Method for estimating fetal fraction in free DNA from maternal sample

Abstract

Digital amplification methods and kits provide the ability to estimate the fetal fraction of cfDNA in a maternal sample, such as plasma or serum, by analyzing target sites differentially methylated in fetal and maternal free DNA (cfDNA).

Description

[0001] Cross - reference to related patent applications

[0002] This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 406,194, filed on September 13, 2022, and U.S. Provisional Patent Application No. 63 / 472,183, filed on June 9, 2023, each of which is incorporated by reference for all purposes. Background of the Invention

[0003] Accurate diagnosis of chromosomal aneuploidies (e.g., trisomies of chromosomes 13, 18, and 21) requires accurate estimation of the fetal fraction of cell - free DNA (cfDNA) in maternal plasma. Cell - free DNA is present in maternal whole blood at low levels of 0 - 100 ng / mL, and although the median fetal fraction of cfDNA is approximately 11% in the first trimester of pregnancy, it may be less than 4% in the early stages of pregnancy when non - invasive prenatal testing (NIPT) is recommended (Wang et al., Prenat. Diagn. 33:662 - 666, 2013). Additionally, fetal cfDNA must be distinguishable from maternal cfDNA, despite the high sequence similarity of the genetic sequences. Summary of the Invention

[0004] In one aspect, the present disclosure provides a digital PCR (dPCR) method for quantifying the fetal fraction of cfDNA in maternal plasma using methylation-sensitive restriction enzyme (MSRE) digestion. In some embodiments, the present disclosure provides a method for estimating the fraction of fetal DNA in a cfDNA sample obtained from a blood sample of a pregnant human subject. In some embodiments, the method comprises a digital amplification reaction method, the digital amplification reaction method comprising: (a) partitioning (e.g., dispensing) an amplification reaction mixture comprising cfDNA from a cfDNA sample, amplification reagents, and a plurality of amplification sets into partitions, the plurality of amplification sets comprising primer and probe sets, wherein each amplification set comprises primers and probes for multiplex amplification and each amplification set generates an amplification product, and when a target is present, each amplification set comprises a different label distinguishable from the label of every other amplification set; and wherein the plurality of amplification sets comprises: (i) an amplification set targeting a site that is hypermethylated in fetal DNA and hypomethylated in maternal DNA, and (ii) an amplification set targeting a site that is hypermethylated in maternal DNA and hypomethylated in fetal DNA, and optionally one or more of (iii), (iv), and (v); (iii) an amplification set targeting total cfDNA comprising a methylation-insensitive region from a chromosome that is unlikely to exhibit aneuploidy; (iv) an amplification set targeting a site that is hypermethylated in both fetal DNA and maternal DNA; (v) an amplification set targeting a site that is hypomethylated in both fetal DNA and maternal DNA; (b) incubating the cfDNA with a methylation-sensitive restriction enzyme (MSRE) mixture comprising at least one methylation-sensitive restriction enzyme that cleaves unmethylated (e.g., hypomethylated) DNA; (c) amplifying, if present, the target nucleic acid sequences in the partitions to obtain amplification products; (d) detecting signals from each different label of the amplification products in the partitions; and (e) quantifying the signals for each different label. In some embodiments, the plurality of amplification sets comprises (iii) an amplification set targeting total cfDNA comprising a methylation-insensitive region from a chromosome that is unlikely to exhibit aneuploidy. In some embodiments, each of the amplification sets of (i) and (ii) comprises primers and probes for targeting at least three sites. In some embodiments, each of the amplification sets of (i)-(v) comprises primers and probes for targeting at least three sites or 6-10 sites. In some embodiments, the method further comprises employing an amplification set targeting a methylation-insensitive region of the Y chromosome. In additional embodiments, the method may further comprise employing an amplification set targeting a site that is hypomethylated in both fetal and maternal cfDNA; and / or an amplification set targeting a site that is hypermethylated in both fetal and maternal cfDNA.In some embodiments, the amplification reaction mixture further comprises a control target fully methylated synthetic DNA sequence and / or the fully unmethylated form of the same synthetic DNA sequence. In some embodiments, the digital amplification reaction method is a digital PCR method, such as droplet digital PCR method. In some embodiments, the amplification reaction mixture in the partition contains the MSRE mixture and is incubated after partitioning and before amplification. In some embodiments, step (b) is performed before partitioning and the digested cfDNA is added to the amplification reaction mixture. In some embodiments, the MSRE mixture comprises at least two, at least three or at least four methylation-sensitive restriction enzymes; and / or wherein the MSRE mixture comprises restriction enzymes selected from HhaI, HpaII, AciI, HpyCH4IV and BsaHI. In some embodiments, the MSRE mixture comprises at least two or three of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV and BsaHI. In some embodiments, the MSRE mixture comprises at least HhaI and HpyCH4IV. In some embodiments, the cfDNA sample is obtained from plasma or serum. In some embodiments, each label is a fluorescent label. In some embodiments, the probe is a molecular beacon probe comprising a fluorescent label. In some embodiments, each probe is an oligonucleotide hybridized to a complementary oligonucleotide, the complementary oligonucleotide comprising a label that provides a detectable signal. In some embodiments, the incubation of cfDNA with the MSRE mixture occurs in the partition. In some embodiments, the incubation of cfDNA with the MSRE mixture is performed in the bulk solution before dispensing (a). In some embodiments, the method further comprises, for example, based on the number of targets (N i ) in the amplification set to determine the normalized copy concentration of each target. In some embodiments, the method further comprises determining the corrected concentration of fetal cfDNA in the cfDNA sample (fetal 校正 ) and / or the corrected concentration of maternal cfDNA in the cfDNA sample (maternal 校正 ), wherein determining the corrected concentration of fetal cfDNA comprises calculating:

[0005] Wherein [Total] is the total cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting total cfDNA, the total cfDNA including methylation-insensitive regions of chromosomes that are unlikely to exhibit aneuploidy; [Hypermethylated] is the hypermethylated reference copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in fetal DNA and maternal DNA; [Hypomethylated] is the hypomethylated reference copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypomethylated in fetal DNA and maternal DNA; and [Fetal] is the fetal cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in fetal DNA and hypomethylated in maternal DNA; and determining the corrected concentration of maternal cfDNA includes calculating Wherein [Maternal] is the maternal cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in maternal DNA and hypomethylated in fetal DNA. In some embodiments, the method further includes determining the fetal fraction (FF) in the cfDNA sample, wherein determining the fetal fraction includes at least one of the following calculations (a)-(d):

[0006] (a)

[0007]

[0008] (b)

[0009]

[0010] (c)

[0011]

[0012] or

[0013] (d)

[0014]

[0015] In some embodiments, the method further includes calculating an estimated fetal fraction at least partially based on the fetal fraction in the cfDNA sample and a model. In some embodiments, the model is a generalized additive model (GAM), a linear model, or a second-order polynomial model that is at least partially based on a set of clinical fetal fraction data and a corresponding set of fetal fraction measurements using next-generation sequencing (NGS).

