Array-based methods and kits for determining copy number and genotype of pseudogenes

Abstract

Provided herein are methods, as well as associated compositions, kits, systems, devices, and instruments, useful for genetic analysis in which one or more sequences similar to a gene of interest in a sample are present, e.g., for determining the state of a spinal muscular atrophy (SMA) vector. In the methods, the combined copy number of related genes (e.g., genes of interest as well as their pseudogenes, such as SMN1 and SMN2) can be determined by analytical assays. In addition, the relative amount of the related genes, i.e. The ratio of the related genes, can be determined by the analytical assay. Using the data of the combined copy number and the ratio of the related genes, the genotype of the gene of interest (and one or more pseudogenes thereof, if desired) can be determined highly accurately.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980048995.X. Technical Field

[0002] The present disclosure provides methods and related compositions, kits, systems, devices and apparatus for genetic analysis, including genotyping and copy number analysis of nucleic acids. Background Art

[0003] The analysis of the DNA and RNA samples obtained from biological samples or organisms to nucleotide sequences has caused great interest in research and health care circles.Use appropriate methods, the set of nucleotide sequences can be analyzed to distinguish various genetic information, such as genotype and copy number variation, which can be important for the source of diagnosis or screening nucleic acid and the disease or symptom of its family member.If there are other nucleotide sequences (for example, pseudogenes) highly similar to actual related genes, the analysis of some nucleotide sequences (for example, clinically relevant genes or genes associated with pathogenic symptom or disease) may be very difficult.The challenge part existing in this analysis (for example, based on array or based on sequencing) is because the signal detected from the analysis corresponds to more than one gene.In some cases, the signal is assigned to its corresponding gene and the signal is subjected to statistical analysis to determine the genetic information of each gene respectively and is usually very complicated in technology.

[0004] Therefore, there is a need to develop improved methods (and associated compositions, kits, systems, devices, and apparatus) that utilize genetic analysis to generate data with high accuracy that can be used to both genotype a given locus or chromosome and estimate the copy number of a given locus or chromosome. Summary of the invention

[0005] Described herein are methods and systems for analyzing nucleic acid samples to detect copy number differences in target polynucleotides, such as detecting copy number variants comprising deletions and insertions, and methods for genotyping such target polynucleotides, which are particularly useful when there are other sequences that have substantial sequence similarity to the target polynucleotides.

[0006] In one aspect, the disclosure provided herein relates to a method for genotyping a nucleic acid of a sample. The method may include (a) providing the nucleic acid or amplified product thereof of the sample to an array having a first set of probes and a second set of probes hybridized to a first target polynucleotide and a second target polynucleotide, wherein the first set of probes hybridizes to a first region having different sequences in the first target polynucleotide and the second target polynucleotide, and the second set of probes hybridizes to a second region that is the same in the first target polynucleotide and the second target polynucleotide, and wherein the first target polynucleotide and the second target polynucleotide have at least 50% sequence identity; (b) detecting a signal indicating that the first set of probes hybridizes to the nucleic acid or amplified product thereof of the sample; (c) detecting a signal indicating that the second set of probes hybridizes to the nucleic acid or amplified product thereof of the sample; and (d) determining the genotype of the nucleic acid of the sample by analyzing the signal.

[0007] In some embodiments, the first region has one or more base positions that differ in the first target polynucleotide and the second target polynucleotide and a sequence that is identical in the first target polynucleotide and the second target polynucleotide and surrounds the one or more differing positions.

[0008] In some embodiments, the first set of probes hybridizes to sequences immediately 5' or 3' of the one or more distinct positions.

[0009] In some embodiments, the first set of probes terminate at adjacent bases of the one or more distinct positions.

[0010] In some embodiments, the first set of probes have sequences that are complementary to the one or more distinct positions.

[0011] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes.

[0012] In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0013] In some embodiments, the analyzing step comprises one or more of: (a) determining the combined copy number of the first target polynucleotide and the second target polynucleotide in the nucleic acid of the sample; and (b) determining the ratio of the amount of the first target polynucleotide to the amount of the second target polynucleotide in the nucleic acid of the sample.

[0014] In some embodiments, the first target polynucleotide and the second target polynucleotide have at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity.

[0015] In some embodiments, the nucleic acid of the sample has a genomic DNA sequence obtained from the sample.

[0016] In some embodiments, the method further comprises amplifying the genomic DNA sequence obtained from the sample.

[0017] In some embodiments, the method further comprises amplifying the first target polynucleotide and the second target polynucleotide prior to hybridizing the first probe set and the second probe set to the nucleic acid of the sample.

[0018] In some embodiments, the method further comprises fragmenting the nucleic acid or an amplified product thereof.

[0019] In some embodiments, fragmented nucleic acids or amplification products thereof are provided to the array.

[0020] In another aspect, the disclosure provided herein relates to a method for determining the carrier status of an individual for an autosomal recessive condition. The method may include (a) providing a nucleic acid obtained from the individual or an amplified product thereof to an array having a first set of probes and a second set of probes that hybridize to a first target polynucleotide and a second target polynucleotide, wherein the first set of probes hybridizes to a first region having a different sequence in the first target polynucleotide and the second target polynucleotide, and the second set of probes hybridizes to a second region that is the same in the first target polynucleotide and the second target polynucleotide, and wherein the first target polynucleotide and the second target polynucleotide have at least 50% sequence identity; (b) detecting a signal indicating that the first set of probes hybridizes to the nucleic acid of the individual or the amplified product thereof; (c) detecting a signal indicating that the second set of probes hybridizes to the nucleic acid of the individual or the amplified product thereof; (d) genotyping the nucleic acid of the individual by analyzing the signal; and (e) determining the carrier status of the individual based on the genotype.

[0021] In some embodiments, the first region has one or more base positions that differ between the first gene and the second gene, and a sequence surrounding the one or more differing positions.

[0022] In some embodiments, the first set of probes hybridizes to sequences immediately 5' or 3' of the distinct positions.

[0023] In some embodiments, the first set of probes terminate at adjacent bases of the one or more distinct positions.

[0024] In some embodiments, the first set of probes have sequences that are complementary to the one or more distinct positions.

[0025] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes.

[0026] In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0027] In some embodiments, the analyzing step comprises one or more of: (a) determining the combined copy number of the first target polynucleotide and the second target polynucleotide in the nucleic acid of the individual; and (b) determining the ratio of the amount of the first target polynucleotide to the amount of the second target polynucleotide in the nucleic acid of the individual.

[0028] In some embodiments, the first target polynucleotide and the second target polynucleotide have at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity.

[0029] In some embodiments, the nucleic acid obtained from the individual comprises genomic DNA.

[0030] In some embodiments, the method further comprises amplifying the genomic DNA.

[0031] In some embodiments, the method further comprises amplifying nucleic acid of the first target polynucleotide and the second target polynucleotide.

[0032] In some embodiments, the method further comprises fragmenting the nucleic acid obtained from the individual or an amplification product thereof, thereby generating fragmented nucleic acids.

[0033] In some embodiments, the method further comprises providing fragmented nucleic acids to the array.

[0034] In some embodiments, the method further comprises determining the presence or absence of a mutation, insertion and / or deletion in the first target polynucleotide in the genome of the individual so as to determine the presence or absence of a functional copy of the first target polynucleotide in the individual.

[0035] In some embodiments, the method further comprises determining that the individual is a carrier of the autosomal recessive condition if the copy number of the functional first target polynucleotide from the individual is 1.

[0036] In another aspect, the disclosure provided herein relates to a kit for genotyping nucleic acids of a sample. The kit may contain an array having a first set of probes and a second set of probes that hybridize to a first target polynucleotide and a second target polynucleotide, wherein the first set of probes hybridizes to a first region that has a different sequence in the first target polynucleotide and the second target polynucleotide, and the second set of probes hybridizes to a second region that is the same in the first target polynucleotide and the second target polynucleotide, and wherein the first target polynucleotide and the second target polynucleotide have at least 50% sequence identity.

[0037] In some embodiments, the first region contains one or more base positions that differ in the first target polynucleotide and the second target polynucleotide, and a sequence surrounding the one or more differing positions.

[0038] In some embodiments, the first set of probes hybridizes to sequences immediately 5' of the distinct positions.

[0039] In some embodiments, the first set of probes terminate at adjacent bases of the one or more distinct positions.

[0040] In some embodiments, the first set of probes have sequences that are complementary to the one or more distinct positions.

[0041] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes.

[0042] In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0043] In some embodiments, the first target polynucleotide and the second target polynucleotide have at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity.

[0044] In some embodiments, the kit further includes instructions, including code for receiving data indicating hybridization of the first set of probes and the second set of probes to the nucleic acid of a sample or its application product, code for determining the combined copy number of the first target polynucleotide and the second target polynucleotide in the nucleic acid of the sample, code for determining the ratio of the amount of the first target polynucleotide to the second target polynucleotide in the nucleic acid from the sample, and code for determining the genotype of the first target polynucleotide and the second target polynucleotide in the nucleic acid from the sample.

[0045] In still another aspect, the disclosure provided herein relates to a method of making an array for genotyping a nucleic acid having a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide have at least 50% sequence identity. The method may comprise: (a) providing a first set of probes to a substrate, wherein the first set of probes hybridizes to a first region having a different sequence in the first polynucleotide and the second polynucleotide; and (b) providing a second set of nucleic acid sequences to the substrate, wherein the second set of probes hybridizes to a second region that is identical in the first polynucleotide and the second polynucleotide.

[0046] In some embodiments, the first set of probes and the second set of probes are synthesized on a substrate or attached to the substrate after synthesis.

[0047] In some embodiments, the first region contains one or more base positions that differ in the first polynucleotide and the second polynucleotide, and sequences surrounding the one or more differing positions.

[0048] In some embodiments, the first set of probes hybridizes to sequences immediately 5' of the distinct positions.

[0049] In some embodiments, the first set of probes terminate at adjacent bases of the one or more distinct positions.

[0050] In some embodiments, the first set of probes contains sequences that are complementary to the one or more distinct positions.

[0051] In some embodiments, the first polynucleotide and the second polynucleotide are from different genes.

[0052] In some embodiments, the first polynucleotide and the second polynucleotide are not allelic variants of a gene.

[0053] In some embodiments, the first polynucleotide and the second polynucleotide have at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity.

[0054] In still another aspect, the disclosure provided herein relates to a computer-implemented method for genotyping a nucleic acid mixture, the mixture having a first target polynucleotide and a second target polynucleotide having at least 50% sequence identity with the first target polynucleotide. The method may include: obtaining first data of intensity measurements from a first set of probes by a computer having a processor, wherein the first set of probes targets sequences that are different in a first target polynucleotide sequence and a second target polynucleotide sequence; obtaining second data of intensity measurements from a second set of probes by the computer, wherein the second set of probes targets sequences that are the same in a first target polynucleotide sequence and a second target polynucleotide sequence; determining by the processor a ratio of the first target polynucleotide to the second target polynucleotide in the mixture based on the first data; determining by the processor a combined copy number of the first target polynucleotide and the second target polynucleotide in the mixture based on the second data; and determining by the processor a genotype of at least one of the first target polynucleotide and the second target polynucleotide.

[0055] In some embodiments, the first set of probes and the second set of probes are provided in an array.

[0056] In some embodiments, the first set of probes and the second set of probes hybridize to target polynucleotides on the array.

[0057] In some embodiments, the ratio of the first target polynucleotide to the second target polynucleotide is a ratio of the first target polynucleotide to the second target polynucleotide in a human genome.

[0058] In some embodiments, the combined copy number of the first target polynucleotide and the second target polynucleotide is the combined genomic copy number of the first target polynucleotide and the second target polynucleotide in a human genome.

[0059] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes.

[0060] In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0061] In some embodiments, the target polynucleotide is the survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) genes or a portion thereof.

[0062] In some embodiments, the first target polynucleotide is found in the SMN2 gene and in a variant of the SMN1 gene having a mutation in and around exon 7.

[0063] In some embodiments, the second target polynucleotide is found in the SMN1 gene.

[0064] In some embodiments, the first set of probes has at least four probe sets, and each probe set corresponds to a different sequence in the SMN1 and SMN2 genes.

[0065] In some embodiments, the at least four probe sets targeting variants of the SMN1 gene in and around exon 7 target the following regions: a region containing a chromosome 5:70,247,773C>T site, a region containing a chromosome 5:70,247,921A>G site, a region containing a chromosome 5:70,248,036A>G site, and a region containing a chromosome 5:70,248,501G>A.

[0066] In some embodiments, the nucleotide sequence is a human sequence.

[0067] In some embodiments, the method further comprises receiving signal data from the array, wherein the first target polynucleotide is reported in the first group of probes; calculating the average intensity value of the probe group and determining the standard deviation between the average intensity values; calculating the raw frequency of the target polynucleotide; calculating the median frequency of the target polynucleotide based on the corresponding raw frequency; calculating the scaled median frequency of the target polynucleotide based on the corresponding median frequency; calculating the median frequency of the target polynucleotide based on the affinity value of each probe group of the target polynucleotide and the predicted copy number (CN); depicting a hyperplane corresponding to the absence of a copy of the target polynucleotide in the mixture, the presence of one copy of the target polynucleotide gene in the mixture, and the presence of two copies of the target polynucleotide in the mixture; and associating the number of probe group clusters within the hyperplane as a statistical indication of the copy number of the target polynucleotide in the mixture.

[0068] In some embodiments, the method further comprises: scaling the scaled centered frequency by: corresponding to the case where the scaled centered frequency is greater than 1, setting the scaled centered frequency to 1; and corresponding to the case where the scaled centered frequency is less than 0, setting the scaled centered frequency to 0; and determining the direction of the frequency by subtracting the median frequency of the first target polynucleotide and using the median frequency value of the second target polynucleotide.

[0069] In some embodiments, calculating the raw frequency of the probe set further comprises dividing the intensity of the second target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0070] In some embodiments, calculating the raw frequency of the probe set further comprises dividing the intensity of the first target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0071] In some embodiments, calculating the median frequency of the probe set based on the raw frequency further comprises subtracting the standard deviation from the raw frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being a frequency between the first target polynucleotide and the second target polynucleotide.

[0072] In some embodiments, calculating the scaled center frequency of the probe group based on the center frequency further includes: corresponding to the case where the center frequency is less than the first alpha cutoff value, multiplying the difference between the center frequency and the first alpha cutoff value by a first scaling factor and then subtracting this value from the first alpha cutoff value; corresponding to the case where the center frequency is greater than the second alpha cutoff value, multiplying the difference between the center frequency and the second alpha cutoff value by a second scaling factor and then adding this value to the second alpha cutoff value; and corresponding to the case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within the range formed by the first alpha cutoff value and the second alpha cutoff value, determining the center frequency as the scaled center frequency.

[0073] In some embodiments, the method further comprises: plotting the scaled centered frequency of the probe set to its predicted copy number; depicting in the plot a hyperplane corresponding to no copies of the target polynucleotide present in the mixture, one copy of the target nucleoid present in the mixture, and two copies of the target nucleotide present in the mixture; and associating the number of probe set clusters within the hyperplane as the statistical indication of the copy number of the target nucleotide in the mixture.

[0074] In some embodiments, the method further comprises normalizing the raw frequencies for each of the probe sets.

