developed to use reference criteria to correct for possible amplification bias between different target loci. For example, due to differences in amplification efficiency between target loci due to factors such as GC content, the amount of PCR product at the target locus that should actually be produced in the same amount may be different. By using reference criteria similar to the target locus, such amplification bias can be detected and corrected during quantification of the target locus.

During sequencing of PCR products, artifacts such as primer dimers are detected, resulting in inhibition of detection of target amplification products. Due to this limitation, microarrays with hybridization probes are often used for detection. The reason is that the sensitivity of the primer dimer to interference is low. High levels of multiplexing with minimal non-target amplification products that are currently achievable make PCR and subsequent sequencing available as an alternative to microarrays.

The multiplex PCR method of the present invention can be used for various purposes. For example, genotyping, detection of chromosomal abnormalities (eg, fetal chromosomal aneuploidy), gene mutations and polymorphisms (eg, single-base polymorphism, SNP) analysis, gene deletion analysis, paternal identification, genetic within a population. It can be used for aneuploidy analysis, forensic analysis, predisposition to disease, quantitative analysis of mRNA, and detection and identification of infectious agents (eg, bacteria, parasites, and viruses). Multiplex PCR can also be used for non-invasive prenatal genetic testing, such as paternity testing or detection of fetal chromosomal abnormalities.

Typical Primer Design Methods Advanced multiplex PCR can often produce very high proportions of product DNA resulting from non-productive side reactions such as primer dimer formation. In one embodiment, certain primers that are most likely to cause unproductive side reactions are removed from the primer library to obtain a primer library that yields a high percentage of amplified DNA that is mapped to the genome. be able to. The step of removing problematic primers, especially those that may stabilize the dimer, unexpectedly enabled very high PCR multiplexing levels for subsequent sequencing analysis. .. In systems such as sequencing where performance is significantly degraded by primer dimers and / or other adverse products, they are more than 10 times, more than 50 times, and more than 100 times more sophisticated than the multiplexing described elsewhere. Multiplexing has been achieved. Note that this is in contrast to probe-based detection methods, such as microarrays, TAQMAN, PCR, where excess primer dimers do not perceptibly affect results. It should also be noted that the general idea in the art is that multiplexed PCR for sequencing is limited to about 100 assays in the same well. Fluidim and Rain Dance provide a platform for performing 48 or 1000 PCR assays in parallel reactions on a single sample.

There are a number of ways to select primers for libraries that minimize the amount of non-mapping primer dimers or other adversely affecting primer products. Empirical data have shown that a small number of "bad" primers are involved in large numbers of non-mapping primer dimer side reactions. Removal of these "bad" primers can increase the percentage of sequence reads that position the target locus. One way to identify "bad" primers is to examine the sequencing data of DNA amplified by targeted amplification, removing the most frequently found primer dimers and mapping them to the genome. It can result in a primer library that is significantly less likely to result in unsuccessful by-product DNA. There are also publicly available programs that can calculate the binding energies of different combinations of primers, and removing the combination of primers with the highest binding energies also results in by-product DNA that is not mapped to the genome. A primer library is created that is significantly less likely.

In some embodiments for primer selection, an initial candidate primer library is made by designing one or more primers or primer pairs for a candidate target locus. The set of candidate target loci (eg, SNPs) can be selected based on publicly available information about the desired parameters of the target loci, such as the frequency of SNPs within the target population or the heterozygous rate 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 by reference in its entirety). If necessary, the primer produces a target amplification product in a specific size range, such as annealing within a specific annealing temperature range, a specific range of GC content, and a specific size range. And / or can be designed to have other parameter characteristics. Starting with multiple primers or primer pairs per candidate target locus increases the likelihood that primers or primer pairs will remain in the library for most or all target loci. In one embodiment, the selection criterion may be that at least one primer pair per target locus needs to remain in the library. As such, by using the final primer library, almost or all target loci will be amplified. This is desirable for applications such as selection of deletions or replication at multiple sites in the genome or selection of multiple sequences (eg, polymorphisms or other mutations) associated with an increased risk of disease or disease. If a primer pair from the library produces a target amplification product that overlaps with a target amplification product produced by another primer pair, one of the primer pairs can be removed from the library to prevent interference.

In some embodiments, an "undesignability score" (a higher score represents the least desirability) is calculated for most or all possible combinations of two primers from the candidate primer library (computer). (By calculation etc.). In various embodiments, the undesignability score is calculated for at least 80, 90, 95, 98, 99, or 99.5% possible combinations of candidate primers in the library. Each undesignability score is at least partially based on the likelihood of dimer formation between the two candidate primers. If desired, the undesignability score also includes heterozygotes at the target locus, prevalence associated with the sequence at the target locus (eg, polymorphism), sequence at the target locus (eg, polymorphism). From disease permeability associated with, specificity of candidate primers for 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. It may be based on one or more other parameters selected from the group of. When considering multiple factors, the undesignability score may be calculated based on weighted averages of various parameters. The parameters can be assigned different weights based on their importance to the particular application in which the primer is used. In some embodiments, the primer with the highest undesignability 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 and removal process can be repeated as needed. In some embodiments, the selection method is performed until the undesignability scores of all combinations of candidate primers remaining in the library are below the minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to the desired number.

In various embodiments, candidate primers that are part of the largest number of combinations of two candidate primers with an undesignability score above the first minimum threshold after the undesignability score has been calculated. Is removed from the library. This step ignores interactions below the first minimum threshold. The reason is that these interactions are less important. 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 process of removing the primer can be repeated as needed. In some embodiments, the selection method is performed until all the undesignability scores for the combination of candidate primers 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 the second minimum threshold and repeating the process of removing the primers. If the number of candidate primers remaining in the library is less than required, increase the first minimum threshold to a larger second minimum threshold, thereby removing the primers using the original candidate primer library. The process can be repeated, thereby continuing the process by leaving more candidate primers in the library. In some embodiments, the undesignability score of the combination of complementary primers remaining in the library is all below the second minimum threshold, or the number of candidate primers remaining in the library is desired. The selection method is executed until the number is reduced.

If desired, a primer pair that produces a target amplification product that overlaps with a target amplification product produced by another primer pair can be split into different amplification reactions. Multiplex PCR amplification reactions may be preferably applied in applications where it is desirable to analyze all candidate target loci (rather than removing candidate target loci from the analysis due to overlapping target amplification products).

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 a smaller number of candidate primers from the library, the resulting primer library can be used to amplify more (or all) target loci.

Multiplexing a large number of primers imposes considerable restrictions on the assays that can be included. Assays that interact unintentionally result in sham amplification products. The size constraints of miniPCR can introduce additional constraints. In certain embodiments, it is possible to start with a large number of potential SNP targets (between about 5 and over 1 million) and attempt to design primers to amplify each SNP. If a primer can be designed, the likelihood of forming a fake primer double chain between all possible primer pairs, and the published thermodynamic parameters for DNA double chain formation. By using and evaluating, it is possible to attempt to identify primer pairs that may form fake products. Primer interactions can be ranked by the score function associated with the interaction and the primers with the worst interaction scores can be excluded until the desired number of primers is met. If a potentially heterozygous SNP is most useful, it is possible to similarly rank the list of assays and select the assay that best suits the heterozygotes. Experiments have verified that primers with high interaction scores are most likely to form primer dimers. With a high degree of multiplexing, it is not possible to eliminate all false interactions, but the primer or primer pair with the highest interaction score in in silico occupies the predominance of the overall reaction and is the intended target. It is essential to remove them as they significantly limit the amplification from. This procedure was performed to create a set of multiplexed primers that reached, and in some cases exceeded, 10,000 primers. The improvement with this procedure is substantive and, as determined by sequencing of all PCR products, more than 80% of the target product compared to 10% from the reaction that did not remove the worst primer. Allows amplification of greater than 90%, greater than 95%, greater than 98%, and even greater than 99%. Combined with the partially semi-nested approach described previously, it is possible to map up to over 90% and up to over 95% amplification products to the target sequence.

Note that there are other methods for determining which PCR probe may form a dimer. In certain embodiments, analysis of a pool of DNA amplified using a non-optimized set of primers may be sufficient to determine the problematic primer. For example, the analysis can be performed using sequencing and the largest number of dimers present can be determined to be the most likely to form a dimer and removed.

This method has a number of potential applications, such as SNP genotyping, heterozygosity rate determination, copy counting, and other targeted sequencing applications. In certain embodiments, the method of designing primers 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.

The use of tags as primers can reduce the amplification and sequencing of primer dimer products. In some embodiments, the primer comprises an internal region that forms a loop structure with tags. In certain embodiments, the primer comprises a 5'region specific for the target locus, an internal region that forms a loop structure that is not specific for the target locus, and a 3'region specific for the target locus. In some embodiments, the loop region may be located between two binding regions designed so that the two binding regions 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 7-20 nucleotides, eg, 7-15 nucleotides, or 7-10 nucleotides. In various embodiments, the primer is a non-target locus-specific 5'region (eg, a tag or universal primer binding site), followed by a target locus-specific region, a target locus-specific region. It contains an internal region that forms no loop structure, and a 3'region specific to the target locus. Tag-primers can be used to shorten the required target-specific sequences 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 accidentally discovered with standard primer design if the target sequence is fragmented within the primer binding site, or can be planned for primer design. The advantages of this method are that it increases the number of assays that can be designed for a particular maximum amplification product length and that it shortens the sequencing of "informative" primer sequences. Can be mentioned. The method can also be used in combination with internal tagging (see elsewhere in this document).

In certain embodiments, the relative amount of unproductive product in multiple target PCR amplification can be reduced by increasing the annealing temperature. When amplifying a library with the same tag as the target-specific primer, the annealing temperature can be increased compared to genomic DNA because the tag contributes to primer binding. Some embodiments use significantly lower primer concentrations than previously reported, along with longer annealing times than reported elsewhere. In some embodiments, the annealing time is 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. It can be more than 480 minutes, and even more than 960 minutes. In some embodiments, a longer annealing time than previously reported is used, which allows for lower primer concentrations. In various embodiments, longer than usual elongation times of, for example, greater than 3, 5, 8, 10, or 15 minutes are used. 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 highly multiplexed reactions such as 1,000 plex reaction, 2,000 plex reaction, 5,000 plex reaction, 10,000 plex reaction, 20,000 plex reaction, 50, Robust performance is provided for 000 plex reactions and even 100,000 plex reactions. In one embodiment, amplification uses 1 cycle, 2 cycles, 3 cycles, 4 cycles or 5 cycles performed over a long annealing time, followed by PCR cycles with more than normal annealing times using tagged primers. I do.

To select a target location, start with a pool of candidate primer pairs and designs to create a thermodynamic model of potentially harmful interactions between the primer pairs, and then use this model. Designs that are incompatible with other designs in the pool can be eliminated.

Primers remaining in the library after the selection process can be used in any of the methods of the invention.

Representative Primer Library In one aspect, the invention features a library of primers, eg, primers selected from a candidate primer library using any of the methods of the invention. In some embodiments, the library is at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40 in one reaction volume. Primers that simultaneously hybridize (or simultaneously hybridize) or simultaneously amplify (or simultaneously amplify) 100,000 different target loci, 000; 50,000; 75,000; or 100,000 different target loci. include. In various embodiments, the library is 1,000 to 2,000; 2,000 to 5,000; 5,000 to 7,500; 7,500 to 10,000; 10,000 to 20,000; 20,000 to 25,000; 25,000 to 30,000; 30,000 to 40,000; 40,000 to 50,000; 50,000 to 75,000; 75,000 to 100,000 different Includes primers that simultaneously amplify (or simultaneously amplify) the target locus in one reaction volume. In various embodiments, the library has 1,000 to 100,000 different target loci in one reaction volume, eg, 1,000 to 50,000; 1,000 to 30,000; 1,000. ~ 20,000; 1,000 ~ 10,000; 2,000 ~ 30,000; 2,000 ~ 20,000; 2,000 ~ 10,000; 5,000 ~ 30,000; 5,000 ~ 20 000; or contains primers that simultaneously amplify (or simultaneously amplify) 5,000 to 10,000 different target loci. In some embodiments, the library comprises primers that simultaneously amplify (or simultaneously amplify) the target locus in one reaction volume, thereby 60, 40, 30, 20, 10, the amplification products. 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or less than 0.5% is the primer dimer. In various embodiments, the amount of primer dimer amplification product is 0.5-60%, eg, 0.1-40%, 0.1-20%, 0.25-20%, 0. It is 25 to 10%, 0.5 to 20%, 0.5 to 10%, 1 to 20%, or 1 to 10%. In some embodiments, the primer simultaneously amplifies (or can simultaneously amplify) the target locus in one reaction volume, thereby at least 50, 60, 70, 80, 90, 95, 96, 97. , 98, 99, or 99.5% amplification products are target amplification products. In various embodiments, the amount of amplification product that is the target amplification product is 50-99.5%, for example 60-99%, 70-98%, 80-98%, 90-99.5%, or 95. It is ~ 99.5%. In some embodiments, the primer simultaneously amplifies (or can simultaneously amplify) the target locus in one reaction volume, thereby at least 50, 60, 70, 80, 90, 95, 96, 97. , 98, 99, or 99.5% of target loci are amplified. In various embodiments, the amount of amplified target locus is 50-99.5%, eg 60-99%, 70-98%, 80-99%, 90-99.5%, 95. It is ~ 99.9%, or 98-99.99%. In some embodiments, the primer library is 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 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 hybridizes to different target loci at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; It contains 30,000; 40,000; 50,000; 75,000; or 100,000 individual primers, and the individual primers are not part of a primer pair.

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 1 uM to 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 30-80%, for example 40-70%, or 50-60%. In some embodiments, the range of GC content of the primer is less than 30, 20, 10, or 5%. In some embodiments, the range of GC content of the primer is 5-30%, eg 5-20% or 5-10%. In some embodiments, the melting temperature ( _Tm ) of the test primer is 40-80 ° C, for example 50-70 ° C, 55-65 ° C, or 57-60.5 ° C. In some embodiments, _Tm is calculated in the primer3 program (libprimer3 release 2.2.3) using the built-in SantaLucia parameter (worldforce web of primer3.sourceforge.net). In some embodiments, the melting temperature range of the primer is less than 15, 10, 5, 3, or 1 ° C. In some embodiments, the melting temperature range of the primer is 1-15 ° C, for example 1-10 ° C, 1-5 ° C, or 1-3 ° C. In some embodiments, the primer length is 15-100 nucleotides, eg, 15-75 nucleotides, 15-40 nucleotides, 17-35 nucleotides, 18-30 nucleotides, 20-65 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, eg, 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides. In some embodiments, the length of the target amplification product is 50-100 nucleotides, for example 60-80 nucleotides, or 60-75 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, eg, 5 to 25 nucleotides, 5 to 15 nucleotides, or 5 to 10 nucleotides.

These primer libraries can be used by any of the methods of the invention.

Representative Primer Kit In one aspect, the invention features a kit comprising any of the primer libraries of the invention (eg, a kit that amplifies a target locus in a nucleic acid sample). In some embodiments, kits comprising multiple primers designed to implement the methods described in the present disclosure can be formulated. Primers may be the outer forward and reverse primers, inner forward and reverse primers disclosed herein, and as disclosed in the Primer Design section, for other primers in the kit. The primers may be designed to have low binding affinity and may be hybrid capture probes, pre-cyclization probes or some combination thereof as described in the relevant section. In one embodiment, a kit designed for use in the methods disclosed herein for determining the polymorphic state of a target chromosome in a pregnant fetus, with multiple inner forward primers. , And optionally multiple inner reverse primers, and optionally outer forward and outer reverse primers, each of which is a target chromosome and, if desired, yet another chromosome. Kits can be constructed that include primers, which are designed to hybridize to regions of DNA immediately upstream and / or downstream of one of the above target sites (eg, polymorphic sites). In certain embodiments, the primer kit can be used in combination with the diagnostic boxes described elsewhere in this document. In some embodiments, the kit comprises instructions for amplifying a target locus using a library.

Representative Multiple PCR Methods In one aspect, the invention features a method of amplifying a target locus in a nucleic acid sample, wherein (i) the nucleic acid sample is at least 1,000; 2,000; 5,. Primers that simultaneously hybridize to 000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci. It comprises contacting with a library to generate a reaction mixture, and (ii) subjecting the reaction mixture to primer extension reaction conditions (eg, PCR conditions) to produce an amplification product, including a target amplification product. In some embodiments, the method also targets at least one target amplification product (eg, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5%). It also includes a step of determining the presence or absence of an amplification product). In some embodiments, the method also targets at least one target amplification product (eg, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5%). It also includes the step of determining the sequence of the amplification product). In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% of 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% amplification products It is a primer dimer.

In certain embodiments, the methods disclosed herein use highly efficient highly multiplex target PCR to amplify DNA and then perform high-throughput sequencing to perform alleles at each target locus. Determine the frequency. It is novel and non-trivial that more than about 50 or 100 PCR primers can be multiplexed in one reaction volume so that the majority of the resulting sequence reads are mapped to the target locus. One technique that allows highly multiplex target PCR to be performed in a highly efficient manner involves the design of primers that are unlikely to hybridize with each other. PCR probes, commonly referred to as primers, are 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. Potentially harmful interactions between 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 potential primer pairs, or intent between primers and sample DNA. A thermodynamic model of the non-existent interaction is created and then selected by using this model to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplex target PCR to be performed in a highly efficient manner is to perform targeted PCR using partial or complete nesting techniques. Multiplexing at least 300, at least 800, at least 1,200, at least 4,000, or at least 10,000 primers in a single pool by using one or a combination of these techniques. The resulting amplified DNA contains the majority of DNA molecules that, upon sequencing, are mapped to the target locus. By using one or a combination of these techniques, it is possible to multiplex multiple primers in a single pool and the resulting amplified DNA is> 50%,> 60%, 67. Over%, over 80%, over 90%, over 95%, over 96%, over 97%, over 98%, over 99%, or over 99.5%, DNA molecules that are mapped to the target locus. include.

In some embodiments, the detection of the target genetic material can be performed in a multimodal fashion. The number of target sequences of genes that can be executed in parallel is 1 to 10, 10 to 100, 100 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 10,000. Obtained in the range of 1,000,000 or 1,000,000 to 10,000,000. Previous attempts to multiplex more than 100 primers per pool have created serious problems with unwanted side reactions such as primer dimer formation.

Target PCR
In some embodiments, PCR can be used to target specific locations in the genome. In plasma samples, the original DNA is highly fragmented (generally less than 500 bp, average length less than 200 bp). In PCR, both the forward and reverse primers anneal to the same fragment to allow amplification. Therefore, if the fragment is short, the PCR assay must also amplify the relatively short region. If the position of the polymorphism is too close to the polymerase binding site, as in MIPS, there will be a bias in amplification from different alleles. Currently, PCR primers targeting polymorphic regions, such as those containing SNPs, generally such that the 3'end of the primer hybridizes to the base immediately adjacent to one or more polymorphic bases. Designed. In one embodiment of the present disclosure, the 3'ends of both the forward and reverse PCR primers hybridize with a base that is separated from the mutation location (polymorphic site) of the target allele by one or a few positions. Design to soy. The number of bases between the polymorphic site (SNP or other type) and the base designed for the 3'end of the primer to hybridize may be 1 base or 2 bases. It may be 3 bases, 4 bases, 5 bases, 6 bases, 7-10 bases, 11-15 bases, or It may be 16 to 20 bases. Forward and reverse primers can be designed to hybridize to different numbers of bases away from the polymorphic site.

Although a large number of PCR assays can be generated, the interaction between different PCR assays makes it difficult to multiplex them beyond about 100 assays. Various complex molecular techniques can be used to increase the level of multiplexing, but may still be limited to less than 100, perhaps 200, or possibly 500 assays per reaction. A sample with a large amount of DNA can be divided into multiple side reactions and then recombined prior to sequencing. For samples where either the entire sample of DNA molecules or some subpopulations is limited, dividing the sample will introduce statistical noise. In certain embodiments, a small or limited amount of DNA can refer to an amount of 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. This method is particularly useful for small amounts of DNA, where other methods involving the step of splitting into multiple pools can cause serious problems associated with the introduction of stochastic noise. It should be noted that when performed on any amount of DNA sample, it provides the benefit of minimizing bias. In these situations, a universal preamplification step can be used to increase the overall sample volume. Ideally, this preamplification step should not change the allele distribution to a perceptible level.

In certain embodiments, the methods of the present disclosure allow a large number of targets to be sequenced or locused by some other locus method from a limited sample, such as a single cell or DNA derived from body fluids. Loci, specifically 1,000-5,000 loci, 5,000-10,000 loci, or more than 10,000 loci-specific PCR products can be produced. Currently, performing multiplex PCR reactions of more than 5 to 10 targets presents a major challenge, often with primer by-products such as primer dimers and other artifacts interfering. When detecting target sequences using microarrays with hybridization probes, primer dimers and other artifacts are not detected and can be ignored. However, when sequencing is used as the method of detection, the majority of sequencing reads sequence such artifacts and do not sequence the desired target sequence in the sample. By the methods described in the prior art used to multiplex and then sequence more than 50 or 100 reactions in one reaction volume, generally more than 20%, and often more than 50%, often 80%. It results in ultra-and in some cases> 90% off-target sequence reads.

In general, in order to perform targeted sequencing on a large number of (n) targets (> 50,> 100,> 500 or> 1,000) in a sample, the sample is divided into a number of parallel reactants. One individual target can be amplified. This is performed in PCR multi-well plates or on commercial platforms such as FLUIDIGM ACCESS ARRAY (48 reactions per sample in microfluidic chips) or DROPLET PCR from RAIN DANCE TECHNOLOGY (100-thousands of targets). ) Can be executed. Unfortunately, these split-and-pool methods often ensure that a copy of the genome present is a copy of each region of the genome in each well. There is a problem with samples where the amount of DNA is limited, as it is not sufficient. This is a particularly serious problem when targeting polymorphic loci, and the proportion of alleles present in the original DNA sample due to the probabilistic noise introduced by splitting and pooling. Relative proportions of alleles at the polymorphic locus are needed, as they cause very inadequately accurate measurements. Methods for effectively and efficiently amplifying many PCR reactants, applicable when only a limited amount of DNA is available, are described herein. In certain embodiments, the method is applied to analyze a mixture of single cells, body fluids, DNA, such as floating DNA, biopsy material, environmental samples and / or forensic samples found in maternal plasma. Can be done.

In certain embodiments, target sequencing may include one, multiple, or all of the following steps. a) Generate and amplify a library with adapter sequences at both ends of the DNA fragment. b) After library amplification, divide into multiple reactions. c) Generate a library with adapter sequences at both ends of the DNA fragment and optionally amplify it. d) 1000-10,000 plex amplification of selected targets is performed using one target-specific "forward" primer and one tag-specific primer per target. e) Second amplification from this product using "reverse" target-specific primers and one (or more) universal tag-specific primers introduced as part of the target-specific forward primers in the first round. I do. f) Perform 1000 plex pre-proliferation of the selective target in a limited number of cycles. g) Divide the product into multiple fractions and amplify the target subpools in individual reactions (eg, 50-500 plexes (this method can be used with any plexes up to a single plex)). h) Pool the products of the parallel subpool reaction. i) During these amplifications, the primer can retain a sequencing compatibility tag (partial length or full length), whereby the product can be sequenced.

Highly multiplex PCR
The present specification discloses a method that enables targeted amplification of more than 100,000 to tens of thousands of target sequences (eg, SNP loci) derived from nucleic acid samples such as genomic DNA obtained from plasma. The amplified sample is relatively free of primer dimer products and has less allele bias at the target locus. If sequencing compatibility adapters are added to the products during or after amplification, analysis of these products can be performed by sequencing.

Performing highly multiplex PCR amplification using methods known in the art yields primer dimer products that are in excess of the desired amplification product and are not suitable for sequencing. These can be empirically reduced by eliminating the primers that form these products or by performing in silico selection of the primers. However, the greater the number of assays, the more difficult this problem becomes.

One solution is to divide the 5,000-plex reaction into several low-plex amplifications, such as 100 50-plex reactions or 50 100-plex reactions, or using microfluidics, or even more. Is to divide the sample into individual PCR reactions. However, if sample DNA is limited, such as in non-invasive prenatal diagnosis from pregnancy plasma, splitting the sample between multiple reactions should be avoided as this causes bottlenecking. ..

Here is a method for first amplifying the plasma DNA of a sample as a whole and then dividing the sample into multiple multiplex target enrichment reactions with a more moderate number of target sequences per reaction. Are listed. In certain embodiments, the methods of the present disclosure can be used to preferentially enrich a DNA mixture at multiple loci, said method comprising one or more of the following steps: from a mixture of DNA. A step of creating and amplifying a library, in which the molecules in the library have adapter sequences ligated at both ends of the DNA fragment, a step of dividing the amplified library into multiple reactions, is selected. The first round of multiple amplification of a target is performed using one target-specific "forward" primer per target and one or more adapter-specific universal "reverse" primers. In certain embodiments, the methods of the present disclosure are "reverse" target-specific primers and one or more universal tag-specific primers introduced as part of the target-specific forward primer in the first round. Further comprises the step of performing a second amplification using. In certain embodiments, the method may involve a complete nested PCR technique, a heminated PCR technique, a semi-nested PCR technique, a one-sided complete nested PCR technique, a one-sided hemmened PCR technique, or a one-sided semi-nested PCR technique. In certain embodiments, the methods of the present disclosure are used to preferentially enrich the DNA mixture at multiple loci, the method performing premultiplexing amplification of selected targets in a limited number of cycles. A step of dividing the product into a plurality of fixed amounts, a step of amplifying a target subpool in each reaction, and a step of pooling the product of a parallel subpool reaction. This technique involves allelic bias for 50-500 loci, 500-5,000 loci, 5,000-50,000 loci, or even 50,000-500,000 loci. Note that it can be used to perform targeted amplification to low levels. In certain embodiments, the primer carries a partial or full length sequencing compatibility tag.

The workflow consists of (1) extracting DNA such as plasma DNA, (2) preparing a fragment library having universal adapters at both ends of the fragment, and (3) making the library adapter-specific. Amplification using a universal primer, (4) dividing the amplified sample "library" into multiple aliquots, and (5) multiplexing for a given aliquot (eg, one target per target). Approximately 100 plexes, 1,000 or 10,000 plexes) amplification using specific and tag-specific primers, (6) a step of pooling a fixed amount of one sample, and (7) a sample. It may be accompanied by a step of barcoding with respect to, (8) a step of mixing the sample and adjusting the concentration, and (9) a step of sequencing the sample. The workflow may include multiple substeps containing one of the listed steps (eg, the step of preparing the library of step (2) is three enzymatic steps (blunted, blunt-ended,). dA tailing and adapter ligation) and may involve three purification steps). Workflow steps can be combined, separated, or performed in a different order (eg, sample barcoding and pooling).

It is important to note that library amplification can be performed in a biased manner in amplifying short pieces more efficiently. Thus, shorter sequences, eg, DNA fragments of mononucleosomes, can be preferentially amplified as cell-free fetal DNA (of placental origin) found in the circulation of pregnant women. It should be noted that the PCR assay may have a tag, eg, a sequencing tag (usually a cleavage form of 15-25 bases). After multiplexing, the PCR multiplexing products of the sample are pooled, and then tagging is completed (including barcoding) by tag-specific PCR (which can also be done by ligation). Also, complete sequencing tags can be added to the same reaction as a multiplexing. In the first cycle, the target can be amplified with target-specific primers, after which the tag-specific primers predominate to complete the complete SQ adapter sequence. The PCR primer does not have to carry the tag. Sequencing tags can be added to the amplification product by ligation.

In certain embodiments, fetal aneuploidy can be used for a variety of applications, such as by evaluating amplified material using highly multiplex PCR followed by clonal sequencing. Whereas conventional multiplex PCR evaluates up to 50 loci simultaneously, the techniques described herein are used to simultaneously evaluate over 50 loci, over 100 loci, and over 500 loci at the same time. Simultaneously evaluate 1,000 or more loci, 5,000 or more loci at the same time, 10,000 or more loci at the same time, 50,000 or more loci at the same time, and 100,000 or more loci at the same time. Can be made possible. Experimentally, up to 10,000, including 10,000, and over 10,000 distinct loci were simultaneously evaluated in a single reaction with sufficiently good efficiency and specificity, non-invasively. It has been shown that prenatal aneuploidy diagnosis and / or copy count calls can be made with high accuracy. The assay can be combined in a single reaction with an entire sample, such as a cfDNA sample isolated from maternal plasma, its fraction or a further processed derivative of the cfDNA sample. The sample (eg, cfDNA or derivative) can also be divided into multiple parallel multiple reactions. Optimal sample fragmentation and multiplexing is determined by trade-offs for various performance specifications. Due to the limited amount of material, dividing the sample into multiple fractions can result in increased sampling noise, handling time, and increased likelihood of error. Conversely, as a result of high multiplexing, the amount of false amplification may increase and amplification inequality may increase, both of which reduce inspection performance.

Two very important related considerations in the application of the methods described herein are in limited quantities of the original sample (eg, plasma) and materials for obtaining allelic frequencies or other measurements. The number of original molecules. If the number of original molecules is below a certain level, random sampling noise will be significant and can affect the accuracy of the test. In general, measurements on samples containing 500-1000 original molecule equivalents per target locus may provide sufficient quality data for non-invasive prenatal aneuploidy diagnosis. can. There are several ways to increase the number of separate measurements, eg, to increase the volume of the sample. Each operation applied to the sample also results in potential material loss. In order 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 certain operations or improve their yields as needed.

In certain embodiments, it is possible to reduce potential losses in subsequent steps by amplifying all or a percentage of the original sample (eg, cfDNA sample). Various methods are available to amplify all of the genetic material in the sample, thereby increasing the amount available for downstream procedures. In one embodiment, a ligation-mediated PCR (LM-PCR) DNA fragment is amplified by PCR after ligating either one separate adapter, two separate adapters, or many separate adapters. In one embodiment, a polysubstituted amplification (MDA) fi-29 polymerase is used to isothermally amplify all DNA. In DOP-PCR and its variants, random priming is used to amplify the original material DNA. Each method has specific properties, such as amplification homogeneity over all typical genomic regions, efficiency of capture and amplification of the original DNA, and amplification performance depending on fragment length.

In certain embodiments, LM-PCR can be used with a single heteroduplex adapter having 3'tyrosine. Heteroduplex adapters allow the use of a single adapter molecule that can be converted to two separate sequences on the 5'and 3'ends of the original DNA fragment during the first round of PCR. It will be possible. In certain embodiments, it is possible to fractionate a library amplified by size separation, or a product such as AMPURE, TASS, or other similar method. Prior to ligation, the sample DNA is blunt-ended and then a single adenosine base is added to the 3'end. Prior to ligation, the DNA can be cleaved using restriction enzymes or some other cleavage method. During ligation, the ligation efficiency may 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 in terms of time 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 a 10-50% enrichment of fetal DNA and improved testing performance. A number of reactions were performed using the conditions specified by the commercially available kit, resulting in successful ligation of less than 10% of the sample DNA molecules. A series of optimizations of reaction conditions for this improved ligation to approximately 70%.

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. The loss of characteristic fetal molecules is high due to conventional PCR assay design, but this loss can be significantly reduced by designing a very short PCR assay called the mini-PCR assay. The fetal cfDNA in maternal serum is highly fragmented, the fragment size is distributed in a nearly Gaussian fashion, with an average of 160 bp, a standard deviation of 15 bp, and a minimum size of about 100 bp. The maximum size is about 220 bp. The distribution of fragment start and end positions for a target polymorphism is not necessarily random, but varies widely among individual targets and collectively among all targets, with one particular target gene. The locus polymorphic site can occupy any position from start to finish in the various fragments originating from that locus. It should be noted that the term mini-PCR may refer equally well to conventional PCR without further limitation or limitation.

During PCR, amplification occurs only from template DNA fragments containing both forward and reverse primer sites. Since the fetal cfDNA fragment is short, the likelihood that both primer sites are present and the likelihood of a long L fetal fragment containing both the forward and reverse primer sites is the length and fragment of the amplification product. Is the ratio of lengths. Under ideal conditions, by assay where the amplification product is 45 bp, 50 bp, 55 bp, 60 bp, 65 bp or 70 bp, 72%, 69%, 66%, 63%, 59% or of the available template fragment molecules, respectively. Amplified successfully from 56%. The length of the amplification product is the distance between the 5'ends of the forward priming site and the reverse priming site. Amplification products of shorter length than those commonly used by those of skill in the art can result in more efficient measurements of the desired polymorphic locus with only the required short sequence reads. In certain embodiments, 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.

In the methods known in the prior art, short assays such as those described herein are not required, and for primer design, primer length limitation, annealing properties, and forward and reverse primers. Note that the distance between them imposes considerable constraints and is usually avoided.

It should also be noted that there is a potential amplification bias if the 3'end of any primer is in the range of approximately 1-6 bases at the polymorphic site. This single-base difference at the site where the polymerase initially binds can result in preferential amplification of one allele, which can alter the frequency of alleles observed and reduce performance. There is sex. 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 are compatible with the same multiplexing reaction. In one embodiment, the 3'ends of the inner forward and reverse primers are designed to hybridize to regions of DNA that are upstream of the polymorphic site and separated from the polymorphic site by a small number of bases. Ideally, the number of bases may be between 6 and 10 bases, but equally well, between 4 and 15 bases, between 3 and 20 bases, and between 2 and 30 bases. Alternatively, it may be between 1 and 60 bases, and substantially the same result can be achieved.

Multiplex PCR may involve a single round of PCR in which all targets are amplified, or may involve one 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 internally by at least one base pair than the primers used in the previous round. Nested PCR reduces the number of false amplification targets by amplifying only amplification products from the previous reaction with the correct internal sequence in subsequent reactions. The reduction of sham amplification targets improves the number of useful measurements that can be obtained, especially in sequencing. Nested PCR generally involves designing a primer that is more completely internal than 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 separate cfDNA molecules from which measurements can be obtained. In one embodiment, to offset this effect, one or both of the second round primers partially overlap with the first binding site, which extends some bases inward. Nesting techniques can be used to achieve yet another specificity while minimizing the increase in total assay size.

In one embodiment, the multiple pool of PCR assays is designed to amplify SNPs or other polymorphic or non-polymorphic loci on one or more chromosomes that are potentially heterozygous. , These assays are used in a single reaction to amplify the DNA. The number of PCR assays is 50 to 200 PCR assays, 200 to 1,000 PCR assays, 1,000 to 5,000 PCR assays or 5,000 to 5,000. PCR assays between 20,000 times (50-200 plexes, 200-1,000 plexes, 1,000-5,000 plexes, 5,000-20,000 plexes, 20,000-plus plexes, respectively). good. In one embodiment, multiple pools of approximately 10,000 PCR assays (10,000 plexes) are latent on the X, Y, 13th, 18th, and 21st and 1st or 2nd chromosomes. Designed to amplify the SNP chromosome, which is heterozygous, and using these assays in a single reaction, material plasma samples, chorionic villi samples, sheep's water puncture samples, single or few cells, etc. Amplifies cfDNA obtained from body fluids or tissues, cancer or other genetic material. The SNP frequency of each locus can be determined by clonal methods of sequencing the amplification product or by some other method. Statistical analysis of the allele frequency distribution or ratios of all assays can be used to determine if a sample contains trisomy of one or more of the chromosomes included in the test. In another embodiment, the original cfDNA sample is split into two samples and a parallel 5,000-plex assay is performed. In another embodiment, the original cfDNA sample is divided into n samples and a parallel (about 10,000 / n) plex assay is performed where n is between 2 and 12 or between 12 and 24. Between 24 and 48 or between 48 and 96. Collect data and analyze as described already. It should be noted that this method is equally well applicable to detect translocations, deletions, duplications, and other chromosomal abnormalities.

In certain embodiments, a tail that is not homologous to the target genome can also be added to either the 3'end or the 5'end of the primer. These tails facilitate subsequent operations, procedures or measurements. In certain embodiments, the tail sequence may be the same for the target-specific forward primer and the target-specific reverse primer. In certain embodiments, different tails can be used for target-specific forward and target-specific reverse primers. In certain embodiments, a plurality of different tails can be used for different loci or sets of loci. A particular tail may be shared among all loci or between a subset of loci. For example, forward and reverse tails corresponding to the forward and reverse sequences required for any of the current sequencing platforms can be used for direct sequencing after amplification. In certain embodiments, the tail can be used as a general priming site 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 (eg, a polymorphic locus). In some embodiments, the primer may contain a molecular barcode. In some embodiments, the primer may contain a universal priming sequence designed to allow PCR amplification.

In one embodiment, a 10,000-plex PCR assay pool is provided with forward and reverse primers to the required forward and reverse sequences required for a high-throughput sequencing instrument, eg, HISEQ, GAIIX or MYSEQ available from ILLUMINA. Make to have a corresponding tail. In addition, the 5'side of the sequencing tail contains yet another sequence that can be used as a priming site for adding the nucleotide barcode sequence to the amplification product in subsequent PCR, thereby providing multiple sequences. Samples can be multiplexed and sequenced in a single lane of a high-throughput sequencing instrument.

In one embodiment, a 10,000-plex PCR assay pool is made such that the reverse primer has a tail corresponding to the required reverse sequence required for a high-throughput sequencing instrument. After amplification in the first 10,000-plex assay, subsequent PCR amplification was applied to partially nested forward primers (eg, 6-base nested) for all targets, and to the reverse sequencing tail included in the first round. It can be performed using another 10,000 plex pool with the corresponding reverse primer. In the next round of this partial nested amplification with only one target-specific primer and universal primer, the size required for the assay is limited, sampling noise is reduced, but the number of false amplification products is significantly reduced. .. The sequencing tag can be attached to the attached ligation adapter and / or as part of the PCR probe, thus the tag becomes part of the final amplification product.

The fetal fraction affects the performance of the test. There are several ways to enrich the fetal fraction of DNA found in maternal plasma. The fetal fraction can be increased by the above-mentioned LM-PCR method already discussed, as well as by targeted removal of long maternal fragments. In one embodiment, an additional multiplex PCR reaction is performed prior to the multiplex PCR amplification of the target locus to selectively remove the long and large maternal fragments corresponding to the targeted locus in subsequent multiplex PCR. be able to. Additional primers are designed to anneal to sites that are farther from the polymorphism than would be expected to be present between cell-free fetal DNA fragments. These primers can be used in a one-cycle multiplex PCR reaction prior to multiplex PCR at the target polymorphism locus. These distal primers are tagged with molecules or moieties that may allow selective recognition of tagged pieces of DNA. In certain embodiments, these DNA molecules are covalently modified with a biotin molecule that allows removal of newly formed double-stranded DNA containing these primers after one cycle of PCR. be able to. The double-stranded DNA formed during that first round appears to be of maternal origin. Removal of the hybrid material can be achieved by using magnetic streptavidin beads. There are other methods of tagging that can work equally well. In certain embodiments, the size selection method can be used to enrich the sample for shorter strands of DNA, such as DNA less than about 800 bp, less than about 500 bp, or less than about 300 bp. The amplification of the short pieces is then proceeded normally.

The mini-PCR method described in the present disclosure allows for highly multiplexed amplification and analysis of hundreds to thousands, or even millions of loci from a single sample in a single reaction. At the same time, the detection of amplified DNA can be multiplexed, and by using barcoding PCR, tens to hundreds of samples can be multiplexed in one sequencing lane. This multiplexing detection has been successfully tested up to 49 plexes, allowing a much higher degree of multiplexing. In effect, this allows a single sequence sequence to be genotyped in thousands of SNPs for hundreds of samples. For these samples, the method described above allows the genotype and heterozygotes to be determined, and at the same time, the number of copies, both of which are used to detect aneuploidy. Can be done. This method is particularly useful in detecting fetal aneuploidy during pregnancy from floating DNA found in maternal plasma. This method can be used as part of a method for determining fetal sex and / or predicting fetal paternity. This method can be used as part of the method for determining the amount of mutation. This method can be used for any amount of DNA or RNA, and the target region may be SNPs, other polymorphic regions, non-polymorphic regions, and combinations thereof.

In some embodiments, ligation-mediated universal PCR amplification of fragmented DNA can be used. Plasma DNA can be amplified using ligation-mediated universal PCR amplification, which can then be divided into multiple parallel reactions. Ligation-mediated universal PCR amplification can also be used to preferentially amplify short fragments, thereby enriching the fetal fraction. In some embodiments, tagging the fragment by ligation detects shorter fragments, uses shorter target sequence-specific moieties of the primer, and / or reduces non-specific reactions. It may be possible to anneal at a higher temperature than letting it.

The methods described herein can be used for a number of purposes in the presence of a collection of target DNA mixed with a certain amount of contaminating DNA. In some embodiments, the target DNA and contaminating DNA may be of genetically related individual origin. For example, genetic abnormalities in the fetal (target) can be detected in maternal plasma containing fetal (target) DNA as well as maternal (contaminated) DNA, and the abnormalities include all chromosomal abnormalities (eg, for example). Abnormalities or differences in chromosomal abnormalities (eg, deletions, duplications, inversions, translocations), polynucleotide polymorphisms (eg, STR), monobase polymorphisms, and / or other genes. Can be mentioned. In some embodiments, the target DNA and the contaminating DNA may be from the same individual, but in the case of cancer, for example, the target DNA and the contaminating DNA will differ by one or more mutations (eg,). H. Mamon et al. Priorential Amplification of Apoptotic DNA from Plasma: Potential for Enchanting Detection of Minor DNA Alterations in Circulation2 (Circulating DNA) 54. In some embodiments, DNA can be found in cell culture (apoptotic) supernatants. In some embodiments, it is possible to induce apoptosis in a biological sample (eg, blood) for subsequent library preparation, amplification and / or sequencing. A number of possible workflows and protocols for achieving this goal are set forth elsewhere in this disclosure.

In some embodiments, the target DNA is a single cell, a DNA sample consisting of less than one copy of the target genome, a small amount of DNA, DNA from a mixed origin (eg, pregnancy plasma: placenta and maternal DNA; Cancer Patients Plasma and Tumors: Mixtures of Healthy and Cancer DNA, Transplants, etc.), Other Body Fluids, Cell Cultures, Culture Supernatants, Forensic DNA Samples, Ancient DNA Samples (eg, Insects Captured in Amber), It may be derived from other DNA samples, and combinations thereof.

In some embodiments, short amplification product sizes can be used. The short amplification product size is particularly suitable for fragmented DNA (eg, A. Sikora et al., Detection of increased amounts of cell-free fetal DNA with short PCR amplifiers. Clin Chem. 2010; No. 1)), see pages 136-8).

The use of short amplification product sizes can provide some significant benefits. Short amplification product sizes can provide optimized amplification efficiency. Short amplification product sizes generally result in shorter products, and therefore the likelihood of non-specific priming is low. Shorter products can be clustered more densely on the sequencing flow cell as the clusters become smaller. It should be noted that the methods described herein may work equally well for longer PCR amplification products. The length of the amplification product can be increased if desired, for example, when sequencing on a larger sequence range. Experiments with 146-plex targeted amplification with 100bp-200bp length assays were performed on single cell and genomic DNA as the first step in the nested PCR protocol with positive results.

In some embodiments, the methods described herein are used to SNP, copy count, nucleotide methylation, mRNA levels, other types of RNA expression levels, other gene forms and / or posterior. Synthetic forms can be amplified and / or detected. The mini-PCR method described herein can be used in conjunction with next-generation sequencing, which can be used with other downstream methods such as microarrays, digital PCR counting, real-time PCR, mass spectrometry, and the like. Can be used together.

In some embodiments, the mini-PCR amplification methods described herein can be used as part of a method for accurately quantifying a minority population. The method can be used for absolute quantification using spike calibrators. The method can be used for quantification of mutations / trace alleles by ultra-deep sequencing and can be performed in a highly multiplexed fashion. The method can be used for standard paternality and identity testing of relatives or ancestors in humans, animals, plants or other organisms. The method can be used for forensic examinations. The method can be used for rapid genotyping and copy number analysis (CN) for any type of material, such as amniotic fluid and CVS, sperm, conceptus (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 by targeted sequencing using min-PCR (less than 1 day, 1 day, or 2 days after biopsy).

In some embodiments, the mini-PCR amplification method can be used for tumor analysis: tumor biopsy is often a mixture of health and tumor cells. Targeted PCR enables deep sequencing of SNPs and loci with no nearby background sequences. The method can be used for analysis of copy count and heterozygous loss to tumor DNA. The tumor DNA can be present in many different body fluids or tissues of a tumor patient. The method can be used for detection of tumor recurrence and / or tumor screening. The method can be used for seed quality control tests. The method can be used for breeding or fishing. It should be noted that both of these methods can be used equally well for targeting non-polymorphic loci for polymorphic calls.

Some references describing some of the basic methods underlying this disclosure include (1) Wang HY, Luo M, Tereshchenko IV, Frikker DM, Cui X, LiJ Y, Hu G, Chu. Y, Azaro MA, Lin Y, Shen L, Yang Q, Kambouris ME, Gao R, Shih W, LiH. Genome Res. 2005 Feb; 15 (2): 276-83. Department of Molecular Genetics, Microbiology and Immunology / The Cancer Institute of New Jersey, Robot Wood Jones3, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, New Brunswick, (2) High-throughput genotyping of single nucleic acid 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) Next Patch PCR enables highly multiplexed mutation discovery discovery genes. Varley KE, Mitra RD. Genome Res. 2008 Nov; 18 (11): 1844-50. EPUB 2008 Oct 10 (this document describes a method comprising multiplexing an average of 9 assays for sequencing). It should be noted that the methods disclosed herein allow for multiplexes that are orders of magnitude higher than those in the references above.

Variants of Targeted PCR-There are many workflows possible when performing nesting PCR, and some workflows typical of the methods disclosed herein are described. The steps outlined herein are not intended to rule out other possible steps, and any of the steps described herein is required for the method to function properly. It does not mean that it is. Modifications of many parameters or other modifications are known in the literature and can be done without affecting the core of the invention. One particular general workflow is shown below, followed by a number of possible variants. Variants generally refer to possible secondary PCR reactions, such as different types of nesting (step 3) that can be performed. It is important to note that variants can be made at different times or in different orders than those expressly described herein. Examples using polymorphic loci for illustration can be readily adapted for amplification of non-polymorphic loci, if desired.
1. 1. A ligation adapter, often referred to as a library tag or ligation adapter tag (LT), can be attached to the DNA in the sample, the ligation adapter containing a universal priming sequence, followed by universal amplification. In certain embodiments, this can be done after fragmentation using a standard protocol designed to generate a sequencing library. In certain embodiments, the DNA sample can be blunt-ended and then 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 other than 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. 2. Specific Target Amplification (STA): Hundreds, thousands, tens of thousands, and even hundreds of thousands of targets can be pre-amplified and multiplexed in one reaction volume. The STA is generally performed for 10 to 30 cycles, but can be performed for 5 to 40 cycles, 2 to 50 cycles, and even 1 to 100 cycles. For example, primers can be tailed for a simpler workflow or to avoid sequencing most dimers. Note that in general, dimers of both primers carrying the same tag are not efficiently amplified or sequenced. In some embodiments, PCR can be performed between 1 and 10 cycles, in some embodiments, PCR can be performed between 10 and 20 cycles, and in some embodiments, PCR can be performed. , 20 to 30 cycles of PCR can be performed, in some embodiments 30 to 40 cycles of PCR can be performed, and in some embodiments more than 40 cycles of PCR can be performed. It can be performed. The amplification may be linear amplification. The number of PCR cycles can be optimized to provide the optimum read depth (DOR) profile. Different DR profiles may be desirable for different purposes. In some embodiments, a more uniform distribution of reads across all assays is desired; if the DOR is very small for some assays, the probabilistic noise is high for the data to be very useful. It may be too deep, but if the lead depth is very deep, the marginal usefulness of each additional lead is relatively small.
The primer tail can improve the detection of fragmented DNA from universally tagged libraries. Hybridization can be improved (eg, lowering the melting temperature ( _TM )) if the library tag and primer tail contain homologous sequences, and some of the primer target sequences are within the DNA fragment of the sample. Only if can the primer be extended. In some embodiments, 13 or more target-specific base pairs can be used. In some embodiments, 10-12 target-specific base pairs can be used. In some embodiments, 8-9 target-specific base pairs can be used. In some embodiments, 6-7 target-specific base pairs can be used. In some embodiments, the STA can be performed against pre-amplified DNA, such as MDA, RCA, other whole genome amplification or adapter-mediated universal PCR. In some embodiments, the STA can be performed, for example, on a sample enriched or depleted of a particular sequence and population by size selection, target capture, directional degradation.
3. 3. In some embodiments, it is possible to perform a secondary multiplex PCR or primer extension reaction to increase specificity and reduce unwanted products. For example, complete nesting, semi-nesting, heminesting, and / or subdivision of smaller assay pools into parallel reactions are all techniques that can be used to increase specificity. Experiments have shown that dividing the sample into three 400-plex reactions yields product DNA with higher specificity than a single 1,200-plex reaction with exactly the same primers. Similarly, experiments have shown that splitting a sample into four 2,400 plex reactions yields product DNA with higher specificity than a single 9,600 plex reaction with exactly the same primers. Shown. In certain embodiments, it is possible to use target-specific and tag-specific primers in the same and opposite directions.
4. In some embodiments, the DNA sample (diluted, purified or otherwise) produced by the STA reaction is amplified using tag-specific primers and "universal amplification", i.e., pre-amplified and tagged targets. It is possible to amplify many or all of them. Primers may contain additional functional sequences required for sequencing on high-throughput sequencing platforms, such as barcodes or complete adapter sequences.