[0016] In some embodiments, the method further includes determining the fetal fraction of a male fetus in the cfDNA sample, wherein determining the fetal fraction of the male fetus includes:

[0017] or

[0018] or

[0019]

[0020] where [YChr] is the concentration of the Y-chromosome specific sequence, based on signals in partitions obtained from an amplification set targeting a methylation-insensitive region of the Y chromosome

[0021] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA are sites within one or more or all of the following:

[0022]

[0023] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are sites within one or more or all of the following:

[0024]

[0025] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in both fetal DNA and maternal DNA are sites within one or more or all of the following:

[0026]

[0027] In some embodiments of the methods or kits described herein, the sites that are hypomethylated in both fetal DNA and maternal DNA are sites within one or more or all of the following:

[0028]

[0029] In some embodiments of the methods or kits described herein, the methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy are sites within one or more or all of the following:

[0030]

[0031]

[0032] In some embodiments of the methods or kits described herein, the methylation-insensitive regions of the Y chromosome are within one or more or all of the following:

[0033]

[0034] On the other hand, the present disclosure provides a digital amplification kit for estimating the fetal DNA fraction in a cfDNA sample obtained from a plasma or serum sample of a pregnant human subject, the kit comprising:

[0035] (a) an amplification reaction mixture comprising amplification reagents and a plurality of amplification sets, the plurality of amplification sets comprising primer and probe sets, wherein each amplification set comprises a different label distinguishable from the labels of each of the other sets, and each set comprises primers and probes for multiplex amplification, and wherein the plurality of amplification sets comprises:

[0036] (i) an amplification set targeting a site that is hypermethylated in fetal DNA and hypomethylated in maternal DNA; and

[0037] (ii) an amplification set targeting a site that is hypermethylated in maternal DNA and hypomethylated in fetal DNA; and optionally one or more of (iii), (iv), and (v);

[0038] (iii) an amplification set targeting total cfDNA comprising a methylation-insensitive region from a chromosome that is unlikely to exhibit aneuploidy;

[0039] (iv) an amplification set targeting a site that is hypermethylated in both fetal DNA and maternal DNA;

[0040] (v) An amplification set that targets sites hypomethylated in fetal DNA and maternal DNA. In some embodiments, each of the amplification sets of (i) and (ii) contains primers and probes for targeting at least three sites. In some embodiments, the kit further contains an amplification set that targets the hypomethylated region of the Y chromosome. In some embodiments, the kit further contains a fully methylated synthetic sequence of DNA and / or a fully unmethylated form of the same synthetic sequence. In some embodiments, each label is a fluorescent label. In some embodiments, the probe is a molecular beacon probe containing a fluorescent label. In some embodiments, each probe is an oligonucleotide that hybridizes with a complementary oligonucleotide containing a label that provides a detectable signal. In some embodiments, the kit further contains a methylation-sensitive restriction enzyme (MSRE) mixture that contains at least one MSRE that cuts hypomethylated DNA. In some embodiments, the MSRE mixture contains at least two, at least three, or at least four methylation-sensitive restriction enzymes; and / or wherein the MSRE mixture contains restriction enzymes selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the kit contains a methylation-sensitive restriction enzyme (MSRE) mixture that contains at least two, three, or more of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture contains at least HhaI and HpyCH4IV. In some embodiments, the kit further contains a methylation-sensitive restriction enzyme (MSRE) mixture that contains at least two, three, or more of the restriction enzymes HhaI, HpalI, AciI, HpyCH4IV, and BsaHI.

[0041] In one aspect, a fetal fraction dataset corresponding to the calculated fetal fraction is created based on the disclosed methods and next-generation sequencing (NGS) using different NIPT clinical samples from a human subject. A number of models are developed that can consider not only the calculated fetal fraction and the corresponding NGS measurements, but also parameters such as gestational age and Y chromosome calculations.

[0042] The term

[0043] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which are incorporated herein by reference, and such conventional methods and various general references are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are well known and commonly used in the art.

[0044] The term "cell-free DNA sample" or "cfDNA sample" refers to a nucleic acid sample containing extracellular DNA, which is obtained from any cell-free biological fluid (e.g., whole blood, urine, saliva, or other biological fluids that have been processed to remove cells). In a typical embodiment, cfDNA for analysis is obtained from whole blood that has been processed to remove cells, such as a plasma or serum sample. As used herein, the term "cfDNA" thus refers to DNA that can be recovered from the acellular portion of a body fluid such as blood.

[0045] The methylation state refers to the presence of methyl at a specific DNA sequence. In some embodiments, methylation of DNA refers to the presence or absence of 5-methylcytosine at one or more CpG dinucleotides in a DNA sequence. The terms "methylation state" or "methylation status" with respect to CpG dinucleotide methylation refer to the presence or absence of 5-methylcytosine ("5-mC" or "5-mCyt") at one or more CpG dinucleotides within a DNA sequence. The methylation state at one or more specific methylation sites within a DNA sequence includes "unmethylated", "fully methylated", and "hemimethylated". For the purposes of this application, the term "hypermethylated" refers to a region in which the average methylation frequency of a specific sample subset, such as fetal DNA or maternal DNA, is greater than 80% as determined by methylation-based sequencing. "Hypomethylated" refers to a region in which the average methylation frequency of a specific sample subset, such as fetal DNA or maternal DNA, is less than 20% as determined by methylation-based sequencing.

[0046] A methylation-sensitive restriction enzyme (MSRE) is an enzyme that cuts DNA at specific unmethylated cytosine residues, but does not cut the recognition sequence when the cytosine residue is methylated.

[0047] As used herein, a "methylation-sensitive" genomic region refers to genomic DNA that can be methylated, for example, at CpG sequences such that the site is not cleavable by a methylation-sensitive restriction enzyme in the methylated state and is cleavable in the absence of methylation. As used herein, "cleavable" means that at least 50% of the DNA is digested by a methylation-sensitive restriction enzyme when the recognition sequence is unmethylated as compared to when it is methylated. Thus, detection of an amplification product obtained by amplifying cfDNA that contains a methylated site after digestion with an MSRE means that the cfDNA is methylated at that site.

[0048] The term "amplification reaction" refers to any in vitro means of amplifying copies of a target sequence of a nucleic acid in a linear or exponential manner. Such methods include, but are not limited to: two-primer methods such as polymerase chain reaction (PCR); ligase methods such as DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; "PCR Protocols: A Guide to Methods and Applications" (edited by Innis et al., 1990)) (LCR); Q Beta RNA replicase and RNA transcription-based amplification reactions (e.g., amplification involving T7, T3, or SP6 primed RNA polymerases), such as transcription-based amplification system (TAS), nucleic acid sequence-based amplification (NASBA), and self-sustained sequence replication (3SR); isothermal amplification reactions (e.g., single primer isothermal amplification (SPIA)); and other techniques known to those of skill in the art.