[0075] In some embodiments, normalizing the original frequency for the probe group further includes: calculating the median frequency of the probe group based on the original frequency, i.e., subtracting the standard deviation from the original frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being the original frequency between the first target polynucleotide and the second target polynucleotide; calculating the scaled median frequency of the probe group based on the median frequency, i.e., by: corresponding to the case where the median frequency is less than a first α cutoff value, multiplying the difference between the median frequency and the first α cutoff value by a first scaling factor and then subtracting this value from the first α cutoff value; corresponding to the case where the median frequency is greater than a second α cutoff value, multiplying the difference between the median frequency and the second α cutoff value by a second scaling factor and then adding this value to the second α cutoff value; and corresponding to the case where the median frequency is equal to the first α cutoff value and the second α cutoff value or is within the range formed by the first α cutoff value and the second α cutoff value, determining the median frequency as the scaled median frequency.

[0076] In still another aspect, the disclosure provided herein relates to a method, the method comprising: receiving probe set data of an array having a first set of probes and a second set of probes, the first set of probes targeting a variable sequence of a first target polynucleotide and a second target polynucleotide and the second set of probes targeting the same sequence of the target polynucleotide, the data having an average signal intensity for each probe set of the target polynucleotide, a standard deviation of the average signal intensity of each probe set, a first scaling factor, a second scaling factor, and a copy number region; calculating the original frequency of the target polynucleotide based on the average signal intensity from the probe sets; and calculating the original frequency based on the corresponding original frequency, ideal frequency, and copy number region. The frequency ratio and the standard deviation are used to calculate the median frequency of the target polynucleotide; the scaled median frequency of the target polynucleotide is calculated based on the corresponding median frequency, the first alpha cutoff value, the second alpha cutoff value, the first scaling factor and the second scaling factor; the median frequency of the target polynucleotide is calculated based on the affinity value of each probe group of the target polynucleotide and the predicted copy number (CN); a hyperplane is depicted corresponding to the absence of a copy of the target polynucleotide, the presence of one copy of the target polynucleotide and the presence of two copies of the target polynucleotide; and the number of probe group clusters associated with the hyperplane is used as a statistical indication of the copy number of the target polynucleotide.

[0077] In some embodiments, the copy number of the target polynucleotide is the genomic copy number of the target polynucleotide in the human genome.

[0078] In some embodiments, the first target polynucleotide and the second target polynucleotide have at least 50% sequence identity.

[0079] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes.

[0080] In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0081] In some embodiments, the target polynucleotide is the survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) genes or a portion thereof.

[0082] In some embodiments, the first target polynucleotide is found in the SMN2 gene and in a variant of the SMN1 gene having a mutation in and around exon 7.

[0083] In some embodiments, the second target polynucleotide is found in the SMN1 gene.

[0084] In some embodiments, the first set of probes has at least four probe sets, and each probe set corresponds to a different sequence in the SMN1 and SMN2 genes.

[0085] In some embodiments, the at least four probe sets targeting variants of the SMN1 gene in and around exon 7 target the following regions: a region containing a chromosome 5:70,247,773C>T site, a region containing a chromosome 5:70,247,921A>G site, a region containing a chromosome 5:70,248,036A>G site, and a region containing a chromosome 5:70,248,501G>A.

[0086] In some embodiments, the method further comprises: scaling the scaled centered frequency by: setting the scaled centered frequency to 1 corresponding to a case where the scaled centered frequency is greater than 1; and setting the scaled centered frequency to 0 corresponding to a case where the scaled centered frequency is less than 0; and determining the direction of the original frequency by subtracting the median frequency value of the first target polynucleotide and using the median frequency value of the second target nucleotide.

[0087] In some embodiments, calculating the raw frequency of the probe set further comprises dividing the intensity of the second target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0088] In some embodiments, calculating the raw frequency of the probe set further comprises dividing the intensity of the first target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0089] In some embodiments, calculating the median frequency of the probe set based on the raw frequency further comprises subtracting the standard deviation from the raw frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being the raw frequency between the first target polynucleotide and the second target polynucleotide.

[0090] In some embodiments, calculating the scaled center frequency of the probe group based on the center frequency further includes: corresponding to the case where the center frequency is less than the first α cutoff value, multiplying the difference between the center frequency and the first α cutoff value by the first scaling factor and then subtracting this value from the first α cutoff value; corresponding to the case where the center frequency is greater than the second α cutoff value, multiplying the difference between the center frequency and the second α cutoff value by the second scaling factor and then adding this value to the second α cutoff value; and corresponding to the case where the center frequency is equal to the first α cutoff value and the second α cutoff value or is within the range formed by the first α cutoff value and the second α cutoff value, determining the center frequency as the scaled center frequency.

[0091] In some embodiments, the method further comprises: plotting the scaled centered frequency of the target polynucleotide to its predicted copy number; depicting in the graph the hyperplane corresponding to the absence of a copy of the target polynucleotide, the presence of one copy of the target polynucleotide, and the presence of two copies of the target polynucleotide; and associating the number of probe group clusters within the hyperplane as the statistical indication of the copy number of the target polynucleotide in the human genome.

[0092] In some embodiments, the target polynucleotide is a human sequence.

[0093] In still another aspect, the disclosure provided herein relates to a method for determining a carrier genotype of an autosomal recessive condition of a subject. The method may include: obtaining first data for a first set of probes, the first set of probes targeting a first marker sequence that is different in a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence and the second polynucleotide sequence have at least 50% sequence identity and the autosomal recessive condition is caused by the absence of a functional copy of the first polynucleotide sequence in the genome; obtaining second data for a second set of probes, the second set of probes targeting a second marker sequence that is the same in the first polynucleotide sequence and the second polynucleotide sequence; calculating the number of copies of at least one polynucleotide sequence based on the first data and the second data and calculating a ratio for determining the relative presence of the first polynucleotide sequence and the second polynucleotide sequence; determining a carrier genotype when the number of copies of the first polynucleotide sequence is less than 2, and / or when the ratio indicates that the second polynucleotide sequence has a higher presence relative to the first polynucleotide sequence.

[0094] Incorporated by Reference

[0095] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and in the accompanying drawings:

[0097] Figure 1 is a schematic diagram illustrating autosomal recessive inheritance.

[0098] Figure 2 A spinal muscular atrophy (SMA) phenotypic presentation 100 is shown according to one embodiment.

[0099] Figure 3 Shown is a survival motor neuron 1 (SMN1) genotype 200 according to one embodiment.

[0100] Figure 4 A genome browser 300 is shown according to one embodiment.

[0101] Figure 5 A genome browser 400 is shown according to one embodiment.

[0102] Figure 6An SMN1 base sequence 500 is shown according to one embodiment.

[0103] Figure 7 A sequence alignment according to one embodiment is shown.

[0104] Figure 8 Shown are survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) sequence variation genotypes 700 according to one embodiment.

[0105] Fig. 9 A copy number determination process 800 is shown according to one embodiment.

[0106] Fig.10 A system 900 is shown according to one embodiment.

[0107] Fig.11 Diagram 1400 is shown according to one embodiment.

[0108] Fig.12 Diagram 1500 is shown according to one embodiment.

[0109] Fig.13 is an example block diagram of a computing device 1600 that may incorporate embodiments of the present disclosure.

[0110] Fig.14 The distribution of SMN1 and SMN2 copy numbers for 96 representative samples is shown.

[0111] Fig.15 Results for determining the carrier of SMA are shown.

[0112] Fig.16 An example of a display of copy numbers for both SMN1 and SMN2 is shown. In the example presented here, data shown with a y-axis value of 1.5 or lower may indicate a sample suspected of being a SMA carrier. DETAILED DESCRIPTION

[0113] The present disclosure has many preferred embodiments, and the present disclosure relies on many patents, applications and other references for details known to those skilled in the art. Therefore, when a patent, application or other reference is cited or repeated below, it should be understood that it is incorporated herein by reference in its entirety for all purposes and the claims stated.

[0114] Throughout the present disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description using the range format is only for the purpose of convenience and simplicity, and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Therefore, the description of a scope should be considered to have all possible sub-ranges disclosed and the individual numerical values ​​within the scope. For example, a scope such as a description from 1 to 6 should be considered to have an exact disclosed sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within the scope, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the scope, this applies.

[0115] Unless otherwise stated, the practice of the present disclosure may employ conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, connection, and detection of hybridization using markers. The specific description of suitable techniques may refer to the examples below herein. However, other equivalent conventional procedures may also be used, of course. Such routine techniques and instructions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all published by Cold Spring Harbor Laboratory Press), Stryer, L. (1995), Biochemistry (4th Edition), Freeman, New York, Gait, Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry (1996), 4th Edition, Freeman, New York, Gait, Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry (1996), 4th Edition, Freeman, New York, Gait, Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry (1996), 4th Edition, Freeman, New York, Freeman, New York, NY, and Berg et al., (2002) Biochemistry (5th edition), WH Freeman, New York, NY, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0116] definition

[0117] As used in this application, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "agent" includes a plurality of agents (including mixtures thereof).

[0118] All references cited herein are incorporated by reference in their entirety for all purposes. To the extent any reference includes a definition or use of a claim term in a manner inconsistent with the definitions and disclosure set forth herein, the definitions and disclosure of the present application control.

[0119] As used herein, the singular or plural forms of the terms "one or more nucleic acids", "one or more nucleic acid molecules", "one or more nucleic acid oligomers", "one or more oligonucleotides", "one or more nucleic acid sequences", "one or more nucleic acid fragments" and "one or more polynucleotides" are used interchangeably and are intended to include, but are not limited to, polymeric forms of nucleotides covalently linked together, possibly of various lengths, deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures and may perform various known or unknown functions. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of sequences, isolated RNA of sequences, nucleic acid probes and primers. The polynucleotides that can be used in the methods of the present disclosure may include natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of such sequences.

[0120] The "percentage of sequence identity" or "percentage of sequence similarity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include mutations, additions or deletions (i.e., gaps) compared to the reference sequence (excluding mutations, additions or deletions) to optimally align the two sequences. The percentage is calculated as follows: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the percentage of sequence identity.

[0121] In the context of two or more nucleic acid sequences, the terms "identical" or "identity" percentage and "similar" or "similarity" percentage refer to two or more sequences or subsequences that are identical or have a specified percentage of nucleotides as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters described below or by manual alignment and visual inspection (i.e., about 50% identity over a specified region, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or specified region) (see, e.g., NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / , etc.). Such sequences are then referred to as "substantially identical". This definition also relates to or can be applied to the supplementation of test sequences. The definition also encompasses sequences with deletions and / or additions, and sequences with mutations and / or substitutions. In some embodiments, the preferred algorithm can account for gaps and the like.

[0122] The terms "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form base pairs with another nucleic acid in a second polynucleotide. For example, the sequence AGT is complementary to the sequence TCA. Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, in which all nucleic acids match according to base pairing.

[0123] As used herein, "gene" refers to a sequence of DNA or RNA encoding a functional molecule. Therefore, the sequence of DNA or RNA translated into a polypeptide forms a gene. In addition, any regulatory sequence, such as DNA, introns and many other promoters, enhancers, 5' and 3' regulatory sequences that have any function (including but not limited to the function in DNA replication, transcription and translation) in cells are considered as part of a gene. Similarly, genes such as miRNA and siRNA are not translated, and genes that provide certain functions in cells are also considered as genes.

[0124] As used herein, "allele" refers to a specific form of a nucleic acid sequence (such as a gene) in a cell, individual or population, which is different from other forms of the same gene in the nucleic acid sequence of at least one (usually more than one) variant site in the sequence of the gene. The sequences at these variant sites that are different between different alleles are called "variations", "polymorphisms" or "mutations". Variants in sequences can appear as a result of SNPs, combinations of SNPs, haplotype methylation patterns, insertions, deletions, etc. Alleles can include variant forms of single nucleotides, variant forms of continuous sequences of nucleotides from regions of interest on chromosomes, or variant forms of multiple single nucleotides (not necessarily all continuous) from regions of interest on chromosomes. In each autosomal specific chromosome location or "locus", one is inherited from one of the parents, and the other is inherited from the other of the parents, for example, one is inherited from the mother and one is inherited from the father. If an individual has two different alleles at a locus, the individual is "heterozygous" at the locus. If an individual has two identical alleles at a locus, the individual is "homozygous" at the locus.

[0125] As used herein, "genome" refers to or means the complete single-copy set of genetic instructions of an organism encoded into the DNA of the organism. The genome can be multichromosomal, so that the DNA is distributed among multiple individual chromosomes in the cell. For example, in humans, there are 22 pairs of chromosomes, plus an XX or XY pair associated with gender.

[0126] As used herein, "polymorphism" refers to the occurrence of two or more genetically determined alternative sequences in a population. The alternative sequence may include alleles (e.g., naturally occurring variations) or spontaneous mutations that occur only in one or several separate organisms."polymorphic site" may refer to one or more nucleic acid positions where nucleic acid sequence differences occur. Polymorphism may include one or more base changes, insertions, repetitions or deletions. The polymorphic locus may be as small as a base pair. The polymorphic site includes restriction fragment length polymorphism, variable tandem repeats (VNTR), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats and insertion elements. The first variant or allele form determined is arbitrarily designated as a reference form, and other variants or allele forms are designated as alternatives or variants or mutant alleles. The most frequent variant or allele form in a selected nucleic acid population is sometimes referred to as a wild-type form. When it comes to genes encoding polypeptides, wild-type may refer to the most common gene sequence, which encodes a polypeptide that exhibits an expected activity. Allelic forms of diploid organisms can be homozygous or heterozygous. Diallel polymorphisms have two forms. Triple strand polymorphisms have three forms. Polymorphisms between two nucleic acids can occur naturally or be caused by exposure to or contact with chemicals, enzymes or other agents, or by exposure to agents that damage nucleic acids (e.g., ultraviolet radiation, mutagens or carcinogens). SNPs are positions where two alternative bases occur at a significant frequency (>1%) in the human population and are the most common type of human genetic variation.

[0127] As used herein, "array" or "microarray" includes a support having nucleic acid probes connected to the support. Preferred arrays generally include a plurality of different nucleic acid probes coupled to the substrate surface at different known locations. These arrays are also described as "microarrays" or colloquially referred to as "chips", which have been widely described in the art, such as U.S. Patents Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al., Science, 251:767-777 (1991). For all purposes, each is incorporated herein by reference in its entirety. The probe can be of any size or any sequence, and can include synthetic nucleic acids, and analogs or derivatives or modifications thereof, as long as the resulting array can hybridize with a nucleic acid sample with sufficient specificity under any suitable conditions to distinguish different target nucleic acid sequences of the sample. In some embodiments, the length of the probe of the array is at least 5, 10, 20, 30, 40, 50, 60, 70 or 80 nucleotides. In some embodiments, the length of the probe is no more than 25, 30, 50, 75, 100, 150, 200 or 500 nucleotides. For example, the length of the probe can be between 10 and 100 nucleotides.