These methods can be used to analyze a sample of any DNA, especially if the sample of DNA is particularly small, or if the DNA is a sample of DNA originating from more than one individual. , For example, especially in the case of maternal plasma. These methods are used for DNA samples such as single or few cells, genomic DNA, plasma DNA, amplified plasma library, amplified autogenic supernatant library or other mixed DNA samples. be able to. In certain embodiments, these methods can be used, for example, in cancer or grafts where cells with different genetic makeup can be present in a single individual.

Protocol variants (variants 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 FIG. 101 shows a double-stranded DNA having the polymorphic locus of interest 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 indicates the final PCR product. In some embodiments, the STA is over 100, over 200, over 500, over 1,000, over 2,000, over 5,000, over 10,000, over 20,000, over 50,000, 100. It can be done for more than 000, or more than 200,000 targets. In subsequent reactions, tag-specific primers amplify all target sequences and extend tags containing all sequences required for sequencing, including sampling indexes. In certain embodiments, the primers may not need to be tagged, or only specific primers may be tagged. Sequencing adapters can be added by conventional adapter ligation. In certain embodiments, the first primer may carry the tag.

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

Experiments were performed using this protocol with 1200 plex amplification. Both genomic DNA and pregnancy plasma were used; about 70% of the sequence reads were mapped to the target sequence. Details are given elsewhere in this document. Sequencing of 1042 plexes without assay design and selection resulted in> 99% of the sequences being primer dimer products.

Sequential PCR: After STA1, multiple aliquots of product can be amplified in parallel using a less complex pool with the same primers. The first amplification may give rise to sufficient material for splitting. This method is particularly good for small samples, such as about 6-100 pg, about 100 pg-1 ng, about 1 ng-10 ng or about 10 ng-100 ng. A protocol was carried out in which 1200 plexes were changed to 400 plexes three times. Sequencing read mapping increased from about 60-70% to over 95% on the 1200 plex alone.

Semi-Nested mini-PCR: (see FIG. 2) STA1 followed by a plurality of sets of inner nested forward primers (103B, 105b) and one (or a few) tag-specific reverse primers (103A). Carry out 2 STAs. 101 shows a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a universally amplified single-stranded DNA in which the forward primer B and the reverse primer A are hybridized. 104 indicates the PCR product from 103. 105 shows a product from 104 having a hybridized nested forward primer b and a reverse tag A that is already part of a molecule from PCR generated between 103 and 104. 106 indicates the final PCR product. Using this workflow, more than 95% of the sequence is typically mapped to the intended target. The nested primer may overlap the outer forward primer sequence, but introduces an additional 3'end base. In some embodiments, it is possible to use an extra 3'base between 1 and 20. Experiments have shown that using 9 or more extra 3'bases in a 1200 plex design works well.

Fully nested mini-PCR: (see Figure 3) After STA step 1, a second multiplex PCR (or less complex parallel mp PCR), tagged (A, a, B, b). It is possible to carry out with two nested primers in possession. 101 represents a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a universally amplified single-stranded DNA in which the forward primer B and the reverse primer A are hybridized. 104 indicates the PCR product from 103. 105 shows the product from 104 in which the nested forward primer b and the nested reverse primer a hybridized. 106 indicates the final PCR product. In some embodiments, it is possible to use the complete set of two primers. Using experiments using the fully nested mini-PCR protocol, 146-plex amplification was performed on single cells and three cells with the addition of universal ligation adapters and without step 102 of amplification.

Heminated mini-PCR: (see FIG. 4) It is possible to use target DNA with an adapter at the end of the fragment. An STA consisting of multiple sets of forward primers (B) and one (or a few) tag-specific reverse primers (A) is performed. The second STA can be performed using universal tag-specific forward and target-specific reverse primers. 101 shows a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a universally amplified single-stranded DNA to which the reverse primer A is hybridized. 104 shows the PCR product from 103 amplified using reverse primer A and ligation adapter tag primer LT. 105 shows the product from 104 to which the forward primer B hybridized. 106 indicates the final PCR product. In this workflow, target-specific forward and reverse primers are used in separate reactions, which reduces the complexity of the reaction and prevents the formation of forward and reverse primer dimers. Note that in this example, primers A and B can be considered as the first primer and the primers "a" and "b" can be considered as the inner primers. This method is as good as direct PCR, but it is a great improvement over direct PCR because it avoids primer dimers. After the first round of heminated protocol, generally about 99% of non-target DNA is found, but after the second round, there is generally a significant improvement.

Triple Heminated mini-PCR: (See FIG. 5) It is possible to use target DNA with an adapter at the end of the fragment. An STA consisting of multiple sets of forward primers (B) and one (or minority) tag-specific reverse primers (A) and (a) is performed. The second STA can be performed using universal tag-specific forward and target-specific reverse primers. 101 shows a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a universally amplified single-stranded DNA to which the reverse primer A is hybridized. 104 shows the PCR product from 103 amplified using reverse primer A and ligation adapter tag primer LT. 105 shows the product from 104 to which the forward primer B hybridized. 106 shows the PCR product from 105 amplified using reverse primer A and forward primer B. 107 shows the product from 106 to which the reverse primer "a" was hybridized. 108 indicates 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 a first primer. If desired, both A and B can be considered as the first primer and the "a" can be considered as the inner primer. The names of the reverse primer and the forward primer can be switched. In this workflow, target-specific forward and reverse primers are used in separate reactions, which reduces the complexity of the reaction and prevents the formation of forward and reverse primer dimers. This method is as good as direct PCR, but it is a great improvement over direct PCR because it avoids primer dimers. After the first round of heminated protocol, generally about 99% of non-target DNA is found, but after the second round, there is generally a significant improvement.

One-sided nested mini-PCR: (see FIG. 6) It is possible to use target DNA with an adapter at the end of the fragment. STAs can also be performed using multiple sets of nested forward primers and using ligation adapter tags as reverse primers. A second STA can then be performed using a set of nested forward and universal reverse primers. 101 represents a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a universally amplified single-stranded DNA to which the forward primer A is hybridized. 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 to which the nested forward primer hybridized. 106 indicates the final PCR product. In this method, overlapping primers in the first STA and the second STA can be used to detect shorter target sequences than by standard PCR. The method is generally performed by subtracting a sample of DNA that has already undergone the addition and amplification of the STA step 1-universal tag described above, with the two nested primers on only one side and live on the other side. Use rally tags. The above method was performed on an apoptotic supernatant and a library of pregnant plasma. Using this workflow, about 60% of the sequences were mapped to the intended target. Note that reads containing the reverse adapter sequence are not mapped and therefore this number is expected to be higher when mapping reads containing the reverse adapter sequence.

One-sided mini-PCR: Target DNA with an adapter at the end of the fragment can be used (see Figure 7). STAs can be performed with multiple sets of forward primers and one (or a small number) tag-specific reverse primers. 101 represents a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a single-stranded DNA hybridized with the 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 allows the detection of shorter target sequences than standard PCR. However, it can be relatively non-specific because it uses only one target-specific primer. The effectiveness of this protocol is half that of one-sided nested miniPCR.

Reverse semi-nested mini-PCR: Target DNA with an adapter at the end of the fragment can be used (see Figure 8). STAs can be performed with multiple sets of forward primers and one (or a small number) tag-specific reverse primers. 101 shows a double-stranded DNA having the polymorphic locus of interest at X. 102 shows double-stranded DNA with a ligation adapter added for universal amplification. Reference numeral 103 indicates a single-stranded DNA hybridized with the reverse primer B. 104 shows the PCR product from 103 amplified using reverse primer B and ligation adapter tag forward primer LT. 105 shows the PCR product 104 hybridized with the forward primer A and the inner reverse primer "b". Reference numeral 106 indicates a PCR product amplified from 105 using the forward primer A and the reverse primer "b", which is the final PCR product. This method allows the detection of shorter target sequences than standard PCR.

There may also be additional variants that are merely repetitions or combinations of the above methods, eg, double nested PCR using three sets of primers. Another variant is unilateral half-nested mini-PCR, where STA can also be performed with multiple sets of nested forward primers and one (or a few) tag-specific reverse primers.

Note that the identity of the forward and reverse primers can be exchanged in all of these variants. It should be noted that in some embodiments, the nested variants can be performed equally well without the addition of adapter tags and the preparation of the initial library comprising the universal amplification step. In some embodiments, additional rounds of PCR can be included with additional forward and / or reverse primers and amplification steps, which are the additional steps of the DNA molecule corresponding to the target locus. Note that it can be particularly useful when it is desirable to further increase the percentage.

Nesting Workflow There are many ways to perform amplification with different degrees of nesting and different degrees of multiplexing. In FIG. 9, a flow chart is shown along with some of the 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.

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

In one embodiment, the adapter has complementary terminal regions, a region tagged with a forward primer (LFT), a region tagged with a reverse primer (LRT), and a cleavage site between the two. It may be a loop of DNA contained (see FIG. 10). 101 refers to the double-stranded blunt-ended target DNA. 102 refers to the target DNA having the A tail. Reference numeral 103 refers to a loop ligation adapter having a T overhang "T" and a cutting site "Z". Reference numeral 104 refers to the target DNA to which the loop ligation adapter is added. Reference numeral 105 refers to the target DNA to which the ligation adapter has been added, which has been cleaved at the cleavage site. LFT refers to the ligation adapter forward tag and LRT refers to the ligation adapter reverse tag. Complementary regions may end with a T-overhang, or other form that can be used to ligate the target DNA. The cleavage site can be a series of uracils for cleavage along UNG, or a sequence that can be recognized and cleaved by restriction enzymes or other cleavage methods or simply by 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, eg, the mini-PCR amplification method.

Internally tagged Primers When using sequencing to determine alleles present at a given polymorphic locus, sequence read is generally initiated upstream of the primer binding site (a) and then , Polymorphic site (X) is read. Tags are generally arranged as shown on the left side of FIG. Reference numeral 101 refers to a single-stranded target DNA having the target polymorphic locus "X" and the primer "a" to which the tag "b" is added. To avoid non-specific hybridization, the primer binding site (the region of target DNA complementary to "a") is generally 18-30 bp in length. The sequence tag "b" is generally about 20 bp, and in theory they may be of any length longer than about 15 bp, but many people are primer sequences sold by companies on sequencing platforms. To use. The distance "d" between "a" and "X" may be at least 2 bp to avoid allele bias. If W multiplex PCR amplification is performed using the methods disclosed herein or other methods that require careful primer design to avoid excessive primer-to-primer interactions, it is acceptable. The window of the distance "d" between "a" and "X" can vary considerably: 2bp to 10bp, 2bp to 20bp, 2bp to 30bp, or even more than 2bp to 30bp. Therefore, when using the primer arrangement shown on the left side of FIG. 11, the sequence read must be a minimum of 40 bp to obtain a read long enough to measure the polymorphic locus. , "A" and "d" may require sequence reads up to 60 bp or 75 bp, depending on the length. In general, the longer the sequence read, the more cost and time it takes to sequence for a given number of reads, thus saving both time and money by minimizing the required read length. Can be done. In addition, on average, early base reads of reads are more accurate than late reads, thus reducing the length of sequence reads required to increase the accuracy of polymorphic region measurements. You can also.

In one embodiment, as shown in 103 of FIG. 11, a plurality of segments (a', a'', a'''. ...) and place the sequence tag (b) on the central DNA segment of the two primer binding sites. This arrangement allows the sequencer to perform shorter sequence reads. In certain embodiments, 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 length. In some embodiments, a ″ should be at least about 6 bp, and in some embodiments 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 at least as high as 6 bp, 8 bp, 10 bp, 12 bp, 15 bp, or even 20 bp or more. It can be truncated to the same extent as 30 bp. This can result in significant money, time and accuracy benefits. An example of an internally tagged primer is shown in FIG.

Primer with Ligation Adapter Binding Region One problem with fragmented DNA is that its short length makes polymorphisms more likely than long strands near the ends of DNA strands (eg,). 101, FIG. 10). Due to the inadequate overlap between the primer and the target binding site, the polymorph's capture by PCR requires primer binding sites of appropriate length on both sides of the polymorphism. Thus, a significant number of DNA strands with the target polymorphism are captured and compromised. In certain embodiments, the ligation adapter 102 can be attached to the target DNA 101, where the target primer 103 is a region (cr) complementary to the ligation adapter tag (lt) added upstream of the designed binding region (a). ) (See FIG. 13); therefore, if the binding region (101 region complementary to a) is shorter than the 18 bp generally required for hybridization, a primer complementary to the library tag. The region (cr) allows the binding energy to be increased to the point where PCR can proceed. Note that any specificity lost due to shorter binding regions can be supplemented by other PCR primers with appropriately long target binding regions. This embodiment is used in combination with either direct PCR or any of the other methods described herein, eg, nested PCR, semi-nested PCR, hemi-nested PCR, one-sided nested or semi-nested or hemi-nested PCR or other PCR protocols. Keep in mind that you can.

Low read depth alleles when sequencing data are used to determine multiples for various hypotheses in combination with analytical methods involving comparison of observed and predicted allele distributions. Each of the additional reads from will provide more information than a read from an allele with a high read depth. Therefore, ideally, it is desired to have a uniform read depth (DOR) where each locus has a similar number of representative sequence reads. Therefore, it is desirable to minimize the variance of DOR. In certain embodiments, it is possible to reduce the coefficient of variation of DOR, which can be defined as the standard deviation / mean DOR of 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, and greater than 1 hour or even longer. Since annealing is an equilibrium process, there is no limit to the improvement in DOR dispersion with increasing annealing time. In certain embodiments, increasing the primer concentration reduces the dispersion of DOR.

Representative Whole Genome Amplification Methods In some embodiments, DNA amplification, such as a whole genome application, can be included to amplify a nucleic acid sample before amplifying only the target locus. DNA amplification is the process of converting a small amount of genetic material into a larger amount of genetic material containing a similar collection of genetic data, including, but not limited to, the polymerase chain reaction (PCR). It can be done by the method. One method of amplifying DNA is whole genome amplification (WGA). There are several methods available for WGA: ligation-mediated PCR (LM-PCR), degenerate oligonucleotide primer PCR (DOP-PCR), and polysubstituted amplification (MDA). In LM-PCR, a short DNA sequence called an adapter is ligated to the blunt end of the DNA. These adapters contain a universal amplification sequence that is used to amplify DNA by PCR. In DOP-PCR, random primers also containing universal amplification sequences are used in the first round annealing and PCR. Second round PCR is then used to further amplify the sequence with universal primer sequences. MDA uses the phi-29 polymerase, a highly processive and non-specific enzyme that replicates DNA and is used for single cell analysis. The major limitations to the amplification of single cell-derived materials are (1) the need to use extremely diluted DNA concentrations or very small volumes of reaction mixtures, and (2) ensuring that DNA is derived from proteins throughout the genome. It is difficult to dissociate. Nevertheless, single-cell whole-genome amplification has been successfully used for many years of various applications. There are other ways to amplify DNA from a sample of DNA. In DNA amplification, the first sample of DNA is converted into a sample of DNA with a similar set of sequences but a much larger amount. In some cases, amplification may not be necessary.

In some embodiments, the DNA can be amplified using universal amplification, such as WGA or MDA. In some embodiments, the DNA can be amplified by using targeted amplification, eg, targeted PCR or cyclized probe. In some embodiments, the DNA is preferentially enriched using a targeted amplification method or a method that results in complete or partial separation of the desired and undesired DNA, eg, capture by hybridization techniques. can do. In some embodiments, the DNA can be amplified by using a combination of universal amplification methods and preferred enrichment methods. A more complete description of some of these methods can be found elsewhere in this document.

Representative enrichment and sequencing methods In certain embodiments, the methods disclosed herein are each target locus from a collection of target loci (eg, polymorphic loci) in a sample of the original DNA. Selective enrichment techniques are used to preserve the relative allele frequencies present at (eg, each polymorphic locus). Although enrichment is particularly useful as a method for analyzing polymorphic loci, these enrichment methods can also be easily adapted to non-polymorphic loci, if desired. 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 cyclization probes. In some embodiments, the method for amplification or selective enrichment is that when exactly hybridized to the target sequence, the 3'or 5'end of the nucleotide probe is allelic with a small number of nucleotides. It may be accompanied by the use of a probe that is separated from the polymorphic site. This gap reduces the preferential amplification of one allele, called allele bias.
This is an improvement over methods involving the use of probes such that the 3'or 5'end of the precisely hybridized probe is directly adjacent to or very close to the allele polymorphic site. In certain embodiments, probes where the hybridization region may or does not contain a polymorphic site are excluded. The presence of polymorphic sites at the site of hybridization can cause heterogeneous hybridization in some alleles, or the hybridization may be totally inhibited, resulting in specific alleles. It is preferentially amplified. These embodiments better preserve the original allelic frequency at each polymorphic locus of the sample, whether the sample is a pure genomic sample from a single individual or a mixture of individuals. In that respect, it is an improvement over other methods with targeted amplification and / or selective enrichment.

A number of unexpected results by enriching a sample of DNA in a set of target loci and then using sequencing techniques as part of a method for non-invasive prenatal allelic or polyploid calls. Benefits can be granted. In some embodiments of the present disclosure, the method comprises measuring an informatics-based method, eg, genetic data for use in PARENTAL SUPPORT ™ (PS). Some final outcomes of the embodiments are ready-to-use genetic data for embryos or fetuses. As part of the embodied methods, there are many methods that can be used to measure genetic data for individuals and / or related individuals. In certain embodiments, methods for enriching the concentration of a set of target allelic genes are disclosed herein, said method comprising one or more of the following steps: targeting a genetic material. Amplification step, adding a gene locus-specific oligonucleotide probe, ligating a specific DNA strand, isolating a desired set of DNA, removing unwanted components of the reaction, A step of detecting a specific DNA sequence by hybridization, and a step of 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 it may refer to the DNA strand. It may refer to the sequences or combinations thereof. These steps can be performed in a number of different orders.

For example, a universal amplification step of DNA prior to targeted amplification may provide some advantages, such as elimination of the risk of bottlenecks and reduction of allele bias. DNA can be mixed with oligonucleotide probes capable of hybridizing to two adjacent regions on either side of the target sequence. After hybridization, the ends of the probe can be ligated by adding a polymerase, which is a means of ligation, and any required reagent to allow cyclization of the probe. After cyclization, exonucleases can be added to digest the non-cyclized genetic material and then the cyclized probe can be detected. The DNA can be mixed with PCR primers that can hybridize to two adjacent regions on either side of the target sequence. After hybridization, the ends of the probe can be ligated by adding polymerase, which is a means of ligation, and any required reagent to complete PCR amplification. Amplified or unamplified DNA may be the target of a hybrid capture probe that targets a collection of loci, after hybridization, the probe is localized, separated from the mixture, and at the target sequence. It can result in a mixture of enriched DNA.

Targeting a particular locus as part of the method of allelic or polyploid call and then using the method of sequencing can provide a number of unexpected benefits. Several methods that can target or preferentially enrich DNA include hybridization-based capture methods such as cyclized probes, linked inverted probes (LIPs, MIPs), and SURESELECT. And include using targeted PCR or ligation-mediated PCR amplification strategies.

In some embodiments, the methods of the present disclosure include informatics-based methods, such as measuring genetic data for use in PARENTAL SUPPORT ™ (PS), further described herein. .. PARENTAL SUPPORT ™ is an informatics-based technique for manipulating genetic data, the embodiments of which are described herein. Some final outcome of the embodiment is a clinical decision based on the ready-to-use genetic data of the embryo or fetus, followed by the ready-to-use data. The background algorithm of the PS method obtains measured genetic data of the target individual, often embryo or fetus, and genetic data measured from related individuals to determine the accuracy of the genetic status of the target individual. Can be raised. In certain embodiments, the measured genetic data is used in situations where polyploidy determinations are made during prenatal genetic diagnosis. In certain embodiments, the measured genetic data is used in situations where polyploidy determinations or allelic calls are made to embryos during in vitro fertilization. There are many methods that can be used to measure genetic data for individuals and / or related individuals in the situations described above. Different methods involve a number of steps, often simply the steps of amplifying a genetic material, adding an oligonucleotide probe, ligating a particular DNA strand, the desired set of DNA. It involves a step of separation, a step of removing unwanted components of the reaction, a step of detecting a specific DNA sequence by hybridization, and a step of detecting the sequence of one or more DNA strands by a DNA sequencing method. .. In some cases, a DNA strand means a target genetic material, in some cases, a DNA strand means a primer, and in some cases, a DNA strand means a synthesized sequence, or a combination thereof. do. These steps can be performed in a number of different orders.

Note that it is theoretically possible to target any number of loci in the genome, from one locus to over one million. When a sample of DNA is targeted and then sequenced, the proportion of alleles read by the sequencer is enriched relative to the amount they are naturally present in the sample. The degree of enrichment may be from 1 percent (or even lower) to 10 times, 100 times, 1,000 times or even more 1 million times. There are approximately 3 billion base pairs and nucleotides in the human genome, including approximately 75 million polymorphic loci. The more loci targeted, the less enrichment is possible. The smaller the number of targeted loci, 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.

In certain embodiments of the present disclosure, targeting or priorial can be fully focused on SNPs. In certain embodiments, targeting or prioritization can focus on any polymorphic site. A number of commercial targeting products are available to enrich exons. Surprisingly, targeting SNPs exclusively or exclusively at polymorphic loci is particularly advantageous when using methods for NPD that rely on allelic distribution. There are also published methods for NPDs that use sequencing, for example, US Pat. No. 7,888,017 focuses on counting the number of reads in which the number of reads maps to a given chromosome. With read count analysis applied, the sequence reads analyzed do not focus on regions of the polymorphic genome. These types of methodologies that do not focus on polymorphic alleles are less useful than targeting or preferentially enriching the set of alleles.

In certain embodiments of the present disclosure, it is possible to use SNP-focused targeting methods to enrich genetic samples in polymorphic regions of the genome. In certain embodiments, a small number of SNPs, eg, SNPs between 1 and 100, or more, eg, between 100 and 1,000, between 1,000 and 10,000, and 10,000 to 100. It is possible to focus on SNPs between 000 or over 100,000. In certain embodiments, the focus is on one or a few chromosomes that correlate with birth in a living trisomy, such as chromosomes 13, 18, 21, 21, X and Y, or some combination thereof. It is possible. In certain embodiments, the target SNP has a small factor, eg, between 1.01 and 100 times, or a larger factor, such as between 100 and 1,000,000 times, or 1,000. It is possible to enrich up to more than 000 times. In one embodiment of the present disclosure, it is possible to use a targeting method to prepare a sample of DNA that is preferentially enriched in a polymorphic region of the genome. In certain embodiments, this method can be used to make a mixture of DNA having any of these properties, where the mixture of DNA also contains maternal DNA and floating fetal DNA. .. In certain embodiments, it is possible to use this method to make a mixture of DNA having any combination of these coefficients. For example, using the methods described herein, it contains maternal and fetal DNA, 200 SNPs, all located on either chromosome 18 or chromosome 21, and on average 1,000-fold richer. It is possible to produce a mixture of DNA that is preferentially enriched in the DNA corresponding to the 200 SNPs to be converted. In another example, using the method described above, 10,000 SNPs, all or most of which are located on chromosomes 13, 18, 21, X and Y, and have an average wealth per locus. It is possible to make a mixture of DNA that is preferentially enriched at 10,000 SNPs that are more than 500-fold. Any of the targeting methods described herein can be used to make a mixture of DNA that is preferentially enriched at a particular locus.

In some embodiments, the method of the present disclosure is a step of measuring DNA in a mixed fraction using a high throughput DNA sequencer, wherein the DNA in the mixed fraction is one or an disproportionate number. Further steps are selected from the group containing sequences from multiple chromosomes, where one or more chromosomes include chromosomes 13, 18, 21, 21, X, Y and combinations thereof. include.

Three methods are described herein, multiplex PCR, targeted capture by hybridization, and ligated reverse probe (LIP), which can be used to detect fetal variability in the maternal line. Measurements can be obtained and analyzed from a sufficient number of polymorphic loci derived from plasma samples. This does not preclude other methods of selectively enriching the target locus. Other methods can be used equally well without changing the core of the method. In each case, the polymorphism assayed may include single nucleotide polymorphisms (SNPs), small insertion deletions or STRs. The preferred method involves the use of SNPs. Each technique produces allelic frequency data, which can be analyzed for allelic frequency data for each target locus and / or simultaneous allelic frequency distributions from these loci to determine fetal multiples. can. Each approach has its own considerations due to the limited source material and the fact that maternal plasma consists of a mixture of maternal DNA and fetal DNA. This method can be combined with other methods to result in more accurate decisions. In certain embodiments, this method can be combined with sequence counting techniques such as those described in US Pat. No. 7,888,017. The techniques described can also be used to detect fetal paternality non-invasively from maternal plasma samples. In addition, each method detects segmented copy number variation (CNV) to genotype a large number of SNPs from degraded DNA samples to detect the presence or absence of heterologous chromosomes. Therefore, it can be applied to a mixture of other DNAs or a pure DNA sample to detect the status of other genotypes of interest or some combination thereof.

Accurate measurement of allele distribution in a sample Current sequencing techniques can be used to estimate the distribution of alleles in a sample. One such method involves a step of randomly sampling sequences from pooled DNA, called shotgun sequencing. The proportion of a particular allele in the 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 yields 100 bp reads, the particular allele is measured approximately once for every 30 million sequence reads.

In certain embodiments, the methods of the present disclosure are used to determine the presence or absence of two or more different haplotypes containing the same set of loci in a sample of DNA, a measured allele of the locus derived from that chromosome. Determined from the distribution. Different haplotypes are two different homologous chromosomes from one individual, three different homologous chromosomes from trisomy individuals, and three different homologous haplotypes from the mother and fetus, one of which is with the mother. A haplotype shared between the fetus, three or four haplotypes derived from the mother and the fetus, one or two of which may indicate a haplotype shared between the mother and the fetus, or another combination. .. Alleles that are polymorphic between haplotypes tend to be more informative, but simple through the allele distribution measured by any allele whose mother and father are not homozygous for the same allele. It provides useful information that goes beyond what is available from read count analysis.

However, shotgun sequencing of such samples results in many sequences for non-polymorphic regions, or non-target chromosomes, among different haplotypes in the sample, and thus the proportion of target haplotypes. It is very inefficient because it does not show any information about. Here we describe alleles obtained by sequencing by specifically targeting and / or preferentially enriching a segment of DNA in a sample that is more likely to be polymorphic in the genome. It describes how to increase the yield of information. One allele distribution measured in an enriched sample that would truly represent the actual amount present in the target individual compared to other alleles at a given locus within the target segment. Note that it is important that the preferential enrichment of alleles is small or absent. Current methods known in the art for targeting polymorphic alleles are designed to ensure that at least a portion of any existing allele is detected. However, these methods were not designed for the purpose of measuring the unbiased allele distribution of polymorphic alleles present in the original mixture. Any particular method of target enrichment can produce an enriched sample, any other method of allelic distribution present in the original unamplified sample with the measured allelic distribution. It is not self-evident to show better and more accurately than. In theory, it can be expected that many enrichment methods will serve such objectives, but those skilled in the art have considerable probabilistic theory for current amplification, targeting and other preferred enrichment methods. I understand well that there is a target or deterministic bias. By one embodiment of the method described herein, multiple alleles found in a mixture of DNA corresponding to a given locus in the genome will have approximately the same degree of enrichment of each allele. It becomes possible to amplify or preferentially enrich. In other words, by the method described above, the ratio between alleles corresponding to each locus remains essentially the same as the ratio in the original DNA mixture, with the relative amount of alleles present in the mixture as a whole. It will be possible to increase. Some reported methods can result in over 1%, over 2%, over 5%, and even over 10% allele bias. This preferential enrichment may be small for capture bias when using hybridization techniques, or for each cycle, but becomes greater when assembled over 20, 30 or 40 cycles. It may be due to the bias of the resulting amplification. For the purposes of the present disclosure, the ratio remains essentially the same when the ratio of alleles in the original mixture divided by the ratio of alleles in the resulting mixture is 0.95 to 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. It means that it is between 0.9999 and 1.0001. The calculation of allele ratios presented herein cannot be used to determine the ploidy state of a target individual, but may simply be a metric used to measure allele bias. Please note.

In one embodiment, once the mixture is preferentially enriched in the set of target loci, cloned samples (samples generated from a single molecule; examples include ILLUMINA GAIIx, ILLUMINA HiSeq, Life Technologies SOLiD, 5500XL. Can be sequenced using any one of the previous, current or next generation sequencing instruments that sequence. Ratios can be assessed by sequencing through specific alleles within the target region. These sequencing reads can be analyzed and counted according to the type of allele and the allocation of different alleles thus determined. For variations that are one to several bases in length, allele detection is performed by sequencing and it is essential that the sequencing reads span the alleles in question in order to assess the composition of the alleles in the captured molecule. Is. The total number of captured molecules assayed for genotype can be increased by increasing the length of the sequencing read. Complete sequencing of all molecules ensures the collection of the maximum amount of data available in the enriched pool. However, sequencing is now costly, and methods that allow allelic distribution to be measured using a small number of sequence reads are of great value. In addition, increasing lead length introduces technical and accuracy limits to the maximum possible lead length. The most useful allele is one to several bases in length, but theoretically any allele that is shorter than the length of the sequencing read can be used. Allelic variability occurs in all types, but the examples provided herein focus on SNPs or variants contained in only a few adjacent base pairs. Larger variants, such as segmented copy count variants, are often detected by summing up these smaller variations, as the overall population of SNPs within the segment overlaps. can do. Mutants larger than a few bases, such as STR, require special consideration and some targeting technique studies, but not others.

There are multiple targeting techniques that can be used to specifically isolate and enrich the location of one or more mutants in the genome. In general, they rely on utilizing unmutated sequences that are flanked by mutant sequences. There are reports by other researchers related to targeting in sequencing when the substrate is maternal plasma (see, eg, Liao et al., Clin. Chem. 2011; 57 (1): pp. 92-101). .. However, these methods use targeting probes that target exons and do not focus on targeting polymorphic regions of the genome. In certain embodiments, the methods of the present disclosure include the step of using a targeted probe that focuses exclusively or nearly exclusively on the polymorphic region. In certain embodiments, the methods of the present disclosure include the step of using a targeted probe that focuses exclusively or nearly exclusively on the SNP. In some embodiments of the present disclosure, the target polymorphic site is at least 10% SNP, at least 20% SNP, at least 30% SNP, at least 40% SNP, at least 50% SNP, 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 It consists of SNPs.

In certain embodiments, the methods of the present disclosure can be used to determine the genotype (the basic composition of DNA at a particular locus) and the relative proportions of these genotypes from a mixture of DNA molecules. DNA molecules can originate from one or several genetically distinct individuals. In certain embodiments, the methods of the present disclosure can be used to determine the genotype in a set of polymorphic loci and the relative ratio of the amount of different alleles present at these loci. In certain embodiments, the polymorphic locus may consist entirely of SNPs. In certain embodiments, the polymorphic locus may include SNPs, single tandem repeats, and other polymorphisms. In certain embodiments, the methods of the present disclosure can be used to determine the relative distribution of alleles in a set of polymorphic loci in a DNA mixture, where the DNA mixture is a DNA of maternal origin, And contains DNA originating from the fetus. In certain embodiments, the co-allelic distribution can be determined for a mixture of DNA isolated from blood from a pregnant woman. In certain embodiments, the allelic distribution in a set of loci can be used to determine the ploidy state of one or more chromosomes for a pregnant fetus.

In certain embodiments, the mixture of DNA molecules may be derived from DNA extracted from multiple cells of an individual. In one embodiment, when the individual is a mosaic (germline or somatic cell), the original cell population from which the DNA is derived contains a mixture of diploid cells or haploid cells of the same or different genotypes. Can include. In certain embodiments, the mixture of DNA molecules may be derived from DNA extracted from a single cell. In certain embodiments, the mixture of DNA molecules may be derived from DNA extracted from a mixture of two or more cells of the same individual or two or more cells of different individuals. In certain embodiments, the mixture of DNA molecules may be derived from DNA isolated from a biological material already freed from cells, such as plasma, which is known to contain cell-free DNA. In certain embodiments, the biomaterial may be a mixture of DNA from one or more individuals, as in the case of pregnancy in which fetal DNA has been shown to be present in the mixture. In certain embodiments, the biomaterial may be from a mixture of cells found in the blood of the maternal line, some of which are of fetal origin. In certain embodiments, the biological material may be cells derived from pregnant blood enriched in fetal cells.

Cyclic probe Some embodiments of the present disclosure use a "connected reverse probe" (LIP) previously described in the literature, either prior to amplification with a non-LIP primer in the multiplex PCR method of the invention. Later, it involves amplifying the target locus. LIP is a generic term that means to include techniques involving the production of circular DNA molecules, and probes are designed to hybridize to regions of the target DNA on both sides of the target allele. Thus, with the addition of the appropriate polymerase and / or ligase, and appropriate conditions, buffers and other reagents, complementary reverse regions of DNA across the target allele are completed and found in the target allele. A circular loop of DNA that captures information is created. The LIP is also referred to as a pre-cyclalized probe, a pre-cyclalizing probe or a cyclized probe. The LIP probe may be a linear DNA molecule with a length between 50 and 500 nucleotides, in some embodiments between 70 and 100 nucleotides in length, and in some embodiments. , May be longer or shorter than described herein. Other embodiments of the present disclosure involve different reifications of LIP technology, such as padlock probes and reverse molecular probes (MIPs).

One method of targeting a specific site for sequencing is that the 3'and 5'ends of the probe are close to the target DNA and the target region and are annealed in a reverse fashion at both sites. Therefore, the addition of DNA polymerase and DNA ligase results in extension from the 3'end, adding a base to the single-stranded probe complementary to the target molecule (gap filling), followed by a new 3'. Synthesizing a probe such that the end ligates to the 5'end of the original probe, resulting in a circular DNA molecule that can later be isolated from the background DNA. The probe end is designed to be adjacent to the target area of interest. O One aspect of this technique, commonly referred to as MIPS, is used in conjunction with array technology to determine the nature of the array to be filled. One drawback of using MIP in situations where the ratio of alleles is measured is that the hybridization, cyclization and amplification steps do not occur at equal rates for different alleles at the same locus. As a result, the ratio of alleles that do not represent the ratio of the actual alleles present in the original mixture is measured.

In certain embodiments, the circularized probe is designed to hybridize to a region of the probe that is designed to hybridize upstream of the target polymorphic locus and downstream of the target polymorphic locus. Regions of the probe are constructed to connect covalently through a non-nucleic acid skeleton. The skeleton may be any biocompatible molecule or a combination of biocompatible molecules. Some examples of possible biocompatible molecules are poly (ethylene glycol), polycarbonate, polyurethane, polyethylene, polypropylene, sulfone polymers, silicones, cellulose, fluoropolymers, acrylic compounds, styrene block copolymers, and others. It is a block copolymer of.

In certain embodiments of the present disclosure, the technique has been modified to facilitate sequencing as a means of examining filling within a sequence. At least one important consideration must be taken into account in order to retain the proportion of the original allele in the original sample. The location of variability between different alleles within the gap-filled region should not be too close to the probe binding site, as there may be a bias in initiation by the DNA polymerase that results in the differentiation of the mutant. Another consideration is that there may be additional variability in probe binding sites that correlate with variants within the gap-filled region that can result in uneven amplification from different alleles. In one embodiment of the present disclosure, the 3'and 5'ends of the pre-cyclization probe hybridize to a base that is separated from the mutation location (polymorphic site) of the target allele by one or a few positions. Design to do. The number of bases between the polymorphic site (SNP or other type) and the base designed to hybridize the 3'and / or 5'ends of the pre-cyclization probe is 1 base. It may be 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7-10 bases, 11 It may be up to 15 bases, or 16 to 20 bases, 20 to 30 bases or 30 to 60 bases. Forward and reverse primers can be designed to hybridize to different numbers of bases away from the polymorphic site. A large number of circularized probes can be generated using current DNA synthesis techniques, which allows a large number of probes to be generated and potentially pooled, allowing the simultaneous examination of many loci. Can be done. It has been reported to work with over 300,000 probes. Two papers discussing methods involving circularized probes that can be used to measure genomic data of target individuals include Porreca et al., Nature Methods, 2007, Vol. 4, (No. 11), 931. Pp. 936; and similarly, Turner et al., Nature Methods, 2009, Volume 6 (No. 5), pp. 315-316. The methods described in these papers can be used in combination with the other methods described herein. The specific steps of the method from these two articles can be used in combination with other steps from the other methods described herein.

In some embodiments of the methods disclosed herein, the genetic material of the target individual is amplified as needed, then hybridized with the pre-cyclization probe, and gap filling is performed for hybridization. The probe is filled with a base between the two ends and the two ends are ligated to form a circularized probe, and the circularized probe is amplified using, for example, rolling circle amplification. Once the genetic information of the desired target allele is captured in a well-designed circular oligonucleotide-like probe, eg, a LIP system, the gene sequence of the circularized probe is measured to yield the desired sequence data. Can be done. In certain embodiments, a well-designed oligonucleotide probe can be directly cyclized in the genetic material of the unamplified target individual and then amplified. It should be noted that a number of amplification procedures, including rolling circle amplification, MDA or other amplification protocols, can be used to amplify the original genetic material or to cyclize the LIP. Using different methods, eg, high-throughput sequencing, Sanger sequencing, other sequencing methods, hybridization-based capture, cyclization-based capture, multiplex PCR, other hybridization methods, and combinations thereof. Genetic information on the target genome can be measured.

One or a combination of the above methods, an informatics-based method, eg, the PARENTAL SUPPORT ™ method, is used in conjunction with appropriate genetic measurement to measure an individual's genetic material, which is then used in the individual. Determines the polymorphic state of one or more chromosomes and / or the state of the gene for one or a set of alleles (specifically, alleles that correlate with the state of the disease or gene of interest). be able to. Note that the use of LIP for multiplexing and capturing gene sequences and then genotyping using sequencing has been reported. However, the use of sequencing data generated by LIP-based strategies to amplify genetic material found in single cells, small numbers of cells or extracellular DNA is to determine the ploidy state of the target individual. Is not used.

The application of informatics-based methods for determining an individual's multiple state from genetic data measured by a hybridization array, such as the ILLUMINA INFINIUM array or the AFFYMETRIX gene chip, is described in references elsewhere in this document. ing. However, the methods described herein show an improvement over the methods previously described in the literature. For example, with a LIP-based approach followed by high-throughput sequencing, this approach is expected because it has better multiplexing capabilities, better capture specificity, better uniformity, and less allele bias. Externally, better genotype data is provided. Larger multiplexing allows more alleles to be targeted, resulting in more accurate results. The better homogeneity measures more target alleles, resulting in more accurate results. The lower rate of allele bias reduces the rate of false calls and provides more accurate results. More accurate results improve clinical outcomes and lead to better medical care.

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

Ligation-mediated PCR
Target loci can be amplified using ligation-mediated PCR before or after PCR amplification with unligated primers. Ligation-mediated PCR is a method of PCR used to preferentially enrich a sample of DNA by amplifying one or more loci in a mixture of DNA, wherein the method is a set of primer pairs. Each primer in the pair contains a target-specific sequence and a non-target sequence, preferably the target-specific sequence is the target region, one upstream of the polymorphic site, and one. Designed to anneal to a target region downstream of the polymorphic site, the target-specific sequence is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, from the polymorphic site. Steps that may be separated by 10, 11-20, 21-30, 31-40, 41-50, 51-100, or more than 100, and DNA polymerized from the 3'end of the upstream primer, and The step of filling the single-stranded region between the 5'end of the downstream primer having a nucleotide complementary to the target molecule and the last polymerized base of the upstream primer are the 5'bases of the adjacent downstream primer. It comprises the steps of ligating with and 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 acts as a universal sequence and therefore all successfully polymerized and ligated primer pairs can be amplified with a single pair of amplification primers.

Capturing by Hybridization In some embodiments, the method of the present disclosure comprises the step of using capture by any of the following hybridization methods, in addition to amplifying the target locus using multiplex PCR. be able to. Priority enrichment of a set of specific sequences in the target genome can be achieved in a number of ways. Other parts of this document describe how LIP can be used to target a set of specific sequences, but in all of these applications other targeting and / Alternatively, preferred enrichment methods can be used equally well for the same purpose. One example of another targeting method is capture by hybridization techniques. Some examples of capture by commercial hybridization techniques include Agilent's SURE SELECT and ILLUMINA's TruSeq. Hybridization capture allows the set of oligonucleotides that are complementary or nearly complementary to the desired target sequence to hybridize to the mixture of DNA and then be physically separated from the mixture. Once the desired sequence hybridizes to the targeted oligonucleotide, the action of physically extracting the targeted oligonucleotide will also extract the target sequence. Once the hybridized oligos are removed, they can be heated and amplified to above their melting temperature. Several methods for physically removing the targeted oligonucleotide are by covalently attaching the targeted oligonucleotide to a solid support, such as a magnetic bead or chip. Another method for physically removing a targeted oligonucleotide is by covalently attaching the targeted oligonucleotide to a molecular moiety that has a strong affinity for another molecular moiety. Examples of such molecular pairs are, for example, biotin and streptavidin used in SURE SELECT. Therefore, the target sequence can be covalently attached to the biotin molecule, and after hybridization, a solid support to which streptavidin is added can be used to pull down the biotinylated oligonucleotide to which the target sequence has hybridized. can.

Hybrid capture involves hybridizing a probe complementary to the target of interest to the target molecule. Hybrid capture probes were originally developed to target and enrich most of the genome with relative homogeneity between targets. In its application, it was important that all amplified targets had sufficient homogeneity so that all regions could be detected by sequencing, but retaining the proportion of alleles in the original sample. Was not paid attention to. After capture, alleles present in the sample can be determined by direct sequencing of the captured molecule. These sequencing reads can be analyzed and counted according to the type of allele. However, using current techniques, the measured allelic distribution of the captured sequence generally does not represent the original allelic distribution.

In certain embodiments, allele detection is performed by sequencing. In order to capture allele identity at the polymorphic site, it is essential that the sequencing read span the allele in question in order to assess the composition of the alleles in the captured molecule. Since capture molecules often vary in length, it cannot be guaranteed that mutation positions will overlap unless the entire molecule is sequenced during sequencing. However, due to cost considerations and technical limitations regarding the maximum possible length and accuracy of sequencing reads, whole molecule sequencing cannot be performed. In certain embodiments, a read length that can be increased from about 30 bases to about 50 bases or about 70 bases can significantly increase the number of reads that overlap the location of the mutation in the target sequence.

Another way to increase the number of reads to locate a subject is to reduce the length of the probe as long as it does not result in a bias in the underlying enriched alleles. The length of the synthesized probe is such that the two probes designed to hybridize with two different alleles found at one locus have approximately the same affinity as the various alleles in the original sample. It should be long enough to hybridize by sex. Currently, methods 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 is 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, about 60. It may be less than a base, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases, and this amount is sufficient to ensure equivalent enrichment from all allelics. Is. When the mixture of DNA enriched using hybrid capture techniques is a mixture containing floating DNA isolated from blood, eg maternal blood, the average length of the DNA is fairly short, generally less than 200 bases. be. The use of shorter probes increases the likelihood that the hybrid capture probe will capture the desired DNA fragment. Larger variability may require longer probes. In certain embodiments, the variation of interest is from 1 (SNP) to a few bases in length. In certain embodiments, a region of the target 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 less than 80 bases. It may be less than 70 bases, less than 60 bases, less than 50 bases, less than 40 bases, less than 30 bases or less than 25 bases. In certain embodiments, the length of the probe, which is designed to hybridize with a region flanking the location of the polymorphic allelic gene, to increase the likelihood that the desired allelic gene will be sequenced. It can be reduced from over 90 bases to about 80 bases, or to about 70 bases, or to about 60 bases, or to about 50 bases, or to about 40 bases, or to about 30 bases, or to about 25 bases. ..

There is minimal overlap between the synthesized probe and the target molecule to allow capture. This synthesized probe can be as short as possible, but still greater than this minimum required overlap. The effect of using shorter probe lengths to target polymorphic regions is that more molecules overlap the target allele region. The fragmentation status of the original DNA molecule also affects the number of reads that overlap the target allele. Some DNA samples, such as plasma samples, are already fragmented due to biological processes that occur in vivo. However, for samples with longer fragments, fragmentation prior to the preparation and enrichment of the sequencing library is beneficial. When both the probe and the fragment are short (about 60-80 bp), maximum specificity can be achieved with relatively few sequence reads that cannot overlap the critical region of interest.

In certain embodiments, hybridization conditions can be adjusted to maximize homogeneity in the capture of different alleles present in the original sample. In certain embodiments, the hybridization temperature is lowered to minimize differences in hybridization bias between alleles. In methods known in the art, lowering the temperature has the effect of increasing hybridization between the probe and unintended targets, so it is recommended to use a lower temperature for hybridization. To avoid. However, if the goal is to preserve allelic ratios with maximum fidelity, then methods using lower hybridization temperatures are optimal despite the fact that the teachings of current techniques avoid this method. Provides the exact allele ratio. The hybridization temperature can also be increased to require a larger overlap between the target and the synthesized probe so that only targets with substantial overlap in the target area are captured. .. In some embodiments of the present disclosure, the hybridization temperature is increased from normal hybridization temperature to about 40 ° C, up to about 45 ° C, up to about 50 ° C, up to about 55 ° C, up to about 60 ° C, up to about 65. Alternatively, the temperature is lowered to about 70 ° C.

In one embodiment, the hybrid capture probe is designed so that the region of the capture probe having DNA complementary to the DNA found in the region adjacent to the polymorphic allele is not immediately adjacent to the polymorphic site. Can be done. Instead, the capture probe is designed to hybridize to DNA adjacent to the target polymorphic site, with the portion of the capture probe where the region of the capture probe contacts the polymorphic site by van der Waals. It can be designed to be separated by a small distance of equal length to one or a few bases. In one embodiment, the hybrid capture probe is designed to hybridize to a region that is adjacent to, but not intersecting with, the polymorphic allele, which is referred to as the adjacent capture probe. .. Adjacent capture probes 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, about 50 bases. It may be less than, less than about 40 bases, less than about 30 bases, or less than about 25 bases. The regions of the genome targeted by the adjacent capture probe are 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, and 9 base pairs with the polymorphic locus. It may be separated by base pairs, 10 base pairs, 11 to 20 base pairs, or 20 super base pairs.

A description of a disease screening test based on targeted capture using targeted sequence capture. Custom-made targeted sequence captures such as those currently offered by Agilent (SURE SELECT), ROCHE-NIMBLEGEN or ILLUMINA. Capturing probes can be custom designed to ensure the capture of various types of mutations. For point mutations, one or more probes that overlap with the point mutation should be sufficient to capture and sequence the mutation.

For small insertions or deletions, one or more probes that overlap the mutation may be sufficient to capture and sequence the fragment containing the mutation. Hybridization may be less efficient among probe-limited capture efficiencies that are generally designed to reference genomic sequences. Two probes can be designed to ensure capture of the fragment containing the mutation, one that matches the normal allele and one that matches the mutant allele. Hybridization can be enhanced with longer probes. Capturing can be enhanced by multiple overlapping probes. Finally, placing the probe directly next to it but not overlapping may allow the mutation to have relatively similar capture efficiencies for normal and mutant alleles.

For simple tandem repeats (STRs), probes that overlap these highly variable sites are unlikely to successfully capture the fragment. To enhance capture, the probe can be placed in close proximity to the variable site but not overlapping. The fragments can then be sequenced as usual to indicate the length and composition of the STR.

For large deletions, a series of overlapping probes, a common technique currently used in exon capture systems, may work. However, using this technique, it can be difficult to determine if an individual is heterozygous. Targeting and assessing SNPs within a captured region can potentially indicate a loss of heterozygotes across that region, indicating that the individual is a carrier. In certain embodiments, non-overlapping or singleton probes can be placed over potentially deleted regions and the number of captured fragments can be used as a measure of heterozygosity. If the individual has a large deletion, it is predicted that half the number of fragments will be available for capture compared to the non-deleted (diploid) reference locus. To. Therefore, the number of reads obtained from the deleted region should be approximately half the number of reads obtained from the normal diploid locus. Sequencing read depths from multiple singleton probes across potentially deleted regions can be summed and averaged to enhance the signal and improve diagnostic confidence. It is also possible to combine two methods, targeting SNPs to identify heterozygous losses, and using multiple singleton probes to obtain a quantitative measure of the amount of underlying fragment from that locus. can. Either or both of these strategies can be combined with other strategies to achieve the same results better.