[0049] "Amplifying" refers to the step of placing a solution under conditions sufficient to amplify a polynucleotide if all components of the reaction are intact. The components of an amplification reaction include, for example, primers, polynucleotide templates, polymerases, nucleotides, etc. In some embodiments, "amplifying" refers to PCR amplification using a first amplification primer and a second amplification primer.

[0050] "Primer" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a starting point for nucleic acid synthesis. Primers can have various lengths, and the length is typically less than 100 nucleotides, for example, 18 - 55 nucleotides in length. The length and sequence of primers for amplification reactions (e.g., PCR) can be designed based on principles known to those skilled in the art. Primers can be DNA, RNA, or chimeras of DNA and RNA moieties. In some cases, primers can include one or more modified or non-natural nucleobases. In some cases, primers are labeled. In some cases, primers can also contain nucleic acid sequences that do not involve hybridization to the target for amplification, such as sequences that hybridize to another labeled oligonucleotide, or to a capture oligonucleotide, or a label sequence, such as a barcode.

[0051] A nucleic acid or a portion thereof "hybridizes" to another nucleic acid provided that non-specific hybridization is minimal at a particular temperature in a physiological buffer. In some cases, a nucleic acid or a portion thereof hybridizes to a conserved sequence shared among a set of target nucleic acids. In some cases, a primer or a portion thereof can hybridize to a primer binding site if there are at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive complementary nucleotides, including "universal" nucleotides that are complementary to more than one nucleotide partner. Alternatively, a primer or a portion thereof can hybridize to a primer binding site if there are less than 1 or 2 complementary mismatches over at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 consecutive complementary nucleotides. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is above room temperature. In some embodiments, the defined temperature for specific hybridization is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C, for example, about 45 °C to about 60 °C, for example, about 55 °C - 59 °C. In some embodiments, the defined temperature for specific hybridization is about 5 °C lower than the calculated melting temperature of the primer.

[0052] As used herein, "nucleic acid" means DNA, RNA, single-stranded, double-stranded or more highly aggregated hybridization motifs and any chemical modifications thereof. Modifications include, but are not limited to, modifications that provide chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, attachment points, and functionality to the nucleic acid ligand base or to the entire nucleic acid ligand. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylation, unusual base pairing combinations such as isobases, isocytidine, and isoguanine, etc. Nucleic acids can also include unnatural bases such as nitroindole. Modifications can also include 3' and 5' modifications that include, but are not limited to, capping with a fluorophore (e.g., quantum dots) or another moiety.

[0053] "Polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides (e.g., DNA). The term encompasses both full-length polypeptides and domains having polymerase activity. At least five families of DNA-dependent DNA polymerases are known, although most belong to families A, B, and C. DNA polymerases are well known to those skilled in the art. The DNA polymerase for use in the compositions and methods disclosed herein can be any polymerase capable of replicating a DNA molecule. In some embodiments, the DNA polymerase is a thermostable polymerase. Thermostable polymerases are isolated from a variety of thermophilic bacteria, such as Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), Bacillus sterothermophilus (Bst), Sulfolobus acidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth) and other species. DNA polymerases are known in the art and are commercially available. In some embodiments, the DNA polymerase is Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT TM , DEEPVENT TM or an active mutant, variant, or derivative thereof. In some embodiments, the DNA polymerase is Taq DNA polymerase. In some embodiments, the DNA polymerase is a high-fidelity DNA polymerase (e.g., iProof TM High-fidelity DNA polymerase, High-fidelity DNA polymerase, High-fidelity DNA polymerase, Taq high-fidelity DNA polymerase and High-fidelity polymerase). In some embodiments, the DNA polymerase is a rapid-start or hot-start polymerase (e.g., FastStart TM Taq DNA polymerase, FastStart TM High-fidelity DNA polymerase or iTaq TMDNA polymerase).

[0054] As used herein, the term "partition" or "partitioned" refers to separating a sample into multiple parts or "partitions". The partitioning is typically physical, such that the sample in one partition does not or substantially does not mix with the sample in an adjacent partition. The partition can be solid or fluid. In some embodiments, the partition is a solid partition, e.g., a microchannel or a micropore. In some embodiments, the partition is a fluid partition, e.g., a droplet. In some embodiments, the fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, the fluid partition (e.g., a droplet) is an aqueous droplet surrounded by an immiscible carrier fluid (e.g., oil). Exemplary arrays of pores and pore descriptions can be found, for example, in U.S. Patent Nos. 9,103,754 and 10,391,493. An array of pores (a set of nanopores, micropores, holes) can be used to capture solid-phase carriers, optionally at addressable known positions. Thus, the array of pores can be configured to facilitate bead capture in at least one of a single solid-phase carrier form or optionally a small group of solid-phase carriers. Exemplary micropore arrays and methods for delivering beads to micropores and their analysis are described, for example, in PCT / US2021 / 034152.

[0055] An "oligonucleotide" is a polynucleotide. In many embodiments, the oligonucleotide will have fewer than 250 nucleotides, and in some embodiments, 4 - 200 nucleotides, e.g., 10 - 150 nucleotides.

[0056] As used herein, the term "a / an" or "the" not only includes aspects having one member, but also aspects having more than one member. For example, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a bead" includes a plurality of such beads, and reference to "the sequence" includes reference to one or more sequences known to those skilled in the art, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Shows methylation-sensitive digestion ddPCR and Y chromosome ddPCR fetal fraction estimation. The figure shows the correlation between methylation-sensitive fetal fraction estimation and SRY-based fetal fraction estimation in the illustrative assays detailed herein (including the example section).

[0058] Figures 2A - 2D Shows the analysis of methylation patterns of maternal and fetal methylation sites and ubiquitously methylated or unmethylated sites.

[0059] Figure 3Depicts how a methylation-sensitive restriction enzyme (MSRE) enables ddPCR to quantify fetal and maternal cfDNA based on differences in fetal and maternal cfDNA methylation. As depicted, MSRE digestion occurs in droplets without disrupting the ddPCR workflow. Fetal and maternal cfDNA are quantified simultaneously in the same ddPCR reaction. 1) Quantify hypermethylated fetal cfDNA after MSRE digestion of hypomethylated maternal cfDNA. 2) Quantify maternal cfDNA after MSRE digestion of hypomethylated fetal cfDNA. 3) Quantify total cfDNA from the non-digested region.

[0060] Figure 4A -B depicts a multiplex ddPCR assay. Figure 4A Depicts a general ddPCR assay format. Figure 4B Depicts combining multiple primer pairs targeting the same chromosome in a single fluorescence channel using unique universal probes.