[0128] Arrays can generally be produced using a variety of techniques, such as mechanical synthesis methods or light-guided synthesis methods combining photolithography methods and solid phase synthesis methods. The technology of synthesizing these arrays using mechanical synthesis methods is described in, for example, U.S. Patents No. 5,384,261 and No. 6,040,193, which are incorporated herein by reference in their entirety for all purposes. Although planar array surfaces are preferred, the arrays can be constructed on surfaces or even multiple surfaces that are actually any shape. Arrays can be nucleic acids on three-dimensional substrates, beads, gels, polymer surfaces, fibers (such as optical fibers), glass, or any other suitable substrates. (See U.S. Patents No. 5,770,358, No. 5,789,162, No. 5,708,153, No. 6,040,193, and No. 5,800,992, which are incorporated herein by reference in their entirety for all purposes.)

[0129] In some embodiments, arrays that can be used in conjunction with the methods and systems described herein include arrays commercially available from Thermo Fisher Scientific (formerly Affymetrix) under the brand name Arrays are used for a variety of purposes, including genotyping and gene expression monitoring of a variety of eukaryotic and prokaryotic species. Methods and hybridization conditions for preparing samples for hybridization to the array are disclosed in the manual accompanying the array, for example, the manufacturer's instructions for use with the product (e.g. FFPE Analysis Assay Kits and Related Products).

[0130] As used herein, "genotyping" refers to determining nucleic acid sequence information from a nucleic acid sample at one or more nucleotide positions. The nucleic acid sample may include or be derived from any suitable source (including genome or transcriptome). In certain embodiments, genotyping may include determining which or which alleles an individual carries at one or more polymorphic sites. For example, genotyping may include or determine which or which alleles an individual carries of one or more SNPs in a group of polymorphic sites. For example, in some individuals, the specific nucleotide in the genome may be A, and in other individuals it may be B. The individual with A at the position may have A alleles, and the individual with B may have B alleles. In a diploid organism, an individual may have two copies of a sequence containing a polymorphic position, so the individual may have A alleles and B alleles, or may alternatively have two copies of A alleles or two copies of B alleles. An individual with two copies of an A allele may be homozygous for an A allele, an individual with two copies of a B allele may be homozygous for a B allele, and an individual with a copy of every kind of allele may be heterozygous. Therefore, in some embodiments, genotyping includes determining the allelic composition (for example, AA, BB or AB) of the gene in nucleic acid sample or individual.In certain embodiments, genotyping includes determining the allelic composition of multiple genes (that is, two or more genes).Therefore, in asking two genes (for example, the first gene and the second gene), and the first gene can have A and / or B alleles and the second gene can have C and / or D alleles in the example, the method herein can determine the genotype of the two genes, for example, AACC, AADD, BBCC or BBDD (if both genes are homozygous) or AACD, BBCD, ABCC, ABDD or ABCD (if at least one gene is heterozygous).In certain embodiments, genotyping is included in detecting the single nucleotide mutation that occurs spontaneously in genome under the background of wild-type nucleic acid.In certain embodiments, one or more polynucleotides (or a part or multiple parts of polynucleotides, an amplified product of polynucleotides or the complement of polynucleotides) containing sequence of interest (for example, one or more SNPs or mutations) can be processed by other techniques (such as sequencing). Therefore, in some embodiments, the polynucleotides can be sequenced to perform genotyping or determine whether a polymorphism or mutation exists. Sequencing can be accomplished by various methods available in the art, such as the Sanger sequencing method, which can be performed by the Applied Biosystems (Applied Biosystems) The genomic DNA can be analyzed using a genetic analyzer or by next-generation sequencing (NGS) methods (e.g., Ion Torrent NGS or Illumina NGS from Thermo Fisher Scientific).

[0131] As used herein, "chromosomal abnormalities" can include any genetic abnormality, including mutations, insertions, additions, deletions, translocations, point mutations, trinucleotide repeat disorders and / or SNPs. Although the present disclosure describes certain examples and embodiments related to detecting chromosomal abnormalities in carriers that are not substantially affected by abnormalities, it should be understood that the methods and systems described herein can be used to detect chromosomal abnormalities in patients who are affected by abnormalities or have a high risk of abnormalities.

[0132] As used herein, a "sample" obtained from a biological sample or organism includes, but is not limited to, any number of tissues or body fluids of virtually any organism, such as blood, urine, serum, plasma, lymph, saliva, feces, and vaginal secretions. In some embodiments, the sample obtained from the organism can be a mammalian sample. And in some embodiments, the sample obtained from the organism can be a human sample.

[0133] The term "mPCR" herein may refer to multiplex PCR, a molecular biology technique used to amplify multiple targets in a single PCR experiment. In a multiplex assay, more than one target sequence can be amplified by using multiple primer pairs in the reaction mixture.

[0134] The term "CarrierScan" herein may refer to a genotyping product available from Thermo Fisher Scientific. CarrierScan comprises the CarrierScan analytical assay, which amplifies the precise target DNA of interest, and the CarrierScan array, an allele-specific oligonucleotide array that provides a single color readout.

[0135] The term "annealing" herein may refer to the pairing of complementary sequences of single-stranded DNA or RNA with hydrogen bonds to form a double-stranded polynucleotide.

[0136] The term "carrier" as used herein may refer to a genotype associated with a homozygous recessive trait that is not currently expressed due to the presence of at least one functional allele. When an individual carrying the homozygous recessive trait is crossed with another carrier, 50% of the offspring will express the trait. See Figure 1 .

[0137] The term "exon" herein may refer to a portion of a gene that will encode a portion of the final mature RNA produced by the gene after the introns are removed by RNA splicing. The term exon refers to both the DNA sequence in a gene and the corresponding sequence in the RNA transcript. In RNA splicing, introns are removed and exons are covalently linked to each other as part of the production of mature messenger RNA. Just as the entire set of genes of a species constitutes the genome, the entire set of exons constitutes the exome.

[0138] The term "DNase" herein may refer to a deoxyribonuclease, an enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading the DNA. Deoxyribonuclease is a type of nuclease, a general term for enzymes that can hydrolyze the phosphodiester bonds that connect nucleotides. A variety of deoxyribonucleases are known, which differ in substrate specificity, chemical mechanism, and biological function.

[0139] The term "replication event" herein may refer to a mechanism by which new genetic material is produced during molecular evolution. The replication event may be defined as any replication of a DNA region containing a gene. Gene replication may occur as a product of several types of errors in DNA replication and repair mechanisms and by the accidental capture of selfish genetic elements. Common sources of gene replication include ectopic recombination, retrotransposition events, aneuploidy, polyploidy, and replication slippage.

[0140] The term "circuit system" as used herein may refer to a circuit system having at least one discrete circuit, a circuit system having at least one integrated circuit, a circuit system having at least one application-specific integrated circuit, a circuit system forming a general-purpose computing device configured by a computer program (e.g., a general-purpose computer configured by a computer program that at least partially executes a process or device described herein, or a microprocessor configured by a computer program that at least partially executes a process or device described herein), a circuit system forming a memory device (e.g., various forms of random access memory), or a circuit system forming a communication device (e.g., a modem, a communications switch, or an optoelectronic device).

[0141] The term "firmware" herein may refer to software logic embodied as processor-executable instructions stored in read-only memory or media.

[0142] The term "hardware" herein may refer to logic embodied as analog or digital circuitry.

[0143] "Logic" herein may refer to machine memory circuits, non-transitory machine-readable media, and / or circuitry that includes control and / or program signals and / or settings and values ​​(such as resistance, impedance, capacitance, inductance, current / voltage levels, etc.) that can be applied to affect the operation of a device through its material and / or material-energy configuration. Magnetic media, electronic circuits, electrical and optical memory (both volatile and non-volatile), and firmware are examples of logic. Logic specifically excludes pure signals or software per se (however, it does not exclude machine memory that includes software and thereby forms a configuration of matter).

[0144] The term "software" herein may refer to logic implemented as processor-executable instructions in machine memory (eg, read / write volatile or non-volatile memory or media).

[0145] The various logical function operations described herein can be implemented using logic referenced by nouns or noun phrases that reflect the operation or function. For example, an association operation can be implemented by an "associator" or "correlator". Similarly, switching can be performed by a "switch", selection can be performed by a "selector", etc.

[0146] Genetic analysis

[0147] Genetic analysis is crucial in many health care and medical applications.Genetic analysis can provide information on one or more genes associated with a disease or condition of disease.For example, genetic analysis can provide the genotype of one or more clinically relevant genes (or one or more genes of concern), and the presence or absence of any genetic abnormality, such as copy number variation, deletion, insertion, repetition and chromosome mutation.When there are other sequences highly similar to one or more genes of concern, genetic analysis may be very difficult.In some cases, there are pseudogenes, which are DNA fragments related to genes of concern.In many cases, relative to actual (or true) genes, pseudogenes have lost at least some functions in cell gene expression or protein coding ability.Pseudogenes are usually produced due to the accumulation of multiple mutations in genes, and the product of the gene is not essential for the survival of an organism, but may also be caused by genome copy number variation (CNV), where the fragment is duplicated or missing.Although not completely functional, pseudogenes can be functional, similar to non-coding DNA of other types, and the non-coding DNA can perform regulatory functions. Given the substantial sequence similarity between pseudogenes and actual genes (e.g., clinically relevant genes or genes associated with genetic diseases or conditions), both sequences generate signals in analytical assays such as arrays and sequencing, and processing such mixed signals is technically challenging compared to the case where only the actual gene is present in the genome. The methods, compositions, systems, devices and apparatus provided herein are particularly useful in genetic analysis where multiple related genes are present in the genome.

[0148] In certain embodiments, the disclosure provides a method for genetic analysis. In certain embodiments, the method can be used for genotyping a nucleic acid with two or more related sequences (e.g., a sequence with substantial sequence similarity). For example, the method can be used for genotyping a target gene with one or more pseudogene sequences in a genome. Genotyping and determining copy number in this case may be technically challenging. The analysis and determination for genotyping and copy number determination, such as array-based, sequencing-based or PCR-based methods, usually rely on the inquiry of the region uniquely present in the target sequence. These analysis and determinations usually inquire about multiple regions of the target sequence, so as to provide statistically significant and accurate results. Taking genotyping analysis and determination as an example, multiple polymorphic sites different in the allele of the target gene can be queried based on array-based, sequencing-based or PCR-based methods, and the statistical analysis of multiple data points generated from each polymorphic site can provide a comprehensive and reliable genotype of the target gene. Also in the example determined by copy number, multiple regions unique to the target sequence can be inquired, and a large number of data points can be compared with the data points of the reference chromosome. In these analytical determinations, one or several data points may not be enough to provide reliable results, because the variation of each data point is relatively large. Measuring a sufficient number of data points (for example, 5 or more) and determining the main relationship of multiple data points can provide reliable genotyping and copy number results for the target gene. Therefore, it is important to ensure that each data point represents a single gene of interest for successfully and reliably performing genotyping and copy number determination in the above-mentioned type of analytical determination. However, if there is more than one highly similar sequence to each other, such as a gene and its pseudogene are present in a genome, then the data are explained and genotyping treatment may be technically highly challenging. This is because each data point may be generated by a mixture of two genes, and it is impossible to perform statistical analysis on these mixed data and provide the result of a single gene separately. Therefore, due to this complexity of sequence in the sample, it is usually impossible to use the analytical determination available in the art to determine the genotype or copy number of the target gene. In order to overcome the above challenges and provide reliable genetic analysis results (comprising genotyping and copy number of the gene of interest), the method and associated compositions, test kits, systems, devices and instruments that can be used for genetic analysis are provided herein, particularly in the case of one or more sequences similar to the gene of interest in the sample. In certain embodiments, the copy number of the related gene (e.g., the gene of interest and one or more pseudogenes thereof) is determined by analytical determination, i.e., the "combination" copy number of the related gene. In addition, the relative amount of the related gene, i.e., the ratio of the related gene, is determined by analytical determination. Using the data of the combined copy number and the ratio of the related gene, the genotype of the gene of interest (and one or more pseudogenes thereof, if necessary) can be determined with high accuracy.

[0149] In some embodiments, provided herein is a method for genotyping a plurality of polynucleotides (e.g., a first polynucleotide and a second polynucleotide), the method having the following steps: (a) providing the nucleic acid or amplified product thereof of a sample to an array having a first set of probes and a second set of probes that hybridize to a first target polynucleotide and a second target polynucleotide, (b) detecting a signal indicating that the first set of probes hybridizes to the nucleic acid or amplified product thereof of the sample, (c) detecting a signal indicating that the second set of probes hybridizes to the nucleic acid or amplified product thereof of the sample, and (d) determining the genotype of the nucleic acid of the sample by analyzing the signal. In some embodiments, the first set of probes hybridizes to a first region having different sequences in the first target polynucleotide and the second target polynucleotide. In some embodiments, the second set of probes hybridizes to a second region that is identical in the first target polynucleotide and the second target polynucleotide. The first target polynucleotide and the second target polynucleotide may have at least 50% sequence identity.

[0150] In some embodiments, the method according to the present disclosure is used to genotype nucleic acids having at least two target polynucleotides, such as the first polynucleotide and the second polynucleotide with sequence similarity. In some embodiments, the first polynucleotide and the second polynucleotide have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 99.99% or any intermediate percentage of the aforementioned sequence similarity. In some embodiments, the first polynucleotide and the second polynucleotide are not allelic variants of a single gene. In some embodiments, the first polynucleotide and the second polynucleotide are two separate genes. In some embodiments, the first polynucleotide is a gene with autosomal recessive inheritance, which causes genetic conditions or diseases once two active copies are lost. In some embodiments of such embodiments, the second polynucleotide is a gene, such as a pseudogene, which is similar to the first polynucleotide (or the first gene) in sequence, but has no activity or has less activity than the first gene.

[0151] In some embodiments, two or more target polynucleotides that can be genotyped by the methods of the present disclosure have a common (or identical) region in the target polynucleotide and another region that is different (or varied) in the target polynucleotide. In some embodiments, the common region and the different region are independently about 10 bases to about several hundred bases. In certain embodiments, common region and different region are about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 110 bases, about 120 bases, about 130 bases, about 140 bases, about 150 bases, about 160 bases, about 170 bases, about 180 bases, about 190 bases, about 200 bases, about 250 bases, about 300 bases, about 400 bases, about 500 bases or the bases of any intermediate number of the foregoing independently. In certain embodiments, all bases in the common region in the target polynucleotide are the same. In certain embodiments, in the variable region, some bases in the bases in the target polynucleotide are different, while other bases are the same. In other words, the variable region has at least one or more bases that are different in the target polynucleotide, and sequences near (or around) the same variable bases in the target polynucleotide. In some embodiments where two related genes are genotyped, the variable bases in the variable region contain mutations, deletions, or insertions of one or more bases of a gene in the gene rather than another gene. In certain embodiments, variable bases can be found anywhere in the genome constituting the gene, not only comprising one or more coding regions, but also comprising one or more non-coding regions (e.g., 5' and 3' regulatory regions, comprising promoters, enhancers, and 5' and 3' untranslated regions (UTRs)) and introns. In certain embodiments, the target polynucleotide comprises non-coding sequences, such as microRNA (miRNA) and small interfering RNA (siRNA). Therefore, the method for genotyping provided herein is not limited to coding sequences, but comprises non-coding sequences present anywhere in the interrogation genome.