Detection of male fetal cfDNA, as well as X-linked dominant mutations unaffected by the mother and father or dominant mutations unaffected by the mother, as indicated by the presence of a Y chromosome fragment captured and sequenced in the same test during the test. It is shown that either of these increases the risk to the fetus. The detection of two mutant recessive alleles within the same gene in an unaffected mother means that the fetus inherited the mutant allele from the father and potentially the second mutant allele from the mother. Means. In all cases, follow-up by amniocentesis or chorionic villus collection may also be indicated.

Disease screening tests based on targeted capture can be combined with non-invasive prenatal diagnostic tests based on targeted capture for aneuploidy. There are a number of ways to reduce read depth (DOR) variability: for example, longer targeted amplification probes can be used that can increase primer concentration, or more STA cycles. Many can be performed (eg, more than 25, more than 30, more than 35, or even more than 40).

Representative method for determining the number of DNA molecules in a sample During the first round of DNA amplification, the number of DNA molecules in a sample is determined by generating molecules that are uniquely identified for each of the original DNA molecules in the sample. Methods for determining are described herein. Procedures for achieving the above object, followed by a single molecule or clonal sequencing method, are described herein.

The technique uses steps to target one or more specific loci, and each target locus has a unique tag, and this barcode is sequenced using a clone sequence or a single molecule sequence. Includes the steps of producing a tagged copy of the original molecule so that it can be distinguished from each other when sequenced. Each of the uniquely sequenced barcodes shows a unique molecule in the original sample. At the same time, the sequencing data is used to identify the locus from which the molecule is derived. This information can be used to determine the number of unique molecules in the original sample for each locus.

This method can be used for any application that requires a quantitative assessment of the number of molecules in the original sample. In addition, the number of unique molecules of one or more targets is associated with the number of unique molecules for one or more other targets, relative copy number, allele distribution or allele ratio. Can be determined. 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, insertions and deletions, eg, detection of those found in carriers of Duchenne muscular dystrophy; quantification of deletions or duplications of chromosomal segments, eg, those observed in copy number variants. The number of chromosomal copies of the sample from the born individual; the number of chromosomal copies of the sample from the unborn individual, eg, embryo or fetal.

The method can be combined with an assessment of concomitant variability 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 an assessment of SNP or other sequence variability to determine the number of chromosomal copies of born and unborn individuals; with short sequence variability, among which PCR For the purpose of identifying and quantifying locus-derived copies that can be amplified from multiple target regions, eg, for carrier detection of spinal cord atrophy; various sources of sample-derived molecules consisting of a mixture of different individuals. For the purpose of determining the number of copies of, eg, detecting fetal aneuploidy from floating DNA obtained from maternal plasma.

In certain embodiments, a method involving a single target locus may include one or more of the following steps: (1) a standard pair of oligomers for PCR amplification of a particular locus. Steps to design. (2) During synthesis, a sequence of a specific base having no complementarity to the target locus or genome or having minimal complementarity is added to the 5'end side of one of the target-specific oligomers. Steps to do. This sequence, referred to as the tail, is a known sequence that is used for subsequent amplification, followed by a random nucleotide sequence. These random nucleotides contain random regions. Random regions contain sequences of randomly generated nucleic acids that are stochastically different between each probe molecule. Thus, after synthesis, the tailed oligomer pool consists of a population of oligomers starting with a known sequence, followed by an unknown sequence that differs between molecules, followed by a target-specific sequence. (3) A step of performing one round of amplification (denaturation, annealing, elongation) using only the tailed oligomer. (4) Add exonuclease to the reactants, effectively stop the PCR reaction, incubate the reactants at an appropriate temperature to remove the forward single-stranded oligo that did not anneal to the template, and extend the two strands. Steps to form chain products. (5) Incubate the reactants at high temperatures to denature exonucleases and eliminate their activity. (6) Other targets are new oligonucleotides complementary to the tail of the oligomer used in the first reaction. A step that allows PCR amplification of the product produced in the first round of PCR in addition to the reactants with specific oligomers. (7) A step of continuing amplification to produce sufficient products for downstream clonal sequencing. (8) A step of measuring an amplified PCR product, in which a sufficient number of bases are sequenced, by a number of methods, eg, clonal sequencing.

In certain embodiments, the methods of the present disclosure include the steps of targeting multiple loci in parallel or otherwise. Primers for different target loci can be independently generated and mixed to create a multiplex PCR pool. In certain embodiments, the original sample can be divided into subpools, where each subpool can be recombined and sequenced after targeting different loci. In one embodiment, the pool was subdivided before tagging and a number of amplification cycles were performed to ensure efficient targeting of all targets, followed by subdivision, improvement, and then subdivision. It can be amplified by continuing amplification using a smaller set of primers in the pool.

One example of an application in which this technique becomes particularly useful is non-invasive prenatal aneuploidy diagnosis, using the ratio of alleles at a given locus or the distribution of alleles at multiple loci. , Can help determine the number of copies of chromosomes present in the fetus. In this situation, it is desirable to amplify the DNA present in the first sample while maintaining the relative amount of various alleles. In some cases, in particular, there is a very small amount of DNA present, for example, less than 5,000 copies of the genome, less than 1,000 copies of the genome, less than 500 copies of the genome, and less than 100 copies of the genome. In the case of copying, a phenomenon called bottlenecking can occur. This is because there are a small number of copies of any given allele in the first sample and as a result of the amplification bias, the ratio of those alleles in the pool of amplified DNA is in the first DNA mixture. The ratio of their alleles is significantly different. By applying a unique or nearly unique set of barcodes to each DNA strand prior to standard PCR amplification, n identical molecules of sequenced DNA originating from the same original molecule It is possible to exclude n-1 copies of DNA from the set.

For example, consider a heterozygous SNP in an individual's genome and a mixture of DNA from an individual in which 10 molecules of each allele are present in a sample of the original DNA. After amplification, there may be 100,000 molecules of DNA corresponding to that locus. Due to the stochastic process, the DNA ratio can be any of 1: 2 to 2: 1, but since each of the original molecules was tagged with a unique tag, the amplified pool. It is possible to determine that the DNA within is exactly from 10 molecules of DNA from each allele. Therefore, this method results in a relative amount of each allele with a more accurate measure than the method without this method. This method provides accurate data for methods in which it is desirable to minimize the relative amount of allele bias.

The association of sequenced fragments with target loci can be achieved in a number of ways. In one embodiment, the molecular barcode corresponding to the target sequence, as well as a sufficient number of unique bases, are tied to the target fragment to obtain a sequence of sufficient length to clearly identify the target locus. Enables. In another embodiment, a molecular barcoding primer containing a randomly generated molecular barcode can also contain a locus-specific barcode (locus barcode) that identifies the target with which it is associated. .. This locus barcode is identical among all molecular barcoding primers for each of the individual targets and thus among all of the resulting amplification products, but unlike all other targets. In certain embodiments, the tagging methods described herein can be combined with a one-sided nesting protocol.

In certain embodiments, the design and generation of molecular barcoding primers can be implemented as follows: a molecular barcoding primer is a sequence that is not complementary to the target sequence, followed by a random molecular barcode region, followed by. It may consist of 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 amplification product into a library for sequencing. Random molecular barcode sequences can be generated in a number of ways. The preferred method is to synthesize the molecularly tagged primer to include all four bases for the reaction during the synthesis of the barcode region. All or various combinations of bases can be specified using the IUPACDNA ambiguous code. Thus, the synthesized population of molecules contains a random mixture of sequences within the molecular barcode region. The length of the barcode region determines how many primers contain a unique barcode. The number of unique sequences is associated with the length of the barcode region as N ^L , 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 certain embodiments, the DNA can be measured by a sequencing method and the sequence data indicates the sequence of a single molecule. This is a method of direct sequencing on a single molecule, or amplifying a single molecule to form a clone detectable by a sequencing instrument, but still showing a single molecule, as used herein with clone sequencing. It can include the method referred to.

Typical Methods and Reagents for Quantification of Amplified Products Quantification of specific nucleic acid sequences of interest is usually performed by quantitative real-time PCR techniques such as TAQMAN (LIFE TECHNOLOGIES), INVADER probe (THIRD WAVE TECHNOLOGIES). Such techniques are accurate only in the limited capacity for parallel simultaneous analysis (multiplexing) of multiple sequences and in a narrow range where amplification cycles are possible (eg, the log-to-cycle number of PCR amplification production is within a linear range). It has many disadvantages, such as the ability to generate variable quantitative data. DNA sequencing technology such as that used in MYSEQ (ILLUMINA), HISEQ (ILLUMINA), ION TORRENT (LIFE TECHNOLOGIES), GENOME ANALYZERILX (ILLUMINA), GSFLEX + (ROCHE454), etc., especially high-throughput next-generation sequencing technology. (Often referred to as sequencing techniques) can be used to quantitatively measure the number of copies of a target sequence present in a sample, thereby obtaining quantitative information about the starting material, eg, copy number or transcription level. Can be done. High-throughput gene sequencers use barcoding (ie, sample tagging with characteristic nucleic acid sequences) to identify specific samples from individual populations, thereby allowing multiple samples in a single operation of the DNA sequencer. Allows simultaneous analysis of. The number of times (reads) the genome of a region in a library preparation (or other nucleic acid preparation of interest) is sequenced is the number of copies of that sequence in the genome of interest (or in the case of cDNA-containing preparations). Will be proportional to the expression level). However, gene library preparation and sequencing (and preparations from similar genomes) can introduce many biases that interfere with the acquisition of accurate quantitative reads of the nucleic acid sequence of interest. For example, different nucleic acid sequences may result in different amplification efficiencies during the nucleic acid amplification steps that occur during gene library preparation or sample preparation.

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

Embodiments of the invention can be used for human or non-human nucleic acids and are also applicable to both animal and plant-derived nucleic acids. It is also possible to detect and / or quantify alleles associated with other hereditary disorders characterized by deletions or insertions using embodiments of the invention. Deletion-containing alleles may be detected in suspected carriers of the target allele.

One embodiment of the invention includes criteria that exist in known quantities (relative or absolute). For example, consider a gene library made from a gene source in which chromosome 8 (including locus A) is diploid and chromosome 21 (containing locus B) is triploid. A gene library can be generated from a sample that contains a number of chromosomes present in the sample, eg, an amount of sequence that is functional in 200 copies of locus A and 300 copies of locus B. However, if locus A is amplified much more efficiently than locus B, there may be 60,000 copies of A and 30,000 copies of B amplification product after PCR. In the case of analysis by high-throughput DNA sequencing (or other quantitative nucleic acid detection techniques), it will obscure the number of chromosomal copies of the early true genomic sample. To alleviate this problem, a reference sequence for locus A is adopted, 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. The reference sequence of locus A and the reference sequence of locus B are added to the mixture prior to PCR (or other amplification technique). These reference sequences exist in known quantities as relative or absolute quantities. Thus, under the same set of conditions, if a 1: 1 mixture of reference sequence A and reference sequence B is added to the mixture in the previous example (prior to amplification), 3000 copies of reference A amplification product will be produced. Also, 1000 copies of the reference B amplification product are produced, indicating that locus A is amplified three times more efficiently than locus B.

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

Many high-throughput DNA sequencing techniques require modification of the starting genetic material, such as universal priming sites and / or barcode ligation, for library formation, thereby small nucleic acids prior to subsequent sequencing reactions. Promotes clonal amplification of fragments. In some embodiments, one or more reference sequences are added during gene library formation, or are added to the precursor component of the gene library prior to library amplification. The reference sequence can be selected to mimic the target genomic fragment prepared for sequencing by high throughput gene sequencing techniques (still distinguishable based on the nucleotide sequence). 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 comprises an SNP, the reference sequence may be the same as the SNP other than the nucleotides of the polymorphic base, out of 4 nucleotides not observed at the natural site. It can be selected to be one. The reference sequence can be used for highly multiplexed analysis of multiple target loci (eg, polymorphic loci). The reference sequence is added in a known amount (relative or absolute) during the process of library formation (pre-amplification) and is a reference for higher accuracy in determining the amount of target sequence of interest in the analytical sample. Can provide metrics. Known haploid levels used with previously characterized polyploidy levels, eg, information on the formation of a haploid level library for sequencing formed from a genome known to be diploid in all autosomal chromosomes. The combination of quantity reference sequence information can be used to calibrate the amplification characteristics of each reference sequence for its corresponding target sequence, and to take into account variations between batch mixtures containing multiple reference sequences. Given that it is often necessary to analyze large numbers of loci at the same time, it is useful to generate a mixture containing many reference sequence sets. Embodiments of the invention include mixtures containing multiple reference sequences. Ideally, the amount of each reference sequence in the mixture is known with high accuracy. However, achieving this ideal is extremely difficult. The reason is that, as a practical matter, the amount of each reference sequence in the mixture, in particular the amount of reference sequence in the mixture containing a large number of different synthetic oligonucleotides, varies widely. This variation includes many sources, such as inter-batch variations in in vitro oligonucleotide synthesis antiefficiencies, volumetric inaccuracy, and pipette operation variations. Moreover, this variation can occur between batches containing exactly the same quantity of exactly the same reference sequence set in theory. Therefore, it makes sense to calibrate each reference sequence batch independently. The reference sequence batch can be calibrated to the reference genome of known chromosomal composition. The 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 invention include a mixture of various calibrated reference sequences. Other embodiments of the invention include a method of calibrating a mixture of different reference sequences and a mixture of different calibrated reference sequences produced by the method.

Various embodiments of subject reference sequence mixtures and methods of their use 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 thereof. 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 a smaller number of reference sequences than the number of target regions in the library being constructed. It would be advantageous to avoid reaching the limit of the sequencing capacity of the high throughput DNA sequencer employed with a smaller number. The number of reference sequences is 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, and 10% or less of the number of target regions. , 5% or less of the number of target areas, 1% or less of the number of target areas, and various intermediate numbers. For example, if the gene library is made with 15,000 pairs of primers targeting specific SNP-containing loci, 1500 criteria corresponding to 1500 of the 15,000 target loci. Appropriate mixtures containing sequences can be added prior to the amplification step of library construction.

The amount of reference sequence added during library construction may vary significantly 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 to prepare the library. In other embodiments, the amount of each reference sequence may be greater than or less than the predicted amount of target sequence present in the genomic material sample used to prepare the library. The initial relative amounts of the target and reference sequences are not significant for the purposes of the invention, but are 100 to 100 times greater than the amount of target sequences present in the genomic material sample used to prepare the library. It is preferably in the range of a small amount. Excessive amounts of reference sequences can overuse much of the sequencing capability of the DNA sequencer with a defined number of operations on the device. If too small a quantity of reference sequence is used, the data will be inadequate for use in the analysis of fluctuations in amplification efficiency.

The reference sequence can be selected to have a nucleotide sequence that closely resembles the region of interest to be amplified, preferably the reference sequence has the exact same primer binding site as the genomic region being analyzed, i.e. the "target sequence". Have. The reference sequence needs to be distinguishable from the corresponding target sequence at a given locus. For convenience, this distinguishable reference sequence region is referred to as the "marker sequence". In some embodiments, the marker sequence region of the target sequence may include a polymorphic region, eg, an SNP, with primer binding regions located at both sides. The reference sequence can be selected to exactly match the GC content of the corresponding target sequence. In some embodiments, the primer binding region of the reference sequence is flanked by universal priming sites. These universal priming sites are selected to match the universal priming sites used in the analytical genomic library. In other embodiments, the reference sequence lacks a universal priming site and the universal priming site is added during library formation. The reference sequence is usually provided in a single chain form. A reference sequence is defined for the corresponding target sequence and the target sequence is amplified using sequence-specific reagents. In some embodiments, the target sequence comprises a target polymorphism present in the nucleic acid sample for analysis, such as an SNP, deletion, or insertion. The reference sequence has a nucleotide base sequence similar to that of the target sequence, but is still distinguishable from the target sequence due to the difference of at least one nucleotide base, whereby the amplification product sequence derived from the reference sequence is referred to as the target sequence. It is a synthetic polynucleotide that provides a mechanism to distinguish it from the derived amplification product sequence. The reference sequence is selected to have substantially the same amplification properties as the corresponding target sequence when amplified with the same set of amplification reagents, eg PCR primers. In some embodiments, the reference sequence can have the same primer sequence binding site as the corresponding target sequence. In other embodiments, the reference sequence can have a primer sequence binding site that is different from the corresponding target sequence. In some embodiments, the reference sequence can be selected to produce an amplification product of the same length as the amplification product from the corresponding target sequence. In other embodiments, the reference sequence can be selected to produce an amplification product with a length slightly different than the length of the amplification product from the corresponding target sequence.

After the amplification reaction is complete, the library is sequenced on a high throughput DNA sequencer, in which case the individual molecules are cloned and amplified and sequenced. The number of sequence reads for each allele of the target sequence is counted, and similarly, the number of sequence reads for the reference sequence corresponding to the target sequence is counted. This process is also performed for at least another pair of target sequences and corresponding reference sequences. For example, with respect to locus A, it is assumed that an _XA1 read is generated for the allele 1 at the locus A, an _XA2 read is generated for the allele 2 at the locus A, and an _XAC read is generated for the reference sequence A. For each locus of interest, the ratio of (X _A1 plus X _A2 ) to X _AC is determined. As discussed earlier, this process can be performed on the reference genome, eg, a genome in which all chromosomes are known to be diploid. This process can be repeated many times to obtain a large number of read values and measure the average number of reads and the standard deviation of the number of reads. This process is carried out on a mixture containing a large number of different reference sequences corresponding to different loci. (1) X- _A1 plus X- _A2 corresponds to 2 for a known number of chromosomes, eg, the normal human female genome, and (2) the reference sequence is associated with their corresponding natural loci. By assuming that they have similar amplification (and detection) properties, reference sequences with different relative quantities in the multiple reference mixture can be determined. A calibrated multiplex reference sequence mixture can then be used to regulate the variation in amplification efficiency between different loci in the multiplex amplification reaction.

Other embodiments of the invention include methods and compositions for measuring the number of copies of a subject-specific gene, such as a mutant gene characterized by large deletions that may interfere with replication and sequencing quantification. .. Sequencing can make it difficult to detect alleles with such deletions. An amplification process involving reference sequences can be used to reduce this problem.

In one embodiment of the invention, the analytical target sequence is a mutant gene characterized by wild (i.e., functional) and deletion. A representative such gene is the allele SMN1 with a deletion responsible for the genetic disease spinal muscular atrophy (SMA). It is meaningful to detect individuals carrying mutant genes by high-throughput gene sequencing techniques. The application of such techniques to the detection of deletion mutations is problematic, especially because sequence deficiencies are observed during sequencing (as opposed to detection of simple point mutations or SNPs). There is. In such an embodiment, (1) a pair of amplification primers specific to the target gene, which amplifies the target gene (or a part thereof) and does not significantly amplify the mutation allele, (2) the target gene. A pair of amplification primers specific for a second target sequence that corresponds to a wild allele of (ie, target sequence) but differs in at least one detectable nucleotide base, (3) serves as a reference sequence. , And (4) the reference sequence corresponding to the reference sequence.

In one embodiment of the invention, a method of measuring the number of copies of a gene of interest is provided, the gene of interest having one intentional allele containing a deletion. The method is specific to the gene of interest in that it does not amplify the deletion-containing allele of the gene of interest, but at least part of the gene of interest, or all of the gene of interest, or the region adjacent to the gene of interest. Amplification reagents such as PCR primers can be adopted. Furthermore, the method employs a reference sequence corresponding to the gene of interest, where the reference sequence differs from the gene of interest by at least one nucleotide base (the sequence of the reference sequence is the natural of the subject). To be easily distinguishable from genes). Normally, the reference sequence contains the same primer binding site as the gene of interest, thereby minimizing any amplification difference between the gene of interest and the reference sequence corresponding to the gene of interest. The reaction also includes an amplification reagent specific for the reference sequence. A reference sequence is a sequence of known (or at least assumed to be known) number of copies in the genome being analyzed. The reaction further comprises a reference sequence corresponding to the reference sequence. Normally, the reference sequence corresponding to the reference sequence contains the same primer binding site as the reference sequence, thereby minimizing any amplification difference between the reference sequence and the reference sequence corresponding to the reference sequence.

Representative Nucleic Acid Samples In some embodiments, genetic samples can be prepared and / or purified. There are a number of standard procedures known in the art to achieve such objectives. In some embodiments, the sample can be centrifuged and separated into various layers. In some embodiments, filtration can be used to isolate the DNA. In some embodiments, DNA preparation is known in the art for amplification, separation, chromatographic purification, liquid separation, isolation, preferential enrichment, preferential amplification, targeted amplification or in the art. Or may be accompanied by any of a number of other techniques described herein.

In some embodiments, the methods disclosed herein are fertilized in vitro or in one or a few cells (typically cells 10) in situations where the amount of DNA present is very small. It can be used in forensic situations where less than 20 cells, less than 20 cells or less than 40 cells) are available. In these embodiments, the methods disclosed herein do not contaminate other DNA, but haploid calls from a small amount of DNA when haploid calls are very difficult due to the small amount of DNA. Help to do. In some embodiments, the methods disclosed herein are contaminated with the DNA of another individual in the target DNA, eg, maternal blood or in the context of prenatal testing, paternal and child testing. It can be used in the product of conception testing. Some other situations in which these methods are particularly advantageous are in the case of cancer screening, where only one or a few cells are present in a larger number of normal cells. Genetic measurements used as part of these methods include any sample containing DNA or RNA, such as, but not limited to, blood, plasma, body fluids, urine, hair, tears, saliva, tissue, skin, fingertips, etc. It can be done on blastomere, embryo, amniotic fluid, chorionic villi sample, feces, bile, lymph, cervical mucus, semen or other cells or materials containing nucleic acids. In certain embodiments, the methods disclosed herein can be performed in conjunction with nucleic acid detection methods such as sequencing, microarray, qPCR, digital PCR or other methods used to measure nucleic acids. can. If desired is found to be desirable for any reason, the ratio of the probabilities of the number of alleles at the locus can be calculated and the ratio of alleles is suitable for some of the methods described herein and those methods. The polyploid state can be determined by using them in combination as long as they have sex. In some embodiments, the methods disclosed herein include the step of computerizing the ratio of alleles at multiple polymorphic loci from DNA measurements made on a processed sample. do. In some embodiments, the methods disclosed herein are multiple, from DNA measurements made on processed samples, along with any combination of other improvements described herein. Includes a computerized calculation of the ratio of alleles at a type locus.

In some embodiments, this method is used to use a single cell, a small number of cells, 2-5 cells, 6-10 cells, 10-20 cells, 20-50 cells, 50. ~ 100 cells, 100 ~ 1,000 cells or small amounts, eg 1-10 picograms, 10-100 picograms, 100 picograms-1 nanograms, 1-10 nanograms, 10-100 nanograms or 100 nanograms-1 micron The extracellular DNA of a gram can be genotyped.

Representative RNA Expression Investigations The multiplex PCR method of the present invention can be used to increase the number of target loci that can be evaluated during gene expression profiling experiments. For example, the expression levels of thousands of genes can be monitored simultaneously to determine if a disease (eg, cancer) or a sequence associated with an increased risk of the disease (eg, a polymorphism or other mutation) is present. Sequences relating to an increased or decreased risk of a disease, eg, cancer, by using these methods to compare gene expression (eg, expression of a particular mRNA allele) in a patient-derived sample to the presence or absence of the disease. (For example, polymorphisms or other mutations) can be identified. In addition, the effect of a particular treatment, disease, or developmental stage on gene expression can be determined. Similarly, these methods can be used to compare gene expression in infected and uninfected cells or tissues to identify which gene expression has changed in response to a pathogen or other organism. In these methods, the number of sequencing reads can be adjusted based on the frequency of the polymorphism (if any polymorphism is analyzed) so that sufficient reads are performed for the polymorphism to be detected. ..

In some embodiments, a sample containing RNA (eg, mRNA) is amplified using reverse transcriptase (RT) and the resulting DNA (eg, cDNA) is then used with DNA polymerase (PCR). Is amplified. The RT and PCR steps can be performed sequentially in the same reaction volume or separately. Any of the primer libraries of the invention can be used in this reverse transcription-polymerase chain reaction (RT-PCR) method. In various embodiments, reverse transcription can be performed using oligo dT, random primers, a mixture of oligo dT and random primers, or target locus-specific primers. To avoid amplification of contaminating genomic DNA, some primers for RT-PCR hybridize to the 3'end of one exon and some of the other primers are adjacent exons. It can be designed to hybridize to the 5'end. Such primers anneal to cDNA synthesized from spliced mRNA, but not genomic DNA. To detect amplification of contaminating DNA, the RT-PCR primer pair can be designed to be located beside the region containing at least one intron. Products amplified from cDNA (intron-free) are smaller than those amplified from genomic DNA (intron-containing). The presence of contaminating DNA is detected using the size difference of the product. In some embodiments, if only the mRNA sequence is known, primer annealing sites separated by at least 300-400 base pairs are selected. The reason is that fragments of this size from eukaryotic DNA may contain splice junctions. Alternatively, the sample can be treated with a DNase to degrade the contaminating DNA.

Representative Father-Sontestation Methods Because a large number of target loci can be analyzed at once (see, for example, US Patent Publication No. 2012/0122701 filed December 22, 2011; this patent is in its entirety by reference. The multiple PCR method of the present invention can be used to improve the accuracy of paternity testing. For example, the multiplex PCR method analyzes and analyzes thousands of polymorphic loci (eg, SNPs) for use in the PARENTAL SUPPORT algorithm described herein, and the father is the biological father of the fetus. It is possible to determine whether or not. In some embodiments, the method is (i) at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 15,000; 30,000; 40,000; 50,000; 75,000; or a first amplification product set that simultaneously amplifies multiple polymorphic loci including 100,000 different polymorphic loci. And (ii) simultaneously amplify the corresponding polymorphic loci of a mixed DNA sample containing fetal DNA and maternal DNA from a pregnant mother's blood sample to generate a second amplification product set. And (iii) a computer-based calculation of the probability that a person to be a father is the biological father of a fetus using genotypic measurements based on the first and second amplification product sets. iv) Includes the step of determining if the alleged father is the biological father of the fetal using the calculated probability that the alleged father is the biological father of the fetal. In various embodiments, the method further comprises simultaneously amplifying the corresponding polymorphic loci on the maternally derived genetic material to generate a third amplification product set, in this case the first. The genotyping values based on the second and third amplification product sets are used to calculate the probability that the alleged father is the biological father of the fetal.

Representative Embryo Characterization and Selection Methods By making it possible to analyze thousands of target loci at once using the multiple PCR method of the present invention, the selection of embryos for in vitro fertilization can be improved (eg, May 2008). See US Patent Publication No. 2011/0092763 filed 27th, 22nd December 2011, which is incorporated herein by reference in its entirety). For example, the multiplex PCR method allows the analysis of thousands of polymorphic loci (eg, SNPs) for use in the PARENTAL SUPPORT algorithm described herein to select embryos for in vitro fertilization from embryo populations. It can be carried out.

In some embodiments, the invention provides a method of estimating the relative likelihood of developing each embryo from an embryo set as desired. In some embodiments, the method comprises at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30, The sample comprises steps to generate a mixture of each embryo by contacting with a primer library that simultaneously hybridizes to 000; 40,000; 50,000; 75,000; or 100,000 different target loci. , Each obtained from one or more cells derived from the embryo. In some embodiments, each reaction mixture is subjected to primer extension reaction conditions to produce an amplification product. In some embodiments, the method is a step of computer-based determination of one or more properties of at least one cell from each embryo based on an amplification product, and the development of each embryo as desired. Includes a step of computer-estimating the relative likelihood based on the properties of one or more cells of at least one cell for each embryo. In some embodiments, the method comprises using an informatics-based method to determine at least one characteristic, eg, the PARENTAL SUPPORT algorithm described herein. In some embodiments, the property comprises a ploidic state. In some embodiments, the properties are aneuploidy, positive multiple, mosaic, zero-chromosomal, monosomy, aneuploidy, trisomy, tetrasomy, aneuploidy type, mismatched copy error trisomy, matched copy error trisomy, maternal. Aneuploidy of origin, aneuploidy of paternal origin, presence or absence of disease chain genes, chromosomal identity of all trisomy chromosomes, aberrant gene status, deletion or replication, likelihood of traits, and combinations thereof. Selected from the group consisting of. The characteristics are chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, and chromosome 16. , Chromosome 17, Chromosome 19, Chromosome 20, Chromosome 21, Chromosome 22, X or Y chromosome, and combinations thereof, may be related to a chromosome selected from the group.

Representative Prenatal Diagnostic Methods The multiple PCR methods of the present invention can be used to improve prenatal diagnostic methods such as determining the ploidy state of fetal chromosomes. Considering the large number of target loci that are amplified at the same time, it is possible to make a more accurate determination.

In certain embodiments, the present disclosure is from genotypic data measured from a mixed sample of DNA (ie, DNA from the mother of the fetus, and DNA from the fetus), and optionally with genetic material from the mother. In some cases, it is an exvivo method for determining the chromosomal polymorphism in a pregnant fetus from genotype data also measured from a sample of genetic material derived from the father, and the above determination is based on a co-distribution model. Using, taking into account parental genotype data, a set of predicted allogeneic distributions for possible different multiple states in the fetus was generated and measured in the predicted allogeneic distribution and mixed samples. Provided is a method performed by comparing with an actual allelic gene distribution and selecting a multiple state in which the predicted allelic gene distribution pattern most closely matches the observed allelic gene distribution pattern. In certain embodiments, the mixed sample is derived from maternal blood or maternal serum or plasma. In certain embodiments, a mixed sample of DNA can be preferentially enriched at a target locus (eg, a plurality of polymorphic loci). In certain embodiments, preferential enrichment is performed so that allele bias is minimized. In certain embodiments, the present disclosure relates to the composition of DNA that is preferentially enriched to reduce allele bias at multiple loci. In one embodiment, the allele distribution (s) are measured by sequencing the DNA from the mixed sample. In certain embodiments, joint distribution models assume that alleles are distributed in a binomial fashion. In one embodiment, taking into account the frequency of existing recombination from various sources, for example, using data from the International HapMap Consortium, predictive simultaneous allelic distributions for genetically linked loci. Create a set.

In certain embodiments, the present disclosure observes allogeneic measurements at multiple polymorphic loci in genotype data measured for non-invasive prenatal diagnosis (NPD) methods, specifically DNA mixtures. This is a method for determining the aneuploid state of a fetal, in which certain allogeneic measurements indicate an aneuploid fetal, while other allogeneic measurements indicate a positive multiple fetal. The indicated method is provided. In one embodiment, genotype data is measured by sequencing on a DNA mixture derived from maternal plasma. In certain embodiments, the DNA sample can be preferentially enriched for DNA molecules corresponding to multiple loci for calculating allelic distribution. In certain embodiments, a sample of DNA containing only or substantially only mother-derived genetic material is measured and, in some cases, containing only father-derived genetic material or substantially father-derived. A sample of DNA containing only the genetic material of is also measured. In one embodiment, genetic measurements from one parent or parent are used in conjunction with an estimated fetal fraction to accommodate multiple predicted alleles that correspond to possible different underlying genetic states in the fetal. A gene distribution is created and the predicted allele distribution can be referred to as a hypothesis. In certain embodiments, maternal genetic data is not determined by measuring genetic material that is naturally exclusive or almost exclusively maternal, but maternal plasma containing a mixture of maternal DNA and fetal DNA. Estimated from the genetic measurements made on the. In some embodiments, the hypothesis may include fetal ploidy on one or more chromosomes, which segment of which chromosome of the fetus was inherited from which parent, and combinations thereof. In some embodiments, the fetal ploidy state is a different hypothesis with the observed allelic measurements, comparing and observing hypotheses in which at least some of the hypotheses correspond to different ploidy states. Determined by choosing the ploidy state that corresponds to the hypothesis most likely to be true, taking into account allele measurements made. In certain embodiments, this method involves the use of allelic measurement data from some or all of the measured SNPs, regardless of whether the locus is homozygous or heterozygous, thus. It does not involve the use of alleles at loci that are only heterozygous. This method may not be suitable for situations where genetic data is associated with only one polymorphic locus. This method is particularly advantageous when the genetic data contains data for more than 10 polymorphic loci or more than 20 polymorphic loci with respect to the target chromosome. This method is especially useful if the genetic data contains data for more than 50 polymorphic loci, more than 100 polymorphic loci, or more than 200 polymorphic loci for the target chromosome. It is advantageous. In some embodiments, the genetic data is for more than 500 polymorphic loci, over 1,000 polymorphic loci, over 2,000 polymorphic loci, or for the target chromosome. May contain data for more than 5,000 polymorphic loci.

In certain embodiments, the methods disclosed herein provide a quantitative measure of the number of independent observations of each allele at a polymorphic locus. This provides information about the ratio of the two alleles, such as microarrays or qualitative PCR, but differs from most methods that do not quantify the number of independent observations of either allele. Methods that provide quantitative information about the number of independent observations use only ratios to calculate ploidy, while quantitative information is not useful in itself. To illustrate the importance of retaining information about the number of independent observations, consider the loci of a sample with two alleles, A and B. In the first experiment, 20 alleles A and 20 alleles B are observed, and in the second experiment, 200 alleles A and 200 alleles B are observed. In both experiments, the ratio (A / (A + B)) is equal to 0.5, but the second experiment conveys more information about the certainty of the frequency of the allele A or B than the first experiment. Some methods by another researcher are to average or sum the allele ratios (channel ratios) from individual alleles (ie x _i / y _i ) and compare this ratio to the reference chromosome. Or it involves analyzing either by using rules regarding how this ratio is expected to behave in a particular situation. It is assumed that such a method can ensure approximately the same amount of PCR product for each allele without allele weighting, and that all alleles should behave similarly. To. Such methods have a number of disadvantages and, more importantly, prevent the use of any of the improvements described elsewhere in this disclosure.

In certain embodiments, the methods disclosed herein are the predicted allogeneic frequency distribution in dysomy and nondisjunction between meiosis I, nondisjunction between meiosis II, and / or fetal. It clearly models the frequency distribution of multiple allelic genes that can be predicted in the case of trisomy caused by chromosomal nondisjunction during early mitosis of development. To illustrate why this is important, consider the case of no crossover: chromosomal nondisjunction during meiosis I results in a trisomy in which two different homologues are inherited from one parent. In contrast, chromosomal nondisjunction during meiosis II or during early mitosis of fetal development results in two copies of the same homologue from one parent. Each scenario will result in different frequencies of predicted alleles at each polymorphic locus and at all jointly considered loci due to genetic linkage. Crossovers that result in the exchange of genetic material between homologues complicate the mode of inheritance, and in certain embodiments, the method uses information on the rate of recombination in addition to the physical distance between loci. By adapting to this. In certain embodiments, in order to allow an improvement in the distinction between chromosomal nondisjunction during meiosis I and chromosomal nondisjunction during meiosis II or mitosis, in this method, the model is from a centromere. Incorporate the probability of a crossover that increases as the distance increases. In meiosis II and mitotic nondisjunction, mitotic nondisjunction generally results in the same or nearly identical copy of one homologue, while at meiosis II. The two homologues that exist after a nondisjunction event can often be distinguished by the fact that they differ due to one or more crossovers during mitosis.

In some embodiments, the methods disclosed herein include the step of comparing the observed allelic measurements to the theoretical hypothesis corresponding to the potential fetal locus. , Does not include the step of quantifying the ratio of alleles at the heterozygous locus. If the number of loci is less than about 20, multiple determinations made using methods involving quantifying the ratio of alleles at heterozygous loci and observed allelic measurements are possible. Multiple determinations made using methods involving comparison with the hypothesis of a theoretical allelic distribution corresponding to the genetic status of a fetal can yield similar results. However, if the number of loci is greater than 50, these two methods can give significantly different results, with more than 400, 1,000 or 2,000 loci. If so, these two methods are likely to produce increasingly significantly different results. These differences are simultaneous with methods involving measuring the size of each allele independently and quantifying the ratio of alleles at the heterozygous loci without summing or averaging the ratios. Obstructed from using distribution models, performing chain analysis, using binomial distribution models, and / or using techniques including other advanced statistical techniques, but observed allelic measurements. These techniques can substantially increase the accuracy of the determination by using a method that includes a step of comparing with the hypothesis of the theoretical allele distribution corresponding to the potential genetic status of the fetus. Due to the fact that it can be used.

In certain embodiments, the methods disclosed herein determine whether the distribution of observed allelic measurements indicates a positive polyploid or aneuploid fetus using a joint distribution model. Including steps. The use of joint distribution models is a method that differs from the method of determining heterozygotes by treating polymorphic loci independently in that the decisions obtained are significantly more accurate. It is significantly improved than. One reason for their high accuracy, without being bound by any particular theory, is that in joint distribution models, chains between SNPs and meiosis that give rise to gametes that form embryos that grow into fetuses. It is considered to take into account the likelihood of crossovers that occurred in the meantime. The purpose of using the concept of linkage in creating a predicted distribution of allelic measurements for one or more hypotheses is thereby a predicted conflict that corresponds to a much better reality than without linkage. It is to make it possible to create a gene measurement value distribution. For example, there are two SNPs, 1 and 2 located close to each other, and the mother has SNP1 on one homology A, SNP2 A, and SNP1 on homology 2 B. Yes, and consider that SNP2 is B. If the father is A for both SNPs on both homologues and B is measured for fetal SNP1, this is because the fetus inherited homology 2 and therefore B to the fetal SNP2. Indicates that the probability of existence is much higher. This is expected in models that take chain into account, but not in models that do not take chain into account. Alternatively, if the mother's SNP1 is AB and the nearby SNP2 is AB, there are two hypotheses corresponding to maternal trisomy at that location-matching copy error (meiosis II or chromosomal nondisjunction in early mitosis). ), And those with a mismatched copy error (nondisjunction in meiosis I) can be used. In the case of coincident copy error trisomy, if the fetus inherits AA from the mother in SNP1, the fetus is much more likely to inherit either AA or BB from the mother in SNP2 instead of AB. High. In the case of a mismatched copy error, the fetus will inherit AB from the mother in both SNPs. These predictions are made by the allelic distribution hypothesis made by the haploid calling method, which takes linkage into account, and therefore, to a much greater extent than the haploid calling method, which does not take linkage into account, in fact. Corresponds to allele measurements. Note that linkage techniques are not possible when using methods that rely on calculating allele ratios and summing the ratios of those alleles.

One reason why multiple determinations using methods involving the step of comparing observed allelic measurements to theoretical hypotheses corresponding to possible fetal genetic states may be more accurate. When measuring alleles using sequencing, this method is able to collect more information from the data from the alleles if the total number of reads is smaller than the other methods. For example, methods that rely on calculating and summing allele ratios result in disproportionately weighted probabilistic noise. For example, consider the case where alleles are measured using sequencing and there is a set of loci in which only 5 sequence reads are detected for each locus. In certain embodiments, for each of the alleles, the data can be compared to the assumed allele distribution and weighted according to the number of sequence reads, thus the data from these measurements are appropriately weighted and overall. Incorporated into a decision. This involves quantifying the ratio of alleles at the heterozygous locus, with possible allele ratios of 0%, 20%, 40%, 60%, 80% or 100%. In contrast to the above method, they can only be calculated and none of them can approach the expected allele ratio. In this latter case, the calculated allele ratio must be rejected because of insufficient reads, or it is disproportionately weighted and probabilistic noise is introduced into the decision, thereby the accuracy of the decision. Decreases. In one embodiment, individual allele measurements can be treated as independent measurements, in which case the relationship between measurements made for alleles at the same locus is for alleles at different loci. It does not differ from the relationship between the measurements made.

In certain embodiments, the method disclosed herein is a step (referred to as the RC method) of comparing any metric with allelic measurements observed on a reference chromosome that is expected to be disomy. ) Is not included, but includes the step of determining whether the distribution of observed allelic measurements indicates a positive polyploid or aneuploid fetus. This is a method using shotgun sequencing, which detects aneuploidy by evaluating the proportion of randomly sequenced fragments from a suspicious chromosome compared to one or more inferred disomy reference chromosomes. It is a significant improvement over such methods. This RC method gives inaccurate results if the inferred disomy reference chromosome is not really disomy. This can occur if the aneuploidy is more substantial than the trisomy of a single chromosome, or if the fetus is triploid and all autosomal chromosomes are trisomy. In the case of female triploid (69, XXX) fetuses, in fact, there are no disomy chromosomes. The methods described herein do not require a reference chromosome and can accurately identify trisomy chromosomes in female triploid fetuses. For each of the chromosomes, hypotheses, child fractions and noise levels, the joint distribution model is fitted without any reference chromosome data, overall child proportion estimates or fixed reference hypotheses. be able to.

In certain embodiments, the methods disclosed herein use the allelic distribution at the observed polymorphic loci to be more accurate than prior art methods in fetal ploidy. Demonstrate whether the state can be determined. In certain embodiments, the method first obtains mixed maternal-fetal genotypes in multiple SNPs and, optionally, maternal and / or paternal genotypes using targeted sequencing. Establishing various predicted allogenotype frequency distributions under different hypotheses, then observing quantitative allogeneic information obtained in the maternal-fetal mixture, and which hypothesis is most in the data. Using assessing good fit, we call the state of the gene that corresponds to the hypothesis that best fits the data as the exact state of the gene. In certain embodiments, the methods disclosed herein also use the degree of fit to generate confidence that the state of the called gene is the correct state of the gene. In certain embodiments, the methods disclosed herein use an algorithm that analyzes the distribution of alleles found for different parental loci, and different parental situations (different parental genotypes). Includes the step of comparing the observed allelic distribution to the predicted allelic distribution for different multiple states). This is an improvement over the method without a method that can estimate the number of independent cases of each allele at each locus in a maternal-fetal mixed sample. In certain embodiments, the method disclosed herein uses the observed allelic distribution measured at a locus in which the mother is heterozygous, with the observed distribution of allelic measurements. Includes the steps to determine if a positive polyploid or aneuploid fetus is shown. This is a multiple if the DNA is not preferentially enriched or preferentially enriched for loci that are not known to be informative for that particular target individual. Observed alleles at loci where the mother is heterozygous, as it will be possible to use approximately twice as many gene measurement data from the set of sequence data in the determination, which will lead to more accurate determination. Unlike the non-distribution method, it is an improvement over it.

In certain embodiments, the methods disclosed herein naturally assume that the allele frequency at each locus is a polynomial (hence, a binomial if the SNP is a bi-allele). Use a distribution model. In some embodiments, the joint distribution model uses a beta-binomial distribution. When using measurement techniques such as sequencing that provide a quantitative measure for each allele present at each locus, a binomial model can be applied to each locus to the extent that it forms the basis of allele frequency. And the reliability of its frequency can be confirmed. It is not possible to ascertain the certainty of the observed ratio using methods known in the art for generating ploidy calls from allelic ratios or methods in which quantitative allelic information is rejected. The method calculates the ratio of alleles at a particular locus and then measures the amount of DNA from any given allele or locus in any method involving summing those ratios. It always assumes that the strengths or counts given are distributed in Gaussian fashion, so it is better than that, unlike the method of calculating allele ratios and summing those ratios to make a multiple call. There is. The methods disclosed herein do not involve calculating the ratio of alleles. In some embodiments, the methods disclosed herein may include incorporating into the model the number of observations of each allele at multiple loci. In some embodiments, the method disclosed herein is a step in calculating the predicted distribution itself, thereby being more accurate than any of the models that assume a Gaussian distribution of allelic measurements. It may include steps that make it possible to use a joint binomial distribution model that may be. The likelihood that the binomial distribution model is significantly more accurate than the Gaussian distribution increases as the number of loci increases. For example, when investigating loci less than 20, the likelihood that the binomial distribution model is significantly better is low. However, with more than 100, or especially more than 400, or especially more than 1,000, or especially more than 2,000 loci, the likelihood that the binomial distribution model is significantly more accurate than the Gaussian distribution model is very high. Higher, which leads to a more accurate ploidy determination. The likelihood that the binomial distribution model is significantly more accurate than the Gaussian distribution also increases as the number of observations at each locus increases. For example, when observing distinct sequences less than 10 at each locus, the binomial distribution model is less likely to be significantly better. However, using more than 50 sequence reads, especially more than 100 sequence reads, or especially more than 200 sequence reads, or especially more than 300 sequence reads for each locus, is more than the Gaussian distribution model of the binomial distribution model. The likelihood of being significantly accurate is very high, which leads to a more accurate ploidy determination.

In certain embodiments, the methods disclosed herein use sequencing to measure the number of cases of each allele at each locus in a DNA sample. Each of the sequencing reads can be mapped to a particular locus and treated as a binary sequence read, or the probability of read and / or mapping identity can be incorporated as part of the sequence read. The result is a probabilistic sequence read, an estimated integer or fraction of the sequence read that maps to a given locus. Binomial distributions can be used for each set of measurements using a binary count value or the probability of the count value, thereby calculating the (around the number of counts) confidence interval for the range of count values. Will be possible. The ability to use the binomial distribution allows for more accurate ploidy estimation and more accurate confidence intervals. It uses a method that uses intensity to measure the amount of allelic genes present, such as a microarray, or a fluorescent reader to measure the intensity of fluorescently tagged DNA in the band of electrophoresis. Unlike the method of making measurements, it is better than that.

In certain embodiments, the methods disclosed herein use aspects of this set of data to determine the parameters of the estimated allele frequency distribution for that set of data. This is an improvement over the method of utilizing training data sets or prior data sets to set parameters for this predicted allele frequency distribution or, in some cases, predicted allele ratios. This is due to the existence of a set of different states involved in the collection and measurement of any genetic sample, and therefore the data from that set of data is used to determine the ploidy for that sample. The method of determining the parameters of the joint distribution model, which is intended to do so, tends to be more accurate.

In certain embodiments, the method disclosed herein uses the most probable method to determine whether the distribution of observed allelic measurements indicates a positive polyploid or aneuploid fetus. Including the steps to be taken. Using the maximum likelihood method is significantly improved, unlike the method using the single hypothesis rejection method, in that the accuracy of the decisions obtained is significantly higher. One reason is that in the single hypothesis rejection method, the cutoff threshold is set based on only one measurement distribution rather than two measurement distributions, that is, the thresholds are usually not optimal. Another reason is that the maximum likelihood method does not determine the cutoff threshold used for all samples regardless of their specific characteristics of each sample, but rather for each individual sample. Is to be possible to optimize. Another reason is that by using the maximum likelihood method, it is possible to calculate the reliability for each haploid call. Being able to calculate confidence for each call allows practitioners to know which calls are accurate and which are more likely to be incorrect. In some embodiments, a wide variety of methods can be combined with maximum likelihood estimation to increase the accuracy of haploid calls. In certain embodiments, the maximum likelihood method can be used in combination with the method described in US Pat. No. 7,888,017. In one embodiment, the most probable method is presented in a read counting method, eg, International Congress of Human Genetics 2011 at Montreal, in October 2011, in which the DNA in the mixed sample is amplified using targeted PCR amplification. It can be used in combination with the method of sequencing and analyzing using the TANDEM DIAGNOSTICS. In certain embodiments, the methods disclosed herein include the steps of estimating the fetal fraction of DNA in a mixed sample, and the estimates used to provide both polyploid and polyploid call reliability. Includes steps to calculate. It is a multiple using a single hypothesis rejection method that uses the estimated fetal fraction as a sufficient fetal fraction screening and then does not take into account the fetal fraction and does not result in the calculation of confidence for the call. Note that it is different from and distinct from the method of making a sex call.

In certain embodiments, the methods disclosed herein take into account the tendency of the data to be noisy and error-containing by imparting probabilities to each measurement. Inaccurate measurements are not taken into account by using the maximum likelihood method to select the correct hypothesis from the set of hypotheses made using the measured data with the given probabilistic estimates. It is more likely that accurate measurements will be used in the calculations that lead to multiple calls. Due to its higher accuracy, this method systematically reduces the effect of inaccurately measured data in determining ploidy. This is an improvement over the method in which all data are assumed to be equally accurate or where out-of-range data is arbitrarily excluded from calculations leading to haploid calls. Current methods using channel ratio measurements claim to extend this method to a large number of SNPs by averaging individual SNP channel ratios. By not weighting individual SNPs by the variance of the measurements predicted based on the quality of the SNPs and the observed read depth, the resulting statistics are less accurate, resulting in multiple call accuracy. However, it is significantly reduced, especially at the boundary.

In certain embodiments, the methods disclosed herein do not presuppose a finding of which SNP or other polymorphic locus is heterozygous in the fetus. This method makes it possible to make polyploid calls when paternal genotype information is not available. This is a priori finding of which SNPs are heterozygous for proper selection of target loci or for interpreting gene measurements obtained for mixed fetal DNA / maternal DNA samples. It is an improvement over the method that must be known.