[0061] Figure 5A -D depicts fetal fraction estimation using the developed linear model, polynomial model, and generalized additive model (GAM) described herein (including in Example 2). Figure 5A Depicts fetal fraction estimation using the developed linear model (“LM”) and using a previous method (“FF_calculated”) compared to fetal fraction estimation using next-generation sequencing (NGS). Figure 5B Depicts the fetal fraction calculated relative to the fetal / hypermethylated variable and the maternal / hypermethylated variable. The residual plot indicates a non-linear relationship of fetal / hypermethylated (510) compared to maternal / hypermethylated (512). Figure 5C Depicts fetal fraction estimation using a second-order polynomial model (“poly2LM”) compared to fetal fraction estimation using NGS. Figure 5D Depicts the GAM for fetal fraction estimation compared to fetal fraction estimation using NGS. MPAE, mean percentage error. MSE, mean square error. Detailed Description

[0062] Introduction

[0063] The present disclosure provides a method for estimating the fraction of fetal DNA in a cfDNA sample obtained from a maternal cell-free biological sample. As described herein, the method comprises a digital amplification reaction that comprises evaluating cfDNA obtained from a pregnant subject, typically a human subject, to determine the methylation status of differentially methylated loci in fetal cfDNA versus maternal cfDNA using a methylation-sensitive restriction enzyme. In particular, the method comprises:

[0064] Analyze the methylation status of one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA in fetal and maternal cfDNA; and detect the methylation status of one or more sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA. In some embodiments, the method further comprises amplifying sites targeting total cfDNA, the total cfDNA comprising methylation-insensitive genomic regions from chromosomes that are unlikely to exhibit aneuploidy, which provides the ability to quantify the total concentration of DNA (i.e., both fetal and maternal) in the sample. In some embodiments, the method further comprises detecting methylation-insensitive regions of the Y chromosome, if necessary, to, for example, determine fetal sex. In some embodiments, the method further comprises evaluating sites that are hypomethylated in both fetal and maternal cfDNA and / or evaluating sites that are hypermethylated in both fetal and maternal cfDNA to serve as internal controls for MSRE digestion. Thus, evaluating the levels of these various chromosomal regions with different methylation profiles provides the ability to quantify the fraction of cfDNA in a maternal sample that is derived from the fetus. In non-invasive prenatal testing, estimation of the fetal fraction is important for quality control and prediction of fetal aneuploidy.

[0065] In some embodiments, the fetal fraction calculated / determined according to the methods of the present disclosure can be used in methods for assessing fetal aneuploidy (e.g., trisomies, such as trisomy of chromosome 13, 18, or 21), chromosomal deletions (e.g., microdeletions, such as microdeletion in chromosome 22), or other genomic alterations (e.g., gene mutations associated with diseases such as alpha thalassemia or beta thalassemia, cystic fibrosis, sickle cell anemia, or hemophilia A). For example, the fetal fraction calculated / determined according to the methods of the present disclosure can be used for quality control in such methods.

[0066] Component

[0067] In the present disclosure, cell-free DNA obtained from a pregnant subject is evaluated to determine the amount of fetal cfDNA in maternal blood, i.e., the fraction of cfDNA in maternal blood that is derived from the fetus. A cfDNA sample from a pregnant subject is digested with one or more methylation-sensitive restriction enzymes, followed by amplification of multiple target loci that have different methylation profiles in fetal DNA and maternal DNA. The fraction of fetal cfDNA can be calculated based on the level of differentially methylated DNA.

[0068] CfDNA

[0069] The cell-free DNA for use in the present invention is obtained from a cell-free biological fluid sample, typically a blood sample. Thus, in a typical embodiment, the sample is a plasma or serum sample. The isolation of cfDNA can be achieved using any number of different methods, for example, by employing columns or magnetic beads or other separation procedures. Kits for extracting cfDNA from samples are commercially available, for example, from Qiagen, Beckman (e.g., Apostle TM kit), and ThermoFisher (e.g., MagMax TM kit), etc.

[0070] Methylation-sensitive restriction enzyme

[0071] The cfDNA is subjected to digestion with one or more methylation-sensitive restriction enzymes (MSREs). Such enzymes will digest the unmethylated regions of the DNA but not the methylated regions. Thus, the abundance of amplification products from the hypermethylated regions of the DNA will be greater than that from the hypomethylated regions of the DNA.

[0072] In some embodiments, one restriction enzyme is employed. In alternative embodiments, two MSREs are employed. In other embodiments, at least three MSREs are employed. In other embodiments, at least four MSREs are employed. Exemplary restriction enzymes include AatII, AciI, AclI, AfeI, AgeI, AscI, BmgBI, BsaAI, BsaHI, BspDI, ClaI, EagI, FseI, PauI, HhaI, HpaII, HpyCH4IV, HinPII, MluI, NarI, NotI, NruI, PvuI, SacII, SalI, and SmaI. In some embodiments, one or more of HhaI, HpaII, AciI, and HpyCH4IV are employed in the analysis.

[0073] In some embodiments, digestion is carried out in bulk with one or more MSREs before the reaction mixture is dispensed into partitions as described below. However, in the preferred embodiment, the cfDNA is added to the dPCR reaction mixture together with the PCR reagents for the target amplification sites and one or more restriction enzymes. Then, restriction enzyme digestion can be carried out in the partitions but before amplification.

[0074] In addition, the targets within the cfDNA eluate can be pre-amplified after bulk MSRE digestion, for example, to reduce the amount of assay multiplexing required to achieve sufficient sensitivity and precision.

[0075] Amplifying the target

[0076] Determination of fetal fractions typically involves multiplex amplification of each targeted hypomethylated or hypermethylated site in the genome being evaluated. Thus, in some embodiments, at least two sites, or at least three sites, or at least four sites or more are targeted for each DNA class available for assay, i.e., sites hypermethylated in fetal cfDNA, sites hypomethylated in fetal cfDNA, sites hypermethylated in maternal cfDNA, sites hypomethylated in maternal cfDNA, sites in methylation-insensitive regions of chromosomes unlikely to exhibit aneuploidy, sites in methylation-insensitive regions of the Y chromosome, sites hypomethylated in both fetal and maternal cfDNA, and sites hypermethylated in both fetal and maternal cfDNA.

[0077] Differentially methylated sites in maternal cfDNA compared to fetal cfDNA have been described (see, e.g., Tonnides, Mol. Genet. Genomic Med. 8: e1094, 2020; Hatte et al., PLOS ONE DOT: 10.1371 / journal.pone.012891, 2015; Bunce et al., Prenat. Diagn. 32: 542 - 54, 2012; Xiang et al., Mol Hum Reprod 20: 875 - 884, 2014). See also Hatt et al., PLOS ONE, July 31, 2015, pp. 1 - 12;

[0078] DoI: 10.1371 / journal.pone.0128918. Differentially methylated sites can also be determined. For example, methylation-based sequencing can be used to identify hypermethylated sequences and hypomethylated sequences from a collection of fetal, maternal, and non-pregnant samples. Exemplary target sites are listed in Table 6, and one, some, or all of the target sites listed in Table 6 can be used, optionally in combination with other target sites not listed in Table 6.