[0152] In some embodiments, the method for genotyping a plurality of target polynucleotides (e.g., a first polynucleotide and a second polynucleotide) disclosed herein utilizes an array having a plurality of probes. In some embodiments, the array has a first set of probes and a second set of probes. In some embodiments, the first set of probes is configured to interrogate regions that are different in the target polynucleotides (i.e., variable regions). As described above, the variable regions may have one or more bases that are different in the target polynucleotides (i.e., variable bases). The variable regions may also have the same sequence surrounding the variable bases. In some embodiments, the first set of probes has a region that can hybridize with both the variable bases and the surrounding bases. In some embodiments, the first set of probes has different affinities with each target polynucleotide in the target polynucleotides. In some embodiments, the first set of probes has a sequence that is completely complementary to only one target polynucleotide in the target polynucleotides (e.g., a first target polynucleotide) but not to other target polynucleotides (e.g., a second target polynucleotide). With the sequence 5'-GAATA C -3' ("-" means there are 0, 1 or more nucleotides) As an example, the underlined "C" is a variable base, and the rest of "GAATA" are surrounding bases. In this example, the first target polynucleotide has a "C" at the variable position, and the second target polynucleotide has an "A" at the same position. In one example, the first set of probes can have a sequence that is completely complementary to the first target polynucleotide, such that the probes have 5'- G TATTC-3' (the "G" complementary to the variable position is underlined). The probe has a sequence that is completely complementary to the second target polynucleotide (ie, 5'- TAlternative embodiments of TATTC-3' ("T" complementary to the variable position is underlined)) are also possible. Therefore, in some embodiments, the first group of probes hybridizes with the first target polynucleotide with an affinity higher than the second target polynucleotide, or vice versa. In some embodiments, the first group of probes hybridizes only with the first target polynucleotide and not with the second target polynucleotide, or vice versa. In these embodiments, the signal indicating this hybridization difference is measured and processed to determine the genotype. In some other embodiments, the first group of probes has a sequence that is complementary to the surrounding area but not complementary to one or more variable bases. In some embodiments, the first group of probes is designed to hybridize with the sequence of 5' or 3' of one or more variable bases. In some embodiments, the first group of probes hybridizes with the sequence of the next 5' or 3' of one or more variable bases. In some embodiments, the first group of probes terminates at the next base of one or more variable bases. In some embodiments of these embodiments, hybridization targets (that is, specific target polynucleotides hybridized with each probe) can be distinguished by incorporating a marker molecule. For example, differentially labeled nucleotides (e.g., A or T labeled with a first label molecule and G or C labeled with a second label molecule) can be incorporated into the probe depending on the target sequence to which the probe hybridizes by single base extension or ligation, thereby indicating the identity (or sequence) of the target polynucleotide.

[0153] It should be understood that genotyping can be carried out in any manner of different sites in a plurality of target sequences that can be used for identifying nucleic acid samples. In certain embodiments, the genotyping method that can be used in conjunction with the present disclosure includes those methods that can be used for SNP detection, and the SNP detection is generally used to analyze the allele of the same gene. In some embodiments of inquiring two or more target genes, the SNP of one or more target genes (for example, clinically relevant genes and / or their pseudogenes) can be detected. The platform for SNP detection is well-known in the art, and such platform can be applicable to the method that is provided herein for analyzing and inquiring two or more target sequences that are not from the same gene. The suitable method for genotyping of this paper method includes the variation of single nucleotide extension, the use of target-specific probes (for example, only with the probe of single gene hybridization), the target distinction based on connection, etc.

[0154] In some embodiments, the array further comprises a second set of probes, which are configured to interrogate common or identical regions in the target polynucleotides. Thus, the second set of probes hybridizes to regions in which all bases in the target sequence are unchanged.

[0155] In the example of two target genes inquired by the method provided by this paper, the second group of probes can be designed to hybridize with the same region in two target genes.In certain embodiments, "same region in two target genes" refers to the same nucleotide sequence in two genes when both genes are wild-type and do not have any sudden change.However, in some cases, if there is sudden change, disappearance and / or insertion in the genome of such individuality, this region may be different between two target genes of some individuals.In these examples, this region can still be inquired by the second group of probes, so as to determine genotype and the copy number of one or two target genes.

[0156] In certain embodiments, the genotyping method according to the present disclosure is configured to determine the combined copy number of target polynucleotides in the nucleic acid of the sample. In certain embodiments, the total copy number of the target polynucleotides is determined based on the hybridization spectrum of the second group of probes and the target polynucleotides. In some instances where the sample has two related genes (e.g., actual genes and pseudogenes), based on the signal indicating the second group of probes and the nucleic acid hybridization in the sample, the combined (or total) copy number of the two genes is determined. These signals related to the abundance of the two target genes can be measured, and these signals are normalized to the signal from the reference sample. If the signal ratio between the test sample and the reference sample is different from the expected ratio, this may indicate the change in the copy number of the two genes. The reference signal can be a signal measured from a sample known as a normal diploid. The reference signal can be measured simultaneously with the test sample. Alternatively, a reference signal or the data indicating the reference signal can be provided electronically, for example. In certain embodiments, there can be more steps to normalize other variable factors, such as hybridization background and nucleic acid quality. In certain embodiments, the measurement of the signal and the data associated with the measurement are processed by a computer as described elsewhere in the present disclosure by certain algorithms.

[0157] In some embodiments, the genotyping method according to the present disclosure is configured to determine the ratio of the amount between each target polynucleotide. For example, if two target genes are inquired for genotyping, the method determines the relative amount (i.e., ratio) of the two genes, such as 1: 1, 2: 0, 3: 2 or greater. The relative amount of the target gene is determined based on the signal indicating the first group of probes and nucleic acid hybridization in the sample. These signals from the first probe group are related to the relative abundance of a target gene relative to another target gene in the nucleic acid sample. In some embodiments, the signal from the first target gene and the signal from the second target gene are measured and compared with each other to determine the ratio of the two genes. In some other embodiments, the ratio refers to the amount of a target gene relative to the total amount of two target genes. Therefore, in an example, the relative amount of the first target gene can be determined by dividing the signal from the first target gene by the sum of the signals from the first target gene and the second target gene. The relative amount of the second target gene can be determined in the same manner, except that the signal from the second target gene is divided by the sum of the signals. In some embodiments, the relative amount of a target gene (e.g., as a first target gene of a clinically relevant gene such as SMN1) is used, and it is sufficient to determine the genotype and copy number. In some other embodiments, the relative amounts of two target genes (e.g., a clinically relevant gene and its pseudogene, such as SMN1 and SMN2) are utilized. In some embodiments, the measurement of the signal and the data associated with the processing and measurement are processed by certain algorithms, by a computer as described elsewhere in this disclosure.

[0158] In the context of array-based analysis and determination, a variety of genotyping methods can be used. In certain embodiments, the array surface is divided into a plurality of features, each feature comprising a plurality of sites, and these sites include copies of substantially identical oligonucleotides configured to be bonded to a specific target nucleic acid sequence. Hybridization of different locations on the array can be detected and quantified. A suitable method is to use any array containing a target-specific probe, which is selectively bonded to a certain or certain target and is not bonded to other targets. In other embodiments, the array contains probes that are non-selectively bonded to target sequences of all different forms, but then expands or otherwise modifies to generate target-specific products in a target-specific manner. For example, the probe of the array can be extended by template-dependent nucleotide polymerization. Alternatively, the probe can be extended by the sequence-dependent connection of a label oligonucleotide, and the label oligonucleotide can contain a signal-generating portion. Target-specific products (for example, target-specific nucleotide extension products or connection products) can still be produced outside the array, and then hybridized with an array containing a probe that is distinguished between various extension products. The signal of the hybridization of the indication nucleic acid molecule and the specific array probe emitted from the array can be detected and quantified. Examples of genotyping array products include Affymetrix Arrays, Affymetrix OncoScan arrays and Affymetrix CytoScan arrays (Thermo Fisher Scientific) and Illumina and Arrays. Suitable array-based genotyping methods are described in, for example, Hoffman et al., Genomics 98(2):79-89 (2011) and Shen et al., Mutation Research 573:70-82 (2005), both of which are incorporated herein in their entirety.

[0159] In certain embodiments, the length of the probe used in the method provided herein is about 10 or more bases. In certain embodiments, the length of the probe is about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases or the base of any intermediate quantity mentioned above. In certain embodiments, the length of the probe is 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, 30 bases, 31 bases, 32 bases, 33 bases, 34 bases and 35 bases.

[0160] In certain embodiments, the nucleic acid for genotyping by the disclosed method comprises DNA and RNA obtained from a biological source (or biological sample) or an individual. The biological sample or source can be, for example, any amount of tissue or body fluid such as blood, urine, serum, plasma, lymph, saliva, feces, and vaginal secretions of almost any organism. The nucleic acid for genotyping can be genomic DNA, cell-free DNA, and any type of RNA, including mRNA.

[0161] In certain embodiments, the nucleic acid interrogated by the method of the present disclosure is amplified, and the amplified product is used for hybridization with an array. In embodiments using genomic DNA as a nucleic acid sample, the whole genome sequence can be amplified before hybridization with the array. In an embodiment, random primers are used to complete whole genome amplification by polymerase chain reaction (PCR).

[0162] In some embodiments, the step of target amplification is included according to the genotyping method disclosed herein. In some embodiments, multiplex PCR (mPCR) is used for selective amplification of target genes. In some embodiments, in a target gene comprising a clinically relevant gene and a closely related pseudogene thereof, only a clinically relevant gene or a part thereof is selectively amplified. In some alternative embodiments, multiple target genes comprising a clinically relevant gene (or a part thereof) and a related gene (or a part thereof) are selectively amplified. In some embodiments, before hybridization with an array, a multiplex PCR product that can be optionally diluted is added to a nucleic acid sample, such as a whole genome DNA or its amplified product. Alternatively or in combination, a sequence-specific probe associated with a collectable means (e.g., biotin beads or antibodies) is used to separate target polynucleotides. The sequence-specific probe bound to the target sequence can be separated by pulling biotin beads or antibodies using any suitable capture means (e.g., affinity chromatography).

[0163] In certain embodiments, the genotyping method according to the present disclosure comprises the step of fragmenting nucleic acid samples or their amplified products. It should be understood that fragmentation (or cracking) can be completed according to any method known in the art that is applicable to the present disclosure (e.g., physical methods, such as shearing, ultrasonic treatment, heat treatment, etc., and chemical methods such as enzyme treatment). In certain embodiments, one or more sequence-specific or sequence-nonspecific enzymes are used to fragment nucleic acid samples or their amplified products. In certain embodiments, one or more restriction enzymes can be used to fragment nucleic acids for inquiry. In certain embodiments, one or more enzymes, such as nucleases such as DNA enzymes and / or restriction enzymes, can be added to catalyze the step of fragmentation. Suitable restriction enzymes include, but are not limited to, AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, Bb vI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpul0I, BpuE I, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BscRI, BscYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI , BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, Bs sHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, Bts I, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdcI, DpnI, DpnII, DraI, DraIII, DrdI, EacI, EagI, EarI, EciI, Ec o53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI,HincII, HindIII, HinfI, HinPlI, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, Hp yCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, M spAlI, MspI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI , NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspO MI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqαI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, and ZraI. In some embodiments, the fragmented nucleic acid or its amplified product is provided to an array for genotyping.

[0164] In certain embodiments, the method described in the present disclosure includes the step of genotyping. Genotyping can include determining the sequence of at least one nucleotide in the target nucleic acid sequence. In certain embodiments, the step of genotyping involves analyzing multiple (for example, one, two or more) target polynucleotides from a sample, and the sample can be obtained from a biological source or an organism. In certain embodiments, the target polynucleotides are different genes. In certain embodiments, the target nucleic acid includes one or more other nucleic acid sequences of clinically relevant genes and sharing some sequence identities, for example, one or more related genes, such as pseudogenes. In some embodiments of asking two or more target genes, the method described herein is used for genotyping one of the target genes, such as one or more clinically relevant genes. In certain embodiments, the method described herein is used for genotyping one of the clinically not (or less) related genes. In certain embodiments, the method described herein is used for genotyping one or more clinically relevant genes and one or more clinically not (or less) related genes related thereto.

[0165] In one aspect, the disclosure herein provides a computer-implemented method for genotyping a nucleic acid mixture. The mixture may have a first target polynucleotide and a second target polynucleotide having at least 50% sequence identity with the first target polynucleotide. The method may include: obtaining first data of intensity measurements from a first set of probes by a computer containing a processor; obtaining second data of intensity measurements from a second set of probes by the computer and determining, by the processor, a ratio of the first target polynucleotide to the second target polynucleotide in the mixture based on the first data. The method then determines, by operation of the processor, a combined copy number of the first target polynucleotide and the second target polynucleotide in the mixture based on the second data. The method then determines, by operation of the processor, a genotype of at least one of the first target polynucleotide and the second target polynucleotide.

[0166] In some embodiments, a first set of probes targets sequences that are different in the first target polynucleotide sequence and the second target polynucleotide sequence, and a second set of probes targets sequences that are the same in the first target polynucleotide sequence and the second target polynucleotide sequence.

[0167] In some embodiments, the first set of probes and the second set of probes can be provided in an array. The first set of probes and the second set of probes can hybridize with a target polynucleotide on the array. The nucleotide sequence can be from a human.

[0168] In some embodiments, the ratio of the first target polynucleotide to the second target polynucleotide can be the ratio of the first target polynucleotide to the second target polynucleotide in a human genome. The combined copy number of the first target polynucleotide and the second target polynucleotide can be the combined genomic copy number of the first target polynucleotide and the second target polynucleotide in a human genome.

[0169] In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes. The first target polynucleotide and the second target polynucleotide may also not be allelic variants of the same gene. The target polynucleotide may correspond to the survival of motor neuron 1 (SMN1) and survival of motor neuron 2 (SMN2) genes or a portion thereof. The first target polynucleotide may be found in the SMN2 gene and in variants of the SMN1 gene having mutations in and around exon 7. The second target polynucleotide may be found in the SMN1 gene. Alternatively, the second target polynucleotide may be found in the SMN2 gene and in variants of the SMN1 gene having mutations in and around exon 7 and the first target polynucleotide may be found in the SMN1 gene. In some embodiments, the first set of probes may include at least four probe sets, and each probe set corresponds to a different sequence in the SMN1 and SMN2 genes. In some embodiments, the at least four probe sets targeting variants of the SMN1 gene in and around exon 7 target the following region: containing chromosome 5:70,247,773C>T site ( Figure 7 27,012) in the region containing chromosome 5:70,247,921A>G site ( Figure 7 The region at position 27,160) contains the chromosome 5:70,248,036A>G site ( Figure 7 position 27,275) and a region containing chromosome 5:70,248,501G>A( Figure 7 In some embodiments, the probe set may also include one or more probes targeting polymorphic regions or sites of SMN1. For example, a region containing the g.27134T>G site (chromosome 5: 70,247,901, Figure 727,134 in SMN1), a region genetically linked to a silent carrier mutation of SMN1. In some embodiments, the copy number of SMN1 can be called by a doubly normalized depth at a single intronic base that distinguishes SMN1 from SMN2. When a chromosome 5:70,247,773C>T SNP is called in SMN1, only those fragments containing the intronic base that distinguishes SMN1 can be populated with the read pileup used to call chromosome 5:70,247,773C>T, and the copy number of SMN1 can define the expected allelic balance to be considered (e.g., with three copies of SMN1, 0%, 33%, 66%, or 100% allelic balance is expected). All genomic locations cited above are in GRCh37 / hg19 coordinates.