The methods described herein are particularly advantageous when used for samples with a small amount of available DNA or with a low percentage of fetal DNA. This occurs when the allele dropout rate is correspondingly high when only a small amount of DNA is available, and / or when the percentage of fetal DNA in the mixed sample of fetal DNA and maternal DNA is low. This is due to the correspondingly high gene dropout rate. The high allele dropout rate, that is, the lack of measurement of most of the alleles for the target individual, resulted in inadequately accurate fetal fraction calculations and inadequately accurate ploidy determinations. Brought to you. The methods disclosed herein can use joint distribution models that take into account linkages in the mode of inheritance between SNPs, allowing for significantly more accurate ploidy determinations. When the percentage of fetal DNA molecules in the mixture is less than 40%, less than 30%, less than 20%, less than 10%, less than 8%, and even less than 6% by the methods described herein. It will be possible to make accurate ploidy determinations.

In certain embodiments, it is possible to determine the ploidy state of an individual based on measurements when the individual's DNA is mixed with the associated individual's DNA. In certain embodiments, the DNA mixture is a floating DNA found in maternal plasma, which may include DNA from a mother with a known karyotype and a known genotype, and is unknown. It may be mixed with fetal DNA having karyotype and unknown genotype. Using information on known genotypes from one parent or parents, the state of multiple potential genes of DNA in a mixed sample, different ploidy states, different chromosomal contributions from each parent to the fetus, and If desired, it is possible to predict the proportion of different fetal DNA in the mixture. Each of the potential compositions can be referred to as a hypothesis. The ploidy state of the fetus can then be determined by examining the actual measurements and taking into account the observed data to determine which potential composition is most likely.

Further consideration of the above points can be found elsewhere in this document.

Non-invasive prenatal diagnosis (NPD)
The non-invasive prenatal diagnosis process involves several steps. Some of the steps include (1) obtaining the genetic material from the fetus, (2) enriching the genetic material of the fetus that may be present in the mixed sample with Exvivo, and (3) the genetic material. Steps to amplify with Exvivo, (4) Steps to preferentially enrich specific loci of genetic material with Exvivo, (5) Steps to measure genetic material with Exvivo, and (6) Genetic type data. , Exvivo, can be mentioned as a step of computer analysis. Methods for reducing the implementation of these six and other related steps are described herein. At least some of the steps in the method do not apply directly to the body. In certain embodiments, the present disclosure relates to methods of treatment and diagnosis applied to tissues and other biological materials isolated and isolated from the body. At least some of the steps in the method are performed on a computer.

Some embodiments of the present disclosure allow the clinician to non-invasively determine the genetic status of the fetus during pregnancy, thereby allowing the infant to collect genetic material from the fetus. Health is not compromised and the mother does not have to undergo invasive procedures. In addition, in certain embodiments, the present disclosure screens the genetic status of the fetus with high accuracy, eg, a triple test, non-invasive maternal serum analysis that is widely used in prenatal care. It will be possible to determine with significantly higher accuracy than.

The high accuracy of the methods disclosed herein is the result of the informatics techniques described herein for analyzing genotype data. Modern technological advances have made it possible to measure large amounts of genetic information from genetic samples using methods such as high-throughput sequencing and genotyping arrays. The methods disclosed herein allow clinicians to make greater use of the large amount of data available and to make more accurate diagnoses of fetal genetic status. Details of a number of embodiments are shown below. Different embodiments may include different combinations of steps described above. Various combinations of different embodiments of different steps can be used interchangeably.

In one embodiment, a blood sample is taken from a pregnant mother and floating DNA in the plasma of the mother's blood containing a mixture of both maternal and fetal origin DNA is isolated and is a multiple of the fetus. Used to determine sexual status. In certain embodiments, the methods disclosed herein show little change in the DNA sequence in the mixture of DNA corresponding to the polymorphic allele, the allele ratio and / or the allele distribution upon enrichment. As it is, it includes the steps of preferential enrichment. In certain embodiments, the methods disclosed herein involve highly efficient target PCR-based amplification such that a very high percentage of the resulting molecule corresponds to the target locus. In certain embodiments, the methods disclosed herein include sequencing a mixture of DNA containing both maternal and fetal origin DNA. In certain embodiments, the methods disclosed herein include the step of using the measured allelic distribution to determine the ploidy state of the fetus in which the mother is pregnant. In certain embodiments, the methods disclosed herein include the step of reporting the determined ploidy status to the clinician. In certain embodiments, the methods disclosed herein are steps of taking clinical measures, such as performing follow-up of invasive villus collection or amniocentesis invasive examination, the birth of an individual of trisomy. Includes steps to prepare, or selective abortion of a trisomy fetus.

This application is filed in US Practical New Application No. 11 / 603,406 filed on November 28, 2006 (US Patent Application Publication No. 20070184467); US Practical New Application Application No. 12/076 filed on March 17, 2008, 348 (US Patent Application Publication No. 20080243398); PCT Application No. PCT / US09 / 52730 filed August 4, 2009 (PCT Publication No. WO / 2010/017214); PCT filed September 30, 2010 Application No. PCT / US10 / 050824 (PCT Publication No. WO / 2011/041485), US Practical New Application Nos. 13 / 110,685 filed May 18, 2011, and PCT filed October 3, 2012. See application No. PCT / US12 / 58578. These patents are incorporated herein by reference in their entirety. Some of the vocabularies used in this application may have their precedents in these references. Some of the concepts described herein can be better understood in the light of the concepts found in these references.

Screening of Maternal Blood Containing Floating Fetal DNA Using the methods described herein, a target genetic material is found in the presence of a certain amount of other genetic material, offspring, fetus or other target individual. Can assist in determining the genotype of. In some embodiments, genotype may refer to a polyploid state of one or more chromosomes, or may refer to one or more disease chain alleles or some combination thereof. In this disclosure, the discussion is focused on determining the genetic status of the fetus when fetal DNA is found in the blood of the maternal line, but this example may be applicable to this method. It does not indicate that it is limited to a certain situation. Further, the method is applicable when the amount of target DNA is present in any proportion to the non-target DNA, for example, the target DNA is 0.000001% to 99.999999% of the present DNA. Any of the spaces may be configured. Furthermore, the non-target DNA does not necessarily have to be derived from one individual, as long as genetic data from some or all of the relevant non-target individuals (s) are known, and even related individuals. It does not have to be of origin. In certain embodiments, the methods disclosed herein can be used to determine fetal genotype data from maternal blood containing fetal DNA. The method is also used when there are multiple fetuses in the womb of a pregnant woman, or when other contaminated DNA, such as DNA from other already born sibs, may be present in the sample. be able to.

This technique can use the phenomenon of fetal blood cells entering the maternal circulation through placental villi. Normally, only a very small number of fetal cells enter the maternal circulation in this way (not enough to be positive on the Kleihauer-Betke test for intermaternal hemorrhage). Fetal cells can be screened and analyzed by a variety of techniques to find specific DNA sequences, but without the inherent risks of invasive procedures. This technique can also be used when the placental tissue in question contains DNA of the same genotype as the fetus, and the floating fetal DNA enters the maternal circulation due to DNA release after the placental tissue apoptosis. Floating DNA found in maternal plasma has been shown to contain fetal DNA in proportions comparable to 30-40% fetal DNA.

In certain embodiments, blood can be drawn from a pregnant woman. Studies have shown that maternal blood can contain small amounts of fetal-derived floating DNA in addition to maternal-derived floating DNA. Furthermore, in addition to many blood cells of maternal origin, enucleated fetal blood cells containing fetal origin DNA may also be present, which generally do not contain nuclear DNA. There are many methods known in the art for isolating fetal DNA or producing fractions enriched with fetal DNA. For example, chromatography has been shown to produce specific fractions enriched with fetal DNA.

A maternal line that is relatively non-invasively extracted and contains a certain amount of fetal DNA, either cellular or floating, enriched in proportion to its maternal DNA, or in its original proportion. Once you have a sample of blood, plasma or other body fluid, you can genotype the DNA found in the sample. In some embodiments, blood can be withdrawn using a needle for collecting blood from a vein, eg, the basilic vein. The methods described herein can be used to determine fetal genotype data. For example, the method can be used to determine polyploid states in one or more chromosomes, and the method can be used to include a single SNP or set of SNPs, including insertions, deletions, and translocations. Can determine the identity of. The method can be used to determine one or more haplotypes, including the parent from which the form of one or more genotypes originated.

This method uses any nucleic acid that can be used for any genotyping and / or sequencing method, eg, ILLUMINA INFINIUM ARRAY platform, AFFYMETRIX GENECHIP, ILLUMINA GENOME ANALYZER or LIFE TECHNOLGIES'SOLID SYSTEM. Please note. This includes floating DNA extracted from plasma or an amplification thereof (eg, whole genome amplification, PCR); genomic DNA from other cell types (eg, human lymphocytes from whole blood) or a amplification thereof. For preparing DNA, any extraction or purification method that produces genomic DNA suitable for one of these platforms will work as well. This method can work equally well with RNA samples. In certain embodiments, storage of the sample may be performed with minimal degradation (eg, under freezing at a temperature of about −20 ° C. or lower).

Parental Assistance Some embodiments may be used in combination with the PARENTAL SUPPORT ™ (PS) method, and the plurality of embodiments of the PARENTAL SUPPORT ™ (PS) method are herein by reference in their entirety. US Patent Application No. 11 / 603,406 (US Patent Application Publication No. 20070184467), US Patent Application No. 12 / 076,348 (US Patent Application Publication No. 20080243398), US Patent Application No. 13 / 110,685, PCT Application No. PCT / US09 / 52730 (PCT Publication No. WO / 2010/017214), and PCT Application No. PCT / US10 / 050824 (PCT Publication No. WO / 2011/041485). ing. These patents are incorporated herein by reference in their entirety. PARENTAL SUPPORT ™ is an informatics-based technique that can be used to analyze genetic data. In some embodiments, the methods disclosed herein can be considered part of the PARENTAL SUPPORT ™ method. In some embodiments, the PARENTAL SUPPORT ™ method provides the genetic data of a target individual with high accuracy, the genetic data of one or a few cells from that individual, or the DNA and one or more from the target individual. To determine genetic data for a mixture of DNA consisting of DNA from multiple other individuals, in particular, disease-related alleles in the target individual, alleles of other subjects, and / or one or more. A set of methods that can be used to determine the polymorphic state of a chromosome. PARENTAL SUPPORT ™ can refer to any of these methods. PARENTAL SUPPORT ™ is an example of an informatics-based method. Representative embodiments of the PARENTAL SUPPORT ™ method are illustrated in FIGS. 29-31G and described in Experiment 19.

In the PARENTAL SUPPORT ™ method, known parental genetic data, ie, maternal and / or paternal haplotype and / or diploid genetic data, is used for incomplete measurement of meiosis mechanism and target DNA, and optionally. With the findings of one or more related individuals, along with population-based crossover frequency, genotypes in multiple allelic genes and / or polyploid states of the embryo or any target cell (s). , And target DNA locating for important genotypes is used to reconstruct with a high degree of confidence. The PARENTAL SUPPORT ™ method can reconstruct not only poorly measured single nucleotide polymorphisms (SNPs), but also insertions and deletions, as well as all regions of SNPs or DNA that have not been measured at all. In addition, the PARENTAL SUPPORT ™ method allows both measurement of multiple disease chain loci and screening for aneuploidy from a single cell. In some embodiments, one or more embryo-derived embryos biopsied during the IVF cycle to determine the genetic status of one or more cells using the PARENTAL SUPPORT ™ method. Individual cells can be characterized.

The PARENTAL SUPPORT ™ method makes it possible to clean genetic data with noise. This can be done by estimating the exact gene allele in the target genome (embryo) using the genotype of the relevant individual (parent) as a reference. PARENTAL SUPPORT ™ is inherently noisy when only small amounts of genetic material are available (eg, PGD) and due to the limited amount of genetic material, direct genotyping is inherently noisy. May be particularly suitable when accompanied. PARENTAL SUPPORT ™ is a direct measurement of genotype when only a small portion of the available genetic material is from the target individual (eg, NPD) and due to contaminated DNA signals from another individual. May be particularly suitable when is inherently noisy. Although the PARENTAL SUPPORT ™ method allows traditional unordered diploid measurements to be characterized by high rates of allele dropouts, drop-ins, variability amplification bias and other errors. A very accurate and regular diploid allelic sequence can be reconstructed on the embryo, along with the number of copies of the chromosomal segment. Both the underlying genetic model and the underlying measurement error model can be used in the method. The genetic model can determine both the probability of alleles in each SNP and the probability of crossover between SNPs. Allele probabilities can be modeled based on data obtained from parents at each SNP, and crossover probabilities between SNPs can be modeled based on data obtained from the HapMap database developed by the International HapMap Project. Can be done. Given the appropriate underlying genetic and measurement error models, maximal posterior (MAP) estimation is used with modifications to make it computationally efficient, accurate, and regular alleles in each embryonic SNP. The value can be estimated.

The techniques outlined above can, in some cases, determine the genotype of an individual, taking into account very small amounts of DNA from that individual. It may be DNA from one or a few cells, or it may be from a small amount of fetal DNA found in maternal blood.

Hypothesis In the context of this disclosure, a hypothesis refers to a possible genetic state. A hypothesis can refer to a possible ploidic state. A hypothesis can refer to a possible allelic state. A set of hypotheses can refer to a set of possible genetic states, a set of possible allelic states, a set of possible ploidic states, or a combination thereof. In some embodiments, a set of hypotheses can be designed such that one hypothesis from the set corresponds to the actual genetic state of any given individual. In some embodiments, a set of hypotheses can be designed such that the state of any possible gene can be described by at least one hypothesis from the set. In some embodiments of the present disclosure, one aspect of the method is to determine which hypothesis corresponds to the actual genetic state of the individual in question.

In another embodiment of the present disclosure, one step comprises the step of making a hypothesis. In some embodiments, the hypothesis may be a copy number hypothesis. In some embodiments, the hypothesis includes a hypothesis as to which segment of the chromosome from each of the related individuals, if any, genetically corresponds to which segment of the other related individual. Creating a hypothesis can refer to the act of setting the limits of a variable so that the complete set of genetic states that may be under consideration is included in those variables.

The "copy number hypothesis", also known as the "haploid hypothesis" or "haploid state hypothesis," is a hypothesis about a possible haploid state for a given chromosomal copy, chromosomal type, or section of a chromosome in a target individual. Can point. This may refer to a ploidic state in two or more chromosomal types of an individual. The set of copy number hypotheses may refer to a set of hypotheses, where each hypothesis corresponds to possible different ploidic states in the individual. The set of hypotheses concerns a set of possible ploidy states, a set of possible parental haplotype contributions, a set of possible fetal DNA percentages in a mixed sample, or a combination thereof. You may.

A normal individual contains one of each chromosomal type from each parent. However, due to errors in meiosis and mitosis, an individual may have 0, 1, 2, or more of a given chromosomal type from each parent. In practice, it is rare to find more than one given parental chromosome. The present disclosure considers only possible hypotheses that, in some embodiments, 0, 1 or 2 copies of a given chromosome are of parental origin, and there is some possibility of parental origin. Considering copying is a self-evident extension. In some embodiments, there are nine possible hypotheses for a given chromosome: 0 chromosomes of maternal origin, 3 possible hypotheses for 1 or 2 chromosomes. Multiplied by three possible hypotheses about zero chromosomes of paternal origin, one chromosome or two chromosomes. Let (m, f) refer to the hypothesis that m is the number of given chromosomes inherited from the mother and f is the number of given chromosomes inherited from the father. Therefore, the nine hypotheses are (0,0), (0,1), (0,2), (1,0), (1,1), (1,2), (2,0), ( 2, 1), and (2, 2). These can also be described as H ₀₀ , H ₀₁ , H ₀₂ , H ₁₀ , H ₁₂ , H ₂₀ , H ₂₁ and H ₂₂ . .. Different hypotheses correspond to different ploidic states. For example, (1, 1) refers to a normal disomy chromosome, (2, 1) refers to maternal trisomy, and (0, 1) refers to paternal monosomy. In some embodiments, if two chromosomes are inherited from one parent and one chromosome is inherited from the other parent, it can be further divided into two cases: the two chromosomes are identical. In some cases (matched copy error) and when the two chromosomes are homologous but not identical (mismatched copy error). In these embodiments, there are 16 possible hypotheses. It should be understood that it is possible to use a set of other hypotheses, and a different number of hypotheses.

In some embodiments of the present disclosure, the ploidy hypothesis refers to the hypothesis as to which of the chromosomes from other related individuals corresponds to the chromosome found in the genome of the target individual. In some embodiments, the key to the method is the fact that related individuals can be expected to share a haplotype block, which haplotype block targets genetic data measured from the relevant individual. When used in conjunction with the finding of agreement between an individual and a related individual, it is possible to infer accurate genetic data for the target individual with higher reliability than using the genetic measurements of the target individual alone. Is. Thus, in some embodiments, the ploidy hypothesis may relate not only to the number of chromosomes, but also to which chromosomes of the associated individual are identical or nearly identical to one or more chromosomes of the target individual.

Once the set of hypotheses has been defined, when the algorithm operates on the input genetic data, a determined statistical probability can be output for each of the hypotheses under consideration. The probabilities of the various hypotheses are such that for each of the various hypotheses, the probabilities indicated by one or more of the techniques, algorithms, and / or methods described elsewhere in this disclosure are equal. , Can be determined by mathematical calculation using relevant genetic data as input.

Once the probabilities of different hypotheses have been estimated, as determined by multiple techniques, they can be combined. This may require multiplying each hypothesis by the probabilities determined by each technique. The product of hypothetical probabilities can be normalized. One ploidy hypothesis refers to one possible ploidy state for a chromosome.

The "combination of probabilities" process, also referred to as the "combination of hypotheses" or the combination of the results of specialized techniques, is a concept that should be familiar to those skilled in the art of linear algebra. One possible combination of probabilities is as follows: When using specialized techniques to evaluate a set of hypotheses considering a set of genetic data, the output of the method is each in the set of hypotheses. It is a set of probabilities that are one-to-one related to the hypothesis. A set of probabilities determined by a first technician, each associated with one of the hypotheses in the set, with a probability determined by a second technician, each associated with the same set of hypotheses. When combined with a set of, multiply by two sets of probabilities. This means that for each hypothesis in the set, the two probabilities associated with that hypothesis, determined by two specialized methods, are multiplied and the corresponding product is the output probability. This process can be extended to any number of specialized techniques. When using only one technique, the output probability is the same as the input probability. When using three or more specialized techniques, related probabilities can be multiplied at the same time. The product can be normalized so that the probabilities of the hypotheses in the set of hypotheses are 100% in total.

In some embodiments, if the compound probability for a given hypothesis exceeds the compound probability for any of the other hypotheses, then that hypothesis can be considered to be determined to be most likely. In some embodiments, if the normalized probability exceeds a threshold, the hypothesis can be determined to be most likely, and a polyploid state or the state of another gene can be called. In certain embodiments, this can mean that the number and identity of chromosomes associated with that hypothesis can be called as a haploid state. In certain embodiments, this can mean that the allelic identity associated with that hypothesis can be called as the allelic state. In some embodiments, the threshold can be between about 50% and about 80%. In some embodiments, the threshold can be between about 80% and about 90%. In some embodiments, the threshold can be between about 90% and about 95%. In some embodiments, the threshold can be between about 95% and about 99%. In some embodiments, the threshold can be between about 99% and about 99.9%. In some embodiments, the threshold can be greater than about 99.9%.

Parental status Parental status refers to the genetic status of each given allele of two related chromosomes for one or both of the two targeted parents. Note that in certain embodiments, the parental situation does not refer to the state of the target allele, but to the state of the parental allele. The parental situation for a given SNP may consist of two paternal and two maternal four base pairs, which may be the same as or different from each other. It is common to write "m ₁ m ₂ | f ₁ f ₂ ", where m ₁ and m ₂ are the genetic states of a given SNP on two maternal chromosomes, f ₁ and f. ₂ is the genetic state of a given SNP on two paternal chromosomes. In some embodiments, the parental situation can be written as "f ₁ f ₂ | m ₁ m ₂ ". Note that the subscripts "1" and "2" indicate genotypes in a given allele of the first and second chromosomes. It should also be noted that the choice of which chromosome is "1" and which chromosome is "2" is optional.

It should be noted in this disclosure that A and B are often used to generally indicate base pair identity; A or B is C (cytosine), G (guanine), A ( Adenine) or T (thymine) can be shown equally well. For example, in an allele based on a given SNP, the mother's genotype is T at that SNP on one chromosome, G at that SNP on a homologous gene, and the father's genotype at that allele. If both of the homologous chromosomes are G in that SNP, then the allele of the target individual can be said to have the parental status AB | BB, and the allele can also be said to have the parental status AB | AA. In theory, all four possible nucleotides may be present in a given allele, thus, for example, in a given allele, the mother has genotype AT and the father has genotype GC. Please note that you may have. However, empirical data show that in most cases only two of the four possible base pairs are observed in a given allele. For example, when using a single tandem repeat, it is possible to have more than two, more than four, and even more than ten parental situations. The discussion of this disclosure assumes that only two possible base pairs are observed in a given allele, but the embodiments disclosed herein assume that this assumption does not apply. It can be modified to take into account.

A "parental situation" can refer to a set or subset of target SNPs that have the same parental situation. For example, when measuring 1000 allogens on a given chromosome of a target individual, situation AA | BB means that the genotype of the target mother is homozygous and the genotype of the target father is homozygous. It may indicate a set of all allelic genes within a group of 1,000 allelic genes that are sex but not similar in maternal and paternal genotypes at that locus. If no phase is specified for the parental data, and therefore AB = BA, there are nine possible parental situations: AA | AA, AA | AB, AA | BB, AB | AA, AB | AB, AB | BB, BB | AA, BB | AB, and BB | BB. Phases are identified for parental data, and therefore if AB ≠ BA, there are 16 possible different parental situations: AA | AA, AA | AB, AA | BA, AA | BB, AB | AA, AB | AB, AB | BA, AB | BB, BA | AA, BA | AB, BA | BA, BA | BB, BB | AA, BB | AB, BB | BA, and BB | BB. All SNP alleles on the chromosome, with the exception of some SNPs on the sex chromosome, have one of these parental situations. A set of SNPs in which the parental situation for one parent is heterozygous can be referred to as the heterozygous situation.

Use of Parental Situation in NPD Non-invasive prenatal diagnosis can be used non-invasively, eg, to determine the genetic status of a fetus from genetic material obtained by drawing blood to a pregnant mother. Technique. Blood can be separated, plasma isolated, and then plasma DNA isolated. Size selection can be used to isolate DNA of appropriate length. DNA can be preferentially enriched in a set of loci. The DNA can then be hybridized to a genotyping array and measured by a number of means, either by measuring fluorescence or by sequencing on a high throughput sequencer.

When using sequencing for fetal ploidy calls in the context of non-invasive prenatal diagnosis, there are several ways to use sequence data. The most common method in which sequence data can be used is simply to count the number of reads mapped to a given chromosome. For example, consider trying to determine the ploidy state of the fetal chromosome 21. Furthermore, it is considered that 10% of the DNA in the sample is composed of fetal origin DNA and 90% is composed of maternal origin DNA. In this case, we examined the average number of reads on chromosomes that could be predicted to be disomy, eg, chromosome 3, and adjusted the reads for the number of base pairs on the chromosomes that were part of the unique sequence. Compare with the number of reads on chromosome 21. If the fetus was positive polyploid, the amount of DNA per unit of the genome is expected to be about the same everywhere (susceptible to stochastic variability). On the other hand, if the fetus was trisomy on chromosome 21, it is expected that there will be slightly more DNA per genetic unit from chromosome 21 than elsewhere in the genome. Specifically, DNA from chromosome 21 in the mixture is expected to be about 5% higher. When measuring DNA using sequencing, it is expected that there will be approximately 5% more uniquely mapable reads from chromosome 21 per unique segment than those from other chromosomes. Observations of DNA from a particular chromosome in an amount greater than a certain threshold can be used as the basis for diagnosing aneuploidy when adjusted for the number of sequences that can be uniquely mapped to that chromosome. can. Another method that can be used to detect aneuploidy is similar to that described above, except that the parental situation can be taken into account.

When considering which allele to target, the likelihood that some parental situations may be more informative than others can be taken into account. For example, the AA | BB and symmetric situations BB | AA are the most informative situations as it is known that the fetus carries a different allele than the mother. For reasons of symmetry, both the AA | BB situation and the BB | AA situation can be referred to as AA | BB. Another set of informative parental situations is AA | AB and BB | AB, because in these cases the fetus is 50% likely to carry an allele that the mother does not have. be. For reasons of symmetry, both the AA | AB situation and the BB | AB situation can be referred to as AA | AB. The third set of informative parental situations is AB | AA and AB | BB, which in these cases the fetus carries a known paternal allele, which is also present in the maternal genome. Because it does. For reasons of symmetry, the AB | AA situation and the AB | BB situation can be referred to as AB | AA. The fourth parental situation is AB | AB, where the fetus has an unknown allele state, whatever the allele state is, that the mother has the same allele. The fifth parental situation is AA | AA, where the mother and father are heterozygous.

Methods for determining the ploidy state of different execution target individuals disclosed herein are disclosed herein. The target individual may be a blastomere, embryo or fetus. In some embodiments of the present disclosure, the method for determining the ploidy state of one or more chromosomes in a target individual may include any of the steps described herein, or a combination thereof. :

In some embodiments, the source of the genetic material used in determining the genetic status of the fetus may be fetal cells such as fetal nucleated red blood cells isolated from maternal blood. The method may include the step of obtaining a blood sample from a pregnant mother. The method uses a visual technique to uniquely associate a particular combination of colors with nucleated red blood cells and not a similar combination of colors with any other cell present in the blood of the maternal line. It may include the step of isolating fetal erythrocytes based on the idea. The color combinations associated with nucleated red blood cells may include the red color of hemoglobin around the nucleus, which can be made more distinguishable by staining, and the color of the nuclear material, which can be stained, for example, blue. Identifying the location of nucleated red blood cells by isolating cells from maternal blood, spreading them on slides, and then identifying the presence of both red (from hemoglobin) and blue (from nuclear material). Can be possible. These nucleated red blood cells can then be extracted using a micromanipulator and genotyping and / or sequencing techniques can be used to determine the genotyping aspect of the genetic material of these cells.

In certain embodiments, nucleated red blood cells are stained with a dye that fluoresces only in the presence of fetal hemoglobin and does not fluoresce in the presence of maternal hemoglobin, thus the nucleated red blood cells are derived from the mother. The ambiguity of whether it is derived from the fetus or from the fetus can be excluded. Some embodiments of the present disclosure may involve marking the nuclear material by staining or other methods. Some embodiments of the present disclosure may involve specifically marking fetal nuclear material with an antibody specific for fetal cells.

There are many ways to isolate fetal cells from maternal blood, or to isolate fetal DNA from maternal blood, or to enrich a sample of fetal genetic material in the presence of maternal genetic material. be. Some of these methods are listed here, but this is not intended to be an exhaustive list. For convenience, some suitable techniques are listed here: antibodies tagged by fluorescence or otherwise, size exclusion chromatography, magnetic or otherwise labeled affinity tags, posterior. Differences, eg, differential methylation between maternal and fetal cells in a particular allelic gene, CD45 / 14 depletion following density gradient centrifugation and CD71 positive selection from CD45 / 14 negative cells, different. Single or double Percoll gradient or galactose-specific lectin method with weight osmol concentration.

In one embodiment of the present disclosure, the target individual is a fetus and different genotyping is performed on a plurality of fetal-derived DNA samples. In some embodiments of the present disclosure, the fetal DNA sample is derived from isolated fetal cells, which may be mixed with maternal cells. In some embodiments of the present disclosure, the fetal DNA sample is derived from floating fetal DNA, which may be mixed with floating maternal DNA. In some embodiments, the fetal DNA sample can be obtained from maternal plasma or maternal blood containing a mixture of maternal DNA and fetal DNA. In some embodiments, the fetal DNA is 99.9: 0.1% to 99: 1%; 99: 1% to 90:10%; 90: 10% to 80: 20%; 80 with the maternal DNA. : 20% to 70:30%; 70:30% to 50:50%; 50:50% to 10:90%; or 10:90% to 1:99%; 1:99% to 0.1:99 Maternal: fetus ratio in the range of 9.9% may be mixed.

Genetic data for target individuals and / or related individuals is not limited to these, but using tools and / or techniques selected from the group including genotyping microarrays and high-throughput sequencing to determine the appropriate genetic material. By measuring, it is possible to convert from a molecular state to an electronic state. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing, ILLUMINA SOLEXA platform, ILLUMINA GENOME ANALYZER or APPLIED BIOSYSTEM 454 sequencing platform, HELICOS TRUE SINGLE MOLECULE SEQUEL Sequencing methods or any other sequencing method can be mentioned. All of these methods physically convert the genetic data stored in the DNA sample into a set of genetic data that is generally stored and processed in a memory device along the way.

Genetic data for relevant individuals is not limited to these, but is limited to bulk diploid tissue of the individual, one or more diploid cells from the individual, one or more diploids from the individual. Cells, one or more blastomeres from a target individual, extracellular genetic material found in an individual, extracellular genetic material from an individual found in maternal blood, cells from an individual found in maternal blood , One or more embryos made from gametes (s) from related individuals, one or more blastomeres obtained from such embryos, extracellular genetic material found in related individuals , Genetic substances known to originate from related individuals, and substances selected from the group including combinations thereof can be measured.

In some embodiments, it is possible to generate at least one set of polyploid state hypotheses for each of the target chromosomal types of the target individual. Each ploidy state hypothesis can refer to one possible ploidy state of a target individual's chromosome or chromosomal segment. The set of hypotheses may include some or all of the possible ploidic states that can be predicted to be possessed by the chromosomes of the target individual. Some of the possible polyploid states are zero-chromosomal, monosomy, aneuploidy, uniparental disomy, positive ploidy, trisomy, matched trisomy, mismatched trisomy, maternal trisomy, paternal trisomy, tetrasomy, equilibrium (2: 2). It may include trisomy, unbalanced (3: 1) tetrasomy, pentasomy, hexasomy, other aneuploidies, and combinations thereof. Any of these aneuploidy states may be mixed or partially aneuploidy, eg, unbalanced translocation, equilibrium translocation, Robertsonian translocation, recombination, deletion, insertion, crossover. , And combinations thereof.

In some embodiments, the knowledge of the determined ploidy state can be used to make clinical decisions. This finding is generally stored in a memory device as a physical array of matters and can then be converted into a report. The report can then be carried out. For example, the clinical decision may be an abortion, or the clinical decision may be to continue the pregnancy. In some embodiments, the clinical decision is an intervention designed to reduce the severity of the expression of the phenotype of the genetic disorder, or relevance to prepare for a special needs child. May be accompanied by a decision to take a certain step.

In certain embodiments of the present disclosure, any method described herein is to allow multiple targets to be derived from multiple blood draws from the same target individual, eg, the same pregnant mother. Can be modified. This can improve the accuracy of the model, as multiple gene measurements can provide more data that can determine the target genotype. In one embodiment, one set of target gene data serves as the reported primary data, and the other set of target gene data serves as data for reconfirming the primary target gene data. .. In one embodiment, multiple sets of genetic data, each measured from genetic material obtained from the target individual, are considered in parallel, thus both sets of target genetic data of the parent measured with high accuracy. It serves as an aid in determining which section of genetic data constitutes the fetal genome.

In certain embodiments, the method can be used for a parent-child test. Given, for example, SNP-based genotype information from a mother and from a man who may or may not be a genetic father, as well as genotype information measured from a mixed sample, that man's genotype. It is possible to determine if the information in the above actually represents the actual genetic father of the pregnant fetus. A simple way to do this is simply to test for situations where the mother is AA and the possible father is AB or BB. In each of these cases, it can be expected that the father will contribute half a time (AA | AB) or always (AA | BB). Taking into account the predictive ADO, it is easy to determine whether the observed fetal SNP correlates with the possible paternal SNP.

One embodiment of the disclosure may be as follows: A pregnant woman wishing to know if her fetus has Down's syndrome and / or cystic fibrosis. And the woman does not want to give birth to a child suffering from any of these conditions. The doctor takes the woman's blood and stains hemoglobin with one marker for a distinct red color and fertile material with another marker for a clear blue color. Maternal erythrocytes are generally anucleated, but it is known that a high proportion of fetal cells contain nuclei, so doctors can identify cells that show both red and blue, in a number of ways. Nucleated red blood cells can be visually isolated. The doctor removes these cells from the slide with a micromanipulator and sends them to the laboratory where 10 individual cells are amplified and genotyped. By using genetic measurements, the PARENTAL SUPPORT ™ method can be used to determine that 6 out of 10 cells are maternal blood cells and 4 out of 10 cells are fetal cells. can. If the pregnant mother already has offspring, PARENTAL SUPPORT ™ makes reliable allelic calls to fetal cells to show that they are not similar to the offspring's alleles. It can also be used to determine that the fetal cells are distinct from the offspring cells that were born. It should be noted that this method is similar in concept to the patrilineal test embodiment of the present disclosure. Genetic data measured from fetal cells can be of very poor quality and contain many allelic dropouts due to the difficulty of single cell genotyping. Clinicians use the measured fetal DNA in conjunction with the parent's reliable DNA measurements and use PARENTAL SUPPORT ™ to estimate the fetal genomic aspect with high accuracy, thereby derived from the fetus. The genetic data contained in the genetic material can be converted into the predicted fetal genetic state stored on the computer. The clinician can determine both the ploidy state of the fetus and the presence or absence of multiple disease chained genes. The fetus is found to be positive polyploid and not a carrier of cystic fibrosis, and the mother decides to continue her pregnancy.

In one embodiment of the disclosure, a pregnant mother wants to determine if her fetus suffers from any chromosomal abnormality. The woman goes to her doctor and provides a sample of her blood, and the woman and her husband provide a sample of their DNA by cheek swab. Laboratory researchers use the MDA protocol to amplify the parent's DNA and the ILLUMINA INFINIUM array to measure the parent's genetic data in multiple SNPs to genotype the parent's DNA. conduct. Researchers then centrifuge the blood, collect plasma, and use size-exclusion chromatography to isolate a sample of floating DNA. Alternatively, researchers use one or more fluorescent antibodies, eg, antibodies specific for fetal hemoglobin, to isolate fetal nucleated red blood cells. Researchers then obtain isolated or enriched fetal genetic material, which is appropriately adapted so that the two ends of each oligonucleotide correspond to adjacent sequences on either side of the target allele. Amplify using a designed library of 70-mer oligonucleotides. Upon addition of polymerase, ligase, and appropriate reagents, the oligonucleotide underwent gap-filled cyclization, capture of the desired allele. Exonucleases were added, heat inactivated, and the product was used directly as a template for PCR amplification. The PCR product was sequenced by ILLUMINA GENOME ANALYZER. Sequence reads were used as inputs for the PARENTAL SUPPORT ™ method, which in turn predicted the ploidy state of the fetus.

In another embodiment, a couple whose mother is pregnant and has advanced maternal age knows if the pregnant fetus has Down's syndrome, Turner's syndrome, Prader-Willi syndrome or some other chromosomal abnormality. I want. Obstetricians collect blood from mothers and fathers. Blood is sent to the laboratory where the technician centrifuges the maternal sample to isolate plasma and buffy coat. The DNA in the buffy coat and the blood sample of the paternal line are converted by amplification, and the genetic data encoded by the amplified genetic material is electron-generated from the genetic data stored molecularly by subjecting the genetic material to a high-throughput sequencer. The genotype of the parent is measured by further conversion to the conserved genetic data. Plasma samples are preferentially enriched at the locus set using the 5,000-plex heminestized target PCR method. A mixture of DNA fragments is prepared in a DNA library suitable for sequencing. The DNA is then sequenced using a high-throughput sequencing method, eg, ILLUMINA GAIIx GENEOME ANALYZER. Sequencing converts information that is molecularly encoded in DNA into information that is electronically encoded by computer hardware. Informatics-based techniques, including the embodiments disclosed herein, can be used to determine the ploidy state of the fetus, eg, PARENTAL SUPPORT ™. This is a computerized step in calculating the probability of alleles at multiple polymorphic loci from DNA measurements made on the prepared sample, each with a possible different multiple state in the chromosome. A computer-based step to create related multiple multiple hypotheses, and a computer-based co-distribution model for the expected number of alleles at multiple polymorphic loci on the chromosome for each multiple hypothesis. Using the co-distribution model and the number of alleles measured in the prepared sample, the steps to computerize the relative probabilities of each of the multiple hypotheses, and the polymorphic states corresponding to the hypotheses with the highest probabilities. It may include steps to call the chromosomal state of the fetal by selection. It is determined that the fetus has Down's syndrome. Print the report or electronically send it to the obstetrician in charge of the pregnant woman, who will convey the diagnosis to the woman. The woman, her husband, and the doctor sit down to discuss options. The couple decides to have an abortion based on the finding that the fetus suffers from a trisomy condition.

In certain embodiments, the entity may decide to provide diagnostic techniques designed to detect aneuploidy in a pregnant fetus from maternal blood draws. The product may require the mother to visit an obstetrician in charge who can collect the mother's blood. Obstetricians can also collect genetic samples from the father of the fetus. The clinician can isolate the plasma from the mother's blood and purify the DNA from the plasma. The clinician can also isolate the buffy coat layer from the mother's blood and prepare the DNA from the buffy coat. The clinician can also prepare DNA from the father's genetic sample. Clinicians can use the molecular biology techniques described in the present disclosure to add a universal amplification tag to DNA in DNA derived from plasma samples. Clinicians can amplify universally tagged DNA. Clinicians can preferentially enrich DNA by a number of techniques, including hybridization capture and targeted PCR. Targeted PCR may involve nesting, heminesting or semi-nesting or any other technique that results in efficient enrichment of plasma-derived DNA. Targeted PCR can be extensively multiplexed, for example, with 10,000 primers in one reaction volume, where the primers are on chromosomes 13, 18, 21, and X. Target loci common to both SNP and X and Y, and optionally to other chromosomes. Selective enrichment and / or amplification may involve tagging each individual molecule with a different tag, molecular barcode, amplification tag, and / or sequencing tag. The clinician can then sequence the plasma sample and, optionally, sequence the prepared maternal and / or paternal DNA. The molecular biology step can be performed completely or partially by the diagnostic box. Array data can be supplied to a single computer or to another type of computing platform, eg, one that can be found in the "cloud". The computational platform allows the number of alleles at the target polymorphic locus to be calculated from measurements made by the sequencer. Computational platform creates multiple multiple hypotheses related to zero-chromosomal, monosomy, disomy, coincident trisomy, and mismatched trisomy for each of chromosomes 13, 18, 21, X, and Y. can do. The computational platform allows us to build a co-distribution model for the predicted number of alleles at the target locus on the chromosome for each polyploidy hypothesis for each of the five chromosomes being investigated. .. The computational platform allows the probability that each of the polyploidy hypotheses is true using a joint distribution model and the number of alleles measured against preferentially enriched DNA from plasma samples. .. For each of chromosomes 13, 18, 21, X, and Y, the computational platform determines the ploidy state of the fetal by selecting the ploidy state that corresponds to the appropriate hypothesis with the greatest probability. You can call. A report containing the called ploidy state can be produced and sent electronically to the obstetrician, displayed on an output device, or a hard copy of the printed report can be delivered to the obstetrician. .. Obstetricians can inform the patient and, if necessary, the father of the fetus, who can determine which clinical options are acceptable and which is most desirable.

In another embodiment, a pregnant woman, hereafter referred to as the "mother," may decide that she wants to know if her fetus (s) carry any genetic abnormality or other condition. .. The mother may wish to be confident of continuing pregnancy after ensuring that there are no general abnormalities. The mother can go to her obstetrician, who can also take a sample of her mother's blood. The obstetrician in charge can also take a genetic sample such as a cheek swab from the mother's cheek. The obstetrician in charge can also take a genetic sample, such as a cheek swab, a sperm sample or a blood sample, from the father of the fetus. The obstetrician in charge can send the sample to the clinician. Clinicians can enrich the fraction of floating fetal DNA in maternal blood samples. Clinicians can enrich the fraction of enucleated fetal blood cells in maternal blood samples. The clinician can use various aspects of the methods described herein to determine fetal genetic data. The genetic data may include the ploidy state of the fetus and / or the identity of one or more disease chain alleles in the fetus. A report can be produced that summarizes the results of prenatal diagnosis. The report can be served or mailed to the doctor, who can inform the mother of the genetic status of the fetus. The mother can decide to discontinue pregnancy based on the fact that the fetus has one or more chromosomal or genetic abnormalities or undesired conditions. The mother can likewise decide to continue the pregnancy based on the fact that the fetus does not have any chromosomal or genetic abnormalities or genetic status of any subject.

Another example may be for a pregnant woman who is artificially fertilized by a sperm donor and is pregnant. The woman wants to minimize the risk that her fetus will have a genetic disease. The woman receives blood drawn by a venous phleboder and uses the techniques described in the present disclosure to isolate three fetal nucleated red blood cells, and tissue samples are also taken from the mother and genetic father. Fetal-derived genetic material and mother-and-father-derived genetic material are amplified as needed and genotyped using ILLUMINA INFINIUM BEADARRAY and genotyped by the method described herein. Clean, identify phases with high accuracy, and make multiple calls for the fetus. The fetus was found to be positive polyploid, predicting phenotypic susceptibility from the reconstructed fetal genotype, producing a report and sending it to the mother's physician, thus making any clinical decisions. Can be determined to be the best.

In certain embodiments, the untreated genetic material of the mother and father is converted by amplification into a quantity of DNA that is similar in sequence but in quantity. The genotyping method then converts the genotypic data encoded by the nucleic acid into a genetic measurement that can be physically and / or electronically stored in a memory device such as the one described above. The relevant algorithms that make up the PARENTAL SUPPORT ™ algorithm are translated into computer programs using programming languages, the relevant parts of which are discussed in detail herein. Then, by running the computer program on computer hardware that is organized into patterns that represent raw measurement data rather than physically coded bits and bytes, a highly reliable determination of the fetal polymorphic state is then made. It is converted to the pattern shown. The details of this conversion depend on the data itself and the computer language and hardware system used to perform the methods described herein. The data, physically configured to show the determination of high quality fetal ploidy, is then transformed into a report that can be sent to health care practitioners. This conversion can be done using a printer or computer display. The report may be a copy printed on paper or other suitable medium, or the report may be electronic. In the case of electronic reports, they can be communicated and physically stored on a memory device located on the computer available to health care practitioners. Electronic reports can also be displayed on the screen for readability. In the case of screen display, the data can be converted into a readable format by inducing a physical transformation of the pixels on the display device. The transformation physically changes the transparency of a particular set of pixels on a screen that can be placed in front of a substrate that emits or absorbs photons by physically emitting electrons onto a phosphorescent screen. It can be realized by changing the target charge. This transformation can be achieved by changing the orientation of the nanoscale molecules in the liquid crystal, for example, from a nematic phase to a cholesteric or smectic phase in a particular set of pixels. This transformation can be achieved by a current that causes the emission of photons from a particular set of pixels composed of multiple light emitting diodes arranged in a meaningful pattern. This conversion can be achieved by any other method used to display the information, such as a computer screen or some other output device or information transmission method. Health care practitioners can then act on the basis of the report to translate the data in the report into measures. The measure may be to continue or discontinue the pregnancy, in which case the pregnant fetus with the genetic abnormality is converted to a non-surviving fetus. The transformations listed herein consist of, for example, miscarriage of a pregnant mother and father's genetic material through a number of steps outlined in this disclosure to a fetus with a genetic abnormality. It can be summed up so that it can be transformed into a medical decision consisting of continuing pregnancy. Alternatively, the set of genotype measurements can be translated into reports that help physicians treat pregnant patients.

In certain embodiments of the present disclosure, using the methods described herein, the host mother, a pregnant woman, is a multiple of the fetus, even if it is not the biological mother of the fetus in her possession. The sexual state can be determined. In certain embodiments of the present disclosure, the methods described herein can be used to determine the ploidy state of the fetus using only maternal blood samples and without the need for paternal genetic samples.

Some of the mathematics in the embodiments disclosed herein make hypotheses about a limited number of aneuploidy states. In some cases, for example, only 0, 1 or 2 chromosomes are expected to originate from each parent. In some embodiments of the present disclosure, other forms of aneuploidy, such as three chromosomes originating from one parent, extend the mathematical derivation without altering the basic concepts of the present disclosure. Quadrosomy, pentasomy, and hexasomy can be taken into consideration. At the same time, it is possible to focus only on smaller numbers of ploidic states, such as trisomy and disomy. Note that polyploidy determinations that indicate non-integer chromosomes can indicate a mosaic phenomenon in a sample of genetic material.

In some embodiments, the genetic abnormality has Down syndrome (or trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13), Turner syndrome (45X), Kleinfelder syndrome (with two X chromosomes). Male), Praderwilly Syndrome, and DiGeorge Syndrome (UPD15). Congenital disorders, such as those listed in the preamble, are generally undesirable and are due to the finding that the fetus suffers from one or more phenotypic abnormalities, resulting in an abortion, the birth of a special needs child. It can provide the basis for deciding to take the necessary steps to prepare or to take some therapeutic approach intended to reduce the severity of the chromosomal abnormality.

In some embodiments, the methods described herein are used for very early gestational periods, eg, 4 weeks at the earliest, 5 weeks at the earliest, 6 weeks at the earliest, 7 weeks at the earliest, 8 weeks at the earliest. It can be used at 9 weeks at the earliest, 10 weeks at the earliest, 11 weeks at the earliest, and 12 weeks at the earliest.

In some embodiments, the methods disclosed herein are used in the context of preimplantation genetic diagnosis (PGD) for selecting embryos during in vitro fertilization, where they are targeted. The individual is an embryo and is a multiple for the embryo from sequencing data from a single or two cell biopsy from the embryo on day 3 or from the nutritional ectoderm biopsy material of the embryo on day 5 or 6. Parental genotype data can be used to make sex decisions. In the PGD environment, only the DNA of the offspring is measured and examined for only a few cells, generally 1-5, but at most 10, 20, or 50. The total number of starting copies of alleles A and B (in SNP) is then self-determined by the genotype of the offspring and the number of cells. In NPD, the number of copies at the start is very large, so it is expected that the allele ratio after PCR accurately reflects the ratio at the start. However, the small number of starting copies in PGD means that contamination and incomplete PCR efficiency have a non-trivial effect on the ratio of alleles after PCR. This effect can be more important than the read depth in predicting variance in allele ratios measured after sequencing. Distribution of allele ratios measured taking into account known offspring genotypes can be generated by MonteCarlo simulation of the PCR process based on the efficiency of the PCR probe and the probability of contamination. The likelihood of various hypotheses can be calculated in the same manner as described for NIPD, taking into account the distribution of allele ratios for each of the possible offspring genotypes.

Maximum likelihood estimation The majority of methods known in the art for detecting the presence or absence of a biological phenomenon or medical condition measure a metric that correlates with the condition, where the metric is one of a given threshold. Includes using a single hypothesis rejection test that the state exists if it is on the other side of the threshold and that the state does not exist if the metric is on the other side of the threshold. The single hypothesis rejection test only examines the null distribution when making a decision between the null hypothesis and the alternative hypothesis. Without taking into account the antagonism, the likelihood of each hypothesis cannot be estimated in light of the observed data, and therefore the confidence in the call cannot be calculated. Therefore, a single hypothesis rejection test is used to obtain a yes or no answer without sensitivity to the confidence associated with a particular case.

In some embodiments, the methods disclosed herein can detect the presence or absence of a biological phenomenon or medical condition using maximum likelihood methods. The maximum likelihood method is a substantial improvement over the method using the single hypothesis rejection method, as the threshold for calling the absence or existence of the state can be adjusted appropriately for each case. It is intended to determine the presence or absence of aneuploidy in a pregnant fetus from genetic data available from a mixture of fetal and maternal DNA present in the floating DNA found in maternal plasma. It is particularly relevant to the diagnostic technique used. This is due to the fact that when a portion of fetal DNA in plasma induces a fraction change, the optimal threshold for calling aneuploidy vs. haploidy changes. As the fetal fraction descends, the distribution of data associated with aneuploidy becomes more and more similar to the distribution of data associated with positive ploidy.

The maximum likelihood estimation method uses the distribution associated with each hypothesis to estimate the likelihood of the data conditioned for each hypothesis. These conditional probabilities can then be converted into hypothetical calls and confidence. Similarly, maximum a posteriori estimation uses the same conditional probabilities as maximum likelihood estimation, but selects the best hypothesis and incorporates the previous population in determining confidence.