[0079] Sites from chromosomes unlikely to exhibit aneuploidy can be from autosomes other than chromosome 21, chromosome 13, or chromosome 18. In some embodiments, the sites are from chromosome 3. A methylation-insensitive region refers to a region of a chromosome that does not contain CpG sites and is thus unlikely to be methylated in any cell and lacks the recognition sequence of the MSRE employed in the method, meaning that even if these sites are not methylated, they will not be cleaved by the MSRE.

[0080] Sites in methylation-insensitive regions of the Y chromosome refer to methylation-insensitive sequences unique to the Y chromosome such that their detection indicates the presence of the Y chromosome, i.e., a male fetus.

[0081] For the purposes of this application, a differentially methylated site is a site at which the methylation pattern between fetal DNA and maternal DNA is detected to be statistically different (p < 0.015) by a two-sample Kolmogorov-Smirnov test. Additionally, to select methylated sites in fetal cfDNA, sites with an average methylation frequency greater than 80% (i.e., hypermethylated) in fetal cfDNA and less than 20% (i.e., hypomethylated) in maternal DNA are selected. Similarly, to select methylated sites in maternal cfDNA, sites with an average methylation frequency greater than 80% (i.e., hypermethylated) in maternal cfDNA and less than 20% (i.e., hypomethylated) in fetal DNA are selected.

[0082] To select hypermethylated target sites as reference sites, sites with an average methylation frequency greater than 80% in both fetal cfDNA and maternal cfDNA are selected.

[0083] To select hypomethylated target sites as reference sites, sites with an average methylation frequency less than 20% in both fetal cfDNA and maternal cfDNA are selected.

[0084] For example, Figures 2A - 2D Such selection results for the analysis based on the differences in fetal and maternal methylation are provided separately for the selection of fetal hypermethylated sites, maternal hypermethylated sites, reference hypermethylated sites, and reference hypomethylated sites. In some embodiments, the target sites from Table 6 are determined according to the methods described herein.

[0085] Primers and probes

[0086] Primer and probe sequences for detecting the amplified product of a desired target can be designed based on known principles. The amplified product is detected with a detectable label. Those skilled in the art understand that there are any number of label configurations for detecting the amplified product. In some embodiments, the oligonucleotide is labeled with a detectable reagent such as a fluorophore, a phosphorescent agent, a chemiluminescent agent, etc.

[0087] In some embodiments, the probe is labeled, for example, with a fluorescent label. In alternative embodiments, at least one of a pair of amplification primers is labeled with a detectable label, such as a fluorescent label. In some embodiments, a complementary oligonucleotide that hybridizes to a non-target region of the primer or probe is labeled with a detectable label, such as a fluorescent label.

[0088] In some embodiments, the probe is a TAQMAN TM probe, a SCORPION TM probe, an ECLIPSE TMProbes, molecular beacon probes, double-stranded probes, dual hybridization probes, or dual quenching probes.

[0089] In some embodiments, oligonucleotides such as primers or probes are labeled with a detectable label, such as a fluorescent label. In some embodiments, the reagent is a fluorophore. A large number of fluorophores are available for use, including those from commercial suppliers. Non-limiting examples of fluorophores include cyanines (e.g., Cy3, Cy5), indocyanines (e.g., 570, 670, and 705), fluoresceins (e.g., 5′-carboxyfluorescein (FAM), 6-carboxyfluorescein (6-FAM), 5- and 6-carboxyfluorescein (5,6-FAM), 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy-fluorescein (JOE), 4,7,2′,4′,5′,7′-hexachloro-6-carboxy-fluorescein (HEX), 4,7,2′,7′-tetrachloro-6-carboxy-fluorescein (TET), 2′-chloro-5′-fluoro-7′,8′-benzo-1,4-dichloro-6-carboxyfluorescein (NED), Oregon Green, and Alexa 488), rhodamines (e.g., N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) and 5- and 6-carboxy-X-rhodamine (ROX), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)), Atto dyes, eosin, coumarin, pyrene, tetrapyrroles, arylmethines, and oxazines. In some embodiments, the dyes are selected from Cy3, Cy5, Cy4.4, ROX, Atto, FAM, HEX, JOE, QUASAR, rhodamine, TAMRA, TET, Texas Red, TYE, and VIC.

[0090] In some embodiments, detection of the amplification product is via a reporter-quencher pair. Reporter-quencher pairs can be selected from xanthene dyes, including fluorescein and rhodamine dyes. Many suitable forms of these compounds are commercially available, having substituents on the phenyl group that can serve as sites for bonding or as bonding functional groups for attachment to the oligonucleotide. Another group of fluorescent compounds used as reporters are naphthylamines, having an amino group in the α or β position. Such naphthylamine-based compounds include 1-dimethylaminonaphthalene 1-5 sulfonate, 1-anilinonaphthalene-8-sulfonate, and 2-p-toluidino-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin; acridine, such as 9-isothiocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazole; stilbene; pyrene, etc.

[0091] Suitable examples of quenchers can be selected from 6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo)benzoic acid (DABYL), tetramethylrhodamine (TAMRA), BHQ-OTM, BHQ-1TM, BHQ-2TM, and BHQ-3TM (each available from Biosearch Technologies, Inc., Novato, Calif.), Qy7TM, QSY-9TM, QSY-21TM, and QSY-35TM (each available from Molecular Probes, Inc.), and ZEN TM and TAO TM Dual quenching probes (obtained from Integrated DNA Technologies). For example, in R.W. Sabnis, H ANDBOOK OF F LUORESCENT D YES AND P ROBES , John Wiley and Sons, New Jersey, 2015, fluorescent quenchers and dark quenchers from which exemplary reporter-quencher pairs can be selected and their related optical properties are listed and described.

[0092] The recognition sequences of one or more MSREs that can be considered for the reaction can be used to design primers. Thus, for example, primers and target regions to be amplified are selected to avoid the presence of one or more MSRE recognition sequences in the primers and / or in the amplicons generated during the amplification reaction.

[0093] In some embodiments, multiple amplicons can be detected with probes of the same color. For example, in some embodiments, 2-20, such as 6-10 amplicons, are detected with monochromatic probes. In some embodiments, multiple color signals are used. For example, in some embodiments, one color signal is used to detect regions that are hypermethylated in fetal cfDNA compared to maternal cfDNA, a second color signal is used to detect regions that are hypermethylated in maternal cfDNA compared to fetal cfDNA, and optionally, if present, a third color can be used to detect Y chromosome-specific sequences. Additional colors can be used to detect regions that are hypermethylated in both fetal cfDNA and maternal cfDNA, regions that are hypomethylated in both fetal cfDNA and maternal cfDNA, or controls for measuring assay functionality.