[0170] In certain embodiments, the method relates to the data of receiving signals from the array. The first group of probes can report the first target polynucleotide. The mean intensity value of the probe group can be calculated, and the standard deviation between the mean intensity values ​​can be determined. The method can calculate the original frequency of the target polynucleotide. The original frequency can be used to calculate the center frequency of the target polynucleotide. The center frequency can be used to calculate the scaling center frequency of the target polynucleotide. The median frequency of the target polynucleotide can be calculated according to the affinity value of each probe group of the target polynucleotide and the predicted copy number (CN). The hyperplane corresponding to the copy of the target polynucleotide not existing in the mixture, a copy of the target polynucleotide gene and two copies of the target polynucleotide in the mixture can be described according to the data. The quantity of the probe group cluster in the hyperplane can then be associated as a statistical indication of the copy number of the target polynucleotide in the mixture.

[0171] In some embodiments, the method may perform a scaling operation to further scale the scaled centered frequency by setting the scaled centered frequency to 1, corresponding to the case where the scaled centered frequency is greater than 1. The scaling operation may also set the scaled centered frequency to 0, corresponding to the case where the scaled centered frequency is less than 0. The scaling operation may then determine the direction of the frequency by subtracting the median frequency of the first target polynucleotide and using the median frequency value of the second target polynucleotide.

[0172] In certain embodiments, the raw frequency of the probe group can be calculated by dividing the intensity of the second target polynucleotide by the intensity of the first target polynucleotide and the intensity of the second target polynucleotide. In certain embodiments, the calculation is completed with the data obtained from the first group of probes. In certain embodiments, the calculation is completed with the data obtained from the second group of probes.

[0173] In some cases, calculating the raw frequency of the probe set comprises dividing the intensity of the first target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide. In some embodiments, this calculation is completed with the data obtained from the first group of probes. In some embodiments, this calculation is completed with the data obtained from the second group of probes.

[0174] In some embodiments, calculating the median frequency of the probe set based on the raw frequency can further involve subtracting the standard deviation from the raw frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being a frequency between the first target polynucleotide and the second target polynucleotide.

[0175] In some embodiments, calculating the scaled center frequency of the probe group based on the center frequency may involve, corresponding to the case where the center frequency is less than a first alpha cutoff value, multiplying the difference between the center frequency and the first alpha cutoff value by a first scaling factor and then subtracting this value from the first alpha cutoff value. Corresponding to the case where the center frequency is greater than a second alpha cutoff value, the difference between the center frequency and the second alpha cutoff value may be multiplied by a second scaling factor and then added to the second alpha cutoff value. Corresponding to the case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within the range formed by the first alpha cutoff value and the second alpha cutoff value, the center frequency may be determined as the scaled center frequency.

[0176] In certain embodiments, the method is related to the described scaling centering frequency of the probe group and its predicted copy number is mapped.Then the hyperplane corresponding to the copy of the target polynucleotide not existing in the mixture, a copy of the target nucleotide existing in the mixture and two copies of the target nucleotide existing in the mixture can be described in the figure.Then the quantity of the probe group cluster in the hyperplane can be associated by the statistical indication of the copy number of the target nucleotide in the mixture.

[0177] In some embodiments, the method involves normalizing the original frequency for the probe group. In some embodiments, normalizing the original frequency for each probe group in the probe group involves calculating the center frequency of the probe group according to the original frequency, that is, subtracting the standard deviation from the original frequency and then adding the ideal frequency ratio 0.5, the ideal frequency is the original frequency between the first target polynucleotide and the second target polynucleotide. In some embodiments, normalization can also involve calculating the scaling center frequency of each probe group in the probe group according to the center frequency. In some embodiments, calculating the scaling center frequency can involve corresponding to the situation where the center frequency is less than the first α cutoff value, multiplying the difference between the center frequency and the first α cutoff value by a first scaling factor and then subtracting this value from the first α cutoff value. In some embodiments, calculating the scaling center frequency can involve corresponding to the situation where the center frequency is greater than the second α cutoff value, multiplying the difference between the center frequency and the second α cutoff value by a second scaling factor and then adding this value to the second α cutoff value. In some embodiments, calculating the scaled center frequency may further involve and corresponding to the case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within a range formed by the first alpha cutoff value and the second alpha cutoff value, determining the center frequency as the scaled center frequency.

[0178] Carrier screening

[0179] In some embodiments, the disclosure provided herein can be used to diagnose the carrier status of an individual of a pathological condition or disease. For example, the methods, compositions, kits, systems, devices and apparatus provided herein can be used to determine whether an individual is a carrier of an autosomal recessive disease, so that the risk of an individual's children being affected by the disease can be obtained.

[0180] Autosomal recessive inheritance is a condition that only occurs in individuals who have received two copies of the altered gene (one from each parent). The parent is a carrier of only one copy of the gene and, because the gene is recessive relative to its normal counterpart, does not exhibit the trait. Figure 1 As shown, if both parents are carriers, the child has a 25% chance of inheriting two abnormal genes and thus developing the disease. The child has a 50% chance of inheriting only one abnormal gene and, like both parents, being a carrier, and a 25% chance of the child inheriting two normal genes.

[0181] A genetic carrier (or simply a carrier) is a person or other organism that has inherited a recessive allele for a trait or mutation but does not display the trait or show symptoms of the disease. Carriers are able to pass the allele to their offspring, who can then express the gene if the offspring inherit the recessive allele from both parents. The chance of a child of two carriers developing the disease is 25%.

[0182] There are a variety of diseases or conditions that are determined by autosomal recessive inheritance. Some examples include cystic fibrosis, sickle cell anemia, fanconi anemia, pyruvatedehydrogenase deficiency, xeroderma pigmentosum, Hartnup's disease, Kartagener's Syndrome, Tay-Sachs disease, and spinal muscular atrophy (SMN). Although the diagnosis of these diseases or conditions (i.e., determining whether an individual is a patient of the disease or condition or has a risk of affecting it) is crucial, it is also important to screen individuals who plan to have children soon or in the future and determine whether the individual is a carrier of the disease or condition. Such screening may be particularly useful, for example, during in vitro fertilization (IVT).

[0183] In some embodiments, the disclosure herein provides a method for determining the carrier status of an individual's autosomal recessive condition. The method may include the step of providing an array with a nucleic acid obtained from an individual or an amplified product thereof. The array may have a first set of probes and a second set of probes that hybridize to a first target polynucleotide and a second target polynucleotide. The first set of probes hybridizes to a first region having different sequences in the first target polynucleotide and the second target polynucleotide, and the second set of probes hybridizes to a second region that is the same in the first target polynucleotide and the second target polynucleotide. The first gene and the second gene may have at least 50% sequence identity. The method may include the step of detecting a signal indicating that the first set of probes hybridizes to the nucleic acid or the amplified product thereof of the individual. The method may also include the step of detecting a signal indicating that the second set of probes hybridizes to the nucleic acid or the amplified product thereof of the individual. The method may further include the step of genotyping the nucleic acid of the individual by analyzing the signal and determining the carrier status of the individual based on the genotype.

[0184] In certain embodiments, the first region of the method inquiry for carrier screening provided by this paper has one or more bases different (variable) in the target polynucleotide and the sequence near or around one or more variable bases.In certain embodiments, the first group of probes and the sequence hybridization of the next-door neighbor 5' or 3' of the one or more variable bases.In certain embodiments, the first group of probes terminates at the next-door neighbor base of the one or more variable bases.In certain embodiments, the first group of probes comprises a sequence complementary to the one or more variable bases.

[0185] In some embodiments, the target polynucleotides interrogated by the carrier status method herein are from different genes. In some embodiments, the target polynucleotides are not allelic variants of a gene. In some embodiments, the method interrogates at least two genes, such as a clinically relevant gene and its related gene (e.g., a pseudogene). An example of this pair of genes includes survival of motor neuron 1 (SMN1) and SMN2 genes. Therefore, the method provided herein can be used to screen for carriers of spinal muscular atrophy (SMA) associated with the SMN 1 gene.

[0186] In certain embodiments, the method for determining carrier state provided herein further comprises the step of determining the combined copy number of the first target polynucleotide and the second target polynucleotide in the nucleic acid of the individual. In certain embodiments, the method further comprises the ratio of the amount of the first target polynucleotide and the second target polynucleotide in the nucleic acid of the individual. In certain embodiments, the method further comprises determining the amount of the target polynucleotide relative to the total amount of total target nucleotides. Therefore, for example, the relative amount of the first target polynucleotide can be determined by the signal from the first target polynucleotide divided by the sum of the signals from the first target polynucleotide and the second target polynucleotide. The relative amount of the second target polynucleotide can be determined in the same manner, except that the signal from the second target polynucleotide is divided by the sum of the signals.

[0187] In some embodiments, the target polynucleotides interrogated by the vector screening methods provided herein have a sequence identity of at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.99%, or any intermediate percentages of the foregoing.

[0188] In certain embodiments, the nucleic acid interrogated by the carrier screening method herein has genomic DNA obtained from an individual. In certain other embodiments, other types of nucleic acid, such as floating DNA (e.g., cell-free DNA) or RNA (e.g., mRNA, siRNA or miRNA) can be used as the nucleic acid sample of the method.

[0189] In certain embodiments, the method for determining carrier state provided herein further comprises the step of amplifying the target polynucleotide. This amplification step may comprise a nucleic acid that amplifies the target polynucleotide. As described elsewhere in the present disclosure, amplification may be accomplished by, for example, a polymerase chain reaction (PCR) with sequence-specific primers. Alternatively or in combination, a sequence-specific probe associated with a collectable means (e.g., biotin beads or antibodies) is used to separate the target polynucleotide. The sequence-specific probe bound to the target sequence may be separated by pulling the biotin beads or antibodies using any suitable capture means (e.g., affinity chromatography).

[0190] In certain embodiments, the method for determining carrier status provided herein further comprises fragmenting the nucleic acid obtained from an individual or its amplification product, thereby generating a step of fragmented nucleic acid. This fragmentation can be accomplished according to any method known in the art that is applicable to combining the present disclosure. In certain embodiments, one or more sequence-specific or sequence-nonspecific enzymes are used to fragment nucleic acid samples or their amplification products. In certain embodiments, one or more restriction enzymes can be used to fragment nucleic acids. In certain embodiments, one or more enzymes, such as nucleases such as DNA enzymes or restriction enzymes, can be added to catalyze the step of fragmentation. In certain embodiments, two or more enzymes can be used to fragment nucleic acids or their amplification products. In certain embodiments, the fragmented nucleic acids or their amplification products are provided to an array for carrier status screening.

[0191] In some embodiments, the method for determining carrier status provided herein further comprises determining the presence or absence of mutation, insertion and / or deletion in the target polynucleotide (e.g., clinically relevant gene), so as to determine the presence or absence of a functional copy of the target polynucleotide in the individual. The functional copy of a gene can refer to a gene copy of at least about 30% of the activity of the wild-type copy of a gene. In some embodiments, the functional copy of a gene comprises a gene copy, and the gene copy has at least about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, about 100% or the activity of the wild-type copy of any intermediate percentage of the gene mentioned above. Various methods for determining the functionality (or activity) of a gene copy are available in the art. For example, there are a variety of computational prediction methods in the art, such as the Virtual Gene Oncology (VIRGO) service (Naveed Massjouni, Corban Rivera, and T. M. Murali, "VIRGO: computational prediction of gene functions," Nucleic Acids Research (2006), Vol. 34, pp. W340-W344) and the SynFPS system (Jason Li, Saman Halgamuge, Christopher Kells, and Sen-Lin Tang, "Gene function prediction based on genomic context clustering and discriminative learning: an application to bacteriophages," BMC Bioinformatics (2007), 8(Suppl 4):S6, which are incorporated herein in their entireties). In addition, various experimental methods can be used in the art to test and / or measure the function of a specific form of a gene, including enzyme activity assays, binding affinity assays, reporter-based assays, or complementation assays, etc. Therefore, in some embodiments, once the structure of a specific copy of a target gene in a test sample is analyzed by the methods provided herein, the function (or activity) of the specific copy of the gene can be predicted computationally or tested experimentally.

[0192] In some embodiments, the method for determining carrier status provided herein further comprises the step of determining whether an individual is a carrier of an autosomal recessive condition of concern. In some embodiments, if the copy number of a target polynucleotide (e.g., a gene clinically relevant to a condition of concern) from an individual is 1, the individual is determined to be a carrier. In some embodiments, if he or she has a functional copy of a target gene, such as a copy of the function of a wild-type target gene with at least about 30% to about 100%, the individual is determined to be a carrier. In some embodiments, the individual tested has two or more copies of a target gene, wherein only one copy is a functional copy, and another or other copies are non-functional copies of the target gene. In this case, the individual tested can still be considered to be a carrier with only one functional copy of the target gene.

[0193] On the other hand, the disclosure herein provides a method for operating a carrier detection algorithm, the method may be related to receiving probe group data of an array with a first group of probes and a second group of probes, the first group of probes targeting the variable sequence of the first target polynucleotide and the second group of probes targeting the same sequence of the target polynucleotide, the data include the average signal intensity for the target polynucleotide of each probe group, the standard deviation of the average signal intensity of each probe group, the first scaling factor, the second scaling factor and the copy number region. In certain embodiments, the method is related to calculating the original frequency of one or two target polynucleotides in the target polynucleotide according to the average signal intensity from the probe group. In certain embodiments, the center frequency of the target polynucleotide can be calculated according to the corresponding original frequency, the ideal frequency ratio and the standard deviation. In certain embodiments, the scaling center frequency of the target polynucleotide is calculated according to the corresponding center frequency, the first α cutoff value, the second α cutoff value, the first scaling factor and the second scaling factor. In certain embodiments, the median frequency of the target polynucleotide is calculated according to the affinity value and and the predicted copy number (CN) of each probe group of the target polynucleotide. In some embodiments, a hyperplane corresponding to the absence of a copy of the target polynucleotide, the presence of a copy of the target polynucleotide, and the presence of two copies of the target polynucleotide is depicted. In some embodiments, the number of probe group clusters in the hyperplane is correlated to a statistical indication of the copy number of the target polynucleotide. In some cases, the target polynucleotide is a human sequence.

[0194] In some embodiments, the copy number of the target polynucleotide can be the genomic copy number of the target polynucleotide in the human genome. The first target polynucleotide and the second target polynucleotide can have at least 50% sequence identity. In some embodiments, the first target polynucleotide and the second target polynucleotide are from different genes. In some embodiments, the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

[0195] In some embodiments, the target polynucleotide may be a survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) gene or a portion thereof. In some embodiments, the first target polynucleotide is found in the SMN2 gene and in variants of the SMN1 gene having mutations in and around exon 7. In some embodiments, the second target polynucleotide is found in the SMN1 gene. Alternatively, the second target polynucleotide may be found in the SMN2 gene and in variants of the SMN1 gene having mutations in and around exon 7 and the first target polynucleotide may be found in the SMN1 gene. The first set of probes may comprise at least four probe sets, and each probe set corresponds to a sequence that may be different in the SMN1 and SMN2 genes.

[0196] In some embodiments, the at least four probe sets targeting variants of the SMN1 gene in and around exon 7 target the following regions: a region containing a chromosome 5:70,247,773C>T site, a region containing a chromosome 5:70,247,921A>G site, a region containing a chromosome 5:70,248,036A>G site, and a region containing a chromosome 5:70,248,501G>A.