Therefore, using maximum likelihood estimation (MLE) techniques or closely related maximum a posteriori (MAP) techniques offers two advantages, first of all, increasing the likelihood of an accurate call and trusting each call. It becomes possible to calculate the degree. In one embodiment, the step of selecting the ploidy state corresponding to the hypothesis with the highest probability is performed using maximum likelihood estimation or maximum a posteriori estimation. In certain embodiments, a method for determining the ploidy state of a fetus during pregnancy, which uses any method currently known in the art using a single hypothesis rejection method, is the MLE technique or the MAP technique. Disclosed are methods that include reformalization to use. Some examples of methods that can be significantly improved by applying these techniques are US Pat. No. 8,008,018, US Pat. No. 7,888,017 or US Pat. No. 7,332. It can be found in No. 277.

In one embodiment, a method for determining the presence or absence of fetal aneuploidy in a maternal chromosomal sample comprising fetal and maternal chromosomal DNA, the step of obtaining the maternal plasma sample, and the plasma. A step of measuring DNA fragments found in a sample using a high-throughput sequencer, a step of mapping sequences to chromosomes and determining the number of sequence reads mapped to each chromosome, and the proportion of fetal DNA in a plasma sample. And the predicted distribution of the amount of target chromosome that is predicted to be present if the second target chromosome is positive multiple, and one that is predicted if that chromosome is aneuploid. Or a step to calculate multiple predicted distributions using the number of sequence reads mapped to one or more reference chromosomes predicted to be fetal fractions and positive multiples, and MLE or MAP. Is used to determine which distribution is most likely to be accurate, thereby describing a method comprising a step indicating the presence or absence of fetal aneuploidy. In certain embodiments, the step of measuring plasma-derived DNA may include performing large-scale parallel shotgun sequencing. In one embodiment, the step of measuring DNA from a plasma sample comprises sequencing DNA that is preferentially enriched at multiple polymorphic or non-polymorphic loci, eg, by targeted amplification. obtain. Multiple loci can be designed to target one or a few suspected aneuploidy chromosomes and one or a few reference chromosomes. The purpose of preferential enrichment is to increase the number of informative sequence reads to determine ploidy.

Informatics Method of Ploidy Calls This specification describes a method for determining the ploidy state of a fetus in consideration of sequence data. In some embodiments, this sequence data can be measured with a high throughput sequencer. In some embodiments, sequence data can be measured for DNA originating from floating DNA isolated from maternal blood, where the floating DNA is DNA of some maternal origin, and. Contains some fetal / placental origin DNA. This section describes one embodiment of the present disclosure that determines the ploidy state of the fetus assuming that the proportion of fetal DNA in the analyzed mixture is unknown and is estimated from the data. The proportion of fetal DNA in the mixture (“fetal fraction”) or the percentage of fetal DNA can be measured by another method, and it is assumed that it is known in determining the ploidy state of the fetus. The morphology is also described. In some embodiments, the fetal fraction can be calculated using only genotyping measurements made on the maternal blood sample itself, which is a mixture of fetal DNA and maternal DNA. In some embodiments, the proportion is calculated using the genotype of the mother measured or otherwise known and / or the genotype of the father measured or otherwise known. You can also do it. In another embodiment, the ploidy state of the fetus is simply compared to the proportion of fetal DNA calculated for the reference chromosome that is supposed to be disomy, based on the proportion of fetal DNA calculated for the chromosome in question. Can be decided.

In a preferred embodiment, when observing and analyzing N SNPs for a particular chromosome,
-A set of NR floating DNA sequence measured values S = (s ₁ , ..., s _NR ). Since this method uses SNP measurements, all sequence data corresponding to non-polymorphic loci can be ignored. In the simplified form, a count value (A, B) is obtained for each SNP, and if A and B correspond to two alleles present at a given locus, then S = ((a). It can be written as ₁ , b ₁ ), ..., (a _N , b _N )), where a _i is the A count value on the SNP _i and bi is the B count value on the SNP i. Σ _{i = 1: N} ( _ai + bi) = NR, and ・ The parent data is
〇 Genotypes from SNP microarrays or other intensity-based genotyping platforms: mother M = (m ₁ , ..., m _N ), father F ₌ (f ₁ , ..., f _N ), mi , _Fi ∈ (AA, AB, BB), and / or 〇 Sequence data measurements: NRM mother's measurements SM = (sm ₁ , ..., sm _nrm ), NRF father's measurements SF = (Sf ₁ , ..., sf _nrf ). Similar to the above simplification, if a count value (A, B) is obtained for each SNP, SM = ((am ₁ , bm ₁ ), ..., (am _N , bm _N )), SF = (((am 1, bm 1)). af ₁ , bf ₁ ), ..., (af _N , bf _N )).

Collectively, mother, father, and child data are indicated by D = (M, F, SM, SF, S). It should be noted that acquisition of parental data is desirable, which increases the accuracy of the algorithm, but parental data, especially paternal data, is not always necessary. This means that it is possible to obtain highly accurate copy count results even in the absence of mother and / or father data.

同様に事後仮説尤度は、データを考慮すると：

と書くことができ、式中、ｐｒｉｏｒｐｒｏｂ（Ｈ）はモデル設計および以前の知見に基づいて各仮説Ｈに割り当てられた事前確率である。
事前確率を用いて最大事後推定値を得ることも可能である：

It is possible to derive the best copy count estimate (H ^{† *} ) by maximizing the log-likelihood LIK (D | H) of the data over all possible hypotheses (H). In particular, the relative probabilities of each of the ploidy hypotheses should use the joint distribution model and the number of alleles measured in the prepared sample to determine the hypothesis most likely to be accurate as follows: Is possible:

Similarly, the posterior hypothesis likelihood is:

In the equation, the priorprob (H) is the prior probability assigned to each hypothesis H based on the model design and previous findings.
It is also possible to obtain the maximum a posteriori estimate using prior probabilities:

In one embodiment, the copy number hypothesis that can be taken into account is:
・ Monosomy:
○ Maternal H10 (one copy from the mother)
○ Patriline H01 (one copy from the father)
Dysomy: H11 (one copy for each mother and father)
・ Simple trisomy, crossover is not considered:
○ Matrilineal: H21_match (two identical copies from the mother, one copy from the father), H21_mismatch (both copies from the mother, one copy from the father)
○ Patriline: H12_match (one copy from the mother, two identical copies from the father), H12_mismatch (one copy from the mother, both copies from the father)
・ Consider compound trisomy and crossover (using joint distribution model):
○ Matrilineal H21 (two copies from the mother, one copy from the father),
○ Patrilineal H12 (one copy from the mother, two copies from the father)

In other embodiments, other polyploid states such as zero chromosomal (H00), uniparental disomy (H20 and H02), and tetrasomy (H04, H13, H22, H31 and H40) can be considered.

In the absence of crossover, each trisomy, whether of mitosis, meiosis I or meiosis II, is either a matched trisomy or a mismatched trisomy. Due to the crossover, the true trisomy is usually a combination of the two. First, a method for deriving the likelihood of a hypothesis for a simple hypothesis is described. Next, a method for deriving the likelihood of a hypothesis for a composite hypothesis in which individual SNP likelihoods are combined with crossover is described.

この仮説では、いかなるＳＮＰ間の連鎖も仮定せず、したがって、同時分布モデルを利用しない。 LIK (D | H) about the simple hypothesis
In one embodiment, for a simple hypothesis, LIK (D | H) can be determined as follows. For the simple hypothesis H, the log-likelihood LIK (H) of the hypothesis H for the entire chromosome can be calculated as the sum of the log-likelihoods of the individual SNPs, assuming a known or derived child ratio cf. can. In certain embodiments, it is possible to derive cf from the data.

This hypothesis does not assume a chain between any SNPs and therefore does not utilize a joint distribution model.

と定義され、式中、ｍは可能性のある真の母親の遺伝子型であり、ｆは可能性のある真の父親の遺伝子型であり、ｍ，ｆ∈｛ＡＡ，ＡＢ，ＢＢ｝であり、ｃは、仮説Ｈを考慮した、可能性のある子の遺伝子型である。特に、モノソミーについてはｃ∈｛Ａ，Ｂ｝であり、ダイソミーについてはｃ∈｛ＡＡ，ＡＢ，ＢＢ｝であり、トリソミーについてはｃ∈｛ＡＡＡ，ＡＡＢ，ＡＢＢ，ＢＢＢ｝である。 In some embodiments, the log-likelihood can be determined for each SNP. Assuming hypothesis H and percent fetal DNA cf for fetal ploidy for a particular SNP i, the log-likelihood of the observed data D is

In the formula, m is the genotype of the possible true mother, f is the genotype of the possible true father, and m, f ∈ {AA, AB, BB}. , C are genotypes of possible offspring, taking into account Hypothesis H. In particular, c ∈ {A, B} for monosomy, c ∈ {AA, AB, BB} for disomy, and c ∈ {AAA, AAB, ABB, BBB} for trisomy.

Genotype Prior Frequency: p (m | i) is the general prior probability of the maternal genotype m in SNP i based on the known population frequency in SNP I and is represented by pAi. In particular,
p (AA | pA _i ) = (pA _i ) ² , p (AB | pA _i ) = 2 (pA _i ) * (1-pA _i ), p (BB | pA _i ) = (1-pA _i ) ²
The probability of the paternal genotype, p (f | i), can be determined in the same way.

True child probability: p (c | m, f, H) is the probability that the true child genotype = c is obtained in consideration of the parent m, f, and the hypothesis H. Can be easily calculated. For example, p (c | m, f, H) for H11, H21 match and H21 disagreement are shown below.

Data likelihood: P (D | m, f, c, H, i, cf) is the genotype m of the true mother, the genotype f of the true father, the genotype c of the true child, the hypothesis H and The probability of a given data D in SNP i taking into account the proportion cf of the offspring. It can be decomposed into probabilities of mother, father and child data as follows:
P (D | m, f, c, H, cf, i) = P (SM | m, i) P (M | m, i) P (SF | f, i) P (F | f, i) P (S | m, c, H, cf, i)

である。 Probability of maternal SNP array data: Assuming that the SNP array genotype is accurate, the probability mi of the maternal SNP array genotype data in SNP _i compared to the true genotype m is simply:

Is.

Probability of mother's sequence data: The probability of mother's sequence data in SNP _i is P (SM | m) without extra noise or bias when the count value S _i = (ami _i , bmi). , I) = binomial probability defined as PX _{| m} (am _i ), where X | _m to Binom (pm (A), am _i + _{bm i} ), pm (A). Is the value shown in the table below.

Likelihood of father's data: A similar formula applies to the likelihood of father's data. Note that it is possible to genotype the offspring without using parental data, especially paternal data. For example, if paternal genotype data F is not available, then simply P (F | f, i) = 1 can be used. If the father's sequence data SF is not available, then simply P (SF | f, i) = 1 can be used.

In some embodiments, the method comprises constructing a joint distribution model for the expected number of alleles at multiple polymorphic loci on the chromosome for each polyploid hypothesis, for such purpose. One method for achieving this is described here. Probability of free fetal DNA data: P (S | m, c, H, cf, i) is the probability of free fetal DNA sequence data in SNP i, true maternal genotype m, true offspring. Considering the genotype c of the gene and the copy number hypothesis H of the offspring, the proportion cf of the offspring is assumed. This is, in fact, the probability P (S | m, c, H, cf, i) of the sequence data S in SNP I considering the true probability μ (m, c, cf, H) of the A content in SNP i = P (S | μ (m, c, cf, H), i).
With respect to the count value, if S _i = ( _ai , bi ₎ and the data is not accompanied by extra noise or bias.
P (S | μ (m, c, cf, H), i) = P _x ( _ai )
In the equation, X to Binomi (p (A), _ai + bi), and p (A) ₌ μ (m, c, cf, H). If the exact alignment and (A, B) counts per SNP are unknown and more complex, then P (S | μ (m, c, cf, H), i) is a combination of integrated binomials. Is.

と定義され、式中、＃Ａ（ｇ）＝遺伝子型ｇにおけるＡの数であり、ｎ_ｍ＝２は、母親のソミーであり、ｎ_ｃは仮説Ｈの下での子の倍数性である（１はモノソミーであり、２はダイソミーであり、３はトリソミーである）。 Probability of true A content: The true probability μ (m, c, cf, H) of A content in SNP i in this mother / child mixture is true mother genotype = m, true child genotype = Assuming c, and the overall proportion of children = cf,

In the formula, # A (g) = the number of A in genotype g, nm = 2 is the mother's trisomy, and _{n c} is the polyploidy of the offspring under hypothesis H. (1 is monosomy, 2 is disomy, and 3 is trisomy).

Use of joint distribution model: LIK (D | H) for compound hypothesis
In some embodiments, this method involves constructing a joint distribution model of the predicted allele numbers at multiple polymorphic loci on the chromosome for each ploidy hypothesis, with such objectives. One method for achieving this is described here. In many cases, trisomy is not purely a match or mismatch, usually due to a crossover, so in this section, a composite of matched and mismatched trisomy, taking into account possible crossovers. Results are derived for the hypotheses H21 (maternal trisomy) and H12 (paternal trisomy).

In the case of trisomy, if there is no crossover, the trisomy is simply a matching trisomy or a disagreeing trisomy. Trisomy coincidence is when the offspring inherits two copies of the same chromosomal segment from one parent. Mismatch trisomy is when the offspring inherits one copy of each homologous chromosomal segment from the parent. By crossover, some segments of the chromosome may have a matching trisomy and other parts may have a mismatched trisomy. This section describes how to build a joint distribution model of the predicted number of alleles at multiple loci for a set of alleles, for heterozygotes, that is, for one or more hypotheses. Are listed.

式中、ＬＩＫ（Ｄ｜Ｅ，１：Ｎ）は、ＳＮＰ １：Ｎについての仮説Ｅの最終尤度である。Ｅ＝最後のＳＮＰの仮説であり、Ｅ∈（Ｈｍ，Ｈｕ）である。再帰的に、以下を算出することができる：

式中、～ＥはＥ以外の仮説（非Ｅ）であり、考慮される仮説はＨ_ｍおよびＨ_ｕである。詳細には、１：ｉ ＳＮＰの尤度を、１から（ｉ－１）までのＳＮＰの尤度に基づいて、同じ仮説で乗換えなしか、または逆の仮説で乗換えありのいずれかを用い、ＳＮＰ ｉの尤度を乗じて算出することができる：
ＳＮＰ １に対し、ｉ＝１、ＬＩＫ（Ｄ｜Ｅ，１：１）＝ＬＩＫ（Ｄ｜Ｅ，１）、
ＳＮＰ ２に対し、ｉ＝２、ＬＩＫ（Ｄ｜Ｅ，１：２）＝ＬＩＫ（Ｄ｜Ｅ，２）＋ｌｏｇ（ｅｘｐ（ＬＩＫ（Ｄ｜Ｅ，１））＊（１－ｐｃ（２））＋ｅｘｐ（ＬＩＫ（Ｄ｜～Ｅ，１））＊ｐｃ（２））、
また、ｉ＝３：Ｎについても同様である。 For SNP i, LIK (D | Hm, i) is a fit for the concordance hypothesis H _m , _LIK (D | Hu, i) is a fit for the disagreement hypothesis Hu, pc (i) = SNP i. It is assumed that it is the probability of crossover between -1 and SNP i. At this time, the complete likelihood can be calculated as follows:

In the equation, LIK (D | E, 1: N) is the final likelihood of hypothesis E for SNP 1: N. E = the last SNP hypothesis, E ∈ (Hm, Hu). Recursively, the following can be calculated:

In the equation, ~ E is a hypothesis other than E (non-E), and the hypotheses considered are H _m and _Hu . Specifically, the likelihood of 1: i SNP is crossed based on the likelihood of SNPs from 1 to (i-1), either with or without crossover with the same hypothesis or with crossover with the opposite hypothesis. Can be calculated by multiplying the likelihood of SNP i:
For SNP 1, i = 1, LIK (D | E, 1: 1) = LIK (D | E, 1),
For SNP 2, i = 2, LIK (D | E, 1: 2) = LIK (D | E, 2) + log (exp (LIK (D | E, 1)) * (1-pc (2)) + Exp (LIK (D | ~ E, 1)) * pc (2)),
The same applies to i = 3: N.

In some embodiments, the fraction of children can be determined. The proportion of offspring may refer to the proportion of sequences originating from offspring in a mixture of DNA. In the context of non-invasive prenatal testing, the proportion of offspring may refer to the proportion of sequences originating from the fetal or part of the placenta with the fetal genotype in the maternal plasma. The proportion of offspring may refer to a fraction of offspring in a sample of DNA prepared from maternal plasma and may be enriched with respect to fetal DNA. One purpose of determining the proportion of offspring in a sample of DNA is for use in algorithms that can make multiple calls with respect to the fetus, and therefore the proportion of offspring is a non-invasive prenatal diagnosis. It may refer to a sample of any DNA analyzed for sequencing.

Some of the algorithms presented in this disclosure, which are part of a non-invasive method of prenatal aneuploidy diagnosis, assume a known proportion of offspring, but this is not always the case. .. In certain embodiments, it is possible to find the most likely proportion of offspring by maximizing the likelihood of disomy on selected chromosomes, with or without the presence of parental data. be.

である。
最も可能性が高い子の割合（ｃｆ^†＊）は、ｃｆ^＊＝ａｒｇｍａｘ_ｃｆＬＩＫ（ｃｆ）として導かれる。 Specifically, for the disomy hypothesis and for the proportion cf of the offspring on the chromosome chr, we assume LIK (D | H11, cf, chr) = log-likelihood as described above. The complete likelihood for the chromosomes selected in Cset (usually 1:16), assumed to be positive polyploidy:

Is.
The most likely proportion of offspring (cf ^{† *} ) is derived as cf ^* = argmax _cf LIK (cf).

It is possible to use any set of chromosomes. It is also possible to derive the proportion of offspring without assuming positive ploidy in the reference chromosome. Using this method, it is possible to determine the proportion of offspring for any of the following situations: (1) have array data for the parent and shotgun sequencing data for the maternal plasma; (2) for the parent. Has array data and targeted sequencing data for maternal plasma; (3) has targeted sequencing data for both parental and maternal plasma; (4) targeted sequences for both maternal and maternal plasma fractions. Has decision data; (5) has targeted sequence determination data for the maternal plasma fraction; (6) other combinations of parental and child fraction measurements.

In some embodiments, data dropouts can be incorporated by informatics methods. This makes it possible to obtain a ploidy determination result with higher accuracy. Elsewhere in the disclosure, the probability of A occurring is assumed to be a linear function of the true mother's genotype, the true offspring genotype, the proportion of offspring in the mixture, and the number of copies of offspring. .. For example, instead of measuring the true offspring AB in the mixture, the mother or offspring allele may drop out, which can occur if only the sequence mapped to the allele A is measured. The parent dropout rate for genomic illumination data can be indicated by _dpg , the parental dropout rate for sequence data can be indicated by _dps , and the child dropout rate for sequence data can be indicated by _dcs . In some embodiments, it can be assumed that the mother's dropout rate is 0 and the child's dropout rate is relatively low, in which case the results are not significantly affected by the dropout. In some embodiments, the potential for allelic dropouts may be large enough to have a significant effect on the predicted ploidy call. In such cases, incorporate the allele dropout into the algorithm here:

で、式中、上記と同様に、

であり、Ｐ（ｍ_ｄ｜ｍ）は、ドロップアウト率ｄについて以下の通り定義される真の遺伝子型ｍを考慮した可能性のあるドロップアウト後の遺伝子型ｍ_ｄの尤度である

同様の式が父親のＳＮＰアレイデータにも当てはまる。 Parental SNP array data dropout: For the mother's genome data M, let the genotype after the dropout be _md .

So, in the formula, in the same way as above

P (md | m) is the likelihood of post-dropout genotype _md that may take into account the true genotype m defined below for the dropout rate _d .

A similar formula applies to the father's SNP array data.

式中、Ｐ（ｍ_ｄ｜ｍ）は前のセクションで定義された通りであり、二項分布からのＰ_ｘ｜ｍｄ（ａｍ_ｉ）確率は、親のデータの尤度セクションにおいて上記の通り定義される。同様の式が父系の配列データにも適用できる。 Parent sequence data dropout: For the mother sequence data SM

In the equation, P ( _md | m) is as defined in the previous section, and the P _{x | md} ( _ami ) probability from the binomial distribution is defined above in the likelihood section of the parent data. Will be done. Similar equations can be applied to patrilineal sequence data.

式中、Ｐ（Ｓ｜μ（ｍ_ｄ，ｃ_ｄ，ｃｆ，Ｈ），ｉ）は浮動性のデータの尤度に関するセクションにおいて定義されている通りである。 Floating DNA Sequence Data Dropout:

In the equation, P (S | μ ( _md , _cd , cf, H), i) is as defined in the section on the likelihood of floating data.

である。 In one embodiment, p (md | m) is the probability of the observed maternal genotype _md , taking into account the true maternal genotype m and assuming a dropout rate _{d ps} , p ( c _d | c) is the probability of the observed offspring genotype cd, taking into account the true offspring genotype c and assuming a dropout rate _{d cs} . nA _T = the number of alleles A in the true genotype _c , nA _D = the number of alleles A in the observed genotype cd, nA _T ≥ nA _D , and similarly _nBT =. Suppose that the number of alleles B in the true genotype c, nB _D = the number of alleles B in the observed genotype c _d , _nBT ≥ _nBD , and d = dropout rate. ,

Is.

In some embodiments, the informatics method may incorporate random and consistent biases. Ideally, there is no consistent sampling bias or random noise (in addition to binomial distribution variation) per SNP in the sequence counts. Specifically, in SNP i, X is for the genotype m of the mother, the genotype c of the true offspring and the proportion cf of the offspring, and the number of A in the set of (A + B) reads at X = SNP i. Acting as X to Binomial (p, A + B), p = μ (m, c, cf, H) = true probability of A content.

である。 In some embodiments, the informatics method may incorporate random bias. In most cases, we assume that the measurements are biased, so the probability of an A occurring in this SNP is equal to q, which is slightly different from p as defined above. How different p and q are depends on the accuracy of the measurement process and the number of other factors and can be quantified by the standard deviation of q away from p. In certain embodiments, q can be modeled with parameters α, β, and some specific standard deviations s, depending on the average of the distributions concentrated on p as having a beta distribution. Specifically, this results in X | q-Bin (q, _Di ), q-Beta (α, β). If E (q) = p and V (q) = s ² , the parameters α and β can be derived as α = pN and β = (1-p) N.

Is.

This is the definition of a beta-binomial distribution, sampled from a binomial distribution with the parameter q of variability, where q follows a beta distribution with mean p. Therefore, in an unbiased setup, in SNP i, the true mother genotype (m) is assumed, and the mother's sequence A count value (am _i ) on SNP i and the sequence B count value of the mother on SNP i. The parental sequence data (SM) probabilities considering (bm _i ) can be calculated as follows: P (SM | m, i) = PX _{| m} (am _i ), where X | m ~. _Binom (pm (A), am _i + bm _i ).

Here, including a random bias with standard deviation s:
X | _m to BetaBinom (pm (A), am _i + bm _i , s).

If there is no bias, the true mother genotype (m), true child genotype (c), and child proportion (cf) are assumed, the child hypothesis H is assumed, and floating on SNP i. The maternal plasma DNA sequence data (S) probability considering the sex DNA sequence A count value (ai) and the floating sequence B count value (bi) on SNP i can be calculated as follows:
P (S | m, c, cf, H, i) = Px ( _ai )
In the formula, X to _Binom (p (A), _ai + bi), p (A) = μ (m, c, cf, H).

を用いて推定することが可能である。データの挙動に応じて、Ｎを、リード深度ａ_ｉ＋ｂ_ｉまたはａ_ｉ＋ｂ_ｉの関数に関係なく一定になるようにし、これにより、より大きなリード深度に対して偏りをより小さくすることができる。 In one embodiment, including a random bias with standard deviation s, this is X- _BetaBiom (p (A), _ai + bi, s) and the amount of extra variation is the deviation parameter s or equivalent. Specified by N. The smaller the value of s (or the larger the value of N), the closer this distribution is to the standard binomial distribution. Estimating the amount of bias, i.e., estimating the above N from the obvious situations AA | AA, BB | BB, AA | BB, BB | AA:

It is possible to estimate using. Depending on the behavior of the data, N can be constant regardless of the function of read depth _ai + _bi or _ai + _bi , which can result in less bias for larger read depths. ..

In certain embodiments, the informatics method may incorporate a consistent per-SNP bias. Due to the artifacts in the sequencing process, some SNPs may have consistently low or high counts, regardless of their true A content. It is assumed that SNP _i consistently adds a wi percent bias to the A count value. In some embodiments, this bias can be estimated from a set of training data derived under the same conditions and returned to the parent sequence data estimate as follows:
P (SM | m, i) = PX _{| m} (am _i ) <Here, X | _m to BetaBinom (pm (A) + wi, am _i + bm _i , s ₎ >,
Also, the floating DNA sequence data probability estimate is:
P (S | m, c, cf, H, _i ) = _PX ( _ai ) <Here, X to _BetaBinom (p (A) + wi, _ai + bi, s)>
Will be.

In some embodiments, the method can be written specifically taking into account yet another noise, differential sample quality, differential SNP quality, and random sampling bias. An example of this is shown here. This method has been shown to be particularly useful in situations where data was generated using a extensively multiplexed mini-PCR protocol, which was used in Experiments 7-13. Each of these methods involves several steps to introduce different types of noise and / or bias into the final model:
(1) The first sample containing a mixture of maternal DNA and fetal DNA contains size = _N0 molecules, usually in the range 1,000-40,000, p = amount of original DNA with true% ref. Then assume.
(2) In amplification using a universal ligation adapter, it is assumed that N1 molecules are sampled; usually, N1 to _{N0 / 2} molecules and a random sampling bias are introduced due to sampling. The amplified sample may contain a number of molecules N ₂ and is N ₂ >> N ₁ . X ₁ indicates the amount of reference loci (per SNP) in the N ₁ sampled molecule, with a variation of p ₁ = X ₁ / N ₁ that introduces a random sampling bias throughout the rest of the protocol. .. This sampling bias is included in the model by using a beta binomial (BB) distribution instead of using a simple binomial distribution model. The beta-binomial distribution parameter N can later be estimated from the training data after adjusting for leakage and amplification bias for SNPs of 0 <p <1 for each sample. Leaks tend to cause SNPs to be read inaccurately.
(3) The amplification step amplifies any allele bias and thus introduces an amplification bias with possible non-uniform amplification. Assuming that one allele at the locus is amplified f-fold and the other allele at that locus g-fold, then f = ge ^b and b = 0 indicates that there is no bias. .. The bias parameter b concentrates on 0 and indicates how much or less allele A is amplified in contrast to allele B in a particular SNP. The parameter b may differ depending on the SNP. The bias parameter b can be estimated for each SNP, for example, from training data.
(4) The sequencing step includes the step of sequencing the sample of the amplified molecule. In this step, a leak may be present, which is a situation in which the SNP is read incorrectly. The leak can be due to a variety of problems, and as a result of the leak, the SNP is not read as the exact allele A, but as another allele B found at that locus, or generally the gene. Read as an allele C or D not found in the locus. It is assumed that the sequencing will measure the sequence data of several DNA molecules from the amplified sample of size N ₃ , N ₃ <N ₂ . In some embodiments, N3 is _{20,000-100,000} ; 100,000-500,000; 500,000-4,000,000; 4,000,000-20,000,000; or It may be in the range of 20,000,000 to 100,000,000. Each sampled molecule has an accurately read probability _pg , in which case it is exactly shown as the allele A. The sample is inaccurately read as an allele unrelated to the original molecule with a probability of 1-pg, like an allele A with a probability _pr , like an allele B with a probability pm, or a probability _po . It seems to be allele C or allele D, and _pr + _pm + _po = 1. The parameters p _g , _pr , p _m , and _po are estimated from the training data for each SNP.

Different protocols may include similar steps and may be accompanied by variations in molecular biology steps, resulting in different amounts of random sampling, different levels of amplification and bias due to different leaks. The following models can be applied equally well to each of these cases. A model of the amount of sampled DNA is shown for each SNP by:
X ₃ to Beta Binomial (L (F (p, b), _pr , _pg ), N * H (p, b))
In the equation, p = true amount of reference DNA, b = bias per SNP, as described above, _pg is the probability of an accurate read, and _pr is, as described above, of bad reads. If it was read incorrectly, but by chance it is the probability of a read that appears to be the correct allele:
F (p, b) = pe ^b / (pe ^b + (1-p)), H (p, ^b ) = (e bp + (1-p)) ² / e ^b , L (p, _pr , p _g ) = p * p _g + _pr * (1- _pg ).

In some embodiments, the method uses a beta-binomial distribution instead of a simple binomial distribution. This addresses the random sampling bias. The parameter N of the beta-binomial distribution is estimated for each sample as needed. The bias correction F (p, b), H (p, b) is used instead of simply p to deal with the amplification bias. The bias parameter b is estimated in advance from the training data for each SNP.

In some embodiments, the method simply uses a leak correction L (p, _pr , _pg ) instead of p, which addresses the bias due to leakage, i.e., variation in SNP and sample quality. In some embodiments, the parameters _pg , _pr , _po are pre-estimated from the training data for each SNP. In some embodiments, the parameters _{pg, pr , po} can be updated with the current sample being performed to reveal variations in sample quality.

The models described herein are fairly general and can reveal both differential sample quality and differential SNP quality. Different samples and SNPs are treated differently, as illustrated by the fact that in some embodiments the mean and dispersion use the quantity of the original DNA, as well as the beta-binomial distribution, which is a function of the quality of the samples and SNPs. Will be done.

Platform Modeling Take into account a single SNP (based on maternal and fetal genotypes) where the ratio of predicted alleles present in plasma is r. The predicted allele ratio is defined as the predicted proportion of allele A in the combination of maternal and fetal DNA. For the genotype g _m of the maternal line and the genotype g _c of the offspring, the predicted allelic ratio is given by Equation 1, assuming that the genotype is also indicated by the allele ratio.
r = fg _c + (1-f) g _m (1)

Observations in the SNP consist of the number of reads, _{na and n b mapped} in each of the alleles present, summing up to the read depth d. It is assumed that the thresholds have already been applied to the mapping probabilities and phred scores and therefore mapping and allele observations can be considered accurate. The phred score is a numerical measure of the probability that a particular measurement at a particular base is erroneous. In one embodiment, when the base is measured by sequencing, the phred score can be calculated from the ratio of the intensity of the dye corresponding to the called base to the intensity of the dye of the other base. The simplest model for observing likelihood is a binomial distribution, assuming that each of the d-reads is independently extracted from a large pool with the allele ratio r. Equation 2 describes this model.

を用いて、予想外の対立遺伝子を少数にすることが可能である。ある実施形態では、トレーニングデータを用いて、各ＳＮＰ上に現れる予想外の対立遺伝子の比率をモデリングすること、およびこのモデルを使用して予測される対立遺伝子の比を補正することが可能である。予測される対立遺伝子の比が０または１ではない場合、観察された対立遺伝子の比は、増幅の偏りまたは他の現象に起因して、予測される対立遺伝子の比に十分に高いリード深度に収束し得ない。次いで、対立遺伝子の比を、予測される対立遺伝子の比に集中したベータ分布としてモデリングし、二項分布よりも分散が大きいＰ（ｎ_ａ，ｎ_ｂ｜ｒ）についてのベータ二項分布を得ることができる。 The binomial model can be extended in a number of ways. If the maternal and fetal genotypes are either all A or all B, the expected allele ratio in plasma will be 0 or 1, and the binomial probability will be well defined. Not done. In practice, sometimes unexpected alleles are observed in practice. In one embodiment, the corrected allele ratio

Can be used to reduce the number of unexpected alleles to a small number. In certain embodiments, training data can be used to model the proportion of unexpected alleles appearing on each SNP, and the model can be used to correct the predicted allele proportions. .. If the predicted allele ratio is not 0 or 1, the observed allele ratio will have a sufficiently high read depth to the predicted allele ratio due to amplification bias or other phenomena. It cannot converge. The allele ratio is then modeled as a beta distribution concentrated on the predicted allele ratio to obtain a beta-binomial distribution for P ( _{na, n b |} r), which has a greater variance than the binomial distribution. be able to.

A platform model for a response in a single SNP is defined as F (a, b, g _c , g _m , f) (3), or the observed probability of n _a = a and n _b = b. Also depends on the fetal fraction according to Equation 1, considering the genotype of the maternal line and the genotype of the fetus. The function form of F may be a binomial distribution, a beta-binomial distribution or a function similar to the above.
F (a, b, g _c , g _m , f) = P ( _{na = a, n b =} b | g _c , g _m , f) = P ( _{na = a, n b =} b | r ( g _c , g _m , f)) (3)

In certain embodiments, the proportion of offspring can be determined as follows. Maximum likelihood estimates of the fetal fraction f for prenatal testing can be derived without the use of paternal information. This can be relevant if paternal genetic data is not available, for example if the father of the record is not really the genetic father of the fetus. The fetal fraction is estimated from the set of SNPs when the maternal genotype is 0 or 1, resulting in a set of only two possible fetal genotypes. S ₀ is defined as a set of SNPs having a genotype of 0 in the maternal line, and S ₁ is defined as a set of SNPs having a genotype of 1 in the maternal line. The possible fetal genotypes at S ₀ are 0 and 0.5, resulting in a set of possible allele ratios R ₀ (f) = {0, f / 2}. Similarly, R ₁ (f) = {1-f / 2,1}. This method can be trivially extended to include SNPs with a maternal genotype of 0.5, but these SNPs are informative due to the larger set of possible allele ratios. Low.

は式４によって定義される。

N _a0 and N _b0 are defined as vectors formed by na _as and n _bs for the SNP in S ₀ , and N a ₁ and N _{b 1} are similarly defined for S ₁ . Maximum likelihood estimation of f

Is defined by Equation 4.

Assuming that the number of alleles in each SNP was conditioned independently of the ratio of plasma alleles in the SNP, the probabilities can be expressed as a product for the SNPs within each set (5).

Dependence on f depends on the set of possible allele ratios R ₀ (f) and R ₁ (f). The SNP probability P ( _nas , n _bs | f) can be estimated by assuming the maximum likelihood genotype conditioned on f. Selection of maximum likelihood genotypes at reasonably high fetal fractions and read depths is reliable. For example, consider an SNP in which the mother has zero genotype at a fetal fraction of 10 percent and a read depth of 1000. The expected allele ratios are 0 percent and 5 percent, which are easily distinguishable at sufficiently high read depths. Substituting the estimated genotype of the offspring into Equation 5 gives the complete equation (6) for estimating the fetal fraction.

The fetal fraction must be in the range [0,1], and therefore optimization can be easily performed by conditional one-dimensional retrieval.

In the case of low read depths or in the presence of high noise levels, it may be preferable not to assume maximum likelihood genotypes that can unnaturally result in high reliability. Alternatively, they are summed across the possible genotypes in each SNP, resulting in the following equation (7) for P (na, n _b | f) for the _SNP at _S0 . Prior probabilities P (r) can be assumed to be uniform across R ₀ (f) or may be based on population frequency. _The expansion into group S1 is self-evident.

In some embodiments, the probabilities can be derived as follows. The reliability can be calculated from the data likelihoods of the two hypotheses H _t and H _f . The likelihood of each hypothesis is derived based on the response model, estimated fetal fraction, maternal genotype, population frequency of alleles, and number of alleles.
Define the following notation:
G _m , G _c true maternal genotype and offspring genotype G _af , G _tf true genotype of the alleged father and true genotype of the true father G (g _c , g _m , g _tt f) ) = P (G _c = g _c | G _m = g _m , G _ft = g _ft ) Genetic trait probability P (g) = P (G _tt f = g) Population frequency of genotype g in a particular SNP

Ｈ_ｔの場合には、父親とされる人は真の父親であり、胎児の遺伝子型は、式９に従って母系の遺伝子型および父親とされる人の遺伝子型から遺伝によって受け継がれる。

The likelihood of the paternal hypothesis is the product of the likelihoods in the SNP, assuming that the observations at each SNP are conditioned independently of the ratio of plasma alleles. The following equation derives the likelihood for a single SNP. Equation 8 is a general expression for the likelihood of any hypothesis h, which is then decomposed in certain cases of H _t and H _f .

In the case of _Ht , the alleged father is the true father, and the genotype of the fetal is inherited from the genotype of the maternal line and the genotype of the alleged father according to Equation 9.

In the case of _Hf , the alleged father is not a true father. The best estimates of true paternal genotype depend on population frequency at each SNP. Therefore, the probability of offspring genotype is determined by the known maternal genotype and population frequency, as in Equation 10.

The confidence C _p for the exact patrilineality is calculated from the product of the two likelihood SNPs using Bayes' law (11).

Most likely model using the fetal fraction by percentage Fetal polyploid state by measuring the floating DNA contained in the serum of the maternal line or by measuring the genotypic material in any mixed sample. Determining is a non-trivial task. There are several ways, for example, the speculation is that if the fetus is trisomy on a particular chromosome, the total amount of DNA from that chromosome found in the blood of the maternal line will increase relative to the reference chromosome. There is a method of performing the read number analysis. One method for detecting trisomy in such embryos is to determine the predicted amount of DNA for each chromosome, eg, according to the number of SNPs in the analysis set corresponding to a given chromosome, or independently of the chromosome. Normalize according to the number of mapable parts. Once the measurements are normalized, any chromosome measured for the amount of DNA above a particular threshold is determined to be trisomy. This technique is described in Fan et al. PNAS, 2008; Vol. 105 (No. 42); pp. 16266-16271, and also in Chiu et al., BMJ 2011; Vol. 342: pp. 7401. In Chiu et al.'S paper, normalization was achieved by calculating the Z-score as follows:
Z-score for the percentage of chromosome 21 in the test example = ((percentage of chromosome 21 in the test example)-(average of the percentage of chromosome 21 in the reference control)) / (standard of the percentage of chromosome 21 in the reference control) deviation).
These methods use a single hypothesis rejection method to determine the ploidy state of the fetus. However, they suffer some significant drawbacks. Since these methods for determining ploidy in the fetus are invariant according to the percentage of fetal DNA in the sample, one cutoff value is used, as a result, the accuracy of the determination is not optimal and in the mixture. If the percentage of fetal DNA is relatively low, the accuracy is the worst.

In certain embodiments, the methods of the present disclosure used to determine the ploidy state of the fetus include the steps of taking into account the proportion of fetal DNA in the sample. In another embodiment of the present disclosure, the method comprises the use of maximum likelihood estimation. In certain embodiments, the methods of the present disclosure include the step of calculating the percentage of DNA of fetal or placental origin in a sample. In certain embodiments, the threshold for calling aneuploidy is adaptively adjusted based on the calculated percent fetal DNA. In some embodiments, a method for estimating the percentage of DNA that is of fetal origin in a mixture of DNA is a step of obtaining a mixed sample containing a maternally-derived genetic material, and a fetal-derived genetic material. A step to obtain a gene sample derived from a fetal father, a step to measure DNA in a mixed sample, a step to measure DNA in a father's sample, a DNA measurement value of a mixed sample, and a DNA measurement value of a father's sample. Includes the step of calculating the percentage of DNA of fetal origin in the mixed sample using.

In certain embodiments of the present disclosure, the proportion of fetal DNA or the percentage of fetal DNA in the mixture can be measured. In some embodiments, the proportion can be calculated using only the genotyping measurements made on the maternal plasma sample itself, which is a mixture of fetal DNA and maternal DNA. In some embodiments, the proportion is calculated using the genotype of the mother measured or otherwise known and / or the genotype of the father measured or otherwise known. You can also do it. In some embodiments, measurements obtained for a mixture of maternal DNA and fetal DNA can be used in conjunction with parental situational insights to calculate percent fetal DNA. In certain embodiments, population frequency can be used to calculate the proportion of fetal DNA in order to adjust the model for probabilities for specific allelic measurements.

In certain embodiments of the present disclosure, confidence can be calculated for the accuracy of determining the ploidy state of the fetus. In certain embodiments, the reliability of the hypothesis of maximum likelihood (H _major ) can be calculated as (1-H _major ) / Σ (all H). If the entire distribution of the hypothesis is known, it is possible to determine the reliability of the hypothesis. If the parental genotype information is known, it is possible to determine the entire distribution of the hypothesis. Given the knowledge of the predicted data distribution for positive ploidy fetuses and the predicted data distribution for aneuploidic fetuses, it is possible to calculate the reliability of the ploidy determination. .. If parental genotype data is known, it is possible to calculate these predicted distributions. In one embodiment, the knowledge of the distribution of test statistic around the normal hypothesis and the distribution of the test statistic around the abnormal hypothesis is used to determine the reliability of the call, as well as to improve the threshold. You can make reliable calls. This is especially useful when the amount and / or percentage of fetal DNA in the mixture is low. This is actually aneuploidy because test statistic such as Z statistic does not exceed the threshold set based on the optimized threshold for the presence of high percentage of fetal DNA. Helps avoid situations where the fetus is found to be positive polyploid.

In certain embodiments, the methods disclosed herein are used to determine the number of copies of the maternal and fetal target chromosomes in a mixture of maternal and fetal genetic material. The aneuploidy of can be determined. This method may involve the step of obtaining maternal tissue containing both maternal and fetal genetic material, and in some embodiments, the maternal tissue is isolated from maternal blood. May be plasma or tissue. This method may also involve the step of obtaining a mixture of maternal and fetal genetic material from the maternal tissue by processing the maternal tissue described above. This method distributes the resulting genetic material to multiple reaction samples in order to randomly yield individual reaction samples containing the target sequence derived from the target chromosome and individual reaction samples not containing the target sequence derived from the target chromosome. A step of performing high throughput sequencing on a sample may also be involved. This method analyzes the target sequences of genetic material present or absent in the individual reaction samples to indicate the presence or absence of fetal chromosomes that are presumed to be positive polyploid in the reaction samples. It may involve a first number of results, and a second number of binary results indicating the presence or absence of fetal chromosomes that may be aneuploid in the reaction sample. For example, any of the number of binary results can be calculated by an informatics technique that counts sequence reads that map to a particular chromosome, a particular region of a chromosome, a particular locus or a set of loci. This method may include the step of normalizing the number of binary events based on the length of the chromosome, the length of the region of the chromosome or the number of loci within the population. This method may involve calculating the predicted distribution of the number of binary results using the first number for the fetal chromosomes that are presumed to be positive polyploid in the reaction sample. This method is a step in calculating the predicted distribution of the number of binary results for a fetal chromosome that is presumed to be aneuploid in the reaction sample, and was found in the first number, and in the mixture. The estimated proportion of fetal DNA is, for example, (1 + n / 2) (n is the estimated fetal picture) in the predicted read number distribution of the number of binary results for the chromosomes of the fetus that are presumed to be positive polyploid. May be accompanied by a step of calculation using by multiplying (in minutes). In some embodiments, sequence reads can be processed with probabilistic mapping rather than binary results, which provides higher accuracy but requires more computational power. The fetal fraction can be estimated by multiple methods, some of which are described elsewhere in the disclosure. This method uses maximum likelihood techniques to determine if the second number corresponds to a fetal chromosome that is or may be aneuploid, which is positive polyploid or aneuploid. May be accompanied by. This method may include the step of calling the ploidy state of the fetus, which is the ploidy state corresponding to the hypothesis that is accurate and has the highest likelihood, taking into account the measured data.

Note that the maximum likelihood model can be used to increase the accuracy of any method of determining the ploidy state of the fetus. Similarly, confidence can be calculated for any method of determining the ploidy state of the fetus. Using the maximum likelihood model improves the accuracy of any method of making ploidy determinations using the single hypothesis rejection method. The maximum likelihood model can be used for any method that can calculate the likelihood distribution for both normal and abnormal cases. Using the maximum likelihood model means the ability to calculate confidence for haploid calls.

Further Discussion of Methods In certain embodiments, the methods disclosed herein utilize a quantitative measure of the number of independent observations of each allele at a polymorphic locus, wherein this is an allele. The step of calculating the ratio of is not included. This provides information about the ratio of the two alleles at the locus, but differs from methods such as some microarray-based methods that do not quantify the number of independent observations of any allele. While some methods known in the art can provide quantitative information about the number of independent observations, only allele ratios are used in the calculations that result in the determination of ploidy, and the quantitative information is available. Do not use. To illustrate the importance of retaining information about the number of independent observations, consider the loci of a sample with two alleles, A and B. In the first experiment, 20 alleles A and 20 alleles B are observed, and in the second experiment, 200 alleles A and 200 alleles B are observed. In both experiments, the ratio (A / (A + B)) is equal to 0.5, but the second experiment conveys more information about the certainty of the frequency of the allele A or B than the first experiment. Instead of utilizing allele ratios, the method uses quantitative data to more accurately model the most likely allele frequency at each polymorphic locus.

In one embodiment, the method constructs a genetic model for summing up measurements from multiple polymorphic loci to better distinguish between trisomy and disomy and also determine the type of trisomy. In addition, the method incorporates genetic linkage information to enhance the accuracy of the method. This is in contrast to some methods known in the art for averaging allele ratios across all polymorphic loci on the chromosome. By the methods disclosed herein, the predicted allogeneic frequency distribution in dysomy, as well as nondisjunction of chromosomes during meiosis I, nondisjunction of chromosomes during meiosis II, and early prevalence of fetal development. The trisomy caused by chromosomal nondisjunction during mitosis is clearly modeled. To illustrate why this is important, without crossover, chromosomal nondisjunction between meiosis I results in a trisomy in which two different homologues are inherited from one parent, resulting in meiosis II. Chromosome meiosis during, or during early mitosis of fetal development, results in two copies of the same homologue from one parent. Each scenario results in different frequencies of alleles predicted at each polymorphic locus and at all physically linked loci considered together (ie, loci on the same chromosome). .. Crossovers that result in the exchange of genetic material between homologues complicate the mode of inheritance, but the method uses genetic linkage information, ie, recombination rate information and the physical distance between loci. Adapt to this. To better distinguish between nondisjunction during meiosis I and chromosomal nondisjunction during meiosis II or mitosis, the method incorporates an increased probability of crossover as an increase in distance from the centromere. In meiosis II and mitotic nondisjunction, mitotic nondisjunction generally results in the same or nearly identical copy of one homologue, while at meiosis II. The two homologues that exist after a nondisjunction event can often be distinguished by the fact that they differ due to one or more crossovers during mitosis.

In certain embodiments, the methods of the present disclosure are unable to determine the parental haplotype when assuming disomy. In one embodiment, in the case of trisomy, the method causes the plasma to take two copies from one parent, and the parental phase information is determined by either of the two copies inherited from the parent in question. By using the fact that one cannot make a decision regarding the haplotype of one parent or parents. In particular, a child can inherit either two identical copies of a parent (matched trisomy) or both copies of a parent (mismatched trisomy). At each SNP, the likelihood of matching trisomy and the likelihood of disagreeing trisomy can be calculated. The polyploid call method, which does not use a chain model that considers crossover, calculates the overall likelihood of trisomy as a simple weighted average of matched and unmatched trisomy across chromosomes. However, trisomy can change from coincident to disagreeable on the chromosome due to separation errors and the biological mechanisms that result in crossovers only in the presence of crossovers (and vice versa). The method stochastically takes into account the likelihood of crossover, resulting in a polyploid call that is more accurate than the method that does not take into account the likelihood of crossover.

In certain embodiments, reference chromosomes are used to determine the proportion of offspring and the amount or probability distribution of noise levels. In one embodiment, the proportion of offspring, noise level, and / or probability distribution are determined using only genetic information available from the chromosome where the polyploidy state is determined. The method works without reference chromosomes and without fixation of specific offspring proportions or noise levels. This is a significant improvement and difference from the methods known in the art that require genetic data from the reference chromosome to calibrate the proportion of offspring and the behavior of the chromosome.

参照染色体を用いるアルゴリズムでは、一般には、参照染色体はダイソミーであると仮定し、次いで、（ａ）最も可能性が高い子の割合およびランダムなノイズレベルＮを、この仮定および参照染色体のデータに基づいて固定し：

とし、次いで、ＬＩＫ（Ｄ｜Ｈ）＝ＬＩＫ（Ｄ｜Ｈ，ｃｆｒ^＊，Ｎ^＊）として換算するか、または、
（ｂ）この仮定および参照染色体のデータに基づいて、子の割合およびノイズレベルの分布を推定する。詳細には、ｃｆｒおよびＮについてただ１つの値に固定しないが、確率ｐ（ｃｆｒ，Ｎ）を可能性のあるｃｆｒ、Ｎ値の広い範囲に割り当てる：
ｐ（ｃｆｒ，Ｎ）～ＬＩＫ（Ｄ（ｒｅｆ．ｃｈｒｏｍ）｜Ｈ１１，ｃｆｒ，Ｎ）＊ｐｒｉｏｒｐｒｏｂ（ｃｆｒ，Ｎ）
式中、ｐｒｉｏｒｐｒｏｂ（ｃｆｒ，Ｎ）が特定の子の割合およびノイズレベルの事前確率であり、以前の知見および実験によって決定される。場合によっては、ｃｆｒ，Ｎの範囲にわたって一様にする。次いで、

と書くことができる。上記のどちらの方法も良好な結果をもたらす。 In one embodiment that does not require a reference chromosome to determine the fetal fraction, the hypothesis determination is made as follows:

Algorithms using reference chromosomes generally assume that the reference chromosome is disomy, and then (a) the proportion of the most likely offspring and a random noise level N are based on this assumption and the data on the reference chromosome. Fixed:

Then, convert as LIK (D | H) = LIK (D | H, cfr ^* , N ^* ), or
(B) Based on this assumption and the data of the reference chromosome, the proportion of offspring and the distribution of noise level are estimated. Specifically, we do not fix to a single value for cfr and N, but assign the probabilities p (cfr, N) to a wide range of possible cfr, N values:
p (cfr, N) to LIK (D (ref.chrom) | H11, cfr, N) * priorprob (cfr, N)
In the equation, priorprob (cfr, N) is the prior probability of a particular child proportion and noise level, as determined by previous findings and experiments. In some cases, it is made uniform over the range of cfr and N. Then

Can be written. Both of the above methods give good results.