[0094] Additional amplification reaction components

[0095] The reagent mixture may further comprise additional reagents, such as amplification reagents, including, for example, one or more of a buffer, salts, nucleotides, stabilizers, primers, polymerase, or nuclease-free water. In some embodiments, additives such as tetramethylammonium chloride (TMAC), DMSO, DTT, or betaine may be used to enhance amplification specificity or yield.

[0096] Partitioning

[0097] The partitioning of the reaction mixture (e.g., cfDNA, digital amplification reaction components, and methylation-sensitive restriction enzymes) into partitions can be achieved by any available method. In some embodiments, methods and compositions for delivering reagents to one or more partitions include microfluidic methods using microtiter plates, capillaries, oil emulsions, and arrays of miniaturized chambers for partitioning. In some embodiments, partitioning utilizes droplets. Methods for generating such droplets include droplet or microcapsule coalescence, aggregation, fusion, rupture, or degradation (e.g., as described in U.S. 2015 / 0027,892, US 2014 / 0227,684, WO 2012 / 149,042, and WO2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151,776); and combinations thereof. Thus, for example, in a method where the partition is a droplet, droplets can be formed in the form of an emulsion with an immiscible fluid such as oil, such that the bulk solution forms droplets containing the reaction mixture reagents, including the cfDNA template. For example, methods for forming emulsions are described in patent applications WO2011 / 109546 and WO 2012 / 061444.

[0098] In some embodiments, the amplification reaction is a droplet digital PCR reaction. For example, methods for performing PCR in droplets are described in US 2014 / 0162266, US2014 / 0302503, and US2015 / 0031034, the contents of each of which are incorporated by reference. In some embodiments, a QX600 droplet digital PCR (ddPCR) system (Bio-Rad) is used.

[0099] In some embodiments, any of a variety of detector devices can be used to detect the detection reagent or detectable label in the partition. Exemplary detection methods include optical detection (e.g., fluorescence or chemiluminescence). As a non-limiting example, a detector device equipped with a module that generates excitation light that can be absorbed by a fluorophore and a module that detects the light emitted by the fluorophore can be used to detect a fluorescent label.

[0100] In some embodiments, the detector further includes the ability to process partitioned samples (e.g., droplets), where the individually partitioned samples enter the detector, are detected, and then leave the detector. In some embodiments, the partitioned samples (e.g., droplets) can be continuously detected as they flow. In some embodiments, the partitioned samples (e.g., droplets) are arranged on a surface, and the detector moves relative to the surface to detect signals at each location containing a single partition. Examples of detectors are provided in WO2010 / 036352, the content of which is incorporated herein by reference. In some embodiments, the detectable markers in the partitioned samples can be continuously detected without flowing the partitioned samples (e.g., using a chamber slide).

[0101] After acquiring fluorescence detection data, a general-purpose computer system (referred to herein as the "host computer") can be used to store and process the data. Computer-executable logic can be employed to perform functions such as subtraction of background signals, assignment of target sequences and / or reference sequences, and quantification of data. The host computer can be used to display, store, retrieve, or calculate the fetal fraction in a sample; store, retrieve, or calculate the raw data from nucleic acid detection; or display, store, retrieve, or calculate any sample or source information useful in the method.

[0102] The host computer can be configured with many different hardware components and can be made in many sizes and styles (e.g., desktop PC, laptop computer, tablet PC, handheld computer, server, workstation, mainframe computer). Standard components such as a display, keyboard, disk drive, CD and / or DVD drive, etc. can be included. When the host computer is connected to a network, the connection can be provided via any suitable transmission medium (e.g., wired, optical, and / or wireless media) and any suitable communication protocol (e.g., TCP / IP); the host computer can include suitable networking hardware (e.g., modem, Ethernet card, WiFi card). The host computer can implement any one of a variety of operating systems, including UNIX, Linux, Microsoft Windows, MacOS, or any other operating system.

[0103] The computer code for implementing aspects of the present invention can be written in a variety of languages, including PERL, C, C++, Java, JavaScript, VBScript, AWK, or any other scripting or programming language that can be executed on the host computer or can be compiled to be executed on the host computer. The code can also be written or distributed in a low-level language such as assembly language or machine language.

[0104] Scripts or programs that combine the various features of the present invention can be encoded on various computer-readable media for storage and / or transmission. Examples of suitable media include magnetic disks or tapes, optical storage media such as compact discs (CDs) or digital versatile discs (DVDs), flash memory, and carrier signals suitable for transmission via wired, optical, and / or wireless networks that conform to various protocols including the Internet.

[0105] Calculation of fetal fraction

[0106] Data processing can be used to obtain the copy concentrations of detected fetal-specific or maternal-specific target sites to generate a fetal fraction calculation, optionally along with determination of fetal gender (when desired). Table 3 shows the various copy concentrations provided by exemplary six assays collected in six illustrative fluorescence channels. As described herein, copy concentrations from one or more fetal-specific or maternal-specific target sites, as well as various control target sites, can be determined in partitions from a sample. Thus, for example, fetal cfDNA copy concentration can be determined from one or more target sites that are specifically hypermethylated in fetal cells and hypomethylated in maternal cells, cleaved with one or more MSREs, and detected by probes, for example, in a digital assay. Signals for one or more target sites can be multiplexed such that, for example, probes that detect different fetal hypermethylated target sites have the same color probe, and the sum of the partitions with signals of that color divided by the number of targets indicates the fetal copy concentration.

[0107] In some embodiments, prior to any downstream calculations, each copy concentration (e.g., each of the six copy concentrations) is normalized by dividing by the number of assays in the associated multiplex assay. The number of partitions that are positive for a particular signal (e.g., detectable wavelength or "color") can represent amplicons from multiple targets (each detected with the same color probe). In these embodiments, N i represents the number of multiple targets detected (and, for example, detected with probes having the same color label) in the amplification set. In an illustrative embodiment, the average corrected concentrations of fetal and maternal cfDNA can be found by interpolation within hypermethylated and hypomethylated reference corrections, as shown in Equations 1 and 2.

[0108]

[0109] Wherein [Total] is the total cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting total cfDNA, the total cfDNA comprising methylation-insensitive regions of chromosomes that are unlikely to exhibit aneuploidy; [Hypermethylated] is the hypermethylated reference copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in fetal DNA and maternal DNA; [Hypomethylated] is the hypomethylated reference copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypomethylated in fetal DNA and maternal DNA; [Fetal] is the fetal cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in fetal DNA and hypomethylated in maternal DNA; and [Maternal] is the maternal cfDNA copy concentration, which is based on signals in partitions obtained from an amplification set targeting sites hypermethylated in maternal DNA and hypomethylated in fetal DNA.