[0197] In some embodiments, corresponding to the case where the scaled centered frequency is greater than 1, the scaled centered frequency is scaled by setting the scaled centered frequency to 1. In some embodiments, corresponding to the case where the scaled centered frequency is less than 0, the scaled centered frequency is scaled by setting the scaled centered frequency to 0. In some embodiments, the method then involves determining the direction of the raw frequency by subtracting the median frequency value of the first target polynucleotide and using the median frequency value of the second target nucleotide.

[0198] In some cases, calculating the raw frequency of the probe set involves dividing the intensity of the second target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0199] In some embodiments, calculating the raw frequency of the probe set involves dividing the intensity of the first target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

[0200] In some embodiments, calculating the median frequency of the probe set based on the raw frequency involves subtracting the standard deviation from the raw frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being the raw frequency between the first target polynucleotide and the second target polynucleotide.

[0201] In some embodiments, calculating the scaled center frequency of each probe group in the probe group according to the center frequency involves, corresponding to the case where the center frequency is less than a first alpha cutoff value, multiplying the difference between the center frequency and the first alpha cutoff value by a first scaling factor and then subtracting this value from the first alpha cutoff value. In some embodiments, calculating the scaled center frequency of each probe group in the probe group according to the center frequency involves, corresponding to the case where the center frequency is greater than a second alpha cutoff value, multiplying the difference between the center frequency and the second alpha cutoff value by a second scaling factor and then adding this value to the second alpha cutoff value. In some embodiments, calculating the scaled center frequency of each probe group in the probe group according to the center frequency also involves and corresponding to the case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within the range formed by the first alpha cutoff value and the second alpha cutoff value, determining the center frequency as the scaled center frequency.

[0202] In some embodiments, the method is directed to mapping the predicted copy number of the target polynucleotide by the scaled center frequency. In some embodiments, the method then depicts a hyperplane corresponding to the absence of a copy of the target polynucleotide, the presence of a copy of the target polynucleotide, and the presence of two copies of the target polynucleotide in the figure. In some embodiments, the method then associates the quantity of the probe group clusters in the hyperplane as the statistical indication of the copy number of the target polynucleotide in the human genome.

[0203] On the other hand, the disclosure herein provides a method for determining the carrier genotype of an autosomal recessive condition of a subject. The method may involve obtaining first data for a first set of probes, the first set of probes targeting a first marker sequence that is different in a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence and the second polynucleotide sequence may have at least 50% sequence identity and the autosomal recessive condition is caused by the absence of a functional copy of the first polynucleotide sequence in the genome. The method may also involve obtaining second data for a second set of probes, the second set of probes targeting a second marker sequence that may be the same in the first polynucleotide sequence and the second polynucleotide sequence. The number of copies of at least one polynucleotide sequence and the ratio of the relative presence of the first polynucleotide sequence and the second polynucleotide sequence may be calculated based on the first data and the second data. When the number of copies of the first polynucleotide sequence is less than 2, and / or when the ratio indicates that the second polynucleotide sequence has a higher presence relative to the first polynucleotide sequence, the carrier genotype may be determined.

[0204] In some embodiments, the methods for determining carrier genotype provided herein can be used to obtain SMA risk caused by autosomal inheritance of SMN 1. The human genome sequence has SMN2, which is highly similar to SMN1 in sequence. Figure 4 A genome browser 300 is shown showing an alignment of SMN2 with SMN1 set as the reference sequence. The genome browser 300 shows markers 302 that define the locations of 26 variants of the invariant 28 kilobases in each gene.

[0205] refer to Figure 5 , genome browser 400 shows an enhanced view comparing exon 7 of SMN1 and SMN2. Within the region of exon 7, there are four markers. Marker 402 determines the gene conversion site that distinguishes the functional copy of SMN1 from SMN2. Marker 402 is found at chr5:70,247,773 and is a C>T conversion. Marker 402 also indicates a common carrier variant of SMN1. Marker 404 is another point mutation that distinguishes SMN1 from SMN2. Marker 404 is found at chr5:70,247,921 and is an A>G conversion. Marker 406 is another point mutation that distinguishes SMN1 from SMN2. Marker 406 is found at Chr5:70,248,036 and is an A>G conversion. Marker 408 is another point mutation that distinguishes SMN1 from SMN2. Marker 408 was found at Chr5:70,248,501 and was a G>A transition.

[0206] Figure 6 The SMN1 base sequence 500 is shown. The lowercase blue bases are specific to SMN1. Exon 7 has 54 base pairs (shown in uppercase letters). The exon 7 SNP shown as a red C (marker 502) indicates a gene conversion site that appears as a T in SMN2. Allele-specific primers can be designed to target these differential sequences for the assessment of amplicon size and intensity as a function of SMN1 copy number (CN).

[0207] In some embodiments, one or more primer sets are used to prepare amplicons SMN1 and / or SMN2. Each primer has four different mismatch designs, resulting in a total of 64 different primer combinations for testing. In some embodiments, only SMN1 or a portion thereof is amplified. Alternatively, both SMN1 and SMN2 or a portion thereof are amplified.

[0208] Figure 7 Shown is a sequence alignment between a region of SMN1 upstream of exon 7 and a corresponding region of SMN2. The sequence alignment shows the alignment of the sequences upstream of the exon 7 regions of the two genes. The variation between the two sequences can be used to distinguish the two genes.

[0209] Figure 8 Selected SMN1-SMN2 sequence variant genotypes 700 are shown. SMN1 and SMN2 have nearly identical sequences and would behave similarly to a tetraploid. The selected variants are non-polymorphic in SMN1 and SMN2, and therefore, a typical sample would be 'aabb' and belong to a normal cluster 702. "a" and "b" herein represent copies of SMN1 and SMN2, respectively. Normal cluster 702 includes non-carrier genotypes 214, such as the '1+1' genotype 202, in which SMN1 is found on two copies, and the '2+1' genotype 204, in which one of the DNA strands contains two working versions of the SMN1 gene (see Figure 3). Both non-carrier genotypes 214 meet the requirement of having at least one working copy of the SMN1 gene on each DNA strand. The carrier genotype 216 is distinguished from the non-carrier genotype 214 by having at least one DNA strand without a working copy of the SMN1 gene. For example, the '1+0' genotype 212 is a carrier in which one of the DNA strands lacks the SMN1 gene, or the '1+1*' genotype 208 in which one of the DNA strands contains a non-functional copy of the SMN1 gene. These specific genotypes are considered common carriers. Unlike the '1+0' genotype 212 and the '1+1*' genotype 208, the '2+0' genotype 210 is called a silent carrier because it is able to function similarly to the non-carrier genotype in terms of protein production, but lacks the SMN1 gene on one of its DNA strands, resulting in 50% of the gametes being without the SMN1 gene. Similarly, the '2+1*' genotype 206 shares replication genes on the same DNA strand, but lacks a working copy of SMN1 on the other DNA strand.

[0210] According to the probe used, the '1+1*' genotype 208 with mutated SMN1 (see Figure 3 ) can belong to variant cluster 704 or variant cluster 706 having a copy number of 4. '1+0' genotype 212 having a deleted SMN1 will belong to variant cluster 708 or variant cluster 710 because the copy number of the '1+0' genotype is 3.

[0211] In some embodiments, the system detects genotypes and copy numbers of SMN1 and SMN2 genes in variant clusters. Genotype clusters determine various copy numbers and genotypes. The system can aggregate data on (e.g., 26) variants to establish a consensus on the number of SMN1 and SMN2 genes. An expectation can be set that, for example, 1 in 50 samples will be from a carrier, and therefore, for example, approximately two samples per assay plate will be determined to be carriers. Samples should include high replicate counts to ensure that the clusters are "tight" (low spread). On average, the system should detect one or two samples outside the main cluster (normal cluster 702).

[0212] Fig. 9 An embodiment of a copy number determination process 800 is shown. Based on 26 gene-specific nucleotides, 26 allele-specific probe sets are constructed with 16 repetitions (frame 802). The region is also covered with a non-polymorphic probe (frame 804). The logarithmic ratio of each probe set is calculated (frame 806).

[0213] In some embodiments, log ratios are calculated using non-polymorphic probes.

[0214] In some embodiments, gene-specific median log ratios are calculated from the non-polymorphic probes to calculate copy number of SMN1 and SMN2 (block 804).

[0215] In some embodiments, log ratio calculations generally avoid probes that map to more than one location in the genome. Fig. 9 In one embodiment shown, probes are selected to obtain a "combined" copy number of the SMN1 and SMN2 genes. In some embodiments, the combined copy number of the SMN1 and SMN2 genes refers to the combined genomic copy number of the two genes in the source genome (e.g., the genome of the individual from whom the nucleic acid sample was obtained).

[0216] refer to Fig.10 , system 900 illustrates a system for implementing an SMA carrier detection algorithm according to one embodiment. In system 900, a sample 904 including a target nucleotide sequence 916, a polymerase, a primer, and nucleotides is loaded onto a reaction plate 902. The reaction plate includes multiple arrays for running parallel reactions. A first set of probes 912 and a second set of probes 914 are present in each array and are used to detect the target nucleotide sequence 916. The first set of probes 912 targets sequences that are different in the first target polynucleotide sequence and the second target polynucleotide sequence. The second set of probes 914 targets sequences that are the same in the first target polynucleotide sequence and the second target polynucleotide sequence. The reaction plate 902 with the sample is then loaded into an instrument 908 for several cycles of replication, which includes a high heat stage (94-98°C (201-208°F)), which denatures the DNA strands that break the hydrogen bonds between complementary bases, thereby producing two single-stranded DNA molecules. The denaturation phase is followed by an annealing phase, in which the reaction temperature is reduced to 50-65°C (122-149°F) for 20-40 seconds. The annealing phase allows the probe set to anneal to the target sequence in the DNA. The annealing phase is followed by labeling, for example, by incorporating one or more labeled nucleotides. Information from each probe is detected and reported as first data or second data. In some configurations, the instrument 908 can be operated in a setting in which the first data is reported through a first signal channel 926 and the second data is reported through a second signal channel 924. The first data and the second data report are reported to a computer system 910 including a processor 920 and a memory 918, wherein the memory 918 includes instructions corresponding to an SMA carrier detection algorithm 922. Through the operation of the SMA carrier detection algorithm 922, the system 900 is able to generate a genotype map 928, which indicates the frequency of the first target nucleotide sequence and the second target nucleotide sequence relative to the total predicted copy number of the two target nucleotide sequences. The SMA carrier detection algorithm 922 adjusts the data based on the affinity of each of the probes to the target nucleotide sequence. When the data is plotted, a depiction indicating hyperplane regions can be made between cluster groups based on the frequency of the two target sequences and the predicted total copy number of both. These hyperplane regions indicate specific SMN1 genotypes corresponding to carriers and non-carriers.

[0217] In some embodiments, the first set of probes 912 can target different sequences, such that the first set or probes indicate the presence of the SMN1 gene and the SMN2 gene. In some embodiments, each probe targets a point mutation at exon 7 that distinguishes a functional copy of SMN1 from a copy of SMN2.

[0218] In some embodiments, the SMA carrier detection algorithm utilizes data collected by multiplex PCR reactions. In some embodiments, the SMN1 gene sequence or a portion thereof is amplified in a multiplex PCR reaction. In some embodiments, the SMN2 gene sequence or a portion thereof is amplified in a multiplex PCR reaction. In some embodiments, the SMN1 gene sequence and the SMN2 gene sequence or a portion thereof are amplified in a multiplex PCR reaction.

[0219] PCR multiplexing may have benefits, three of which include increased throughput (potentially more samples per plate), reduced sample usage, and reduced reagent usage (depending on the number of targets in the experiment). For example, if a quantitative experiment consists of only one target assay, running the target assay with a normalizer assay (such as an endogenous control assay) as a duplex will increase throughput, reduce required samples, and reduce reagent usage by half. If a quantitative experiment consists of two target assays, it is possible to combine the two target assays and the normalizer assay in a triple reaction. In such cases, the increase in throughput, reduction in sample, and reduction in reagents will be even greater.

[0220] refer to Fig.11 , Figure 1400 shows the initial distribution of the reported data for the target sequence relative to the predicted copy number of the probe set. A plot indicating the frequency of SMN1 relative to SMN2 is shown, with SMN2 above the plot and SMN1 below the plot. Although the results show distinction between the genes, there is some overlap that may indicate potential carrier variation. Fig.11 and 12 In FIG, the y-axis represents the allele frequency of SMN1 / SMN2, and the x-axis represents the combined SMN1 and SMN2 copy number.

[0221] refer to Fig.12 , Figure 1500 illustrates a clear depiction in the data reported after implementing an SMA carrier detection algorithm of an embodiment. The adjusted data can allow for depictions to be made indicating different carrier genotypes of SMA. The top depiction indicates a low value for SMN1 relative to the ratio of SMN1 and SMN2, which indicates that the top region corresponds to only copies of SMN2 based on the predicted copy number. The middle depicted region indicates the presence of only one copy of SMN1 which may correspond to a '1+1*' or '1+0' carrier genotype.

[0222] Fig.13 is an example block diagram of a computing device 1600 that may incorporate some embodiments of the present disclosure. Fig.13 The machine system that only illustrates various aspects of the technical process described herein does not limit the scope of the claims. Those skilled in the art will recognize other changes, modifications and alternatives. In one embodiment, the computing device 1600 generally includes a monitor or graphical user interface 1602, a data processing system 1620, a communication network interface 1612, one or more input devices 1608, one or more output devices 1606, etc.

[0223] like Fig.13 As depicted, data processing system 1620 may include one or more processors 1604 that communicate with a number of peripheral devices via a bus subsystem 1618. In some embodiments, these peripheral devices include one or more input devices 1608, one or more output devices 1606, a communication network interface 1612, and a storage subsystem such as volatile memory 1610 and non-volatile memory 1614.

[0224] In some embodiments, volatile memory 1610 and / or non-volatile memory 1614 store computer executable instructions, and thus form logic 1622 , which when applied to and executed by one or more processors 1604 implement embodiments of the processes disclosed herein.

[0225] In some embodiments, input device(s) 1608 include devices and mechanisms for inputting information into data processing system 1620. These may include a keyboard, a keypad, a touch screen incorporated into monitor or graphical user interface 1602, audio input devices such as a voice recognition system, a microphone, and other types of input devices. In various embodiments, input device(s) 1608 may be embodied as a computer mouse, a trackball, a trackpad, a joystick, a wireless remote control, a drawing tablet, a voice command system, an eye tracking system, and the like. Input device(s) 1608 typically allow a user to select objects, icons, control areas, text, and the like that appear on monitor or graphical user interface 1602 through commands such as clicking a button, and the like.

[0226] In some embodiments, output device(s) 1606 include devices and mechanisms for outputting information from data processing system 1620. These may include a monitor or graphical user interface 1602, speakers, printer, infrared LEDs, etc. as are well known in the art.

[0227] In some embodiments, communication network interface 1612 provides an interface to communication networks (e.g., communication network 1616) and devices external to data processing system 1620. Communication network interface 1612 can act as an interface for receiving data from other systems and transmitting data to other systems. Embodiments of communication network interface 1612 can include an Ethernet interface, a modem (telephone, satellite, cable, ISDN), a (asynchronous) digital subscriber line (DSL), FireWire, USB, a wireless communication interface such as Bluetooth or WiFi, a near field communication wireless interface, a cellular interface, etc.