ｐ（ｃｆｒ，Ｎ｜Ｈ）は、各染色体について別々に、単に参照染色体についてダイソミーであると仮定するのではなく、仮説Ｈを仮定して、上記の通り決定することができる。この方法を用いて、固定されたノイズのパラメータと子の画分のパラメータ両方を保持すること、いずれかのパラメータを固定すること、または両方のパラメータを各染色体および各仮説について確率的な形態で保持することが可能である。 Note that in some cases it may not be desirable, impossible, or feasible to use a reference chromosome. In such cases, it is possible to derive the best ploidy call separately for each chromosome. In detail:

p (cfr, N | H) can be determined separately for each chromosome, as described above, assuming hypothesis H, rather than simply assuming that it is a disomy for the reference chromosome. Using this method, hold both fixed noise parameters and child fraction parameters, fix one parameter, or both parameters in probabilistic form for each chromosome and each hypothesis. It is possible to hold.

Measurements of DNA, especially when the amount of DNA is low or when DNA is mixed with contaminating DNA, are noisy and / or prone to error. This noise results in inaccurate genotype data and inaccurate haploid calls. In some embodiments, platform modeling or some other noise modeling method can be used to count the adverse effects of noise on ploidy determination. The method uses a simultaneous model of both channels and takes into account random noise due to the quantity of input DNA, the quality of DNA, and / or the quality of the protocol.

This is in contrast to some methods known in the art for making ploidy determinations using the ratio of allele intensities at the locus. This method interferes with accurate SNP noise modeling. In particular, the error in the measurement is generally independent of the measured channel intensity ratio and shrinks the model to use one-dimensional information. Accurate modeling of noise, channel quality and channel interactions requires a two-dimensional simultaneous model that cannot be modeled using allele ratios.

In particular, projecting the two channel information onto a ratio r where f (x, y) is r = x / y is not itself useful for accurate channel noise and bias modeling. The noise in a particular SNP is not a function of ratio, i.e. noise (x, y) ≠ f (x, y), but is actually a joint function of both channels. For example, in the binomial model, the measured ratio of noise has a variance of r (1-r) / (x + y), which is not purely a function of r. In a model that includes any channel bias or noise, in _SNPi , the observed channel X value is assumed to be x _{= ai} X + bi, where X is the true channel value and bi. Is extra channel bias and random noise. Similarly, it is assumed that y = c _i Y + _di . Since (a _i X + bi) / ( _ci Y + _di ) is not a function of X / Y, the observed ratio r = x / _y is an accurate prediction of the true ratio X / Y or the remaining noise. Cannot be modeled.

The methods disclosed herein describe a method of effective modeling of noise and bias using all simultaneous binomial distributions of individual measurement channels. Relevant equations are described elsewhere in the document, consistent bias per SNP that effectively regulates SNP behavior, P (good) and P (ref | bad), P (mut | bad). You can find it in the section you are in. In certain embodiments, the methods of the present disclosure use a beta-binomial distribution that relies solely on the ratio of alleles, avoids implementation limitations, and instead models behavior based on both channel counts. ..

In certain embodiments, the methods disclosed herein call the ploidy of a pregnant fetus from genetic data found in maternal plasma by using all available measurements. Can be done. In certain embodiments, the methods disclosed herein provide ploidy of a pregnant fetus from genetic data found in maternal plasma by using measurements from only a subset of the parental situation. You can call. In some methods known in the art, if the parental situation is from the AA | BB situation, i.e., both parents are homozygous at a given locus, but the alleles are different. Use only the genetic data measured in. One problem with this method is that the proportion of polymorphic loci from the AA | BB situation is small, generally less than 10%. In certain embodiments of the methods disclosed herein, the method does not use maternal plasma genetic measurements made at loci where the parental situation is AA | BB. In certain embodiments, the method uses plasma measurements only for polymorphic loci whose parental status is AA | AB, AB | AA, and AB | AB.

Several methods known in the art are to average the ratio of alleles from SNPs in the AA | BB situation where the genotypes of the parents are present, and from the ratio of the average alleles in these SNPs to the polyploid call. Includes steps to assert a decision. This method is significantly inaccurate due to the differential SNP behavior. Note that this method assumes that the genotypes of the parents are known. In contrast, in some embodiments, the method uses a simultaneous channel distribution model that does not assume the presence of any of the parents and does not assume uniform SNP behavior. In some embodiments, the method takes into account the behavior / weighting of different SNPs. In some embodiments, the method does not require knowledge of the genotype of one parent or parent. Here is an example of how this method achieves this:

と定義され、式中、ｍは可能性のある真の母親の遺伝子型であり、ｆは可能性のある真の父親の遺伝子型であり、ｍ，ｆ∈｛ＡＡ，ＡＢ，ＢＢ｝であり、ｃは、仮説Ｈを考慮した、可能性のある子の遺伝子型である。詳細には、モノソミーについてはｃ｛Ａ，Ｂ｝であり、ダイソミーについてはｃ∈｛ＡＡ，ＡＢ，ＢＢ｝であり、トリソミーについてはｃ∈｛ＡＡＡ，ＡＡＢ，ＡＢＢ，ＢＢＢ｝である。親の遺伝子型データを含めることにより、一般には、より正確な倍数性の決定がもたらされるが、当該方法が良好に機能するために親の遺伝子型データは必須ではないことに留意されたい。 In some embodiments, the log-likelihood of the hypothesis can be determined for each SNP. Assuming hypothesis H and percent fetal DNAcf for fetal ploidy for a particular SNPi, the log-likelihood of the observed data D is:

In the formula, m is the genotype of the possible true mother, f is the genotype of the possible true father, and m, f ∈ {AA, AB, BB}. , C are genotypes of possible offspring, taking into account Hypothesis H. Specifically, for monosomy, c {A, B}, for disomy, c ∈ {AA, AB, BB}, and for trisomy, c ∈ {AAA, AAB, ABB, BBB}. It should be noted that although inclusion of parental genotype data generally results in more accurate ploidy determination, parental genotype data is not essential for the method to work well.

Some methods known in the art are homozygous for mothers, but different alleles are measured in plasma (AA | AB or AA | BB status), averaging the ratio of alleles from the SNP. And it comprises the step of claiming the determination of polyploid call from the ratio of the average alleles in these SNPs. This method is intended when the paternal genotype is not available. Note that it is questionable how accurately plasma can be claimed to be heterozygous at a particular SNP without the presence of a homozygous and opposite paternal BB: of the offspring. At low proportions, the appearance of allele B may simply be the presence of noise, and the absence of B may be the presence of the simple allele of the fetal measurements. It may be a dropout. Furthermore, even if the plasma heterozygotes can actually be determined, this method cannot distinguish paternal trisomy. Specifically, for SNPs where the mother is AA, and for SNPs where some B is measured in plasma, if the father is GG, the genotype of the offspring produced is AGG, with an average ratio of 33% A. (Ratio of children = 100%). However, if the father is AG, the genotype of the resulting offspring may be AGG for matched trisomy, give a ratio of A of 33%, or may be AAG for mismatched trisomy. A, which is closer to 66% of the average ratio, is obtained. Given that many trisomy are on the chromosome with crossover, the overall chromosome can be between no trisomy of mismatch and trisomy of all mismatch, and this ratio is It can vary between 33 and 66%. For normal disomy, the ratio should be about 50%. Without the use of a chained model or an accurate error model of the mean, this method often overlooks paternal trisomy. In contrast, the methods disclosed herein assign a parent genotype probability to each parent genotype candidate based on available genotype information and population frequency, and the parent. Does not explicitly require the genotype of. In addition, the methods disclosed herein allow trisomy to be detected in the absence or even in the presence of parental genotype data, and from possible matching trisomy using a linkage model. It can be compensated by identifying the point of transfer to the discrepancy trisomy.

Several methods known in the art are for averaging the ratio of alleles from SNPs of unknown maternal and paternal genotypes, and polyploid calls from the ratio of the averages in these SNPs. Insist on a method for determining. However, no method has been disclosed to achieve these objectives. The methods disclosed herein allow accurate haploid calls in such situations, using simultaneous probability maximum likelihood methods, and optionally SNP noise and bias models, as well as chaining models. Implementations that utilize are disclosed elsewhere in this document.

Some methods known in the art include averaging allele ratios and claiming the determination of ploidy calls from the average allele ratio in one or a few SNPs. However, such a method does not utilize the concept of chaining. The methods disclosed herein do not have these drawbacks.

Use sequence lengths as a precursor to determine the origin of DNA It has been reported that the distribution of sequence lengths differs between maternal and fetal DNA, with fetal being generally shorter. In one embodiment of the present disclosure, previous findings are used in the form of empirical data to predict the expected length of both maternal DNA (P (X | maternal)) and fetal DNA (P (X | fetal)). It is possible to construct a prior distribution of data. Given the new unidentified DNA sequence of length x, the probability that a given sequence of DNA is either maternal or fetal DNA, based on the pre-likelihood of x considering either maternal or fetal. It is possible to determine. Specifically, if P (x | maternal line)> P (x | fetus), the DNA sequence can be classified as a maternal line, and P (x | maternal line) = P (x | maternal line) / [(P). If (x | maternal line) + P (x | fetus)] and p (x | maternal line) <p (x | fetus), the DNA sequence can be classified as a fetus, and P (x | fetus). = P (x | fetus) / [(P (x | maternal) + P (x | fetus)]. In certain embodiments of the present disclosure, the length of the maternal sequence and that is specific to the sample. The distribution of the length of the fetal sequence can be determined by taking into account the sequence that can be assigned to the maternal line or the fetus with high probability, and then the sample-specific distribution is predicted for the sample. It can be used as a size distribution.

In many clinical trials on variable read depth diagnostics to minimize the cost of sequencing, such as Chiu et al. BMJ, 2011: 342: c7401, set a protocol with a number of parameters. The same protocol is then performed for each patient in the trial with the same parameters. If the mother uses sequencing as a method for measuring genetic material to determine the ploidy state of the fetus during pregnancy, one relevant parameter is the number of reads. The number of reads can refer to the actual number of reads, the intended number of reads, the sequencer's split lanes, the complete lanes, or the complete flow cell. In these tests, the number of reads is generally set at a level that ensures that all or almost all samples achieve the desired level of accuracy. Sequencing is currently a costly technique, costing approximately $ 200 per 5 million mappable reads, while at lower prices, it works with similar levels of accuracy but has fewer reads. Any method that enables diagnostics based on will inevitably save a significant amount of money.

The accuracy of the ploidy determination generally depends on a number of factors, including the number of reads and the proportion of fetal DNA in the mixture. The accuracy is generally higher as the proportion of fetal DNA in the mixture is higher. At the same time, the accuracy is generally higher as the number of reads increases. It is possible to have a situation with two cases that determine the ploidy state with comparable accuracy, the first case having a lower proportion of fetal DNA in the mixture than the second case, the first. In the case of 1, more reads are sequenced than in the second case. The estimated proportion of fetal DNA in the mixture can be used as a guide in determining the number of reads required to achieve a given level of accuracy.

In one embodiment of the present disclosure, a set of samples can be performed when different samples in the set are sequenced at different read depths, and the number of reads performed for each of the samples is each mixture. Considering the proportion of fetal DNA calculated in, choose to achieve a given level of accuracy. In certain embodiments of the present disclosure, this may involve measuring the mixed sample to determine the proportion of fetal DNA in the mixture, and estimation of this fetal fraction is performed using sequencing. Any method that can be done with TAQMAN, can be done with qPCR, can be done with SNP arrays, and can distinguish different alleles at a given locus. Can be done using. The need to estimate the fetal fraction is eliminated by including hypotheses that include all or selected sets of fetal fractions within the set of hypotheses considered when comparing to actual measured data. be able to. After determining the proportion of fetal DNA in the mixture, the number of sequences read for each sample can be determined.

In one embodiment of the present disclosure, 100 pregnant women visit their OBs and each in a blood tube containing an anti-lysant (anti-lysant) and / or inactivated DNAase. Blood is collected. Each of the women brings home a kit for the father of the fetus, who is pregnant, to provide saliva samples. A collection of both genetics for all 100 couples is sent back to the laboratory where the mother's blood is centrifuged to isolate the buffy coat as well as the plasma. Plasma contains a mixture of maternal DNA as well as DNA derived from the placenta. Maternal buffy coats and paternal blood are genotyped using SNP arrays and DNA in maternal plasma samples is targeted using SURESELECT hybridization probes. DNA pulled down with a probe is used to generate 100 tagged libraries for each of the maternal samples, each sample tagged with a different tag. Extract parts from each library, mix each of those parts together and add to the two lanes of the ILLUMINA HISEQ DNA sequencer in multiplex fashion, resulting in approximately 50 million mapable reads from each lane. The result was approximately 100 million mapable leads in 100 multiplexed mixtures, or approximately 1 million reads per sample. Sequence reads were used to determine the proportion of fetal DNA in each mixture. Fifty samples had more than 15% fetal DNA in the mixture and one million reads were sufficient to determine fetal ploidy status with 99.9% confidence.

Of the remaining mixture, 25 have fetal DNA between 10% and 15%, multiplexing each portion of the relevant library prepared from these mixtures into one lane of HISEQ. The sink generated an additional 2 million leads for each sample. Two sets of sequence data for each of the mixtures with fetal DNA between 10% and 15% were added together and 3 million reads per sample produced 99.9 of the ploidy state of those fetuses. % Confidence was sufficient to determine.

Of the remaining mixture, 13 had fetal DNA between 6% and 10%, multiplexing each fraction of the relevant library prepared from these mixtures and one lane of HISEQ. And generated an additional 4 million leads for each sample. Two sets of sequence data for each of the mixtures having fetal DNA between 6% and 10% were added together, and a total read of 5 million per resulting mixture 99. It was enough to make a decision with 9% confidence.

Of the remaining mixture, 8 had fetal DNA between 4% and 6%, and each portion of the relevant library prepared from these mixtures was multiplexed and flushed into one lane of HISEQ. , Generated an additional 6 million leads for each sample. Two sets of sequence data for each of the mixtures having fetal DNA between 4% and 6% were added together, and a total read of 7 million per resulting mixture 99. It was enough to make a decision with 9% confidence.

All of the remaining four mixtures have fetal DNA between 2% and 4%, multiplexing each portion of the relevant library prepared from these mixtures and one lane of HISEQ. And generated an additional 12 million leads for each sample. Two sets of sequence data for each of the mixtures having fetal DNA between 2% and 4% were added together, and a total read of 13 million per resulting mixture 99. It was enough to make a decision with 9% confidence.

This method required 6 lanes for sequencing on a HISEQ machine to achieve 99.9% accuracy over 100 samples. If the same number of runs are required for every sample, 25 lanes are taken for the sequencing to ensure that every ploidy determination is made with 99.9% accuracy, 4 If a% call-free ratio or error rate was tolerated, this could be achieved with a 14-lane sequencer.

Use of Raw Genotyping Data There are a number of ways in which NPD can be achieved using fetal genetic information measured in fetal DNA found in maternal blood. Some of these methods involve making measurements of fetal DNA using SNP arrays, some include non-targeted sequencing, and some include targeted sequencing. do. Targeted sequencing can target SNPs, STRs, other polymorphic loci, non-polymorphic loci or some combinations thereof. Can be targeted. Some of these methods may include the use of commercial or proprietary allele callers that call allelic identity from intensity data provided by the sensor of the measuring machine. For example, the ILLUMINA INFINIUM system or the AFFYMETRIX GENECHIP microarray system includes beads or microchips attached with DNA sequences that can hybridize to complementary segments of DNA, and hybridization changes the fluorescence of the sensor molecule. , It can be detected. There are also sequencing methods, such as the ILLUMINA SOLEXA GENOME SEQUENCER or ABI SOLID GENOME SEQUENCER, which are sequenced for the gene sequence of a fragment of DNA and extended as the DNA strand complementary to the sequenced strand is extended. Nucleotide identity is generally detected via a fluorescent or radioactive tag attached to a complementary nucleotide. In all of these methods, genotyping or sequencing data is generally determined on the basis of fluorescence or other signals or the absence thereof. These systems are typically combined with low-level software packages that make calls for specific alleles (secondary gene data) from the analog output (primary gene data) of fluorescence or other detection devices. For example, in the case of a given allele on an SNP array, the software makes a call that a particular SNP is present or absent if the fluorescence intensity is above or below a certain threshold. conduct. Similarly, the output of the sequencer is a chromatogram showing the level of fluorescence detected for each of the dyes, and the software makes a call that a particular base pair is A or T or C or G. The high-throughput sequencer makes a series of such measurements, commonly referred to as reads, to show the structure of the most likely DNA sequence sequenced. The direct analog output of the chromatogram is defined herein as primary genetic data, and base pair / SNP calls made by the software are considered herein as secondary genetic data. In certain embodiments, the primary data refers to raw intensity data, which is the unprocessed output of the genotyping platform, and the genotyping platform can refer to an SNP array or sequencing platform. Secondary gene data is processed genetic data in which allele calls are made, sequence data is assigned to a base pair, and / or sequence reads are mapped to the genome. Refers to data.

Many higher levels of application utilize these allelic calls, SNP calls and sequence reads, ie, secondary genetic data generated by genotyping software. For example, obtain sequencing reads with DNA NEXUS, ELAND or MAQ and map them to the genome. For example, in the context of non-invasive prenatal diagnosis, complex informatics, such as PARENTAL SUPPORT ™, can affect multiple SNP calls to genotype an individual. It is also possible to obtain a set of sequence reads that are mapped to the genome in the context of preimplantation genetic diagnosis, and to obtain a normalized number of reads that are mapped to each chromosome or section of the chromosome. It may be possible to determine the polyploid state of an individual. In the context of non-invasive prenatal diagnosis, it may be possible to obtain a collection of sequence reads measured in DNA present in maternal plasma and map them to the genome. The normalized number of reads mapped to each chromosome or section of the chromosome can then be obtained and the data used to determine the ploidy state of the individual. For example, a chromosome with a disproportionately large number of reads may be able to conclude that the bleeding mother is trisomy in the pregnant fetus.

However, in reality, the first output from the instrument is an analog signal. When a particular base pair is called by software associated with the sequencing software, such as software capable of calling base pair T, the call is, in fact, considered most likely by that software. It's a call. However, in some cases, the call may be unreliable, for example, due to an analog signal, a particular base pair is only 90% likely to be T and may be A. It may also be shown to be 10%. In another example, the genotype calling software associated with the SNP array reader may call that a particular allele is G. However, in practice, the underlying analog signal may indicate that the allele is only 70% likely to be G and the allele is 30% likely to be T. In these cases, some information is lost when using genotype and sequence calls made by lower level software in higher level applications. That is, the primary genetic data measured directly by the genotyping platform can be more troublesome than the secondary genetic data determined by the attached software package, but it contains more information. Many reads are discarded because some bases are not sufficiently clearly read in the mapping of the secondary gene data sequence to the genome, or because the mapping is not clear. When using primary gene data sequence reads, all or many of the reads that may have been discarded when first converted to secondary gene data sequence reads are used by stochastically processing the reads. be able to.

In certain embodiments of the present disclosure, the higher level software does not rely on allelic calls, SNP calls or sequence reads determined by the lower level software. Instead, higher levels of software base their calculations on analog signals measured directly from genotyping platforms. In certain embodiments of the present disclosure, an informatics-based method, eg, PARENTAL SUPPORT ™, whose ability to reconstruct embryo / fetal / offspring genetic data is measured by a genotyping platform. Modify to be engineered for direct use. In certain embodiments of the present disclosure, informatics-based methods, such as PARENTAL SUPPORT ™, use primary gene data and without secondary gene data, call alleles and / or chromosomal copies. Can make a call. In one embodiment of the disclosure, gene calls, SNP calls, all sequence reads, sequence mappings are measured directly by a genotyping platform rather than converting primary gene data into secondary gene calls. Probabilistically processed by using the raw intensity data produced. In certain embodiments, the DNA measurements from the prepared samples used in the step of calculating the probability of the number of alleles and the step of determining the relative probability of each hypothesis include primary gene data.

In some embodiments, the method can increase the accuracy of the genetic data of a target individual, incorporating the genetic data of at least one related individual, and the method is specific to the genome of the target individual. Steps to obtain primary genetic data and genetic data specific to the genome (s) of the relevant individual (s) and which chromosomal segment from the relevant individual (s) are those within the genome of the target individual. Each of the hypotheses, taking into account the primary genetic data of the target individual and the genetic data of the relevant individual (s), as well as the steps to create a set of one or more hypotheses as to whether they may correspond to a segment of It includes the steps of determining the probability of each hypothesis and the step of determining the most probable state of the genetic material of the actual target individual using the probabilities associated with each hypothesis. In some embodiments, the method can determine the number of copies of chromosomal segments in the genome of a target individual, the method of which is how many copies of chromosomal segments are present in the genome of the target individual. Steps to create a set of hypotheses about the number of copies, and steps to incorporate primary genetic data from the target individual and genetic information from one or more related individuals into the data set, and the platform associated with the data set. Relative to each of the hypotheses about the number of copies, taking into account the steps in which the response characteristics of the platform can fluctuate in one experiment and another, and the response characteristics of the data set and platform. It involves calculating the target probability and determining the number of copies of the chromosomal segment based on the hypothesis about the most likely copy number. In certain embodiments, the methods of the present disclosure can determine the ploidy state of at least one chromosome of a target individual, wherein the method is primary from the target individual and from one or more related individuals. The step of obtaining genetic data, the step of creating a set of hypotheses for at least one ploidy state for each of the chromosomes of the target individual, and the step of using one or more specialized techniques to make the ploidy within the set. One or more for each hypothesis about ploidic states, taking into account the genetic data obtained for each step of determining the statistical probability of each hypothesis about the state and for each of the expertise used. It includes a step of combining statistical probabilities determined by specialized techniques and a step of determining ploidy states based on compound statistical probabilities for each hypothesis about ploidy states for each of the chromosomes of the target individual. .. In certain embodiments, the methods of the present disclosure are allogenes in a set of allelic genes, in the target individual and from one parent or parent of the target individual, and optionally from one or more related individuals. The state of the gene can be determined, the method of obtaining primary genetic data from a target individual and from one parent or parent, and from any related individual, and with respect to the target individual, and one parent or parent. And, optionally, for one or more related individuals, a step of creating a set of hypotheses for at least one allogen, where the hypothesis is a possible alleged set of allogens. For each of the steps to describe the state, the step to determine the statistical probability of the hypothesis for each allelic gene in the set of hypotheses, taking into account the obtained genetic data, and the alligator gene in the set of allelic genes. Steps to determine the status of an allelic gene for a target individual, for one parent or parent, and, optionally, for one or more related individuals, based on their respective statistical probabilities of hypotheses about allogenes. And include.

In some embodiments, the genetic data of the mixed sample may include sequence data, where the sequence data may not be uniquely mapped to the human genome. In some embodiments, the genetic data of the mixed sample may include sequence data, where the sequence data is mapped to multiple locations within the genome, where each of the possible mappings. It involves the probability that a given mapping is accurate. In some embodiments, sequence reads are not assumed to be associated with a particular location in the genome. In some embodiments, sequence reads are associated with multiple positions within the genome, with associated probabilities belonging to that position.

Counting Method for Determining the Number of Chromosome Copies In one aspect, the invention features a method of examining the abnormal distribution of fetal chromosomes by comparing the number of sequence tags aligned on different chromosomes (eg, 2012). See U.S. Pat. No. 8,296,076 filed April 20, which is incorporated herein by reference in its entirety. As is known in the art, the term "sequence tag" can be used, for example, to identify certain larger sequences that map to a chromosome or genomic region or gene (eg, 15). ~ 100) Means a nucleic acid sequence. In some embodiments, the method comprises (i) a sample containing a mixture of maternal and fetal DNA at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; It is a step of contacting with a primer library that simultaneously hybridizes to 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target gene loci to generate a reaction mixture. The target locus is derived from a plurality of different chromosomes, and at least one first chromosome suspected to have an abnormal distribution in the sample and at least presumed to have a normal distribution in the sample. A step containing one second chromosome, (ii) a step of subjecting the reaction mixture to primer extension reaction conditions to produce an amplification product, and (iii) a plurality of sequences in which the amplification product is sequenced at a target loci. To obtain sequence tags long enough to assign specific target loci, (iv) to assign multiple sequence tags to their corresponding target loci on a computer, and (v) first. The step of computer-determining the number of sequence tags aligned with the target locus of the chromosome and the number of sequence tags aligned with the target locus of the second chromosome is compared with the number from (vi) step (v). It includes a step of determining the presence or absence of an abnormal distribution of the first chromosome.

In one aspect, the invention provides a method of detecting the presence or absence of fetal aneuploidy by comparing the relative frequencies of target amplification products between chromosomes (eg, filed January 23, 2012). See WO2012 / 103031; this patent is incorporated herein by reference in its entirety). In some embodiments, the method comprises (i) sampling a sample containing a mixture of maternal and fetal DNA at least 1,000; 2,000; 5,000; 7,500; 10, from a plurality of different chromosomes. Contact with a primer library that simultaneously hybridizes to 000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different non-polymorphic target loci. And (ii) a step of subjecting the reaction mixture to primer extension reaction conditions to produce an amplification product containing a target amplification product, and (iii) target amplification derived from the first and second target chromosomes. The step of quantifying the relative frequency of the product by computer, (iv) the step of comparing the relative frequency of the target amplification product derived from the first and second target chromosomes by computer, and (v) the first and second targets. It includes the step of identifying the presence or absence of aneuploidy based on the relative frequency of the comparison result of chromosomes. In some embodiments, the first chromosome is a chromosome suspected to be a positive polyploid. In some embodiments, the second chromosome is a chromosome suspected to be aneuploidy.

Combination Methods of Prenatal Diagnosis There are many methods that can be used for prenatal diagnosis or prenatal screening for aneuploidy or other genetic defects. Other parts of this document, as well as US Utility Model Application No. 11 / 603,406 filed November 28, 2006; US Utility Model Application No. 12/076 filed March 17, 2008, 1 of a method for increasing the accuracy of known or estimated genetic data of a target individual, such as a fetus, using the genetic data of the relevant individuals in 348 and PCT application PCT / S09 / 52730. One is described. Other methods used for prenatal diagnosis include measuring the levels of specific hormones in the blood of the maternal line that correlate with abnormalities in various genes. An example of this is a triple test, a test that measures the levels of several (generally two, three, four, or five) different hormones in the blood of a maternal line. Multiple methods are used to determine the likelihood of a given outcome, and if none of them are deterministic in their own right, the information produced by these methods is combined to make a more accurate prediction than any of the individual methods. It is possible to do. The triple test can combine information from three different hormones to provide more accurate predictions of genetic abnormalities than can be predicted by individual hormone levels.

The present specification is a method for making a more accurate prediction regarding the state of a gene in a fetus, in particular, the possibility of a gene abnormality in the fetus, and the gene in the fetus is subjected to various methods. Methods are disclosed that include combining anomaly predictions. A "more accurate" method can refer to a method for diagnosing an abnormality with a lower false negative rate at a given false positive rate. In a preferred embodiment of the present disclosure, one or more of the predictions are made based on known genetic data for the fetus and the genetic findings were determined using the PARENTAL SUPPORT ™ method, i.e. to the fetus. The genetic data of the relevant individual was used to determine the genetic data of the fetus with higher accuracy. In some embodiments, the genetic data may include a ploidic state of the fetus. In some embodiments, genetic data can refer to a set of allelic calls in the fetal genome. In some embodiments, some of the predictions were made using triple tests. In some embodiments, some of the predictions were made using measurements of other hormone levels in the maternal blood. In some embodiments, the predictions made by the method considering diagnosis can be combined with the predictions made by the method considering screening. In some embodiments, the method comprises measuring the maternal blood levels of alpha-fetoprotein (AFP). In some embodiments, the method comprises measuring the maternal blood levels of unconjugated estriol (UE ₃ ). In some embodiments, the method comprises measuring the maternal blood levels of beta-human chorionic gonadotropin (beta-hCG). In some embodiments, the method comprises measuring the maternal blood level of an invasive trophoblast antigen (ITA). In some embodiments, the method comprises measuring blood levels of activin maternal lineage. In some embodiments, the method comprises measuring the maternal blood level of pregnancy-related plasma protein A (PAPP-A). In some embodiments, the method comprises measuring the maternal blood levels of other hormones or maternal serum markers. In some embodiments, some of the predictions may be made using other methods. In some embodiments, some of the predictions use a fully integrated test, eg, a combination of ultrasonography and blood tests at about 12 weeks of gestation and a second blood test at about 16 weeks. May be done. In some embodiments, the method comprises the step of measuring fetal neck edema (NT). In some embodiments, the method comprises the step of using the measured hormone levels described above to make a prediction. In some embodiments, the method comprises a combination of the methods described above.

There are many ways to combine predictions, for example, hormone measurements can be converted to multiple of the median (MoM) and then to likelihood ratio (LR). can. Similarly, other measurements can be converted to LR using a mixed model of NT distribution. LR for NT and biochemical markers can be multiplied by age and pregnancy-related risks to derive risks for various conditions such as trisomy 21. The detection rate (DR) and false positive rate (FPR) can be calculated by taking the percentage of the risk that exceeds a given risk threshold.

In certain embodiments, the methods for calling the statistic state are, but are not limited to, the relative probabilities of the joint probabilities and the probabilities of the statistic determined using the probability of the joint distribution model and the number of allogenes. , Read number analysis, heterojunction rate comparison, statistics available only when using parental genetic information, probability of genotype signal normalized to a particular parental situation, first sample or Statistical techniques selected from other methods of determining the risk score for a trisomy of the fetus, including statistics calculated using the estimated fetal fractions in the prepared samples, and combinations thereof. Includes steps to combine with the respective relative probabilities of the hypothesis about multipliers calculated using.

Another method can include situations involving four measured hormone levels, where the probability distribution around these hormones is known: p (x ₁ , x ₂ for positive ploidy). , X ₃ , x ₄ | e), and in the case of aneuploidy, p (x ₁ , x ₂ , x ₃ , x ₄ | a). The probability distributions for the DNA measurements can then be measured, g (y | e) and g (y | a) for positive ploidy and aneuploidy, respectively. These are p (x ₁ , x ₂ , x ₃ , x ₄ | a) g (y | a) and p (x 1, x 2, x 3, x 4 | a) g (y | a), assuming they are independent, taking into account the assumptions of positive ploidy / aneuploidy. They can be combined as x ₁ , x ₂ , x ₃ , x ₄ | e) g (y | e) and then pre-multiplied by p (a) and p (e), taking into account the age of the maternal line. .. Then you can choose the highest one.

In one embodiment, it is possible to elicit a central limit theorem, assume that the distribution of g (y | a or e) is Gaussian, and measure the mean and standard deviation by examining a large number of samples. Is. In another embodiment, considering the outcome, assuming that they are not independent, sufficient samples to estimate the joint distribution p (x ₁ , x ₂ , x ₃ , x ₄ | a or e). Can be collected.

In certain embodiments, the haploid state for determining that the target individual is haploid is associated with the hypothesis with the highest probability. In some cases, one hypothesis has a normalized compound probability of greater than 90%. Each hypothesis is associated with one ploidic state or a set of ploidy states, the normalized composite probability of which is greater than 90% or some other threshold, such as 50%, 80%, etc. The ploidy state associated with the hypothesis above 95%, 98%, 99% or 99.9% can be selected as the threshold required for the hypothesis to be called as the determined ploidy state. can.

DNA from previous pregnancy-derived offspring in maternal blood One difficulty in non-invasive prenatal diagnosis is to distinguish between current pregnancy-derived fetal cells and previous pregnancy-derived fetal cells. Is. Some believe that genetic matter from the previous pregnancy disappears after some time, but no conclusive evidence has been shown. In one embodiment of the present disclosure, the PARENTAL SUPPORT ™ (PS) method and paternal genomic findings are used to inherit paternally-origin fetal DNA present in maternal blood (ie, the fetus is inherited from the father). It is possible to determine the DNA). In this method, the genetic information of the parent whose phase has been identified can be utilized. From genotype information for which the phase has not been identified, using genetic data from the grandparents (eg, genetic data measured from the sperm of the grandfather), or from genetic data from other born offspring, or samples of abortion. It is possible to identify the genotype phase of the parent from. HapMap-based phase identification or haplotyping of paternal cells can also identify the phase of genetic information for which the phase has not been identified. Successful haplotyping has been demonstrated by quiescing cells in the mitotic phase, where chromosomes are tightly bundled, and using microfluidics to put separate chromosomes into separate wells. In another embodiment, phase-identified parental haplotype data can be used to detect the presence of two or more homologues from the father, which are from two or more offspring. It means that the genetic material is present in the blood. By focusing on chromosomes that are predicted to be positive polyploid in the fetus, the possibility that the fetus suffers from trisomy can be ruled out. It is also possible to determine if the fetal DNA is not of current paternal origin, in which case other methods, such as triple tests, can be used to predict genetic abnormalities.

There may be other sources of fetal genetic material available by methods other than blood sampling. In the case of fetal genetics available in maternal blood, there are two main categories: (1) whole fetal cells, such as fetal nucleated red blood cells or erythroblasts, and (2) floating fetal DNA. be. In the case of whole fetal cells, fetal cells can survive for a long time in the blood of the maternal line, thus from pregnant women, cells containing DNA from pre-pregnancy-derived offspring or fetuses. There is some evidence that it is possible to isolate. There is also evidence that floating fetal DNA is removed from the system within a few weeks. One challenge is how to determine the identity of the individual whose genetic material is contained in the cell, i.e., how to ensure that the measured genetic material is not of fetal origin from a previous pregnancy. Is it? In certain embodiments of the present disclosure, the knowledge of maternal genetic material can be used to ensure that the genetic material in question is not the maternal genetic material. As described in this document or any patent referenced herein, there are a number of methods to achieve this goal, including informatics-based methods, such as PARENTAL SUPPORT ™.

In one embodiment of the present disclosure, blood drawn from a pregnant mother can be separated into a fraction containing floating fetal DNA and a fraction containing nucleated red blood cells. Floating DNA can be enriched as needed and information on the genotype of the DNA can be measured. From the genotype information measured from the floating DNA, the genotype knowledge of the maternal line can be used to determine the genotypic aspect of the fetus. These embodiments may refer to a ploidic state and / or the identity of a set of alleles. Individual nucleated red blood cells can then be genotyped using the methods described elsewhere in this document and in other reference patents, in particular the methods described in the first section of this document. Knowledge of the maternal genome makes it possible to determine whether any single blood cell is genetically maternal. Also, the aspects of the fetal genotype determined as described above make it possible to determine whether a single blood cell is genetically derived from the currently pregnant fetus. In essence, by this aspect of the present disclosure, genetic findings of the mother and, in some cases, genetic information from other related individuals, such as the father, were measured from the floating DNA found in the blood of the maternal line. The isolated nucleated cells found in the blood of the maternal lineage, used in conjunction with genetic information, are either (a) genetically maternal or (b) genetically derived from the currently pregnant fetus. It will be possible to determine whether (c) it is genetically derived from a fetus from a previous pregnancy.

Determining Prenatal Sex Chromosome Alinessity In methods known in the art, those who attempt to determine the sex of a pregnant fetus from the mother's blood will find that the fetal floating DNA (fffDNA) is the mother. It uses the fact that it is present in the plasma of the fetus. If the Y-specific locus in maternal plasma can be detected, this means that the pregnant fetus is male. However, when using methods known in the art, in some cases the amount of fffDNA is too low to ensure that the Y-specific locus is detected in the case of a male fetus. The lack of detection of a Y-specific locus in plasma does not necessarily guarantee that the pregnant fetus is female.

Here, a novel method is presented that does not require the measurement of Y-specific nucleic acids, that is, DNAs derived exclusively from paternally derived loci. The previously disclosed Parental Support method uses crossover frequency data, parental genotype data, and informatics techniques for determining the ploidy state of the fetus during pregnancy. Fetal sex is simply the ploidy state of the fetus on the sex chromosomes. The child who is XX is a woman, and XY is a man. The ploidy state of the fetus can also be determined by the method described herein. It should be noted that sex determination is effectively synonymous with determining sex chromosome polyploidy; in the case of sex determination, assumptions are often made assuming that the offspring are positive polyploid, and therefore possible. There are few sexual hypotheses.

胎児由来のＤＮＡが母親由来のＤＮＡと混在しており、混合物中の胎児ＤＮＡの割合がＦであり、混合物中の母系ＤＮＡの割合がＭであり、したがってＦ＋Ｍ＝１００％である場合、以下が認められることが予想される：

ＦおよびＭが既知である場合には、予測比を計算することができ、観察されたデータを、予測データと比較することができる。ＭおよびＦが未知である場合には、閾値を過去のデータに基づいて選択することができる。どちらの場合でも、ＸとＹの両方に特異的な遺伝子座において測定されたＤＮＡの量をベースラインとして用いることができ、胎児の性別の検査は、Ｘ染色体のみに特異的な遺伝子座において観察されたＤＮＡの量に基づいてよい。その量がベースラインよりも、およそ１／２Ｆと等しい量だけ低い、またはそれが予め定義された閾値未満になる量だけ低ければ、胎児は男であることが決定され、その量がベースラインとほとんど等しい、またはそれが予め定義された閾値未満になる量だけ低くなければ、胎児は女であることが決定される。 The methods disclosed herein include examining loci common to both the X and Y chromosomes to create a baseline for the predicted amount of fetal DNA present for the fetus. The region specific to the X chromosome alone can then be examined to determine if the fetus is female or male. In the case of males, it is expected that there will be less fetal DNA from the X chromosome-specific locus than fetal DNA from both the X and Y loci. In contrast, in female fetuses, the amount of DNA in each of these groups is expected to be the same. The DNA in question can be measured by any technique that can quantify the amount of DNA present in the sample, such as qPCR, SNP array, genotyping array or sequencing. For DNA exclusively derived from one individual, the following is expected to be observed:

If the fetal DNA is mixed with the maternal DNA, the proportion of fetal DNA in the mixture is F, the proportion of maternal DNA in the mixture is M, and therefore F + M = 100%, then: Expected to be accepted:

If F and M are known, the prediction ratio can be calculated and the observed data can be compared with the prediction data. If M and F are unknown, the threshold can be selected based on past data. In either case, the amount of DNA measured at both X and Y specific loci can be used as the baseline, and fetal sex testing is observed at the X chromosome-specific locus only. It may be based on the amount of DNA produced. If the amount is lower than the baseline by an amount equal to approximately 1 / 2F, or by an amount that is below a predefined threshold, then the fetus is determined to be male and that amount is with the baseline. A fetus is determined to be female if it is not almost equal or lower by an amount that is less than a predefined threshold.

In another embodiment, only loci common to the X and Y chromosomes, often referred to as the Z chromosome, can be examined. A subset of loci on the Z chromosome is generally always A on the X chromosome and B on the Y chromosome. If the Z chromosome-derived SNP is found to have the B genotype, the fetus is called a man, and if the Z chromosome-derived SNP is found to have only the A genotype, the fetus is called. Is called a woman. In another embodiment, loci found only on the X chromosome can be investigated. Situations such as AA | B are particularly informative as the presence of B indicates that the fetus has a paternally-derived X chromosome. Situations such as AB | B are also informative, as it is often expected that female fetuses will have only half of B as compared to male fetuses. In another embodiment, both the allelic genes A and B are present on both the X and Y chromosomes, and it is known which SNPs are from the paternal Y chromosome and which are from the paternal X chromosome. The SNP on the Z chromosome can be examined.

In certain embodiments, it is possible to amplify a single nucleotide position known to vary between homologous non-recombinant (HNR) regions shared by the Y and X chromosomes. The sequences within this HNR region are almost identical between the X and Y chromosomes. Within this same region, there is a single base position that is unchanged between the X and Y chromosomes within the population, but differs between the X and Y chromosomes. Each PCR assay can amplify sequences from loci present on both the X and Y chromosomes. Inside each of the amplified sequences is a single base that can be detected using sequencing or some other method.

In certain embodiments, the sex of the fetal can be determined from the floating DNA of the fetal found in the plasma of the maternal line, the method comprising some or all of the following steps: 1) X / within the HNR region. Steps to design PCR (either regular PCR or mini-PCR, if desired, and multiplexing if desired) primers that amplify the Y variant single base position, 2) Obtain maternal plasma, 3) Maternal PCR amplification of plasma-derived targets using the HNRX / Y PCR assay, 4) Sequencing of amplification products, 5) Sequencing data inside one or more of the amplified sequences. Steps to test for the presence of the Y allelic gene. The presence of one or more indicates a male fetus. The absence of all Y alleles from all amplification products indicates a female fetus.

In certain embodiments, targeted sequencing can be used to determine the DNA and / or genotype of the parent in maternal plasma. In certain embodiments, all sequences apparently originating from paternally fed DNA can be ignored. For example, in situations AA | AB, the number of A sequences can be counted and all of the B sequences can be ignored. To determine the heterozygous rate for the above algorithm, the number of observed A sequences and the predicted number of total sequences can be compared for a given probe. There are many methods that can calculate the predicted number of sequences for each probe for each sample. In one embodiment, historical data is used to determine which fraction of all sequence reads belongs to each of the specific probes, and then this empirical fraction is combined with the total number of sequence reads. Can be used to estimate the number of sequences in each probe. Another approach is to target some known homozygous alleles and then use historical data to correlate the number of reads in each probe with the number of reads in known homozygous alleles. Can be done. For each sample, the number of reads in homozygous alleles should then be measured and then used in conjunction with empirically derived associations to estimate the number of sequence reads in each probe. Can be done.

In some embodiments, it is possible to determine the sex of the fetus by combining predictions made by multiple methods. In some embodiments, the method is selected from the methods described in the present disclosure. In some embodiments, at least one of the plurality of methods is selected from the methods described in the present disclosure.

In some embodiments, the methods described herein can be used to determine the ploidy state of the fetus during pregnancy. In certain embodiments, the multiple call method uses a locus specific for the X chromosome or a locus common to both the X and Y chromosomes, but does not use any Y-specific locus. In certain embodiments, the multiple call method uses one or more of the following: X-chromosome-specific loci, loci common to both X and Y chromosomes, and Y-chromosome-specific loci. Chromosome. In certain embodiments, if the sex chromosome ratios are similar, eg, 45, X (Turner's syndrome), 46, XX (normal female) and 47, XXX (trisomy X), differentiation is allelic according to various hypotheses. This can be achieved by comparing the gene distribution with the predicted allelic distribution. In another embodiment, this can be achieved by comparing the relative number of sequence reads for a sex chromosome with one or more reference chromosomes that are supposed to be positive polyploid. can. It should also be noted that these methods can be expanded to include aneuploidy cases.

Monogenic Disease Screening In certain embodiments, the method for determining the ploidy state of the fetus can be extended to allow simultaneous testing for monogenic disorders. Diagnosis of monogenic diseases affects the same targeting techniques used for aneuploidy testing and requires more specific targets. In certain embodiments, the single gene NPD diagnosis is by linkage analysis. In many cases, direct testing of cfDNA samples is unreliable because the presence of maternal DNA makes it virtually impossible to determine whether the fetus has inherited the maternal mutation. It is less difficult to detect alleles of unique paternal origin, but the disease is dominant and is completely informative only if it is carried by the father, thereby limiting the usefulness of this technique. .. In certain embodiments, the method comprises PCR or related amplification techniques.

In some embodiments, the method comprises the step of identifying a phase in a parent with information from a first-degree relative for an abnormal allele with a very tightly linked SNP around it. .. A Partal Support can then be performed on the targeted sequencing data obtained from these SNPs to determine whether the fetus has inherited normal or abnormal homologues from the parents. can. As long as the SNPs are well linked, the inheritance of fetal genotypes can be determined with great certainty. In some embodiments, the method (a) adds a set of SNP gene loci for closely adjoining a particular set of routine diseases to our multiple pools for testing variability. And; (b) reliably phase-identify alleles from these added SNPs with normal and aberrant alleles based on genetic data from various close relatives. , (C) Reconstructing a set of fetal haplotypes or phase-identified SNP alleles in the inherited maternal and paternal homologues within the region surrounding the disease locus of the fetal. Includes steps to determine the genotype. In some embodiments, additional probes that reliably bind to the disease chain locus are added to the set of polymorphic loci used for aneuploidy testing.

Since the sample is a mixture of maternal DNA and fetal DNA, it is difficult to reconstruct the fetal diplomat. In some embodiments, the method incorporates information from relatives to identify phases of SNPs and disease alleles, and then SNPs and recombinant data from location-specific recombination likelihood and the maternal lineage. The most probable fetal genotype is obtained, taking into account the physical distance of the data observed from the genotypes of the plasma.

In one embodiment, a number of additional probes per disease-linked locus are included in the set of targeted polymorphic loci; the number of additional probes per disease-linked locus is between 4 and 10, between 11 and 20. It may be between 21 pieces, between 21 pieces, between 41 pieces and 60 pieces, between 61 pieces and 80 pieces, or a combination thereof.

It can be difficult to identify the phase of diploid data from the parent, and there are many ways this can be done. Some are discussed in this disclosure, others are described in more detail in another disclosure (eg, WO 2009105531 filed February 9, 2009, and August 4, 2009). See WO20100017214 of the Japanese filing; these patents are incorporated herein by reference in their entirety). In one embodiment, the parental phase can be inferred and identified by measuring haploid tissue from the parent, for example, by measuring one or more sperms or eggs. In one embodiment, first-degree relatives, such as parental or sibling measured genotype data, can be used to infer and identify the parental phase. In one embodiment, the DNA is diluted in one or more wells until it is predicted that there will be no more than one copy of each haplotype in each well, followed by one or more. The parental phase can be identified by measuring the DNA in the wells. In one embodiment, a computer program that infers the most probable phase using population-based haplotype frequency can be used to identify the phase of the parental genotype. In one embodiment, if unspecified genetic data for one or more phases of a parent's genetic progeny, as well as phase-identified halotype data, is known for the other parent, of the parent. The phase can be specified. In some embodiments, the genetic progeny of the parent may be one or more embryos, one or more fetuses, and / or a child born. Some of these and other methods relating to the identification of one parent or parental phase are described, for example, in US Patent Publication No. 2011/0033862 filed August 19, 2010; filed February 3, 2011. It is disclosed in more detail in No. 2011/017879; No. 2007/018446 filed on November 22, 2006; No. 2008/0243398 filed on March 17, 2008. These patents are incorporated herein by reference in their entirety.

Fetal Genome Reconstruction In one aspect, the invention features a method of determining fetal haplotypes. In various embodiments, the method determines which polymorphic locus (eg, SNP) is inherited in the fetus and reconstructs (and includes recombination events) the homologues present in the fetus. , Thereby interpolating the sequences between polymorphic loci). If desired, virtually the entire fetal genome can be reconstructed. If some ambiguity remains in the fetal genome (eg, crossover intervals), this ambiguity can be minimized by analyzing additional polymorphic loci, if desired. In various embodiments, the polymorphic loci are selected to have a constant density over a range of one or more chromosomes in order to reduce all ambiguity to the desired level. The method is in the fetus to allow detection of subject polymorphisms or other mutations in the fetal genome based on linkage (eg, the presence of linked polymorphic loci in the fetal genome) rather than direct detection. It has important uses for the detection of polymorphisms or other mutations in the subject. For example, if the parent is a carrier of a mutation associated with cystic fibrosis (CF), the fetal DNA will have a CF mutation by analyzing a nucleic acid sample containing maternal DNA from the fetal mother and fetal DNA from the fetus. It can be determined whether or not the haplotype to be contained is included. In particular, the polymorphic locus can be analyzed to determine if the fetal DNA contains a haplotype carrying the CF mutation, without the need to detect the CF mutation itself in the fetal DNA.