[0110] In addition, there are several relationships between independently measured metrics. Thus, compliance with the relationships expressed in Equations 3-5 can be used as a measure of data quality.

[0111] [Total] = [Fetal 校正 + [Maternal 校正 (Equation 3)

[0112]

[0113] 2 * [YChr] = [Fetal 校正 (Equation 5),

[0114] where [YChr] is the concentration of Y chromosome-specific sequences, which is based on signals in partitions obtained from an amplification set targeting methylation-insensitive regions of the Y chromosome.

[0115] A male fetus can be determined by confirming that the Y chromosome copy concentration is not zero and that the relationship shown in Equation 5 is satisfied. Additionally, in the case of Y chromosome sex aneuploidy, the coefficient will be 1 instead of 2.

[0116] In the case of a female or male fetus, the fetal fraction can be calculated, for example, by four methods shown in Equations 6-9, which can be averaged to obtain a more precise value. Additionally, failure of the fetal fraction calculations to converge on an average will indicate a problem with the data quality.

[0117]

[0118] In the case of a male fetus, two additional fetal fraction calculations (Equations 10 and 11) can be performed using data including the copy concentration of the Y chromosome.

[0119]

[0120] Fetal fraction estimation model

[0121] To improve the accuracy of the calculated fetal fraction, a model is developed to take the calculated fetal fraction as input and adjust the fetal fraction based on the model output. The purpose of developing this model is to minimize the average error of the fetal fraction calculated from the same clinical sample through next-generation sequencing (NGS) measurements of the fetal fraction of clinical samples. In one example, the model is designed to reduce the mean percentage error or the mean squared error.

[0122] For example, consider x1, x2, x3, ..., xN representing the calculated fetal fractions, and z1, z2, z3, ..., zN being the corresponding NGS measurements. For example, z1 corresponds to x1, z2 corresponds to x2, etc. Additionally, consider the estimation model being represented as a function f(), which takes the calculated values x1, x2, ..., xN and, for each calculated fraction, calculates the estimated fetal fractions y1, y2, y3, ..., yN, such as y1 = f(x1), y2 = f(x2), ..., yN = f(xN). The error for each estimated value can be defined as e1 = (y1 - z1) = (f(x1) - z1), e2 = (y2 - z2) = (f(x2) - z2), ... eN = (yN - zN) = (f(xN) - zN). Several criteria can be considered when defining the estimation or mapping function f(). For example, when minimizing the mean squared error (MSE), the function f() is designed such that the MSE, such as (e1 2 + e2 2 +... + eN 2 ) / N is minimized. In another example, f() can be designed such that the mean absolute percentage error (MAPE), such as (|e1| / z1 + |e2| / z2 +... + |eN| / zN) / N (where |.| is the absolute value operation) is minimized.

[0123] In one example, once the model is designed using clinical or training data, the model can be used to estimate the fetal fraction based on the calculated fetal fraction. In another example, the model can be dynamic and can be updated using additional training data.

[0124] In one example, the function f() is a linear function. For example, f(x) = a x + b. In another example, random noise can be included in the model, such as f(x) = a x + b + n, where n is a random variable with a predefined distribution.

[0125] Figure 5AShows the impact of applying a linear model to the calculated fetal fraction and its impact on MSE and MAPE. In 501, the calculated fetal fraction is depicted against the corresponding NGS fetal fraction. The calculated fetal fraction data has a MAPE of 28% and an MSE of 6.9×10 -4 . In 503, the same data as in 501 is represented using a box plot. In 502, a linear model is applied to the calculated fetal fraction to obtain the depicted estimated fetal fraction. As shown, the estimates are more consistent with the corresponding NGS values, which is also verified by the decreased MSE and MAPE values. Using the linear model, the MAPE is reduced to 19%, and the MSE is reduced to 2.8×10 -4 . In 504, the same data as in 502 is represented using a box plot. Comparing the box plots in 504 and 503 shows less variation in the data, and the estimated fetal fraction is closer to the measured NGS value compared to the calculated fetal fraction.

[0126] In one example, the function f() can be a polynomial. For example, f(x) = a0 + a1x + a2x 2 + a3x 3 +... + aMx M , where M determines the order of the polynomial. For example, for a second-order polynomial, M = 2, and f(x) = a0 + a1x + a2x 2 . Using an optimization criterion, such as minimizing MSE or MAPE, and applying the model to clinical data, the coefficients a0, a1, and a2 can be calculated.

[0127] Figure 5B Depicts a residual plot. In 510, the calculated fetal fraction is depicted against the fetal / high methylation variable. Compared to the residual plot of fetal / high methylation, in 512, the calculated fetal fraction is depicted against the maternal / high methylation variable. The plot does not indicate a linear relationship between fetal / high methylation and the calculated fetal fraction.

[0128] Figure 5C Shows the impact of applying a linear model to the calculated fetal fraction and its impact on MSE and MAPE. The same calculated fetal fraction data as in 501 is used to develop a second-order polynomial model. The second-order polynomial model (poly2LM) is applied to the calculated fetal fraction to obtain the depicted estimated fetal fraction. As Figure 5C shown, the estimates are more closely distributed along a straight line with the corresponding NGS values, which is also verified by the decreased MSE and MAPE values. Using the second-order polynomial model, the MAPE is reduced to 17%, and the MSE is reduced to 2.5×10 -4 .

[0129] In one example, a Generalized Additive Model (GAM) can be used to estimate the fetal fraction based on the calculated fetal fraction. The GAM can be used to model non-linearity using an additive model. For example, at different NGS values, there may be different relationships between the calculated fetal fraction and the corresponding NGS values. A piecewise model can be developed that defines different estimation functions. For example, the range of NGS can be divided into K regions, such as K = 20, 30, or 40, and each region uses a spline estimation, such as a spline of order 2, 3, 4, or different orders. Additionally, the GAM can incorporate other parameters as variables of the model, such as gestational age, maternal weight, etc.

[0130] Figure 5D Shows the effect of applying the GAM on the calculated fetal fraction and its effect on the MSE and MAPE. The same data for calculating the fetal fraction as in 501 is used. The GAM is applied to the calculated fetal fraction to obtain the depicted estimated fetal fraction. As Figure 5D shown, the estimated values are more aligned with the corresponding NGS values along the line, which is also verified by the reduced MSE and MAPE values. Using the GAM, the MAPE is reduced to 13%, and the MSE is reduced to 1.5×10 -4 .