[0228] In some embodiments, the communication network interface 1612 is coupled to the communication network 1616 via an antenna, cable, etc. In some embodiments, the communication network interface 1612 may be physically integrated on a circuit board of the data processing system 1620, or in some cases may be implemented in software or firmware such as a "soft modem" or the like.

[0229] In some embodiments, computing device 1600 includes logic that allows communication over a network using schemes such as HTTP, TCP / IP, RTP / RTSP, IPX, UDP, etc.

[0230] The volatile memory 1610 and the non-volatile memory 1614 are examples of tangible media that are configured to store computer-readable data and instructions to implement various embodiments of the processes described herein. Other types of tangible media include removable memory (e.g., pluggable USB memory devices, mobile device SIM cards), optical storage media such as CD-ROMS, DVDs, semiconductor memories such as flash memory, non-transitory read-only memories (ROMS), battery-backed volatile memory, networked storage devices, etc. In some embodiments, the volatile memory 1610 and the non-volatile memory 1614 are configured to store basic programming and data structures that provide the functions of the disclosed processes and other embodiments thereof within the scope of the present disclosure.

[0231] Logic 1622 implementing an embodiment of the present disclosure may be stored in the volatile memory 1610 and / or the non-volatile memory 1614. The logic 1622 may be read from the volatile memory 1610 and / or the non-volatile memory 1614 and executed by one or more processors 1604. The volatile memory 1610 and the non-volatile memory 1614 may also provide a repository for storing data used by the logic 1622.

[0232] In some embodiments, volatile memory 1610 and non-volatile memory 1614 include multiple memories including a primary random access memory (RAM) for storing instructions and data during program execution and a read-only memory (ROM) in which read-only non-transitory instructions are stored. In some embodiments, volatile memory 1610 and non-volatile memory 1614 include a file storage subsystem that provides persistent (non-volatile) storage for program and data files. In some embodiments, volatile memory 1610 and non-volatile memory 1614 include a removable storage system, such as removable flash memory.

[0233] In some embodiments, bus subsystem 1618 provides a mechanism for allowing the various components and subsystems of data processing system 1620 to communicate with each other as needed. Although communication network interface 1612 is schematically depicted as a single bus, some embodiments of bus subsystem 1618 may utilize multiple distinct busses.

[0234] It will be readily apparent to those skilled in the art that computing device 1600 may be a device such as a smart phone, a desktop computer, a laptop computer, a rack-mounted computer system, a computer server, or a tablet computer device. As is generally known in the art, computing device 1600 may be implemented as a series of multiple networked computing devices. Further, computing device 1600 will typically include operating system logic (not shown), the type and nature of which are well known in the art.

[0235] Reagent test kit

[0236] In some embodiments, the disclosure herein provides a kit for genotyping the nucleic acid of a sample. The kit may include an array having a first group of probes and a second group of probes hybridized to a plurality of target polynucleotides. In some embodiments, a plurality of target polynucleotides include two or more different target polynucleotides, for example, a first target polynucleotide and a second target polynucleotide. The first group of probes may hybridize to a first region having a sequence different from that in the first target polynucleotide and the second target polynucleotide, and the second group of probes may hybridize to a second region that is identical in the first target polynucleotide and the second target polynucleotide. The first target polynucleotide and the second target polynucleotide may have at least 50% sequence identity.

[0237] In certain embodiments, the first region of the test kit inquiry (or analysis) provided by this paper has one or more bases that are different (variable) in the target polynucleotide and sequences near or around one or more variable bases. In certain embodiments, the first group of probes and the sequence hybridization of the next-door neighbor 5' or 3' of the one or more variable bases. In certain embodiments, the first group of probes terminates at the next-door neighbor base of one or more variable bases. In certain embodiments, the first group of probes includes a sequence complementary to the one or more variable bases.

[0238] In certain embodiments, the target polynucleotides inquired by the test kit herein are from different genes. In certain embodiments, the target polynucleotides are not allelic variants of genes. In certain embodiments, the test kit can be used to inquire at least two genes, such as clinically relevant genes and their related genes (e.g., pseudogenes). In certain embodiments, the target polynucleotides inquired by the test kit herein have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% sequence identity.

[0239] In some embodiments, the kit provided herein further comprises instructions for data collection and its analysis. In some embodiments, the instruction is in a computer-readable medium or in a computer. In some embodiments, the instruction contains a code for receiving data indicating that the first group of probes and the second group of probes are hybridized with the nucleic acid of the sample or its application product. In some embodiments, the instruction also comprises a code for determining the combined copy number of the target polynucleotide, such as the total copy number of the first polynucleotide and the second polynucleotide in the nucleic acid of the sample. In some embodiments, the instruction comprises a ratio for determining the amount of the target polynucleotide, such as the relative amount of the first polynucleotide and / or the second polynucleotide from the nucleic acid of the sample, code. In some embodiments, the ratio refers to the relative amount of two target polynucleotides, such as 1:1, 3:0 or 1:2. In some other embodiments, the ratio refers to the amount of a target polynucleotide relative to the total amount of the target polynucleotide. Therefore, in an example, the relative amount of the first target polynucleotide can be determined by dividing the signal from the first target nucleotide by the sum of the signals from the first target nucleotide and the second target nucleotide. In addition to dividing the signal of the second target polynucleotide by the sum of the signals, the relative amount of the second target polynucleotide can be determined using the same method. In certain embodiments, the relative amount of a target polynucleotide (e.g., clinically relevant gene) is used and is sufficient for carrier screening. In some other embodiments, the relative amount of two or more target polynucleotides (e.g., clinically relevant gene and pseudogene thereof) is used for carrier screening. In certain embodiments, the instruction also includes a code for determining the genotype of the target polynucleotide, such as the genotype of the first target polynucleotide and / or the second target polynucleotide of the nucleic acid from the sample.

[0240] In some embodiments, the disclosure herein provides a method for manufacturing an array for genotyping a nucleic acid having a plurality of target polynucleotides. In some embodiments, a plurality of target polynucleotides comprises two or more different target polynucleotides, for example, a first target polynucleotide and a second target polynucleotide. The first polynucleotide and the second polynucleotide may have at least 50% sequence identity. The manufacturing method may comprise providing a first group of probes to a substrate. The first group of probes may hybridize with a first region comprising a sequence that is different in the target polynucleotide. The method may also comprise providing a second group of probes to a substrate. The second group of probes may hybridize with a second region that is identical in the target polynucleotide. In some embodiments, the first group of probes and the second group of probes are synthesized on a substrate. In an alternative embodiment, the first group of probes and the second group of probes are attached to the substrate after synthesis. In some embodiments, the first region has one or more base positions that are variable in the target polynucleotide, and a sequence surrounding the one or more variable bases. In some embodiments, the first group of probes hybridizes with a sequence that is immediately adjacent to 5' of the one or more variable bases. In some embodiments, the first group of probes terminates at the immediately adjacent base of the one or more variable bases. In some embodiments, the first group of probes has a sequence that is complementary to the one or more variable bases.

[0241] Although preferred embodiments of the present disclosure have been shown and described herein, it should be apparent to those of ordinary skill in the art that such embodiments are provided by way of example only. Without departing from the present disclosure, those of ordinary skill in the art will now expect numerous variants, changes and substitutions. It should be understood that a variety of alternatives to the embodiments of the present disclosure described herein may be used to practice the present disclosure. It is contemplated that the following claims define the scope of the present disclosure and thus cover methods and structures within the scope of these claims and their equivalents.

[0242] Examples

[0243] Screening for carriers of spinal muscular atrophy

[0244] Spinal muscular atrophy (SMA) is a rare but devastating disease that has autosomal recessive inheritance. In some populations, 1 in 50 individuals carry a mutation in the SMN1 gene, which encodes a defective survival motor neuron (SMN) protein. Carrier screening requires accurate determination of the number of functional SMN1 genes in an individual. The presence of a highly homologous but largely non-functional SMN2 gene complicates carrier detection. Of the 28,081 bp of the SMN1 and SMN2 genes, only 27 positions differ (21 single nucleotide substitutions and 6 small indels), accounting for only 38 nucleotides that differ between the SMN1 and SMN2 gene sequences.

[0245] In the examples provided herein, array-based analytical assays for genotyping and screening of SMA carriers according to some embodiments were designed and performed. Specifically, the array used herein has a probe set designed to distinguish the SMN1 gene from the SMN2 gene based on these sequence differences. In addition, the array further contains 1,181 probe sets covering the SMN1 gene and the SMN2 gene for determining the combined gene copy number. The data show that these probe designs can detect the relative number of SMN1 genes and SMN2 genes as well as the total copy number. The combination of these data is used in a novel algorithm to identify individuals carrying SMA mutations, providing highly accurate and improved screening results compared to any other available method in the art.

[0246] Example 1 - Design of probe sets

[0247] Comparison of the SMN1 gene and SMN2 genomic DNA sequences identified 27 positions where there were sequence differences between the two genes. These differences were used to design gene-specific probe sets. Most of these positions were introns, but one was located within exon 7 and one was located within exon 8. (See Figure 5 ) Exon 7 is located both as a sequence difference between SMN1 and SMN2 and as the site of a mutation that converts SMN1 to a non-functional SMN2 gene. This mutation interferes with the exon splice junction and results in a transcript that does not contain exon 7. The most common type of carrier is an exon 7 deletion mutation, but gene conversion mutations can also occur. Figure 2 Four genomic locations with probe sets for detecting relative copy numbers of SMN1 and SMN2 are shown.

[0248] Carrier determination requires an accurate assessment of the total copy number of SMN1 and SMN2. The 1,181 copy number probe sets hybridize equally to the wild type of both genes (SMN1 and SMN2), so the assumed baseline copy number is 4 (two of each gene). Since the most common deletion is in exon 7, the 35 copy number probe sets were designed to focus on exon 7 and its surroundings.

[0249] Example 2 - Sample Preparation

[0250] Example 2.1 - During genome amplification and target amplification

[0251] Typically by following the Thermo Fisher Scientific CarrierScan TM Nucleic acid samples for genotyping and carrier screening of SMA are prepared and processed using the available protocols of the assay kit (Catalog Number: 931931) and the GeneTitan instrument (supra). In short, biological samples (e.g., whole blood, saliva, or cells) and genomic DNA (gDNA) obtained from individuals are separated from the biological sample. The gDNA separated by dilution to 5 μg / μL is used to amplify the gDNA and multiple PCR for amplifying the target polynucleotides is also performed. For the amplification of the gDNA sample, 20 μL of the diluted gDNA and 20 μL of the control DNA are aliquoted to a plate (e.g., Applied Biosciences (Applied Bioscience) gene 96 square well plates) separately. After the plate is sealed and rotated, the PCR reaction is performed with the reagents as indicated in the manufacturer's protocol. For target amplification, 10 μL of the diluted gDNA and 10 μL of the reference DNA are aliquoted to a separate 96-well plate. The plate is sealed, rotated, and the PCR reaction is continued as directed in the protocol. In the mPCR reaction, sequence-specific primers are used to amplify the SMN1 gene and / or the SMN2 gene or portions thereof. Target amplification is performed to amplify regions of the SMN1 gene and the SMN2 gene targeted by the probes. Thus, in one example, certain regions targeted by a first set of probes from both genes are amplified, the first set of probes being used to determine the relative amounts of the two genes. In addition, regions targeted by a second set of probes are also amplified, the second set of probes being used to measure the combined copies of the two genes. In this example, regions covering exon 7 and / or exon 8 of SMN1 and SMN2 are amplified by the mPCR reaction. The amplified DNA samples and mPCR reaction plates are stored at -20°C, if desired.

[0252] Example 2.2 - Fragmentation of amplified DNA

[0253] After the whole genome amplification and mPCR reaction are completed, the 10 μL mPCR reaction products in each well of the 96 plates are carefully transferred to the corresponding wells of the whole genome amplification plate. The sample is fully mixed by up and down pipetting and pulse spin sedimentation. The master mixture for fragmentation containing Axiom Frag enzyme (Thermo Fisher Scientific) is aliquoted into each well of the mixed DNA sample. The sample is incubated at 37 ° C for 45 minutes to carry out the fragmentation reaction. Once the fragmentation reaction is completed, the stop solution according to the manufacturer's protocol is added to the sample plate to terminate the fragmentation reaction. After the fragmentation reaction is completed, the master mixture for precipitating the sample DNA is added to each well of the plate, and 2-propanol is added to each well thereafter. The DNA pellet precipitated in each well is dried and stored until the next step.

[0254] Example 3 - Denaturation of fragmented DNA

[0255] Resuspension buffer was added to each well of the sample plate containing the precipitated DNA. Subsequently, the hybridization master mix was added to each well in which the DNA had been suspended, following the manufacturer's protocol. The sample plate was then subjected to a denaturation step (10 minutes at 95°C and 3 minutes at 48°C) using a thermal cycler as recommended by the manufacturer.

[0256] Example 4 - Hybridization and staining

[0257] The scheme that GeneTitan MC instrument (Thermo Fisher Scientific) and manufacturer provide is used to carry out hybridization, dyeing and connection step.According to the scheme, prepare in advance the master mixture for dyeing, connection and stabilization.In this example presented herein, because the analysis and determination adopts the 2-channel system that utilizes two labeling molecules to dye, therefore the dyeing master mixture has two independent solutions.

[0258] The plate with denatured DNA is loaded into the GeneTitan MC instrument together with the hybridization array with probe. The automation process of the instrument transfers the denatured DNA to the hybridization array plate and incubates the array plate under controlled conditions for hybridization. After hybridization, the array plate is washed several times with washing buffer and two separate dyeing steps (dyeing 1 and dyeing 2) are carried out as a part of the automation process. After hybridization and washing, the master mixture for the first dyeing step (dyeing 1) is added to the array plate, and the connection master mixture is added thereafter. The first dyeing master mixture is marked A / T with the first mark, and if the template has A or T, the first mark is added to the probe. The second dyeing master mixture for dyeing 2 has been marked G / C with the second mark, and if the template has G or C, the G or C of the mark is added to the probe. This template-specific connection will mark the probe hybridized with its corresponding target polynucleotide.

[0259] Example 5 - Scan

[0260] Once the array plate has passed the applied fluidics stage of the process described above, the array plate is moved to the imaging station of the instrument and scanned for data collection.

[0261] Use multiple controls containing reference genomic DNA to assess the quality of each reaction step as well as sample quality.

[0262] Example 6 - Algorithm

[0263] Provided herein is a description of an embodiment of the SMN detection algorithm as a program operation on a computer system. In this particular example, as indicated below, SMN1 and SMN2 (see the last two columns of the following txt input file) are detected by a two-channel system (channel a and b). In this example, the frequency measured and calculated for the target sequence measured in each channel is indicated as allele frequency. For example, the frequency measured from channel B is shown below as B allele frequency (BAF). However, it should be noted that this frequency is for the different genes measured from each channel, rather than for different alleles. Therefore, the allele frequency provided in the disclosure of this paper, such as B allele frequency (BAF) should be considered as false BAF, and the false BAF indicates the frequency of one of the related genes, rather than the allele variant of a single gene.