In some embodiments, the method comprises determining the parental haplotype (eg, fetal mother and father haplotype). In some embodiments, this determination is made without the use of data from relatives of the mother or father. In some embodiments, it is described herein and elsewhere (see, eg, US Patent Publication No. 2011/0033862, filed August 19, 2010, which is hereby in its entirety by reference. The parental haplotype is determined using SNP genotyping or sequencing following the dilution procedure. No more than one haplotype will be present in the same moiety (or tube) due to the dilution of the DNA. This effectively results in the presence of a single DNA molecule in the tube, allowing the haplotype on the single DNA molecule to be determined. In some embodiments, the method divides a DNA sample into multiple portions, wherein at least one portion comprises one chromosome or one chromosomal segment derived from a chromosomal pair, in at least one portion. It comprises the step of performing genotyping of a DNA sample (eg, determining the presence of two or more polymorphic loci), thereby determining the parental haplotype. In some embodiments, genotyping comprises the step of performing sequencing (eg, shotgun sequencing). In some embodiments, genotyping uses SNP arrays to use polymorphic loci, eg, 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. In some embodiments, genotyping comprises the step of using multiplex PCR. In some embodiments, the method comprises at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40, A step of contacting with a primer library that simultaneously hybridizes to 000; 50,000; 75,000; or 100,000 different polymorphic loci (eg, SNPs) to generate a reaction mixture, and the reaction mixture as a primer. It includes a step of obtaining an amplification product that is subjected to elongation reaction conditions and measured with a high-throughput sequencer to generate sequencing data.

In some embodiments, the maternal haplotype is determined by any of the methods described herein using data from the mother's relatives. In some embodiments, the haplotype of the father is determined by any of the methods described herein using data from the father's relatives. In some embodiments, the haplotype for both the father and the mother is determined. In some embodiments, SNP arrays are used to at least 1,000; 2,000; 5,000; 7,500; 10 in DNA samples from mother (or father) and mother (or father) relatives. The presence of 000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different polymorphic loci is determined. In some embodiments, the method comprises at least 1,000; 2,000; 5,000; 7,500; 10 DNA samples from mother (or father) and / or mother (or father) relatives. Primer live that simultaneously hybridizes to 000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different polymorphic loci (eg, SNPs). It comprises contacting with a rally to generate a reaction mixture and subjecting the reaction mixture to primer extension reaction conditions and measuring with a high throughput sequencer to obtain an amplification product that produces sequencing data. The parent haplotype can be determined based on the SNP array or sequencing data. In some embodiments, the phase of the parental data can be identified by methods described in this document or referenced elsewhere in this document.

This parental haplotype data can be used to determine if the fetus inherits the parental haplotype. In some embodiments, nucleic acid samples containing maternal DNA from the fetal mother and fetal DNA from the fetus are analyzed using an SNP array and 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 are detected. In some embodiments, the nucleic acid sample comprising maternal DNA from the fetal mother and fetal DNA from the fetus is at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000. Contact the sample with a primer library that simultaneously hybridizes to 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different polymorphic loci (eg, SNP). Is analyzed by producing a reaction mixture. In some embodiments, the reaction mixture is subjected to primer extension reaction conditions to produce an amplification product. In some embodiments, the amplification product is measured on a high-throughput sequencer to generate sequencing data. In various embodiments, SNP arrays or sequencing data are used to recombined data as can be found in a haplomap database, for example, by using data on chromosome crossover probabilities at different sites in the chromosome. The parental haplotype is determined by modeling the dependence between polymorphic alleles on the chromosome (by generating a recombination risk score for any interval). In some embodiments, the number of alleles at the polymorphic locus is calculated computerized based on the sequencing data. In some embodiments, multiple ploidy hypotheses for different possible ploidy states of the chromosome are computer-generated and a model for the number of alleles predicted at the polymorphic locus on the chromosome (eg, a co-distribution model). ) Is computer-constructed for each ploidy hypothesis, and using the co-distribution model and the number of alleles, the relative probability of each ploidy hypothesis is computer-calculated, and the ploidy corresponding to the hypothesis with the maximum probability. The ploidy state of the fetal is called by selecting the state. In some embodiments, the steps of constructing a joint distribution model for the number of alleles and determining the relative probabilities of each hypothesis are performed using methods that do not require the use of reference chromosomes.

In some embodiments, the fetal haplotype is determined for one or more chromosomes selected from the group consisting of chromosomes 13, 18, 21, X, and Y. In some embodiments, the fetal haplotype is determined for all fetal chromosomes. In various embodiments, the method determines substantially the entire fetal genome. In some embodiments, the haplotype is determined for at least 30, 40, 50, 60, 70, 80, 90, or 95% of the fetal genome. In some embodiments, fetal haplotyping is at least 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; Contains information on which alleles are present for 50,000; 75,000; or 100,000 different polymorphic loci.

DNA Composition Alleles when performing informatics analysis on genomic information about the fetus, eg, sequencing data measured for a mixture of fetal blood and maternal blood to determine the ploidy state of the fetus. It may be advantageous to measure the distribution of alleles in the set of. Unfortunately, in many cases, such as when attempting to determine the ploidy state of a fetal from a DNA mixture found in the plasma of a maternal blood sample, the amount of DNA available is allelic distribution with good fidelity in the mixture. Is not enough to measure directly. In these cases, amplifying the DNA mixture results in a sufficient number of DNA molecules capable of measuring the desired allele distribution with good fidelity. However, current methods of amplification commonly used for amplifying DNA for sequencing are often highly biased, that is, both alleles at the polymorphic locus are not amplified in the same amount. Biased amplification can result in an allelic distribution that is significantly different from the allelic distribution in the original mixture. For most purposes, it is not necessary to measure the relative amount of alleles present at the polymorphic locus very accurately. In contrast, in certain embodiments of the present disclosure, amplification or enrichment methods that specifically enrich polymorphic alleles and conserve the ratio of alleles are advantageous.

A number of methods have been described herein that can be used to preferentially enrich DNA samples at multiple loci so that allelic bias is minimized. In some examples, circularized probes are used to target multiple loci, with the 3'and 5'ends of the pre-cyclized probe being one or a few from the target allele polymorphic site. It is designed to hybridize to distant bases. Another example is to use a PCR probe in which the 3'end PCR probe is designed to hybridize to a base one or a few positions away from the polymorphic site of the target allele. Another example is a mixture of DNA in which the preferentially enriched loci are enriched using the split-and-pool technique with less allele bias and without the drawbacks of direct multiplexing. Is to be produced. Another example is that the capture probe is designed so that the region of the capture probe that is designed to hybridize to the DNA adjacent to the target polymorphic site is separated from the polymorphic site by one or a few bases. It uses the designed hybrid capture method.

When using the measured allele distribution in a set of polymorphic loci to determine the multiple state of an individual, when a DNA sample is prepared for gene measurement, the relatives of the alleles in the DNA sample It is desirable to store a suitable amount. This preparation amplifies the amount of WGA amplification, targeted amplification, selective enrichment techniques, hybrid capture methods, cyclized probes or DNA, and / or selectively the presence of DNA molecules corresponding to specific alleles. It may include other methods intended to be augmented.

In some embodiments of the present disclosure, there is a set of DNA probes designed to target the loci with the highest minor allele frequency. In some embodiments of the present disclosure, there is a set of probes designed to target the loci with the highest likelihood that the fetus has a highly informative SNP at those loci. In some embodiments of the present disclosure, there is a set of probes designed for the probe to target a locus optimized for a given population subgroup. In some embodiments of the present disclosure, there is a set of probes designed for the probe to target a locus optimized for a given mixture of population subgroups. In some embodiments of the present disclosure, the probe targets a locus optimized for a given parent pair from different population subgroups with different minor allele frequency profiles. There is a set of designed probes. In some embodiments of the present disclosure, there is a circularized DNA strand containing at least one base pair annealed with a portion of DNA of fetal origin. In some embodiments of the present disclosure, there is a circularized DNA strand containing at least one base pair annealed with a portion of DNA of placental origin. In some embodiments of the present disclosure, there is a circularized DNA strand that is circularized while at least a portion of the nucleotide is annealed with DNA of fetal origin. In some embodiments of the present disclosure, there is a cyclized DNA strand that is cyclized while at least a portion of the nucleotide is annealed with placental-origin DNA. In some embodiments of the present disclosure, there is a set of probes, some targeting a single tandem repeat and some targeting single nucleotide polymorphisms. In some embodiments, loci are selected for non-invasive prenatal diagnosis. In some embodiments, probes are used for non-invasive prenatal diagnosis. In some embodiments, loci are targeted using methods that may include circularization probes, MIPs, captures with hybridization probes, probes on SNP arrays, or combinations thereof. In some embodiments, the probe is used as a cyclized probe, MIP, capture by a hybridization probe, a probe on an SNP array, or a combination thereof. In some embodiments, loci are sequenced for non-invasive prenatal diagnosis.

For mixed samples, by maximizing the number of sequence reads containing SNPs for which the parental situation is known, if the relative information value of the sequence is greater when combined with the relevant parental situation. The information value of the set of sequencing leads of can be maximized. In certain embodiments, the number of sequence reads containing SNPs whose parental status is known can be enhanced by preferentially amplifying specific sequences using qPCR. In certain embodiments, the number of sequence reads containing SNPs whose parental status is known can be enhanced by preferentially amplifying specific sequences using a circularized probe (eg, MIP). In certain embodiments, the number of sequence reads containing SNPs whose parental status is known can be enhanced by preferentially amplifying specific sequences using hybridization capture (eg, SURESELECT). .. Different methods can be used to increase the number of sequence reads containing SNPs whose parental status is known. In certain embodiments, targeting can be achieved by extended ligation, non-extended ligation, hybridization capture or PCR.

In a sample of fragmented genomic DNA, one fraction of the DNA sequence is uniquely mapped to each chromosome and the other DNA sequence is found on a different chromosome. It should be noted that the DNA found in plasma, whether of maternal or fetal origin, is generally fragmented to lengths below 500 bp. In a typical genomic sample, approximately 3.3% of the mappable sequence is mapped to chromosome 13 and 2.2% of the mappable sequence is mapped to chromosome 18 and 1.35 of the mappable sequence. % Is mapped to chromosome 21, 4.5% of the mappingable sequences are mapped to the X chromosome in women, 2.25% of the mappingable sequences are mapped to the X chromosome (in men), and mapping is possible. 0.73% of the sequence is mapped to the Y chromosome (in men). These are the chromosomes most likely to be aneuploid in the fetus. Further, when the SNP contained in the dbSNP is used, about one out of 20 sequences in the short sequence contains the SNP. This percentage can be higher if there can be many undiscovered SNPs.

In one embodiment of the present disclosure, a targeting method is used to capture a portion of the DNA in a sample of DNA that is mapped to a given chromosome, the portion of which is a percentage typical of the genomic samples listed above. Can be enhanced to significantly exceed. In one embodiment of the present disclosure, a targeting method is used such that a percentage of a sequence containing SNP in a portion of DNA in a DNA sample significantly exceeds the percentage that can be typically found in a genomic sample. Can be enhanced to. In certain embodiments of the present disclosure, targeting methods can be used to target chromosome-derived DNA or DNA from a collection of SNPs in a mixture of maternal DNA and fetal DNA for prenatal diagnosis.

Count the number of reads mapped to the suspicious chromosome and compare it to the number of reads mapped to the reference chromosome, and the excess of leads on the suspicious chromosome is the fetal triploid on that chromosome. Note that methods for determining fetal aneuploidy by using the assumption of sex correspondence have been reported (US Pat. No. 7,888,017). These methods for prenatal diagnosis do not use any kind of targeting and do not describe the use of targeting for prenatal diagnosis.

By using targeting techniques in the sequencing of mixed samples, it may be possible to achieve a certain level of accuracy with a small number of sequence reads. Accuracy may refer to sensitivity, accuracy may refer to specificity, or accuracy may refer to some combination thereof. The desired level of accuracy may be between 90% and 95%, the desired level of accuracy may be between 95% and 98%, and the desired level of accuracy may be. It may be between 98% and 99%, the desired level of accuracy may be between 99% and 99.5%, and the desired level of accuracy may be between 99.5% and 99%. It may be between 9.9%, the desired level of accuracy may be between 99.9% and 99.99%, and the desired level of accuracy may be between 99.99% and 99%. It may be between .999% and the desired level of accuracy may be between 99.999% and 100%. A level of accuracy above 95% can be referred to as high accuracy.

A number of published prior art methods demonstrating how the ploidy state of the fetus can be determined from a mixed sample of maternal and fetal DNA, eg: G.M. J. W. Liao et al. Clinical Chemistry 2011; Vol. 57 (No. 1), pp. 92-101. These methods focus on thousands of locations along each chromosome. It can be targeted, while from a mixed sample of NA, for a given number of sequence reads, the number of locations along the chromosome that provides high accuracy in determining ploidy in the fetus is unexpectedly small. In certain embodiments of the present disclosure, accurate by using any method of targeting, eg, qPCR, ligand-mediated PCR, other PCR methods, targeted sequencing using a hybridization-captured or cyclized probe. Multiple determinations can be made, where the number of loci along the chromosome that needs to be targeted may be between 5,000 and 2,000 loci. It may be between 000 and 1,000 gene loci, it may be between 1,000 and 500 gene loci, and it may be between 500 and 300 gene loci. It may be a locus between 300 and 200, a locus between 200 and 150, and a locus between 150 and 100. , 100 to 50 gene loci, 50 to 20 loci, or 20 to 10 loci. Optimally, the number of loci along the chromosome that needs to be targeted can be between 100 and 500. A high level of accuracy can be achieved by targeting a small number of loci and performing an unexpectedly small number of sequence reads. The number of leads may be between 100 million and 50 million, the number of leads may be between 50 million and 20 million, and the number of reads may be from 20 million. The number of leads may be between 10 million, the number of leads may be between 10 million and 5 million, and the number of leads may be between 5 million and 2 million. There may be, the number of reads may be between 2 million and 1 million; the number of reads may be between 1 million and 500,000; the number of reads may be 500, It may be between 000 and 200,000, the number of reads may be between 200,000 and 100,000, and the number of reads may be between 100,000 and 50,000. The number of reads may be between 50,000 and 20,000, and the number of reads may be between 20,000 and 10,000. The number may be less than 10,000. Fewer reads are needed for larger amounts of input DNA.

In some embodiments, the composition comprises a mixture of fetal and maternal DNA, wherein the percentage of sequences uniquely mapped to chromosome 13 is greater than 4%, greater than 5%, greater than 6%. There are compositions that are greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the present disclosure, the composition comprises a mixture of fetal and maternal DNA, wherein the percentage of sequences uniquely mapped to chromosome 18 is greater than 3% and greater than 4%. There are compositions that are greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. .. In some embodiments of the present disclosure, the composition comprises a mixture of fetal and maternal DNA, wherein the percentage of sequences uniquely mapped to chromosome 21 is greater than 2% and greater than 3%. Compositions of more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 12%, more than 15%, more than 20%, more than 25% or more than 30% There are things. In some embodiments of the present disclosure, the composition comprises a mixture of fetal and maternal DNA, wherein the percentage of sequences that are uniquely mapped to the X chromosome is> 6%,> 7%, 8. There are compositions that are greater than%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the present disclosure, the composition comprises a mixture of fetal and maternal DNA, wherein the percentage of sequences that are uniquely mapped to the Y chromosome is greater than 1%, greater than 2%, and 3%. More than%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 12%, more than 15%, more than 20%, more than 25% or more than 30% There is a composition that is.

In some embodiments, the composition is described as comprising a mixture of DNA of fetal origin and DNA of maternal origin, and the percentage of sequences that are uniquely mapped to a chromosome and contain at least one single base polymorphism is More than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, 1% Super, over 1.2%, over 1.4%, over 1.6%, over 1.8%, over 2%, over 2.5%, over 3%, over 4%, over 5%, 6% Super,> 7%,> 8%,> 9%,>> 10%,> 12%,> 15% or> 20%, and chromosomes are selected from the group 13, 18, 21, X or Y. In some embodiments of the present disclosure, a composition comprising a mixture of embryonic and maternal origin DNA, at least one single nucleotide polymorphism from a set of single nucleotide polymorphisms that is uniquely mapped to a chromosome. The percentage of sequences containing molds is greater than 0.15%, greater than 0.2%, greater than 0.3%, greater than 0.4%, greater than 0.5%, greater than 0.6%, greater than 0.7%, More than 0.8%, more than 0.9%, more than 1%, more than 1.2%, more than 1.4%, more than 1.6%, more than 1.8%, more than 2%, more than 2.5%, More than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 12%, more than 15% or more than 20%, and the chromosome is thirteenth. The number of single nucleotide polymorphisms selected from the set of chromosomes, 18th chromosome, 21st chromosome, X chromosome and Y chromosome is between 1 and 10 and 10 to 20 in the set of single nucleotide polymorphisms. Between 20 and 50, between 50 and 100, between 100 and 200, between 200 and 500, between 500 and 1,000, from 1,000 Between 2,000, between 2,000 and 5,000, between 5,000 and 10,000, between 10,000 and 20,000, between 20,000 and 50, There are compositions between 000 and between 50,000 and 100,000.

Theoretically, each cycle of amplification doubles the amount of DNA present, but in practice the degree of amplification is slightly less than double. In theory, amplification, including targeted amplification, results in the amplification of an unbiased DNA mixture, but in practice, different alleles tend to be amplified to a different extent than other alleles. When amplifying DNA, the degree of allele bias generally increases with the number of amplification steps. In some embodiments, the methods described herein include the step of amplifying DNA with low levels of allele bias. Since the allele bias is compounded in each of yet another cycles, the allele bias per cycle can be determined by calculating the nth root of the overall bias, where n is wealth. It is a logarithm with a base of 2 for the degree of conversion. In some embodiments, a composition comprising a mixture of second DNA is present, the mixture of second DNA preferentially enriching multiple polymorphic loci from the mixture of first DNA. The degree of enrichment is at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000 or at least 1,000,000, and each gene in the second DNA mixture. The ratio of allogens at the locus averages less than 1,000%, 500%, 200%, 100%, 50%, 20% with the ratio of allogens at that locus in the first DNA mixture. Only the coefficients of 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02% or 0.01% are different. In some embodiments, there is a composition comprising a mixture of second DNA, which is preferentially enriched with multiple polymorphic loci from the mixture of first DNA. Here, the allele bias for multiple polymorphic loci per cycle is, on average, less than 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0. .1%, 0.05% or 0.02%. In some embodiments, the plurality of polymorphic loci is at least 10 loci, at least 20 loci, at least 50 loci, at least 100 loci, at least 200 loci, At least 500 loci, at least 1,000 loci, at least 2,000 loci, at least 5,000 loci, at least 10,000 loci, at least 20,000 genes Contains loci, or at least 50,000 loci.

Some Embodiments In some embodiments, methods for making a report disclosing a determined multiple state of a chromosome in a pregnant fetus are disclosed herein, said. The method is to obtain a first sample containing DNA from the mother of the fetal and DNA from the fetal, a step to obtain genotypic data from one parent or parent of the fetal, and a prepared sample. The step of preparing the first sample by isolating the DNA in the gene, the step of measuring the DNA in the sample prepared at multiple polymorphic loci, and the DNA measurement value obtained for the prepared sample. From the computerized step of calculating the number of allogens or the probability of allogens at multiple polymorphic loci, and the prediction of possible different multiple states on the chromosome at multiple polymorphic loci on the chromosome. Using genotypic data from one parent or parent of the fetus, for each of the steps to computerize multiple hypotheses about the number of allogens to be made, and for each of the hypotheses about the polymorphism, Multiplier using the steps to computerize a co-distribution model for the probability of allogens at each polymorphic locus on the chromosome, the co-distribution model for the prepared sample, and the calculated probability of allogens. The step of computer-determining the relative probability of each of the hypotheses about the gene and the step of calling the chromosomal state of the fetal by selecting the chromosomal state corresponding to the hypothesis with the highest probability. Includes steps to produce a report in which the polymorphic state is disclosed.

In some embodiments, the method is used to determine the polymorphic state of multiple pregnant fetuses in each of the plurality of mothers, wherein the method is of DNA of fetal origin in each of the prepared samples. Further including a step of determining the percentage, where the steps of measuring the DNA in the prepared sample are performed by sequencing a number of DNA molecules in each prepared sample to obtain a larger fraction of fetal DNA. More sequenced DNA molecules from the prepared sample with a smaller fraction of fetal DNA than the prepared sample with.

In some embodiments, the method is used to determine the polymorphic state of a plurality of pregnant fetuses in each of the plurality of mothers, wherein the step of measuring the DNA in the prepared sample is: For each fetus, the first fraction of the sample prepared with DNA is sequenced to obtain a set of first measurements, the method of which takes into account the set of first DNA measurements. Then, the step of determining the first relative probability for each of the hypothesis about the multipleness of each fetus and the determination of the first relative probability for each of the hypothesis about the polymorphism are aneuploids. The second fraction of the prepared sample from the fetal, showing that the hypothesis about the polypality corresponding to the fetal has a significant but inconclusive probability, is reordered and the second A second relative probability to the hypothesis about fetal multiples, using the step of obtaining a set of measurements and, optionally, a set of second measurements and, if necessary, a set of first measurements. The second sample was reordered by selecting the step of making the determination and the polymorphic state corresponding to the hypothesis with the highest probability as determined by the determination of the second relative probability, the fetus. Further includes a step of calling a state of multiples of.

In some embodiments, the composition comprises a sample of preferentially enriched DNA, wherein the sample of preferentially enriched DNA is a plurality of polymorphic genes from the first sample of DNA. It is preferentially enriched at the locus, and the sample of the first DNA consists of a mixture of maternal DNA and fetal DNA derived from maternal plasma, the degree of enrichment is at least twice that of the first sample. On average, alligator bias between preferentially enriched samples is less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, 0.05%. A composition selected from the group consisting of less than, less than 0.02%, and less than 0.01% is disclosed. In some embodiments, methods for making samples of such preferentially enriched DNA are disclosed.

In some embodiments, it is a method for determining the presence or absence of fetal variability in a maternal tissue sample comprising fetal genomic DNA and maternal genomic DNA, wherein (a) the maternal tissue sample. From the steps of obtaining a mixture of fetal genomic DNA and maternal genomic DNA, (b) selectively enriching the mixture of fetal DNA and maternal DNA in a plurality of polymorphic allogens, and (c) step a. With the step of selectively distributing enriched fragments from a mixture of fetal and maternal genomic DNA in a reaction sample containing a single genomic DNA molecule or an amplification product of a single genomic DNA molecule. , (D) A step of performing large-scale parallel DNA sequencing on a fragment of selectively enriched genomic DNA in a reaction sample in step c) to determine the sequence of the selectively enriched fragment. And (e) the step of identifying the chromosome to which the sequence obtained in step d) belongs, and (f) the data from step d) are analyzed to determine that i) is diploid in both the mother and the fetus. Inferred, the number of pieces of genomic DNA from step d) belonging to at least one first target chromosome, and ii) belonging to a second target chromosome suspected to be aneuploid in the fetal step. The step of determining the number of pieces of genomic DNA from d) and (g) the number determined in step f) part i) for the second target chromosome if the second target chromosome is positive multiple. To calculate the predicted distribution of the number of pieces of genomic DNA from step d), and (h) for the second target chromosome if the second target chromosome is aneuploid. f) A step of calculating the predicted distribution of the number of pieces of genomic DNA from step d) using the first number of part i) and the estimated proportion of fetal DNA found in the mixture of step b). And (i) the number of pieces of genomic DNA determined in step f) part ii) using the most probable or maximal ex post facto method was calculated in step g) distribution or step h). Methods are disclosed that include a step of determining which part of the distribution is more likely to be, thereby indicating the presence or absence of fetal chromosomal chromosomal.

Typical Cancer Diagnostic Methods It should be noted that it has been demonstrated that DNA originating from a living cancer in the host can be found in the host's blood. Just as a genetic diagnosis can be made by measuring the mixed DNA found in the maternal blood, a genetic diagnosis can be made equally well by measuring the mixed DNA found in the host blood. .. Genetic diagnosis can include aneuploidy states or genetic mutations. Any claim in the disclosure that reads in determining fetal ploidy or genetic status from measurements made on maternal blood is the cancer's ploidy or genetic status from measurements on host blood. In determining, it can be read equally well.

In some embodiments, the methods of the present disclosure allow the determination of a multiple state of cancer, wherein the method comprises a mixed sample containing a host-derived genetic material, and a cancer-derived genetic material. Using the steps obtained, the step of measuring the DNA in the mixed sample, the step of calculating the proportion of DNA of cancer origin in the mixed sample, and the measured values obtained and the calculated proportion for the mixed sample. Includes steps to determine the multiple status of the cancer. In some embodiments, the method may further comprise the step of giving cancer treatment based on the determination of the ploidy state of the cancer. In some embodiments, the method may further comprise the step of giving the cancer treatment based on the determination of the multiple state of the cancer, the cancer treatment being a drug, a biotherapeutic agent, and an antibody. Selected from groups that include based treatments and combinations thereof.

Typical Implementation Methods Any of the embodiments disclosed herein are in or on digital electronic circuits, integrated circuits, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, or them. It can be executed in the combination of. The apparatus of the embodiments disclosed herein can be performed in a computer program product materially embodied in a machine-readable storage device for execution by a programmable processor, the embodiment disclosed herein. The steps of the method of embodiment can be performed by a programmable processor that executes a program of instructions for performing the functions of the embodiments disclosed herein by manipulating the input data and producing the output. .. The embodiments disclosed herein can be advantageously executed in one or more computer programs executable and / or interpretable in a programmable system comprising at least one programmable processor, said at least. One programmable processor is coupled with a storage system, at least one input device, and at least one output device for receiving and transmitting data and instructions, which may be special or general purpose. Each computer program can be implemented in a high-level procedural or object-oriented programming language, or in assembly or machine language, if desired, and in any case the language is a compiler or interpreted language. May be. The computer program can be deployed in any form, including as a stand-alone program or as a module, component, subroutine or other unit suitable for use in a computer environment. Computer programs can be deployed to be executed or interpreted on a single computer or multiple computers distributed in one place or across multiple places and interconnected by a communication network.

Computer-readable storage medium, as used herein, refers to, but is not limited to, a physical or tangible storage device (as opposed to a signal), a tangible storage device for information. For example, it refers to volatile and non-volatile, removable and non-removable media implemented in any method or technique of computer-readable instructions, data structures, program modules or other data. Computer-readable storage media include, but are not limited to, RAMs, ROMs, EPROMs, which can be used to substantially store desired information or data or instructions and can be accessed by a computer or processor. EEPROM, flash memory or other solid state storage technology, CD-ROM, DVD or other optical storage device, magnetic cassette, magnetic tape, magnetic disk storage device or other magnetic storage device or any other physical or tangible The medium of.

Any of the methods described herein may include outputting data in physical form, such as printing on a computer screen or paper. The description of any embodiment elsewhere in this document may be combined with the steps described to output ready-to-use data in a format that allows the physician to act on it. Should be understood. In addition, the methods described can be combined with clinical treatment, or the actual execution of a clinical decision that results in the execution of a clinical decision not to take action. Some of the embodiments described in this document for determining genetic data relating to a target individual are required in combination with the decision selected to transfer one or more embryos in the context of IVF. Accordingly, it can be combined with the process of transferring the embryo into the uterus of a future mother. Some of the embodiments described in this document for determining genetic data associated with a target individual are combined with a medical professional's notification of a potential chromosomal abnormality or its absence, and if necessary, prenatal. In the context of diagnosis, it can be combined with the decision to miscarriage or not miscarriage the fetus. Some of the embodiments described herein are combined with the output of readily available data and the execution of clinical decisions that result in the execution of clinical treatments or clinical decisions not to be taken. be able to.

Representative Diagnostic Box In certain embodiments, the present disclosure includes a diagnostic box capable of partially or completely performing any of the methods described in the present disclosure. In certain embodiments, the diagnostic box may be placed in any suitable location reasonably close to the examination room, hospital laboratory or place of care for the patient. The box may allow the entire method to be performed in a fully automated manner, or the box may require one or more steps for the technician to complete manually. In certain embodiments, the box may be able to analyze genotype data measured at least for maternal plasma. In certain embodiments, the box can be coupled with a means of transmitting the genotype data measured in the diagnostic box to an external computing facility that then analyzes the genotype data and, in some cases, also produces reports. The diagnostic box may include a robotic unit capable of transferring an aqueous or liquid sample from one container to another. The diagnostic box may contain a number of reagents, both solid and liquid. The diagnostic box may include a high throughput sequencer. The diagnostic box may include a computer.

Experimental Section The embodiments disclosed herein are described in the following examples, which are described to aid in the understanding of the present disclosure and are defined in the claims that follow. Should not be construed as limiting the scope of. The following examples are presented to those skilled in the art to provide a complete disclosure and description of how the embodiments described herein are used, without limiting the scope of this disclosure to: It does not indicate that this experiment is all or the only experiment performed. Attempts have been made to ensure accuracy with respect to the numbers used (eg, quantity, temperature, etc.), but some experimental errors and deviations should be considered. Unless otherwise specified, parts are by volume and temperature is in degrees Celsius. It should be understood that modifications of the described method can be performed without altering the basic aspects that the experiment is meant to illustrate.

Experiment 1
The goal is to improve the accuracy of non-invasive prenatal trisomy diagnosis compared to published methods by the Bayes maximum likelihood estimation (MLE) algorithm, which calculates the fetal fraction using the parental genotype. It was to show that.
Sequencing data simulated for maternal cfDNA was generated by sampling the reads obtained in trisomy 21 and each maternal cell line. Accurate disomy and trisomy call rates are determined from published methods (Chiu et al. BMJ 2011: 342: c7401) and 500 simulations of various fetal fractions for our MLE-based algorithms. did. Simulations were validated by obtaining 5 million shotgun leads from four pregnant mothers and their respective fathers collected under an IRB-approved protocol. Parental genotypes were obtained on a 290KSNP array (see Figure 14).

In the simulation, the MLE-based method achieved 99.0% accuracy for a 9% low fetal fraction, reporting a reliability that corresponded well to the overall accuracy. We validated these results using four real samples, all with accurate calls and a calculated confidence of over 99%. In contrast, when the published algorithm of Chiu et al. Was run, 18% fetal fraction was required to achieve 99.0% accuracy, and 87.8% with 9% fetal DNA. Only the accuracy of was achieved.

Determining the fetal fraction from the genotype of the parent in conjunction with an MLE-based approach provides higher accuracy than published algorithms for the predicted fetal fractions in early and mid-pregnancy. Will be done. Moreover, the methods disclosed herein give rise to a very important confidence metric in determining the reliability of the results, especially in the low fetal fraction where ploidy is more difficult to detect. The published method uses a less accurate threshold method for calling ploidy based on a large set of disomy training data, which is a method of predefining false positive rates. In addition, without confidence metrics, there is a risk of reporting false-negative results if the fetal cfDNA is insufficient to make calls in a published manner. In some embodiments, confidence estimates are calculated for the called ploidy state.

Experiment 2
The purpose is to use targeted sequencing techniques in combination with parental genotype and HapMap data in the Bayesian maximum likelihood estimation (MLE) algorithm, especially in samples consisting of low fetal fractions, in trisomy 18 and trisomy 21 of the fetus. And to improve the non-invasive detection of trisomy X.

Maternal and maternal samples from four positive polyploid pregnancies and two trisomy-positive pregnancies and their respective paternal samples were obtained from patients with known fetal karyotypes under IRB-approved protocols. Maternal cfDNA was extracted from plasma and preferentially enriched with specific SNPs of the target, after which approximately 10 million sequence reads were obtained. The parent sample was sequenced in the same manner to obtain a genotype.

The algorithm described accurately called the chromosome 18 and chromosome 21 dysomy for all normal chromosomes in the positive and aneuploid samples. The calls for trisomy 18 and trisomy 21 were accurate, and the number of X-chromosome copies in male and female fetuses was also accurate. The confidence generated by the algorithm exceeded 98% in all cases.

All of the chromosomes tested from 6 samples, including samples composed of less than 12% fetal DNA, which account for approximately 30% of early and early pregnancy samples by the methods described. The ploidy was reported accurately for. A crucial difference between the MLE algorithm and the published method is that the MLE algorithm leverages parental genotype and HapMap data to improve accuracy and generate confidence metrics. With a low fetal fraction, the accuracy of all methods is low; it is important to accurately identify samples that do not have sufficient fetal cfDNA and make reliable calls. Others used a Y-chromosome-specific probe to estimate the fetal fraction of a male fetus, but by simultaneously genotyping the parent, the fetal fraction can be estimated for both sexes. .. Another inherent limitation of the published method using untargeted shotgun sequencing is that the accuracy of polyploid calls varies between chromosomes due to different factors such as GC rich. The targeted sequencing technique is largely independent of such chromosomal scale variations, resulting in more consistent performance between chromosomes.

Experiment 3
The purpose is to determine whether trisomy can be reliably detected in triploid fetuses using novel informatics for analyzing the SNP loci of floating fetal DNA in maternal plasma. Met.

After ultrasound abnormalities, 20 mL of blood was withdrawn from the pregnant patient. After centrifugation, maternal DNA was extracted from the buffy coat (DNEASY, QIAGEN) and cell-free DNA was extracted from plasma (QIAAMP QIAGEN). Targeted sequencing was applied to the SNP loci on chromosomes 2, 21, and X in both DNA samples. Maximum likelihood Bayes estimation selected the most probable hypothesis from a set of all possible ploidic states. The method determines fetal DNA proportions, ploidy status and definite confidence in determining ploidy. No assumptions are made regarding the ploidy of the reference chromosome. The diagnosis uses the current state of the art, test statistic independent of the number of sequence reads.

By this method, trisomy of chromosome 2 and chromosome 21 was accurately diagnosed. The proportion of offspring was estimated to be 11.9% [CI 11.7 to 12.1]. The fetus was found to have one maternal copy and two paternal copies of chromosomes 2 and 21 with a confidence of effectively 1 (error probability < ^10-30 ). This was achieved using 92,600 and 258,100 reads on chromosomes 2 and 21, respectively.

This is the first demonstration of a non-invasive prenatal diagnosis that the fetus is triploid, a trisomy chromosome derived from maternal blood, as confirmed by the metaphase karyotype. Existing methods of non-invasive diagnosis do not detect aneuploidy in this sample. Current methods rely on extra sequence reads on the trisomy chromosome compared to the disomy reference chromosome, whereas triploid fetuses do not have a disomy reference. Moreover, existing methods do not provide similarly reliable ploidy determinations using this proportion of fetal DNA and the number of sequence reads. It is easy to extend the technique to all 24 chromosomes.

Experiment 4
Using standard PCR (meaning no nesting) of DNA isolated from maternal plasma from positive polyploid pregnancy, and genomic DNA from 21 triploid cell lines as well. The following protocol was used for the 800-plex amplification. Library preparation and amplification was accompanied by single tube blunting and subsequent A-tailing. Adapter ligation was performed using the ligation kit found in the Agilent SURESELECT kit and PCR was performed for 7 cycles. Then, 15 cycles of STA were performed using 800 different primer pairs targeting SNPs on chromosomes 2, 21 and X (95 ° C for 30 seconds; 72 ° C for 1 minute; 60 ° C. 4 minutes; 1 minute at 65 ° C; 30 seconds at 72 ° C). The reaction was carried out at a primer concentration of 12.5 nM. The DNA was then sequenced using the ILLUMINA IIGAX sequencer. The sequencer output 1.9 million reads, 92% of which were mapped to the genome, and more than 99% of the reads mapped to the genome were mapped to one of the regions targeted by the targeted primers. .. The numbers were basically the same for both plasma DNA and genomic DNA. FIG. 15 shows the ratio of two alleles for about 780 SNPs detected by the sequencer in genomic DNA obtained from a cell line with a known trisomy on chromosome 21. Note that the allele distribution is not easy to read visually, so the allele ratios are plotted here for ease of visualization. Circles indicate SNPs on the disomy chromosome, and stars indicate SNPs on the trisomy chromosome. FIG. 16 is another representation of the same data, as in FIG. X, where the Y-axis is the relative number of A and B measured for each SNP, and the X-axis separates the SNPs by chromosome. The number of SNPs. In FIG. 16, SNPs 1-312 are found on chromosome 2, SNPs 313-605 are found on the trisomy 21st chromosome, and SNPs 606-800 are found on the X chromosome. Data from chromosomes 2 and X show disomy chromosomes, as the relative sequence counts are within the three clusters: AA at the top of the graph and BB at the bottom of the graph. , The center of the graph is AB. Data from chromosome 21, trisomy, show four clusters: AAA at the top of the graph, AAA around the line (2/3) at 0.65 ,. ABB is around the line (1/3) of 35, and BBB is at the bottom of the graph.

17A-D show data for the same 800-plex protocol, measured for DNA amplified from four plasma samples from pregnant women. For these four samples, seven clusters of dots are expected to be found: (1) Along the top of the graph are loci where both the mother and the fetus are AA. (2) Slightly below the top of the graph is the locus where the mother is AA and the fetal is AB, and (3) Slightly above the line 0.5 is the mother is AB. The locus where the fetus is AA and (4) along the line 0.5 is the locus where both the mother and the fetus are AB and (5) slightly of the line 0.5. Below is the locus where the mother is AB and the fetus is BB, and (6) slightly above the bottom of the graph is the locus where the mother is BB and the fetus is AB. 1) At the bottom of the graph is the locus where both the mother and the fetus are BB. The smaller the fetal fraction, the smaller the separation between clusters (1) and (2), between clusters (3), (4) and (5), and between clusters (6) and (7). Separation is expected to be half the fraction of DNA of fetal origin. For example, if 20% of the DNA is fetal and 80% is maternal, (1)-(7) are 1.0, 0.9, 0.6, 0.5, 0.4, respectively. Expected to concentrate on 0.1 and 0.0; see, for example, Figure 17D, POOL1_BC5_ref_late. Instead, if 8% of the DNA is fetal and 92% is maternal, (1)-(7) are 1.00, 0.96, 0.54, 0.50, 0. Expected to concentrate on 46, 0.04 and 0.00; see, for example, Figure 17B, POOL1_BC2_ref_rate. If fetal DNA is not detected, then (2), (3), (5) or (6) is not expected to be present; or the separation can be said to be 0, so (1) and (2) are At the top of each other, (3), (4) and (5), as well as (6) and (7); see, for example, Figure 17C, POOL1_BC7_ref_rate. Note that for FIG. 17A, POOL1_BC1_ref_rate, the fetal fraction is about 25%.

Experiment 5
By the majority of methods of DNA amplification and measurement, two alleles commonly found at loci are detected with intensities or counts that do not represent the actual amount of alleles in the DNA sample. Allele bias occurs. For example, for a single individual, at the heterozygous locus, a 1: 1 ratio of the two alleles, which is the expected theoretical ratio for the heterozygous locus, is expected. , 55:45 or even 60:40 can be observed due to the bias of alleles. It should also be noted that in the context of sequencing, simple stochastic noise can result in significant allele bias in low read depths. In certain embodiments, it is possible to model the behavior of each SNP, and thus if a consistent bias is observed for a particular allele, this bias can be corrected. FIG. 18 shows the percentage of data that can be explained by the binomial variance before and after the bias correction. In FIG. 18, the asterisk indicates the allele bias observed in the raw sequence data for the 800-plex experiment, and the circles indicate the corrected allele bias. Note that the data are expected to follow the x = y line if there is no allele bias. A similar set of data generated by amplifying DNA using 150-plex targeted amplification yielded data contained in the immediate vicinity of the 1: 1 line after bias correction.

Experiment 6
Universal amplification of DNA using adapters ligated with adapter tag-specific primers, where primer annealing and elongation times are limited to minutes, has the effect of enriching the proportion of shorter DNA strands. The majority of library protocols designed to generate DNA libraries suitable for sequencing include such steps, and examples of the protocol are publicly available and well known to those of skill in the art. In some embodiments of the invention, an adapter with a universal tag is ligated to plasma DNA and amplified using primers specific for the adapter tag. In some embodiments, the universal tag may be the same tag used for sequencing, it may be a universal tag solely for PCR amplification, or it may be a collection of tags. This method has the effect of enriching the proportion of fetal DNA in the mixture, as fetal DNA is generally short in nature, while maternal DNA can be both short and long in nature. Floating DNA, which is thought to be of apoptotic cell-derived DNA and contains both fetal and maternal DNA, is short, mostly less than 200 bp. Cellular DNA released by cytolysis, a common phenomenon after venous incision, is generally almost exclusively matrilineal and is also fairly long, mostly above 500 bp. Therefore, blood samples left for more than a few minutes contain a mixture of short (fetal + maternal) DNA and longer (maternal) DNA. By performing universal amplification on maternal plasma with a relatively short elongation time and then targeting amplification, the relative proportion of fetal DNA is compared to plasma amplified using targeted amplification alone. Tends to increase. This is measured when the fetal percent vs. input DNA measured when the input is plasma DNA (vertical axis) is plasma DNA with a library prepared using the ILLUMINA GAIIx library preparation protocol. It can be seen in FIG. 19, which shows the percentage of plasma. All points below the line indicate that the library preparation step enriches the fraction of DNA of fetal origin. The two plasma samples that were red showed hemolysis, thus indicating that cytolysis increased the amount of long maternal DNA present, which was the case when library preparation was performed prior to targeted amplification. It is shown that the fetal fraction is particularly significantly enriched. The methods disclosed herein result in several other situations in which cells with hemolysis or containing relatively long strands of contaminated DNA are lysed and contaminate a sample containing a mixture of short and long DNA. Especially useful if you have one. Generally, the relatively short annealing and extension times are between 30 and 2 minutes, but may be as short as 5 or 10 seconds or less, or as long as 5 or 10 minutes. ..

Experiment 7
A direct PCR protocol for DNA isolated from maternal plasma from a positive polyploid pregnancy, as well as genomic DNA from a 21 triploid cell line, and a 1,200 plex using a semi-nested technique as well. The following protocol was used for amplification. Library preparation and amplification was accompanied by single tube blunting and subsequent A-tailing. Adapter ligation was performed using the modification of the ligation kit found in the Agilent SURESELECT kit, and PCR was performed for 7 cycles. In the target primer pool, 550 assays for SNPs from chromosome 21 and 325 assays for SNPs from chromosome 1 and X, respectively, were performed. Both protocols involved 15 cycles of STA with a primer concentration of 16 nM (95 ° C. for 30 seconds; 72 ° C. for 1 minute; 60 ° C. for 4 minutes; 65 ° C. for 30 seconds; 72 ° C. for 30 seconds). .. The semi-nested PCR protocol was accompanied by a second amplification of the STA15 cycle with a forward tag concentration inside 29 nM and a reverse tag concentration of 1 μM or 0.1 μM (30 seconds at 95 ° C; 1 minute at 72 ° C; 60 ° C. for 4 minutes; 65 ° C. for 30 seconds; 72 ° C. for 30 seconds). The DNA was then sequenced using the ILLUMINA IIGAX sequencer. For direct PCR protocols, 73% of reads are mapped to the genome, and for semi-nested protocols, 97.2% of sequence reads are mapped to the genome. Therefore, it is speculated that the semi-nested protocol provides approximately 30% more information, primarily due to the elimination of the primers most likely to cause primer dimers.

Read depth variability tends to be higher when using the semi-nested protocol than when using the direct PCR protocol (see Figure 20), with diamonds being the read depth for loci performed using the semi-nested protocol. The square indicates the read depth for the locus performed without nesting. The SNPs are arranged by the lead depth for the diamonds, so all the diamonds are placed on the curve, while the squares appear to be loosely correlated; the placement of the SNPs is arbitrary and refers to the lead depth. It is the height of the dot, not the location from left to right of the dot.

In some embodiments, the methods described herein can achieve excellent read depth (DOR) dispersion. For example, in one version of this experiment (FIG. 21) using 1,200-plex direct PCR amplification of genomic DNA in the 1,200 assay, the 1186 assay has a DOR greater than 10 and an average read depth of 400. And in the 1063 assay (88.6%), the read depth is between 200 and 800, and the number of reads for each allele is high enough to obtain meaningful data, but for each allele. The number of reads in these leads had an ideal window, not too high, especially if the limit usage of these leads was small. Only 12 alleles had high read depths, with 1035 reads being the deepest. The standard deviation of the DOR was 290, the mean DOR was 453, the coefficient of variation of the DOR was 64%, there were 950,000 total reads, and 63.1% of the reads were mapped to the genome. In another experiment using the 1,200 plex semi-nested protocol (FIG. 22), the DOR was higher. The standard deviation of DOR was 583, the mean DOR was 630, the coefficient of variation of DOR was 93%, there were 870,000 total reads, and 96.3% of the reads were mapped to the genome. Note that in both of these cases the SNPs are arranged by the lead depth for the mother and therefore the curve indicates the lead depth of the maternal line. The distinction between the child and the father is not important, it is just a trend that is important for this explanation.

Experiment 8
In the experiment, a semi-nested 1,200-plex PCR protocol was used to amplify DNA from one cell and DNA from three cells. This experiment is associated with prenatal aneuploidy testing using fetal cells isolated from maternal blood, or for preimplantation genetic diagnosis using biopsy blastomeres or vegetative ectoderm samples. Is. There were 3 replicas of 1 cell and 3 cells from 2 individuals (46XY and 47XX + 21) for each condition. The assay targeted chromosomes 1, 21 and X. Three different dissolution methods were used: ARCTURUS, MPERv2 and alkaline dissolution. Sequencing was performed on 48 multiplexed samples in one sequencing lane. The algorithm yielded exact ploidy calls for each of the three chromosomes and for each of the replicas.

Experiment 9
In one experiment, four maternal plasma samples were prepared and amplified using the Heminestized 9,600 plex protocol. Samples were prepared as follows: Up to 40 mL of maternal blood was centrifuged to isolate the buffy coat and plasma. Genomic DNA of maternal samples was prepared from buffy coats and paternal DNA was prepared from blood or saliva samples. Cell-free DNA in maternal plasma was isolated using the QIAGEN CIRCULATING nucleoIC ACID kit and eluted with 45 μL of TE buffer according to the manufacturer's instructions. A universal ligation adapter was added to the end of each molecule of 35 μL of purified plasma DNA and the library was amplified over 7 cycles using adapter-specific primers. The library was purified using AGECOUNT AMPURE beads and eluted with 50 μl of water.

3 μl of DNA was amplified using 15 cycles of STA using a primer concentration of 14.5 nM for reverse primers with 9,600 target-specific tags and 500 nM for one library adapter-specific forward primer. (For the first polymerase activation at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds; 72 ° C. for 10 seconds; 65 ° C. for 1 minute; 60 ° C. for 8 minutes; 65 ° C. for 3 minutes and 72 ° C. 30 seconds; 15 cycles, and final elongation at 72 ° C. for 2 minutes).

The heminated PCR protocol is a 15-cycle STA with a dilution of the first STA product, a reverse tag concentration of 1000 nM, and a concentration of 16.6 unM for each of the 9,600 target-specific forward primers. With amplification of 2 (for the first polymerase activation at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds; 65 ° C. for 1 minute; 60 ° C. for 5 minutes; 65 ° C. for 5 minutes and 72 ° C. for 30 seconds. 15 cycles of seconds; and final elongation at 72 ° C. for 2 minutes).

An aliquot of STA product was then amplified by standard PCR over 10 cycles with 1 μM tag-specific forward and barcoded reverse primers to generate a barcoded sequencing library. did. A fixed amount of each library was mixed with libraries of different barcodes and purified using a spin column.

Thus, 9,600 primers were used in the single-well reaction; the primers were found on chromosomes 1, 2, 13, 13, 18, 21, 21, X and Y. Designed to target the SNPs. The amplified product was then sequenced using the ILLUMINA GAIIX sequencer. Approximately 3.9 million reads per sample are generated by the sequencer, 3.7 million reads are mapped to the genome (94%), of which 2.9 million reads (74%) are mapped to the target SNP, averaging. The lead depth was 344 and the median lead depth was 255. Fetal fractions for the four samples were found to be 9.9%, 18.9%, 16.3%, and 21.2%.

Relevant maternal and paternal genomic DNA samples were amplified and sequenced using a semi-nested 9600 plex protocol. The semi-nested protocol differs in that the first STA applies 9,600 outer forward and tagged reverse primers at 7.3 nM. The thermocycling conditions, the composition of the second STA, and the barcoding PCR were the same as for the heminated protocol.

Sequencing data were analyzed using the informatics methods disclosed herein to call for polyploid states on 6 chromosomes for fetuses in which DNA was present in 4 maternal plasma samples. Ploidy calls for all 28 chromosomes in the population were called correctly and confidence was called correctly, but exceeded 99.2% except for one chromosome, which had a confidence of 83%. ..

FIG. 23 shows the lead depth of the 9,600 plex heminesting method together with the lead depth of the 1,200 plex seminested method described in Experiment 7, with lead depths greater than 100, greater than 200 and greater than 400. The number of SNPs was significantly higher than that in the 1,200 plex protocol. The number of reads in the 90th percentile can be divided by the number of reads in the 10th percentile to obtain a dimensionless metric, which shows the uniformity of the lead depth; the smaller the number, the more uniform the lead depth. (Narrow). The average 90th percentile / 10th percentile ratio is 11.5 in the method performed in Experiment 9, but 5.6 in the method performed in Experiment 7. For sequencing efficiency, for a given protocol plexity, as less sequence reads are needed to ensure that the percentage of a particular read exceeds the read count threshold. Narrower lead depths are better.

Experiment 10
In one experiment, four maternal plasma samples were prepared and amplified using a semi-nested 9,600 plex protocol. The details of Experiment 10 were very similar to Experiment 9, with the exception of the nesting protocol and the inclusion of the identity of the four samples. Ploidy calls for all 28 chromosomes in the population were accurately called and confidence exceeded 99.7%. 7.6 million (97%) reads were mapped to the genome and 6.3 million (80%) of the reads were mapped to the target SNP. The average lead depth was 751 and the median lead depth was 396.

Experiment 11
In one experiment, three maternal plasma samples were divided into five even portions, each portion being divided into 2,400 multiplexed primers (4 moieties) or 1,200 multiplexed primers (1). Amplification was performed using any of the moieties (partial), and a total of 10,800 primers were amplified using a semi-nested protocol. After amplification, the moieties were pooled together for sequencing. The details of Experiment 11 were very similar to Experiment 9, with the exception of the nesting protocol and the split-and-pool method. For all 21 chromosomes in the population, polyploid call was called correctly and confidence was over 99.7% except for one with a confidence of 83% and one that did not work. 3.4 million leads were mapped to the target SNP, with an average lead depth of 404 and a median lead depth of 258.

Experiment 12
In one experiment, four maternal plasma samples were divided into four even portions, each portion amplified using 2,400 multiplexed primers for a total of 9,600 primers. Amplified using a semi-nested protocol. After amplification, the moieties were pooled together for sequencing. The details of Experiment 12 were very similar to Experiment 9, with the exception of the nesting protocol and the split-and-pool method. Ploidy calls for all 28 chromosomes in the population were accurately called, and confidence was over 97% with a confidence of 78%, except for one that did not work. 4.5 million leads were mapped to the target SNP, with an average lead depth of 535 and a median lead depth of 412.

Experiment 13
In one experiment, four matrilineal plasma samples were prepared and amplified using a 9,600-plex triple hemmened protocol for a total of 9,600 primers. The details of Experiment 12 are very similar to Experiment 9, with the exception of the nesting protocol with three rounds of amplification; the three rounds are 15 cycles of STA, 10 cycles of STA and 15 cycles, respectively. Accompanied by STA. Ploidy calls on 27 of the 28 chromosomes in the population were called correctly, with a confidence of 94.6%, one that was called correctly, and a confidence of 80.8%. Except for the one that did not go, it exceeded 99.9%. 3.5 million leads were mapped to the target SNP, the average lead depth was 414, and the median lead depth was 249.

Experiment 14 In one experiment, 45 cell aggregates were amplified and sequenced using a 1,200-plex seminested protocol, and ploidy determinations were made on three chromosomes. This experiment is intended to simulate the conditions under which preimplantation genetic diagnosis is performed in day 3 embryo-derived single cell biopsy material or day 5 embryo-derived ectodermal germ layer biopsy material. Please note that there is. Fifteen individual single cells and thirty aggregates of three cells were placed in 45 individual reaction tubes for a total of 45 reactions, each reaction containing cells from only one cell line. However, different reactions contained cells from different cell lines. Cells were prepared in 5 μl of wash buffer, lysed by adding 5 μl of ARCTURED BIOSYSTEMS, and incubated at 56 ° C for 20 minutes and 95 ° C for 10 minutes.

DNA from a single cell / 3 cells was amplified with 1200 target-specific forward and tagged reverse primers using a primer concentration of 50 nM using 25 cycles of STA (first polymerase). 25 ° C for 10 minutes at 95 ° C, then 95 ° C for 30 seconds; 72 ° C for 10 seconds; 65 ° C for 1 minute; 60 ° C for 8 minutes; 65 ° C for 3 minutes and 72 ° C for 30 seconds; Cycle, and final elongation for 2 minutes at 72 ° C.).

The semi-nested PCR protocol consists of 20 cycles of STA (95 ° C. for initial polymerase activation) using reverse tag-specific primers at a concentration of 1000 nM and 400 target-specific nested forward primers at a concentration of 60 nM each. 10 minutes, then 95 ° C. for 30 seconds; 65 ° C. for 1 minute; 60 ° C. for 5 minutes; 65 ° C. for 5 minutes and 72 ° C. for 30 seconds; 15 cycles, and 72 ° C. for 2 minutes final elongation). Along with three parallel second amplifications of the dilution of the first STA product. Therefore, in the three parallel 400-plex reactions, a total of 1200 targets amplified in the first STA were amplified.

An aliquot of STA product was then amplified by standard PCR over 15 cycles with 1 μM tag-specific forward and barcoded reverse primers to generate a barcoded sequencing library. did. A fixed amount of each library was mixed with libraries of different barcodes and purified using a spin column.

Thus, 1,200 primers were used in the single cell reaction; the primers were designed to target SNPs found on chromosomes 1, 21 and X. The amplified product was then sequenced using the ILLUMINA GAIIX sequencer. Approximately 3.9 million reads per sample were generated by the sequencer and 500-800 billion reads were mapped to the genome (74% -94% of all reads per sample).

Relevant maternal and paternal genomic DNA samples from the cell line, using the same semi-nested 1200-plex assay pool, using similar protocols, with fewer cycles and a second STA of 1200 plexes. Was analyzed and sequenced.

Sequencing data was analyzed using the informatics method disclosed herein to call the polyploid state on three chromosomes for the sample.

FIG. 24 shows the ratio of normalized read depths (vertical axis) on three chromosomes (1 = chromosome 1; 2 = chromosome 21; 3 = chromosome X) for the six samples. The ratio was set to be equal to the number of reads mapped to that chromosome, normalized and divided by the number of reads mapped to that chromosome, each averaging over 3 wells containing 3 46XY cells. The set of three data points corresponding to the 46XY reaction is expected to have a 1: 1 ratio. The set of three data points corresponding to 47XX + 21 cells is expected to have a ratio of 1: 1 for chromosome 1, 1.5: 1 for chromosome 21, and 2: 1 for chromosome X. ..

FIG. 25 shows the ratio of alleles plotted for three chromosomes (1, 21, X) for the three reactions. The reaction in the lower left shows the reaction in three 46XY cells. The region on the left is the allele ratio for chromosome 1, the central region is the allele ratio for chromosome 21, and the region on the right is the allele ratio for chromosome X. For 46XY cells, it is expected that for chromosome 1, ratios of 1, 0.5 and 0 corresponding to SNP genotypes AA, AB and BB will be observed. For 46XY cells, it is expected that for chromosome 21, ratios of 1, 0.5 and 0 corresponding to SNP genotypes AA, AB and BB will be observed. For 46XY cells, it is expected that for the X chromosome, ratios of 1 and 0 corresponding to SNP genotypes A and B will be observed. The reaction in the lower right shows the reaction in three 47XX + 21 cells. Allele ratios are separated by chromosome as in the lower left graph. For 47XX + 21 cells, it is expected that for chromosome 1, ratios of 1, 0.5 and 0 corresponding to SNP genotypes AA, AB and BB will be observed. For 47XX + 21 cells, it is expected that for chromosome 21, ratios of 1,0.67, 0.33 and 0 corresponding to SNP genotypes AAA, AAA, ABB and BBB will be observed. For 47XX + 21 cells, for the X chromosome, ratios of 1, 0.5 and 0 corresponding to SNP genotypes AA, AB, and BB are expected. The upper right plot was made for a reaction containing 1 ng of genomic DNA from the 47XX + 21 cell line. FIG. 26 shows the same graph as in FIG. 25, but for a reaction performed on only one cell. The graph on the left was a graph for a reaction containing 47XX + 21 cells, and the graph on the right was a graph for a reaction containing 46XX cells.

From the graphs shown in FIGS. 25 and 26, there are two clusters of dots for chromosomes for which a ratio of 1 and 0 is expected to be observed, and a ratio of 1, 0.5, and 0 is observed. There are 3 point clusters for chromosomes that are expected to be, and 4 point clusters for chromosomes that are expected to have ratios of 1, 0.67, 0.33 and 0. Is visually clear. The partial support algorithm allowed accurate calls to all three chromosomes in all 45 reactions.

Experiment 15
In one experiment, maternal plasma samples were prepared and amplified using the Heminestized 19,488 plex protocol. Samples were prepared as follows: Up to 20 mL of maternal blood was centrifuged to isolate the buffy coat and plasma. Genomic DNA of maternal samples was prepared from buffy coats and paternal DNA was prepared from blood or saliva samples. Cell-free DNA in maternal plasma was isolated using the QIAGEN CIRCULATING nucleoIC ACID kit and eluted with 50 μL of TE buffer according to the manufacturer's instructions. A universal ligation adapter was added to the end of each molecule of 40 μL of purified plasma DNA and the library was amplified over 9 cycles using adapter-specific primers. The library was purified using AGECOURT AMPURE beads and eluted with 50 μl DNA suspension buffer.

6 μl of DNA was amplified using 15 cycles of STAR1 using a primer concentration of 7.5 nM for the reverse primer with 19,488 target-specific tags and 500 nM for one library adapter-specific forward primer. (For the first polymerase activation at 95 ° C. for 10 minutes, then at 96 ° C. for 30 seconds; 65 ° C. for 1 minute; 58 ° C. for 6 minutes; 60 ° C. for 8 minutes; 65 ° C. for 4 minutes and 72 ° C. 30 seconds; 15 cycles, and final elongation at 72 ° C. for 2 minutes).

The heminated PCR protocol is a second of 15 cycles (STAR2) with a dilution of the first STAR1 product, a reverse tag concentration of 1000 nM, and a concentration of 20 nM for each of the 19,488 target-specific forward primers. With amplification (for the first polymerase activation at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds; 65 ° C. for 1 minute; 60 ° C. for 5 minutes; 65 ° C. for 5 minutes and 72 ° C. for 30 seconds; 15 cycles, and final elongation at 72 ° C. for 2 minutes).

An aliquot of STAR2 product was then amplified by standard PCR over 12 cycles with 1 μM tag-specific forward and barcoded reverse primers to generate a barcoded sequencing library. did. A fixed amount of each library was mixed with libraries of different barcodes and purified using a spin column.

Thus, 19,488 primers were used in the single-well reaction. Primers were designed to target SNPs found on chromosomes 1, 2, 13, 13, 18, 21, 21, X and Y. The amplified product was then sequenced using the ILLUMINA GAIIX sequencer. For plasma samples, approximately 10 million reads are generated by the sequencer, 9.4-9.6 million reads are mapped to the genome (94-96%), of which 99.95% are mapped to the target SNP. The average lead depth was 460 and the median lead depth was 350. Calculated for comparison, a perfectly even distribution is 10 million reads / 19,488 targets = 513 reads / targets. For the primer dimer, 30,000 reads (0.3% of the reads generated by the sequencer) were derived from the sequenced primer dimer. For genomic samples, 99.4-99.7% of the reads are mapped to the genome, 99.99% of which is mapped to the target SNP, and 0.1% of the reads generated by the sequencer are primers. It was a dimer.

For plasma samples of 10 million sequencing reads, 19,350 (99.3%) of at least 19,488 target SNPs are typically amplified and sequenced. For DNA samples of 2 million sequencing reads, at least 19,000 target SNPs (97.5%) are typically amplified and sequenced. The small numbers may be due to random sampling noise. The reason is that the number of reads is small and the sequencer misses some amplification products. If necessary, the number of sequencing reads can be increased to increase the number of targeted SNPs to be amplified and sequenced.

Relevant maternal and paternal genomic DNA samples were amplified using 7.5 nM semi-nested 19,488 outer forward and tagged reverse primers in STAR1. The thermocycling conditions, the composition of STAR2, and the barcoding PCR were the same as for the heminated protocol.

The average fetal fraction for 407 samples was found to be 14.8%. Sequencing data was analyzed using the informatics method disclosed herein and the four chromosomes of the fetal (13, 18, 21, Y) in which DNA was present in 378 of the 407 maternal plasma samples. , And the ploidy state of chromosome X in 375 of the 407 maternal plasma samples. Ploidy calls for all 1,887 chromosomes in the population were accurately called and confidence exceeded 90%. Of the 1887 calls, 1882 had a confidence of over 95%, and of the 1,887 calls, 1,862 had a confidence of over 99%.

A similar control experiment was performed by plasma PCR protocol using water instead of DNA extracted from plasma. In 6 such trial experiments, 5-6% of the sequencing leads were primer dimers. The other arranging leads were due to background noise. In this experiment, a small amount of primer dimer (without hybridizing to other primers to form an amplified primer dimer) even in the absence of a nucleic acid sample with a target locus to which the primer hybridizes. Is formed.

Experiment 16
The following experiments show a representative design and selection method of a primer library that can be used in any of the multiplex PCR methods of the invention. The goal is to select from the initial candidate primer library a primer that can be used to simultaneously amplify large numbers of target loci (or subsets of target loci) in a single reaction. For early candidate target locus sets, it is not necessary to design or select primers for each target locus. It is preferred that primers be designed and selected for as many desirable target loci as possible.

Step 1
A set of candidate target loci (eg, SNPs) can be referred to as the desired parameter of the target loci, eg, SNP frequency within the target population or the heterozygous rate of SNPs (ncbi.nlm.nih.gov/projects/SNP/worldweb; Shery ST, Ward MH, Khorodov M, et al. DBSNP: NCBI Database of Genetic Variations, Nucleic Acids Res. 2001 Jan 1; 29 (1): 308-11. Selected based on publicly available information about (included in the book). For each candidate locus, one or one using the Primer3 program (worldwide web of primer3.sourceforge.net; libprimer3 release 22.3, which are incorporated herein by reference in their entirety). Multiple PCR primer pairs have been designed. If there was no viable design for the PCR primer for a particular target locus, that target locus was excluded from further review.

Calculated based on "target locus score" (high score represents high desirability), eg, weighted average of desirable parameters for various target loci, as needed for almost or all target loci The target locus score may be calculated. The parameters can be assigned different weights depending on their importance to the particular application in which the primer is used. Typical parameters include heterozygosity of the target locus, prevalence associated with the sequence of the target locus (eg, polymorphism), disease penetration associated with the sequence of the target locus (eg, polymorphism), Includes the specificity of the candidate primer used to amplify the target locus, the size of the candidate primer used to amplify the target locus, and the size of the target amplification product.

Step 2
Thermodynamic interaction scores were calculated between each primer and all primers for all other target loci from step 1 (eg, Allawi, HT & SantaLucia, J., Jr. (1998), "Thermodys of International C-T Mismatches in DNA", Nucleic Acids Res. 26, 2694-2701; Peyret, N., Seneviratne, PA, Allawi, H. Jr. (1999), "Nearest-Neigbor Thermodynamics and NMR of DNA Sequences with Internal A-A, CC, GG, and T-T Missitches, Bioche. & SantaLucia, J., Jr. (1998), "Nearest-Neigbor Thermodynamics of International AC Mismatches in DNA: Sequence Depensence J., Jr. (1998), "Nearest Negativer Thermodynamic Parameters for International GA Mismatches in DNA", Biochemistry 37, 2170-2179; and Allawi, H. et al. T. & Santa Lucia, J. Santa. , Jr. (1997), "Thermodynamics and NMR of International GT Missatures in DNA", Biochemistry 36, 10581-10594; MultiPLX2.1 (Kaplinski L, Andoleson R, Puru) Bioinformatics. 2005 Apr 15; 21 (8): 1701-2, all of which are incorporated herein by reference). This step produces a 2D matrix of interaction scores. The interaction score predicted the likelihood of a primer dimer containing two interaction primers. The score was calculated as follows:
Interaction_score = max (-deltaG_2,0.8 * (-deltaG_1))
During the ceremony
deltaG_1 = Gibbs energy for a dimer where the 3'end of each primer anneals to the other primer (energy required to break the dimer); and deltaG_1 = at least one end Gibbs energy for a dimer that can be extended by PCR.

Step 3:
If there are more than one primer pair design for each target locus, one design is selected using the method below:
Find the worst (highest) interaction score for each primer pair design of one locus, for the two primers of that design and for all primers of all designs for all other target loci.
2 Select the design with the best (lowest) score among the worst interaction scores.

Step 4
The graph was constructed so that each node represents one locus and its associated primer pair design (eg, the maximum creek problem). One edge was formed between each node pair. Each edge was assigned a weight equal to the worst (highest) interaction score of the primers associated with the two nodes linked by the edge.

Step 5
If necessary, one primer from one design and one primer from another design for all design pairs of two different target loci that are likely to anneal to overlapping target regions. Added an additional edge between the nodes for the design of. The weights of these edges were set to be the same as the maximum weights assigned in step 4. Therefore, step 5 anneals to overlapping target regions and thus prevents the library from including primers that may interfere with each other during the multiplex PCR reaction.

Step 6
The initial interaction score threshold was calculated by:
Weight_threshold = max (edge_weight) -0.05 * (max (edge_weight) -min (edge_weight))
During the ceremony
max (edge_weight) is the maximum edge weight in the graph.
min (edge_weight) is the minimum edge weight in the graph.
The starting point of the threshold was set as follows:
max_weighted_threshold = max (edge_weighted)
min_weighted_threshold = min (edge_weighted)

Step 7
A new graph was constructed with only weighted edges that exceed the weighted_threshold value and consisting of nodes with the same settings as the graph in step 5. Therefore, this step ignores the interaction of scores below the weighted _threshold.

Step 8
Nodes (and all edges attached to the removed node) were removed from the flag in step 7 until there were no remaining edges. Each of the following steps was iteratively applied to remove the node:
1 Find the node with the highest angle (with the highest number of edges). If there are two or more, select any one.
2 Define a set of nodes consisting of the nodes selected above and all the nodes connected to them (excluding nodes with angles smaller than those selected above).
3 Select a node from the set with the lowest target locus score from Step 1 (a smaller score represents lower desirability). Remove that node from the graph.

Step 9
If the number of nodes remaining in the graph meets the number of target genes required for the multiplex PCR pool (within acceptable error), the method was continued in step 10.

If there was too much or too little left in the graph, a binary search was performed to determine what threshold generated the desired number of nodes remaining in the graph. If there were too many nodes in the graph, the weighted threshold boundaries were adjusted as shown below:
max_weight_threshold = weighted_threshold If not (if there are too few nodes in the graph), the weighted threshold boundary is adjusted as shown below:
min_weighted_threshold = weighted_threshold value Next, the weighted threshold was adjusted as shown in the following equation:
Weighted_threshold = (max_weighted_threshold value + min_weighted_threshold value) / 2
Steps 7-9 were repeated.

Step 10
The primer pair design associated with the remaining nodes in the graph was selected for the primer library. This primer library can be used by any of the methods of the invention.

If desired, this primer design and selection method can also be performed on primer libraries where only one primer (rather than a primer pair) is used to amplify the target locus. In this case, the node indicates the target locus per primer (rather than the primer pair).

Experiment 17
FIG. 27 is a graph comparing two primer libraries designed using the method of the invention. This graph shows the number of loci with a particular minor allele frequency targeted by each primer library. More primers were retained during the selection of the "new pool" library. This library contains more target loci, especially target loci with more relatively large minor allelic frequencies, for some methods of the invention, eg, detection of fetal chromosomal abnormalities. On the other hand, it enables amplification of alleles with higher information value).

These primer libraries were used in the following multiplex PCR method. Blood (20-40 mL) was collected from each subject into 2-4 CELL-FREE ™ DNA tubes (Streck). Plasma (minimum 7 mL) was isolated from each sample by a double centrifugation protocol at 2,000 g for 20 minutes followed by 3,220 g for 30 minutes and the supernatant was transferred after the first centrifugation. CfDNA was isolated from 7-20 mL plasma using the QIAGEN QIAamp Circulating Nucleic Acid kit and eluted with 45 uL of TE buffer. Pure maternal genomic DNA was isolated from the buffy coat obtained after the first centrifugation and pure paternal genomic DNA was similarly prepared from blood, saliva or buccal samples.

Using the 11,000 target-specific assay, maternal cfDNA, maternal genomic DNA, and paternal genomic DNA samples were pre-amplified 15 cycles and aliquots were transferred to a 15-cycle second PCR reaction using nested primers. Finally, samples were prepared for sequencing by adding barcoded tags in a third 12-cycle round of PCR. Therefore, one reaction amplified 11,000 targets, which contained SNPs found on the 13, 18, 21, X, and Y chromosomes. The amplification products were then sequenced using an ILLUMINA GAIIx or HISEQ sequencer. Parental genotypes were sequenced at a read depth shallower than the fetal genotype (about 20% of the cfDNA read depth).

Experiment 18
If desired, standard methods such as Agilent Technologies 2100 Bioanalyzer (FIG. 28A-M) can be used to analyze the size and quantity of PCR products. For example, the direct PCR method without nesting described herein was used for 2,400 plexes (FIGS. 28B-28G) and 19,488 plex experiments (FIGS. 28H-28M). The amounts of primers for FIGS. 28B-28D and 28H-28J were 10 nM. The amounts of primers in FIGS. 28E-28G and 28K-28M were 1 nM. At FIGS. 28B, 28E, 28H, and 28K, the amount of input DNA was 24 ng; at FIGS. 28C, 28F, 28I, and 28L, 80 ng; and at FIGS. 28D, 28G, 28J, and 28M, 250 ng. With more input DNA, a larger proportion of the desired 180 base pair products was produced. The peak at the 140 base pair position is the primer dimer product.

Experiment 19
Principle proof studies have demonstrated the detection of T13, T18, T21, 45, X, and 47, XXY with equal high accuracy across all chromosomes.

Pregnant couples were enrolled in a special prenatal care center based on a protocol approved by the Institutional Review Board in accordance with the Patient Region Act. The selection criteria were at least 18 years of age, a gestation period of at least 9 weeks, a single pregnancy, and a sign of informed consent. Blood samples were taken from the pregnant mother and blood or buccal samples were collected from the father. 2 pregnancies with T13 (Pateau syndrome), 2 pregnancies with T18 (Edwards syndrome), 2 pregnancies with T21 (Down syndrome), 2 pregnancies with 45, X, 2 cases 47 Pregnancy with XXY, and 90 normal pregnancies, were selected from a cohort of approximately 500 women prior to the test to test which chromosomal abnormality the method would detect. If postnatal child tissue was available, the karyotype of the normal fetus was confirmed by molecular karyotype analysis on the sample. Positive polyploid samples were taken prior to invasive testing of low-risk women. Positive polyploid samples were taken for at least 7 days after invasive testing and confirmed aneuploidy by cytogenetic karyotyping or fluorescent insights hybridization in separate laboratories.

Sample preparation and multiplex PCR
For the data in FIGS. 30A-E, 30G, 30H, and 31A-31G, sample preparation and 19,488 plex PCR were performed as described in Experiment 15. For the data in FIG. 30F, sample preparation and 11,000 plex PCR were performed as described in Experiment 17.

Methods and data analysis algorithms take into account parental genotype and transfer frequency data (eg, data from the Hapmap database), a large number of possible fetal multiple states, and various fetal cfDNA fractions. The predicted allelic distribution at the 19,488 polymorphic locus for is calculated (FIGS. 29A-29C). Unlike methods based on allelic ratios, the algorithm also takes into account linkage disequilibrium and uses a non-Gaussian distribution data model to show the predicted distribution of allelic measurements for SNPs with observation platform characteristics and amplification bias. The algorithm then compares the various predicted allele distributions to the actual allele distribution measured on the cfDNA sample (Fig. 29C) and based on the sequencing data to each hypothesis (monosomy, disomy, or trisomy, these). For that, there are many hypotheses based on various possible crossovers). The algorithm sums the likelihoods of each individual monosomy, disomy, or trisomy hypothesis (FIG. 29D) and calls a ploidy state with maximum total likelihood as a copy count and fetal fraction (FIG. 29E). Lab researchers were not blind to the sample karyotype, but the algorithm calls the ploidy state without human intervention, so it remains blind to the truth.

Interpretation of data Graph display of generated data To determine the ploidy state of the target chromosome, the algorithm is based on the number of sequences of two possible alleles at each of the 3,000-4,000 SNP positions per chromosome. Consider the distribution. It is important to note that the algorithm makes ploidic calls using techniques that are not useful for visualization. Therefore, for illustration purposes, the data are presented herein in a simplified manner as the ratio of the two most probable alleles labeled as A and B, thereby tending to be related. Can be visualized more easily. This simplified illustration does not take into account some features of the algorithm. For example, two important aspects of the algorithm that cannot be explained by the method of visualization that presents allele ratios are: 1) Linkage disequilibrium, that is, measurements at one SNP give possible unique properties of adjacent SNPs. The use of a non-Gaussian distribution data model to explain the ability to harness the effects and 2) the distribution of predicted allelic measurements for SNPs with platform characteristics and amplification bias. It should also be noted that the algorithm only considers the two most frequent alleles at each SNP position and ignores the other possible alleles.

The graphs of FIGS. 30A-30H include samples with 2, 1, or 3 fetal chromosomes. Usually, they show positive ploidy (FIGS. 30A-30C), monosomy (FIGS. 30D), and trisomy (FIGS. 30E-30H), respectively. In all plots, each spot represents a single SNP, and the target SNPs are plotted sequentially along the horizontal axis from left to right for a single chromosome. The vertical axis shows the number of reads for the A allele as the ratio of the total number of reads for both A and B alleles to the SNP. Measurements are made on total cfDNA isolated from maternal blood, cfDNA containing both maternal and fetal cfDNA, so each spot represents a combination of fetal and maternal DNA contributions to that SNP. Please note. Therefore, an increase in the proportion of maternal cfDNA from 0% to 100% will gradually move some spots up and down in the plot depending on the genotype of the maternal and fetal.

Where visualization needs to be facilitated, maternal genotypes contribute significantly to the localization of each spot, and most trisomy is maternally inherited, so spots are color-coded according to maternal genotype. It can, which is useful for visualization of ploidic states. Specifically, SNPs with a maternal genotype of AA can be shown in red, SNPs with a maternal genotype of AB can be shown in green, and SNPs with a maternal genotype of BB are shown in blue. be able to.

In all cases, SNPs that are homozygous (AA) from both the mother and the fetus to the A allele have a high A allele read ratio because the B allele should not be present, which is perfect for the upper limit of the plot. It is recognized that it is stuck. Conversely, SNPs that are homozygous for both the mother and fetus to the B allele should stick to the lower end of the plot because the A allele read ratio is small because only the B allele should be present. It is admitted that there is. Spots that do not stick to the upper and lower bounds of the plot indicate SNPs that are heterozygous for the mother, fetus, or both. While these spots are useful in identifying fetal ploidy, they can also be informative for determining paternal vs. maternal inheritance. These spots are separated based on both maternal and fetal genotypes and fetal fractions, so the detailed location of individual spots along each y-axis is both stoichiometric and fetal fractions. Depends on. For example, loci in which the mother is AA and the fetus is AB have different A allele read ratios and are therefore expected to take different positions along the y-axis depending on the fetal fraction.

Presence of Two Chromosomes Figures 30A-30C show when the sample is completely maternal (no fetal cfDNA: FIG. 30A), contains a medium fetal cfDNA fraction (FIG. 30B), or is high. Data showing the presence of two chromosomes when the fetal cfDNA fraction is included (FIG. 30C).

FIG. 30A shows data obtained from cfDNA isolated from the blood of non-pregnant women. If there is no fetal cfDNA and the sample contains only maternal cfDNA, the plot represents purely single nucleotide polymorphism, the whole mark pattern contains spot "clusters" and the red cluster is at the top of the plot. (SNPs with maternal genotype AA), blue clusters snugly attached to the bottom edge of the plot (SNPs with maternal genotype BB), and a single green cluster Centrally located (SNPs with maternal genotype AB) (not shown in color).

If fetal cfDNA is present, the spot location shifts so that the clusters separate into discrete "bands". For a sample of 0% fetal fraction, a group of spots is called a "cluster" (as in Figure 30A) and for all samples of a fetal fraction greater than 0%. It should be noted that a group of spots is called a "band" (as in FIGS. 30B-30J). If the fetal fraction is large enough, these discrete bands will be readily visible. Specifically, FIGS. 30B and 30C show characteristic patterns associated with two fetal chromosomes, each with a medium and high fetal fraction. This pattern is located at both the top (red) and bottom (blue) plots corresponding to the heterozygous SNPs in the mother and the homozygous SNPs in the mother, respectively. Includes two "peripheral" bands (not shown in color in the figure).

FIG. 30B shows data from cfDNA derived from a plasma sample containing a 12% fetal cfDNA fraction from a female carrying a positive polyploid fetus. Here, the clusters of spots that stick tightly to the top and bottom of the plot are: one red and one blue outer peripheral band that remains tightly stuck to the top or bottom of the plot, as well as the edge of the plot. Separated into two discrete bands, one red and one blue inner peripheral band away from (color not shown in the figure). These inner peripheral bands centered around 0.92 and 0.08 are SNPs with a maternal genotype of AA and a fetal genotype of AB (shown in red), and a fetal gene with a maternal genotype of BB. Represents SNPs (shown in blue) of type AB, respectively. The central cluster of green spots is widespread, but in this fetal fraction, the separation into clear bands is not readily visible.

In the high fetal cfDNA fraction, a typical pattern showing the presence of two chromosomes (three sets of green bands, and two red and two blue peripheral bands) is readily visible (colors shown in the figure). figure). FIG. 30C shows data obtained from a plasma sample from a female carrying a positive polyploid fetus for a 26% fetal cfDNA fraction. Here, the peripheral band separates the inner band and moves towards the center of the plot due to the altered level of the B allele from the increased fetal cfDNA fraction. It is significant that the separation of the central green cluster into three separate bands in the high fetal fraction is fairly easily visible at this point. The three central bands are clustered near 0.37, 0.50 and 0.63 in this case, with the maternal genotype AB and the fetal genotypes BB (bottom) and AB (center), respectively. ) And SNPs that are AA (upper).

These whole mark patterns, namely the three green bands and the four peripheral bands (two red and two blue), are as in the case of autosomal haploid or the X chromosome in a female (XX) fetus. Shows the existence of two chromosomes.

Existence of One Chromosome If the fetus inherits only a single chromosome and therefore only a single allele, then fetal heterozygosity is not possible. Therefore, the only possible fetal SNP identification information is A or B. Thus, the maternally inherited monosomy chromosome is the only one that remains tightly attached to the top and bottom of the plot, showing two central green bands showing the mother's heterozygous SNP and the mother's homozygous SNP, respectively. Has a characteristic pattern of red and blue peripheral bands (lead ratios of 1 and 0) (FIG. 30D) (colors not shown in the figure). Note that there is no inner peripheral band. This pattern indicates the presence of a single chromosome, as in the case of maternally inherited autosomal monosomy or the X chromosome of a male (XY) fetus.

Existence of 3 Chromosomes Trisomy chromosomes have 3 characteristic patterns. The first pattern shows maternally inherited meiotic trisomy (meiotic error) in which the fetus inherits two homologous, non-identical chromosomes from the mother (Fig. 30E). This pattern contains two central green bands, each with two red and blue peripheral bands (colors not shown in the figure). The second pattern shows meiotic trisomy inherited from the father, where the fetus inherits two homologous, non-identical father-derived chromosomes (Fig. 30F). This pattern contains four central green bands and three red and blue peripheral bands, respectively (colors not shown in the figure). The third pattern shows mitotic trisomy (mitotic error) inherited from the mother (Fig. 30G) or father (Fig. 30H), where the fetus has two identical mother or father-derived chromosomes. It is inherited. This pattern contains four central green bands, each with two red and two blue peripheral bands. Mitotic trisomy inherited from the mother and father can be identified by the placement adjacent to the red and blue bands. In the mitotic trisomy inherited from the father, the red and blue medial peripheral bands (bands not attached to the edges of the plot) are closer to the center (color not shown in the figure). This is due to the paternal contribution of the same chromosome. Note that our previous results show that at the cleavage stage, 66.7% of maternal trisomy is meiotic and only 10.2% of trisomy is paternally inherited.

For the Y chromosome, the PS method considers the existence of a separate hypothesis set: zero, one, or two chromosomes. If there is no maternal contribution to the sequence reads at each locus, heterozygous loci are not possible (two Y chromosome cases always contain two identical chromosomes), so the band is in the plot. It remains tightly attached to the upper end (A allogeneic gene) or the lower end (B alligator gene) (data not shown), making the analysis extremely simple and dependent on quantitative alligator number data. Note that since the method examines SNPs, homologous non-recombinant SNPs from the Y chromosome are used, thus obtaining data on both X and Y for a single probe pair.

Identification of Aneuploidy As mentioned above, identification of autosomal aneuploidy using this plot-based visualization method directly identifies plots with sufficient fetal fractions and an abnormal number of chromosomes. You just need to do it. By summing the information on the number of copies of the X and Y chromosomes, it is possible to identify whether or not sex chromosome aneuploidy exists. Specifically, plots representing fetuses with the 47, XXX genotype have a typical "three-chromosome" pattern, and plots representing fetuses with the 47, XXY genotype are typical for the X chromosome. It has a "two-chromosome" pattern, but also has an allogeneic read indicating the presence of one Y-chromosome. Similarly, the method can also call 47, XYY, where the "chromosome" pattern indicates the presence of a single X chromosome and the allelic read is the presence of two Y chromosomes. Is shown. 45, Fetuses with the X genotype have a "chromosome 1" pattern typical for the X chromosome, and the data show a zero Y chromosome.

Effects of fetal fractions As discussed above, the number of fetal-derived sequence reads gives the exact position of each spot along the y-axis of the plot. Since the fetal fraction affects the proportion of fetal and maternal leads, it also affects the positioning of each spot. At high proportions of fetal cfDNA in FIGS. 30C-30E and 30G and 30H (usually about 20%), spot clusters are primarily based on the maternal genotype, but are derived from alleles whose genotype is different from the maternal genotype. It is easy to see that the presence of fetal DNA in the cluster transforms the cluster into multiple distinct bands. However, as the fetal fraction decreases (as in FIGS. 30B and 30F), the spots return towards the ends and center of the plot, producing more packed clusters. Specifically, the set of red peripheral bands with maternal genotype AA returns toward the top of the plot, the set of blue peripheral bands with maternal genotype BB returns toward the bottom, and the mother is heterozygous. The set of green center bands in is compressed into a single cluster in the center of the plot (compare FIGS. 30B and 30C) (colors not shown in the figure). Aneuploidy is not easily visible when using this visualization technique for low fetal fractions, but the algorithm is polyploidy for very small fetal fractions, eg, 3% fetal fractions. The state can be identified. Using statistical techniques to compare observational data to a highly accurate data model that predicts allelic distribution for a given sample parameter set (eg, copy count, parent genotype, and fetal fraction, etc.) This makes this possible. In the case of small fetal fractions, the accuracy of the data model is important because the differences between allelic distributions for different ploidy states are proportional to the fetal fraction. In addition, the algorithm can determine if the dataset does not contain enough data to make a reliable fetal ploidy determination.

Results The mapped sequencing reads of the target SNP were considered informative and used in the algorithm. In the sequencing results, over 95% of target loci were observed. Plots that visualize significant ploidy calls are shown in FIGS. 31A-31G. FIG. 31A shows a positive polyploid sample. Here, chromosomes 13, 18, and 21 have a typical "two-chromosome" pattern (as described herein). It contains three sets of green central bands, and two red bands and two blue peripheral bands. It shows a positive polyploid XY genotype (not shown in color in the figure), along with the presence of two green central bands for the X chromosome and a Y chromosome band along the edge of the plot.

The most common autosomal trisomy T13, T18, and T21 are shown in plots in FIGS. 31B, 31C, and 31D, respectively. Specifically, FIG. 31B shows a T13 sample. Here, chromosomes 18 and 21 show a typical "two-chromosome" pattern, chromosome X shows a typical "one-chromosome" pattern, and there are reads from the Y chromosome. Overall, it shows disomy on chromosomes 18 and 21 and identifies the fetal XY genotype. However, specifically, chromosome 13 exhibits a typical "three-chromosome" pattern. Similarly, FIG. 31C shows a T18 sample and FIG. 31D shows a T21 sample.

The method can also detect sex chromosome aneuploidy, including 45, X (FIG. 31E), 47, XYY (FIG. 31F), and 47, XYY (FIG. 31G). Note that the method calls the number of copies on chromosomes 13, 18, 21, X, and Y, and the total number of chromosomes is reported assuming a disomy on the remaining chromosomes. .. 45 of the plot, the X chromosome region showing the X sample, indicates the presence of a single chromosome. However, in addition to the "two-chromosome" pattern for chromosomes 13, 18, and 21, the lack of reads from the Y chromosome points to the 45, X genotype. Conversely, the 47, XXY sample produces a plot showing the presence of the 2X chromosome. The data also showed reads for alleles from the Y chromosome. In addition to the two copies of chromosomes 13, 18, and 21, this points to the 47, XXY genotype. 47, XYY genotype is indicated by a "chromosome 1" pattern for the X chromosome and the presence of a read that represents the presence of two Y chromosomes.

Discussion The above method non-invasively detected T13, T18, T21, 45, X, 47, XYY, and 47, XYY from maternal blood. The method examines cfDNA from maternal plasma by targeted multiplex PCR amplification and high throughput sequencing of 19,488 SNPs. The method, in combination with the advanced informatics analysis of the method, which takes into account parental genotype information including fetal fraction and DNA quality and many sample parameters, more reliably detects fetal precursors and is the most of the seven species. Highly accurate polyploidy on all five chromosomes associated with the common types of birth aneuploidy (T13, T18, T21, 45, X, 47, XXX, 47, XYY, and 47, XYY). Make a call. The method offers many clinical advantages such as significantly greater clinical coverage and higher sample-specific computational accuracy (as well as individualized risk scores) over previous methods.

Expanding clinical scope Considering the ability to accurately detect autosomal trisomy and sex chromosome aneuploidy, the method is about twice as wide as the clinically available NIPT method. increase. The method presented herein is merely a non-invasive test that calls the ploidy of the sex chromosomes with high accuracy. Previous plasma mixing experiments and another plasma sample analyzed in our experimental assay suggest that the method can detect a larger sex chromosome aberration cohort containing 47,XXX. In addition, the methods presented herein can detect aneuploidies of chromosomes 13, 18, and 21 with high sensitivity and specificity, and with appropriate primer design, copy the rest of the chromosomes as well. It is expected that the number can be detected.

Calculation of Sample-Specific Accuracy Of particular significance is that the method calculates the sample-specific accuracy for the ploidy call of each chromosome in each sample. The accuracy calculated by the method is the wrong call ratio by identifying and marking individual samples with poor quality DNA or low fetal fractions that may yield low accuracy test results. Is expected to be greatly reduced. In contrast, methods based on large-scale shotgun sequencing (MPSS) make positive or negative calls using a single hypothesis rejection test, and their accuracy estimates are published rather than characteristic of individual samples. It is based on a survey cohort and is assumed to have the same accuracy as the cohort. However, the individual accuracy for samples with parameters at the tail of the cohort distribution can vary widely. This phenomenon is exacerbated in low fetal fractions, such as during early pregnancy, or for samples with low DNA quality. These samples are usually not identified and tagged for follow-up, which can result in missed calls. However, the present invention makes each chromosome copy number call taking into account many parameters such as fetal fractions and many DNA quality measures, and calculates sample-specific accuracy for that call. This allows individual samples with low accuracy to be identified and flagged for tracking using the method. This is expected to ensure that most calls are not overlooked, especially in the early stages of pregnancy when the fetal fraction is usually low. The premise is that without a call is much better than missing a call, as simply rebleeding and reanalyzing in the absence of a call.

であり、式中、Ｒ_１は前記方法により計算したリスクスコアで、Ｒ_２は第１期スクリーニング（ｆｉｒｓｔ ｔｒｉｍｅｓｔｅｒ ｓｃｒｅｅｎｉｎｇ）により計算したリスクスコアである。 Converting Calculated Accuracy to a Traditional Risk Score The method can provide a pre-prepared risk of heterogeneous risk for high-risk pregnant women, in which case the pre-risk is taken into account. (BennP, Cuckle H, Pergament E. Non-invasive prenatal diagnosis for Down syndrome: the paradigm will shift, but sluary. Will be incorporated). The method provides computational accuracy tailored to each patient, but for clinical use these accuracy can be changed to traditional risk scores. This also means the risk of ploidy pregnancy, but is expressed as a percentage. Traditional risk scores are provided with a risk score that takes into account various parameters such as maternal age-related risk and biochemical markers of serum levels, above which the mother is considered high risk and the appropriate mother In contrast, invasive diagnostic procedures for follow-up are recommended. The method significantly improves this risk score and thus reduces both false positive and false negative rates, giving a more accurate assessment of individual maternal risk. The computational accuracy used herein is the likelihood that the polyploid call is accurate and is expressed as a percentage, whereas the computational accuracy used in Experiment 19 does not include age-related risk. Since risk score calculations typically include age-related risks, calculation accuracy and traditional risk scores are incompatible and need to be combined to convert to traditional risk scores. The formula for combining age-related risk with computational accuracy is:

In the formula, R ₁ is the risk score calculated by the above method, and R ₂ is the risk score calculated by the first trimester screening.

SNP-based methods solve problems with amplification variability A unique drawback of the counting method used by some other methods is that it maps to the chromosome of interest (eg, chromosome 21) for the number of reads mapped to the reference chromosome. The polymorphic state of the fetal is determined by measuring the ratio of lead numbers. There are large variations in the amplification of chromosomes with high or low GC content, including chromosomes 13, X, and Y. This can result in signal variability as large as the fetal cfDNA signal, which can result in erroneous copy count calls by changing the ratio of target chromosome-derived allelic reads to reference chromosome-derived reads. be. This may result in low accuracy for chromosomes 13, X, and Y. Importantly, this problem is exacerbated in the case of low fetal cfDNA rates, which are more likely to occur during early pregnancy.

In contrast, SNP-based methods do not depend on consistent amplification levels between chromosomes and are therefore expected to give results of equal accuracy across all chromosomes. The method does not require the use of a reference chromosome because, by definition, only one nucleotide tests for the relative number of another allele at a different polymorphic locus, thereby quantifying leads. Prevents problems with interchromosomal amplification variability inherent in number-dependent methods. Unlike quantitative methods that require a positive polyploid reference chromosome, the method is expected to be able to detect triploid and uniparental disomy-like abnormalities that do not involve changes in copy count.

Importance of Early Detection Importantly, the prevalence of complex births of sex chromosome aneuploidy is higher than that of the most common autosomal aneuploidy (Fig. 32). However, there is currently no routine non-invasive selection method that can reliably detect sex chromosome abnormalities. Therefore, sex chromosome abnormalities are usually detected prenatally as a side effect of routine testing for Down's syndrome or other autosomal aneuploidies, and most cases are totally overlooked. Early accurate detection is very important for many of these disorders where early intervention improves clinical outcomes. For example, the overall prevalence of combined births of Turner syndrome is 1 in 2,500 women, but Turner syndrome is often undiagnosed until adolescence. Growth hormone therapy is known to prevent short stature due to the disorder, but if started before the age of four, the treatment will be significantly more effective. In addition, estrogen replacement therapy can stimulate secondary sexual characteristics in patients with Turner syndrome, but again, treatment should be initiated shortly before puberty, before the syndrome is usually detected. All of these underscore the importance of early, routine and safe detection of sex chromosome aneuploidy. The method provides the first method that has the potential to serve as a routine screening for sex chromosome abnormalities.

Additional Uses The method is ready to detect ultramicroscopic anomalies, such as microdeletion and microduplication, in a unique way to take advantage of target amplification. Although non-targeted methods such as MPSS have been shown to be able to detect DiGeorge microdeletion syndrome, the method requires a sufficiently high level of gene coverage to perform difficult procedures. ..
This is the reason why non-targeted amplification is orders of magnitude less efficient for the ultramicroscopic region when very low proportions of sequencing reads appear to be informative. Moreover, the fact that recently available methods have problems in accurately identifying ploidic states for sex chromosomes, for smaller chromosomal segments, those methods also pose a variability amplification problem. It suggests that you are facing.

Similarly, SNP-based methods are detected in current non-invasive methods that are count-dependent or conventional invasive methods such as amniocentesis and in CVS that rely on cytogenetic karyotype analysis and / or fluorescence in situ hybridization. It is possible to detect an abnormal UPD failure that cannot be accompanied by a change in the number of copies. This is because clinically available MPSS-based targeting methods amplify non-polymorphic loci and thus, for example, it is not possible to determine if a subject chromosome is of the same parental origin, whereas SNP-based methods The reason is that individual haplotypes can be specifically identified. This is because microdeletion / microduplication and UPD syndrome such as these Prader-Willi, Angelman, and Beckwith-Wiedemann syndromes are usually not diagnosed prenatally and are misdiagnosed early after birth. Means that there are many. As a result, treatment intervention is significantly delayed. In addition, because the method targets SNPs, the method also facilitates parental haplotype reconstruction and enables detection of fetal inheritance at individual disease chain loci (Kitzman JO, Snyder MW, Ventura). M, et al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012; 4: 137ra76, which is incorporated herein by reference in its entirety).

The results presented herein demonstrate that the scope of the method can be expanded for prenatal aneuploidy identification. Specifically, by amplifying and sequencing 19,488 SNPs, the method can determine the number of copies of chromosomes 13, 18, 21, X, and Y, and other chromosomal abnormalities, such as any other. It is expected to specifically detect triploids and UPDs that are not detected by clinically available non-invasive methods. The increased clinical scope and strong sample-specific computational accuracy suggest that the method can provide a useful adjunct to invasive testing for the detection of fetal chromosomal aneuploidy.

All patents, patent applications and publication references cited herein are incorporated herein by reference in their entirety. It will be appreciated that the methods of the present disclosure are described with particular embodiments thereof, but can be further modified. Further, the present disclosure includes deviations from the present disclosure that fall within the scope of known or customary practice in the art relating to the methods of the present disclosure and within the scope of the appended claims. It shall include any variation, use or adaptation of the method of. For example, any of the methods disclosed herein for DNA can be readily adapted to RNA by introducing a reverse transcription step to convert RNA into DNA. Examples using polymorphic loci for illustration can be easily adapted to amplification of non-polymorphic loci, if desired.

Claims (20)

A method for nested PCR amplification,
Cell-free DNA is isolated from the biological sample and the adapter is ligated to the isolated cell-free DNA, where each of the adapters comprises a universal priming site.
Simultaneously amplify at least 10 target loci by performing a first PCR within the first reaction volume with a first universal primer that binds to the universal priming site and at least 10 target-specific primers. Here, each target-specific primer forms a primer pair with the first universal primer to amplify one of the target loci.
By performing a second nested PCR using the universal primer and at least 10 inner target specific primers, the at least 10 target loci were simultaneously amplified and amplified within the second reaction volume. DNA is obtained, wherein the primer binding site of the inner target-specific primer of the second PCR is present inside the primer binding site of the target-specific primer of the first PCR. Each inner target-specific primer forms a primer pair with the second universal primer to amplify one of the target loci, where at least 80% of the amplified DNA is said to be said. Methods involving mapping to a target locus. The method according to claim 1, wherein the biological sample is a blood, plasma, serum, or urine sample. The method of claim 1, wherein each of the adapters comprises a molecular barcode. The method according to claim 1 , wherein the first universal primer and the second universal primer are the same . The method of claim 1, wherein the PCR is a one-sided nested PCR. The first PCR comprises simultaneously amplifying at least 50 target loci within the first reaction volume using the first universal primer and at least 50 target-specific primers. The method according to 1. The first PCR comprises simultaneously amplifying at least 100 target loci within the first reaction volume using the first universal primer and at least 100 target-specific primers. The method according to 1. The second PCR comprises simultaneously amplifying at least 50 target loci within the second reaction volume using the second universal primer and at least 50 inner target specific primers. Item 1. The method according to Item 1. The second PCR comprises simultaneously amplifying at least 100 inner target loci within the second reaction volume using the second universal primer and at least 100 inner target specific primers. The method according to claim 1. The method of claim 3, wherein the isolated cell-free DNA is tagged with up to 1024 different molecular barcodes. The method of claim 3, wherein the isolated cell - free DNA is tagged with 1024 to 65536 different molecular barcodes. The method of claim 1, wherein the concentration of each target-specific primer in the first and / or second PCR is 20 nM or less. The method of claim 1, wherein the concentration of each target-specific primer in the first and / or second PCR is 10 nM or less. The method of claim 1, wherein the annealing step of the first and / or second PCR has a length of at least 3 minutes. The method of claim 1, wherein the annealing step of the first and / or second PCR has a length of at least 5 minutes. The method of claim 1, wherein at least 90% of the amplified DNA is mapped to the target locus. The method according to claim 1, wherein the target locus is an SNP locus. The method of claim 1, wherein the cell-free DNA comprises DNA from a fetus. The method of claim 1, wherein the cell-free DNA comprises DNA from a tumor. The method of claim 1, wherein the cell-free DNA comprises DNA from a graft.

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