[0264] Sample CarrierScan.SMN.v1.AB_probesets.txt input file:

[0265]

[0266]

[0267] When SMN1 is listed as the A channel, the method will end up calculating 1-BAF=A / (A+B), but this calculation is described in this document as calculating BAF, and then, at the end (after the probe sets for each given marker are calculated), this calculation is added.

[0268] Channel A is the signal obtained from the genotyping profile for Channel A of the probe set.

[0269] Another item in the pseudo code below is that the final BAF is desired to be between 0 and 1. Due to scaling, the final BAF can be below 0 or above 1, in which case the BAF is simply reset.

[0270] The procedure is shown here for six probe sets, corresponding to three markers (affy_snp_id)

[0271] 1. Calculation of raw "B allele frequency" (rBAF)

[0272] a. Read in all probesets from the probeset_id column in *AB_probesets.txt

[0273] b. Find the intensity A and intensity B values ​​from AxiomGT1.summary.a5 (hdf5 format)

[0274] i.RowNames table

[0275] 1. Strength A=<probeset_id> -A

[0276] 2. Strength B =<probeset_id> -B

[0277] ii.ColNames table

[0278] 1. The index of each row will give the cel_file name in the left-right order shown in the data table

[0279] c. For each sample, calculate rBAF using the spreadsheet:

[0280]

[0281] 2. Central original BAF

[0282] a. Find the associated centering factor from the Factor 1 column in *AB_probesets.txt

[0283] b. For each sample: Probe set rBAF center:

[0284] cBAF = Centered original BAF = 0.5 + (rBAF - factor 1)

[0285] In some embodiments, Factor 1 may be the baseline Bi. In some embodiments, rBAF is calculated for samples having 2 copies of SMN1 and 2 copies of SMN2 and thus may be considered to have 2 A alleles and 2 B alleles. In such embodiments, Factor 1 is the median rBAF across these samples. As a result, the median BAF across these samples is 0.5.

[0286] 3. Zoom and center BAF

[0287] a. If cBAF < 0.485, find the factor 2 column in *AB_probesets.txt

[0288] i. For each sample: probe set cBAF scaling: `

[0289] scBAF = scaled centered original BAF = 0.485 - (0.485 - cBAF) x factor 2

[0290] b. If cBAF>0.515, find the factor 3 column in *AB_probesets.txt

[0291] i. For each sample: probe set cBAF scaling:

[0292] scBAF = scaled centered original BAF = (cBAF - 0.515) x factor 3 + 0.515

[0293] c. Otherwise (0.515 ≥ cBAF ≥ 0.485)

[0294] scBAF = scaled centered original BAF = cBAF

[0295] d. Scale scBAF to between 0 and 1:

[0296] i. If scBAF>1, set scBAF to 1,

[0297] ii. If scBAF<0, set scBAF to 0,

[0298] iii. Otherwise, use the calculated scBAF in the following steps.

[0299] Here the algorithm starts to group together probe sets that measure exactly the same marker. Affy_snp_if is the ID that references a given marker. Therefore, after calculating all scBAFs for a given marker, take the median of the measurements for each marker.

[0300] 4. Median scBAF of affy_snp_id

[0301] a. For each probeset_id, find the associated affy_snp_id in *AB_probesets.txt

[0302] b. For each sample = mBAF, calculate the median of affy_snp_id

[0303] In this step below, if the median moves in the opposite direction, the median is complemented.

[0304] 5. Check the channel to determine the "true BAF" direction by checking the smn1_channel column for each affy_snp_id:

[0305] a. If smn1_channel = A:

[0306] i.mBAF_<affy_SNP_ID> =1–mBAF

[0307] b. If smn1_channel = B:

[0308] i.mBAF_<affy_SNP_ID> =mBAF

[0309] ii.

[0310] Here multiple markers were looked at and the median of the median values ​​across the multiple markers in the region was determined. To make the final call, 3 measurements were used.

[0311] 6. Median mBAF of cn_region

[0312] a. For each cn_region, find the associated affy_snp_id in *AB_probesets.txt

[0313] b. By using each sample = mBAF_<cn_region> Each<affy_snp_id> The median BAF is calculated based on the median of each cn_region

[0314] Median (mBAF_<affy_SNP_ID1> 、mBAF_<affy_SNP_ID2> , …)

[0315] In step 7, each affy_snp_is value (calculated for the marker as well as the median across markers) is referred to below as the mBAF of cn_region.

[0316] 7. Report

[0317] a.<analysis_name> .SMN_ABreport.txt(example:mPCR90.SMN_ABreport.txt)

[0318] i.cel_files = cel file name

[0319] ii.mAB_<affy_snp_id> = mBAF of affy_snp_id

[0320] iii.mAB_<cn_region> = median mBAF of cn_region

[0321] Sample analysis report mPCR90.SMN_ABreport.txt

[0322]

[0323] Example 8 - Report call

[0324] The copy number (CN) status (of SMN1+SMN2) was calculated for the regions. Each copy number status had a different threshold at which "SMN1 has less than 2 copies" was called, as shown in the following table: Table 1: Thresholds.

[0325] Table 1

[0326]

[0327] Additional tables (below) show the expected BAF for each CN and state of SMN1. Table 2: Expected values ​​for each CN state.

[0328] Table 2

[0329]

[0330] Note that the thresholds in Table 1 are empirically derived, and while the thresholds are driven by the theoretical values ​​in Table 2, there is no formula to calculate the actual thresholds used based on the theoretical values.

[0331] The threshold was applied as follows, and four possible outcomes are reported.

[0332] 1) Samples were designated as "carriers" when the median mBAF of the cn_region was less than or equal to the value listed in the table above for the corresponding copy number, e.g., CN of SMN1 was 1 or less.

[0333] or

[0334] 2) When the BAF of Affx-206872225 is less than the threshold in the above table, a “conversion event is called”.

[0335] This is interpreted as a "conversion" event. A conversion event is reported - and the sample is also a carrier, SMN1 is present, but the critical allele of the above marker is mutated to the value possessed by SMN2 - thereby inactivating the gene.

[0336] or

[0337] 3) There is a marker inside exon 8 - when the BAF of only that marker is less than the corresponding threshold in the table above, call "Exon 8 deletion". Not sure if the customer interpreted this as a carrier, but the customer requested that it be reported.

[0338] or

[0339] 4) Nothing was reported.

[0340] Parameters / Options

[0341]

[0342]

[0343] Example 9 - Calling SMN1 / SMN2 copy number

[0344] Fig.14 The distribution of copy numbers for 96 representative samples is shown. The peak with a log2 ratio of 0.0 represents an individual with 4 copies of SMN1 and SMN2 combined. The CNVMix algorithm used in the examples herein determined 4 copy number states in this group of samples 2, 3, 4, and 5. Surprisingly, a large number of samples have 3 copies of these genes. The total copy number is important. For example, at a total copy number of 2, a sample with equal amounts of SMN1 and SMN2 is clearly a carrier, but at a total copy number of 4, the ratio means a non-carrier.

[0345] Example 10 - Determination of the carrier of SMA

[0346] The frequency of the SMN1 and SMN2 genes, denoted as BAF (B allele frequency) in the examples presented herein, is a measure that reports the relative amounts of SMN1 and SMN2. Fig.15 The left figure shows that only BAF cannot separate carriers (red dots) from non-carriers. By stratifying the data by total copy number and BAF, there is a clear separation of carriers from non-carriers (dashed lines), which forms the basis of the carrier detection algorithm. The preliminary application of the SMA detection algorithm on the data set of 493 samples has no false negative calls. A certain ratio of false positive calls was generated, but this is acceptable on the screen. The examples presented herein clearly demonstrate that the analytical determination and algorithm provided herein provide highly accurate and significantly improved screening results for carrier screening.

[0347] Example 11 - Displaying the copy number of the SMA gene

[0348] In some embodiments, the copy number calculated according to Example 10 can be, for example, Fig.16 The results are shown in the graph shown in Figure 1 (e.g., SMN1 copy number on the y-axis and SMN2 copy number on the x-axis). Based on the frequency of each gene, the copy number inferred from the frequency is plotted in a hyperplane format. In one example, samples suspected of being SMN1 carriers are plotted on the y-axis as values ​​of 1.5 or below. Thus, samples suspected of being SMN1 carriers are plotted as values ​​of 1.5 or below. Fig.16 The samples marked with triangles in the figure are carriers. Each suspected carrier has a different copy number of SMN2, as shown on the x-axis. By converting the data into a more easily understandable, user-friendly format or interface, the carrier status of the sample can be easily determined.

[0349] In some embodiments, the copy number of one or more target genes in the target gene can be displayed on a screen (locally or remotely created via a network) or in print in any user interface format. This display can be in the form of a table or text.

Claims

1. A computer-implemented method for genotyping a nucleic acid mixture, the mixture comprising a first target polynucleotide and a second target polynucleotide having at least 50% sequence identity with the first target polynucleotide, the method comprising: obtaining, by a computer comprising a processor, first data of intensity measurements from a first set of probes, wherein the first set of probes targets sequences that are different in a first target polynucleotide sequence and a second target polynucleotide sequence, and wherein the first set of probes hybridizes to a first region, the first region having a sequence that is different in the first target polynucleotide and the second target polynucleotide, the first region having one or more base positions that are different in the first target polynucleotide and the second target polynucleotide and a sequence that is the same in the first target polynucleotide and the second target polynucleotide and surrounds the one or more different positions, the sequence being immediately 5' or 3' to the one or more different positions; obtaining, by the computer, second data of intensity measurements from a second set of probes, wherein the second set of probes targets the same sequence in the first target polynucleotide sequence and the second target polynucleotide sequence; determining, by the processor, a ratio of the first target polynucleotide to the second target polynucleotide in the mixture based on the first data; determining, by the processor, a combined copy number of the first target polynucleotide and the second target polynucleotide in the mixture based on the second data; as well as determining, by the processor, a genotype of at least one of the first target polynucleotide and the second target polynucleotide; Wherein the first group of probes includes at least four probe sets, and each probe set corresponds to a different sequence in the SMN1 and SMN2 genes, and wherein at least four probe sets targeting variants in and around exon 7 of the SMN1 gene target the following regions: a region containing a chromosome 5:70,247,773C>T site, a region containing a chromosome 5:70,247,921A>G site, a region containing a chromosome 5:70,248,036A>G site, and a region containing a chromosome 5:70,248,501G>A site.

2. The method of claim 1, wherein the first set of probes and the second set of probes are provided in an array. 3 . The method of claim 1 , wherein the ratio of the first target polynucleotide to the second target polynucleotide is a ratio of the first target polynucleotide to the second target polynucleotide in a human genome.

4. The method of claim 1, wherein the combined copy number of the first target polynucleotide and the second target polynucleotide is the combined genomic copy number of the first target polynucleotide and the second target polynucleotide in a human genome. The method of claim 1 , wherein the first target polynucleotide and the second target polynucleotide are from different genes. The method of claim 1 , wherein the first target polynucleotide and the second target polynucleotide are not allelic variants of a gene.

7. The method of claim 1, wherein the target polynucleotide is the survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) genes or a portion thereof.

8. The method of claim 7, wherein the first target polynucleotide is found in the SMN2 gene and in a variant of the SMN1 gene having a mutation in and around exon 7.

9. The method of claim 7, wherein the second target polynucleotide is found in the SMN1 gene.

10. The method according to claim 1, further comprising: receiving signal data from the array, wherein the first target polynucleotide is reported in the first set of probes; calculating mean intensity values ​​for the probe sets and determining standard deviations between the mean intensity values; calculating the raw frequency of the target polynucleotide; Calculating the median frequency of the target polynucleotide according to the corresponding raw frequency; calculating a scaled median frequency of the target polynucleotide according to the corresponding median frequency; Calculating the median frequency of the target polynucleotide based on the affinity value of each probe set and the predicted copy number (CN) of the target polynucleotide; Describing hyperplanes corresponding to no copies of the target polynucleotide being present in the mixture, one copy of the target polynucleotide being present in the mixture, and two copies of the target polynucleotide being present in the mixture; as well as The number of probe set clusters within the hyperplane is associated as a statistical indication of the copy number of the target polynucleotide in the mixture.

11. The method according to claim 10, further comprising: The copy number of one or more target polynucleotides in the target polynucleotides in the mixture is displayed.

12. The method according to claim 10, wherein the method further comprises: The scaled center frequency is scaled by: Corresponding to the case where the zoom centering frequency is greater than 1, the zoom centering frequency is set to 1; as well as Corresponding to the case where the scaling and centering frequency is less than 0, the scaling and centering frequency is set to 0; as well as The direction of the frequency is determined by subtracting the median frequency of the first target polynucleotide and using the median frequency value of the second target polynucleotide.

13. The method of claim 10, wherein calculating the raw frequency of the probe set further comprises dividing the intensity of the second target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

14. The method of claim 10, wherein calculating the raw frequency of the probe set further comprises dividing the intensity of the first target polynucleotide by the sum of the intensity of the first target polynucleotide and the intensity of the second target polynucleotide.

15. The method of claim 10, wherein calculating the median frequency of the probe set based on the raw frequency further comprises subtracting the standard deviation from the raw frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being a frequency between the first target polynucleotide and the second target polynucleotide.

16. The method of claim 10, wherein calculating the scaled center frequency of the probe group based on the center frequency further comprises: corresponding to the case where the center frequency is less than a first alpha cutoff value, multiplying the difference between the center frequency and the first alpha cutoff value by a first scaling factor and then subtracting this value from the first alpha cutoff value; Corresponding to the case where the center frequency is greater than a second alpha cutoff value, multiplying the difference between the center frequency and the second alpha cutoff value by a second scaling factor and then adding this value to the second alpha cutoff value; as well as Corresponding to a case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within a range formed by the first alpha cutoff value and the second alpha cutoff value, the center frequency is determined as the scaled center frequency.

17. The method according to claim 10, further comprising: Plotting the scaled centered frequencies of the probe sets against their predicted copy numbers; Depicting in said figure the hyperplanes corresponding to the absence of copies of said target polynucleotide in said mixture, the presence of one copy of a target nucleoid in said mixture, and the presence of two copies of a target polynucleotide in said mixture; as well as The number of probe set clusters within the hyperplane is associated as the statistical indication of the copy number of the target nucleotide in the mixture.

18. The method of claim 10, further comprising: The raw frequencies are normalized for each of the probe sets.

19. The method according to claim 18, wherein normalizing the original frequency for the probe set further comprises: Calculating the median frequency of the probe set based on the original frequency, i.e., subtracting the standard deviation from the original frequency and then adding an ideal frequency ratio of 0.5, the ideal frequency being the original frequency between the first target polynucleotide and the second target polynucleotide; The scaled center frequency of the probe set is calculated based on the center frequency, namely by: corresponding to the case where the center frequency is less than a first alpha cutoff value, multiplying the difference between the center frequency and the first alpha cutoff value by a first scaling factor and then subtracting this value from the first alpha cutoff value; Corresponding to the case where the center frequency is greater than a second alpha cutoff value, multiplying the difference between the center frequency and the second alpha cutoff value by a second scaling factor and then adding this value to the second alpha cutoff value; as well as Corresponding to a case where the center frequency is equal to the first alpha cutoff value and the second alpha cutoff value or is within a range formed by the first alpha cutoff value and the second alpha cutoff value, the center frequency is determined as the scaled center frequency.

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