[0131] Kit

[0132] In another aspect, the present disclosure provides a digital amplification kit for estimating the fraction of fetal DNA in a cfDNA sample obtained from a blood sample of a pregnant subject, such as a human (such as a plasma or serum sample). The kit may include any of the components described herein with respect to the method. In some embodiments, the kit contains an amplification reaction mixture that includes amplification reagents and a plurality of amplification sets, the plurality of amplification sets including primer and probe sets, where each amplification set includes a different label distinguishable from the labels of every other amplification set, and each amplification set includes primers and probes for multiplex amplification, and where the plurality of amplification sets includes (i) an amplification set targeting a site that is hypermethylated in fetal DNA and hypomethylated in maternal DNA; and / or (ii) an amplification set targeting a site that is hypermethylated in maternal DNA and hypomethylated in fetal DNA. In some embodiments, the kit further contains an amplification set targeting total cfDNA, the total cfDNA including a methylation-insensitive region from a chromosome that is not likely to exhibit aneuploidy. In some embodiments, the kit contains an amplification set targeting a site that is hypermethylated in both fetal DNA and maternal DNA; and / or an amplification set targeting a site that is hypomethylated in both fetal DNA and maternal DNA. In some embodiments, the kit further contains an amplification set targeting a methylation-insensitive region of the Y chromosome. In some embodiments, one or more sites are selected from Table 6 as described elsewhere herein. In some embodiments, the kit as described in this paragraph contains a fully methylated synthetic sequence of DNA and / or a fully unmethylated form of the same synthetic sequence. In some embodiments, the probe for detection is a molecular beacon probe, such as a fluorescently labeled molecular beacon probe.

[0133] In some embodiments, each of the amplification sets of (i) and (ii) includes primers and probes for targeting at least two sites, at least three sites, or more (such as 1 - 20 sites, such as between 6 - 10 sites).

[0134] In some embodiments, each label of the amplification sets is a fluorescent label. In some embodiments, one or more primers or one probe includes a region that does not hybridize to the target amplification site but is complementary to an oligonucleotide that provides a detectable signal.

[0135] In some embodiments, a kit comprising one or more amplification sets as described in the foregoing paragraphs further comprises at least one methylation-sensitive restriction enzyme that cleaves hypomethylated DNA. In some embodiments, the kit comprises one or more of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV, or BsaHI. In some embodiments, the methylation-sensitive restriction enzyme (MSRE) mixture comprises at least one, two, at least three, or at least four methylation-sensitive restriction enzymes selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the kit comprises a methylation-sensitive restriction enzyme (MSRE) mixture that comprises at least two, three, or more of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture comprises at least HhaI and HpyCH4IV.

[0136] In some embodiments, the reaction mixture is lyophilized.

[0137] In some embodiments, the kit comprises a standard ddPCR kit, a primer and probe set for fetal fraction determination as described herein, and at least one MSRE. In some embodiments, such a kit further comprises at least one PCR enhancer, such as TMAC and / or salts.

[0138] In some embodiments, the kit comprises a standard ddPCR kit, a primer and probe set for fetal fraction determination as described herein, at least one MSRE, and stabilizers (such as trehalose, potassium glutamate, ammonium sulfate) (lyophilized together as a mixture).

[0139] In some embodiments, the kit comprises a standard ddPCR kit, a primer and probe set for fetal fraction determination as described herein, a stabilizing reagent (lyophilized together), and at least one MSRE, which may or may not be lyophilized.

[0140] Examples

[0141] The following examples further illustrate aspects and embodiments of the methods of the present disclosure.

[0142] Example 1: Methylation-based digital PCR assay for quantification of fetal-free DNA in NIPT samples.

[0143] First, cfDNA is isolated from maternal plasma, for example, using a commercially available kit, such as a bead kit designed to preferentially capture and elute cfDNA (e.g., a bead-based extraction kit (Apostle)). The fragment length distribution of the cfDNA eluate can be confirmed, for example, by a commercially available method, such as a high-sensitivity DNA bioanalyzer kit (Agilent).

[0144] Digest cfDNA isolated from a maternal sample with a methylation-sensitive restriction enzyme (MSRE) mixture containing enzymes (such as, but not limited to, HhaI, HpaII, AciI, or HpyCH4IV). These enzymes cut DNA at sites where there are several bases in a specific sequence and cut DNA only when the site is unmethylated (Table 1). For differentially methylated sites (DMSs) that are hypermethylated in fetal cfDNA and hypomethylated in maternal cfDNA, only the fetal cfDNA DMSs remain to be quantified. Conversely, for DMSs that are hypermethylated in maternal cfDNA and hypomethylated in fetal cfDNA, only the maternal cfDNA DMSs remain to be quantified. Although in some embodiments, the digestion of cfDNA can be carried out globally, in this illustrative embodiment, the digestion is carried out after partitioning the sample and the PCR reagents but before PCR amplification.

[0145] Add cfDNA (e.g., the eluate from the sample) to a dPCR reaction mixture containing a dPCR premix (including all reagents required for both PCR and partitioning), the MSRE mixture, and (in this example) a 6-channel assay set (Table 1). This in-partition digestion technique allows for a more simplified procedure and a single thermal cycling run (Table 2).

[0146] The methylation-sensitive digestion strategy described herein is further illustrated below using a 4-channel droplet digital PCR (ddPCR) instrument (QX ONE).

[0147] Twenty-two maternal plasma samples (CerbaXpert, France) and two non-pregnant plasma samples (Stanford Blood Bank) were selected, and as measured by VeriSeq NIPT, the fetal fractions of the twenty-two maternal plasma samples were in the range of 10% to 25%. cfDNA was extracted from the twenty-four samples using the Apostle MiniMax cfDNA Isolation Kit, which was carried out automatically on a KingFisher Flex (ThermoFisher). Before being used with ddPCR, the cfDNA was characterized on a Bioanalyzer platform (Agilent) and was found to contain 62% ± 6% mononucleosomal cfDNA, while the remaining nucleic acid content consisted of high-molecular-weight genomic DNA.

[0148] For each sample, 5.5 μL of the extraction eluate was used in six 22-μL ddPCR reactions (Table 4). All reactions included fetal DMS triplets (FAM channel) targeting sites that are hypermethylated in fetal cfDNA and hypomethylated in maternal cfDNA, maternal DMS triplets (HEX channel) targeting sites that are hypermethylated in maternal cfDNA and hypomethylated in fetal cfDNA, and a Y chromosome target (SRY, Cy5.5 channel). In the remaining Cy5 channel, three reactions contained an X chromosome target (SPIN4), and three reactions contained a chromosome 10 target (RPP30). Additionally, four reactions contained the MSRE mix (HhaI and HpyCH4IV, 10 U per ddPCR reaction), while two reactions did not.

[0149] Fetal and maternal triplets were used to calculate the fetal fraction as shown in Equations 6 - 11, although uncorrected values were used. Copy concentrations of SRY, SPIN4, and RPP30 were used to calculate orthogonal ddPCR fetal fraction estimates independent of the methylation-sensitive digestion protocol.

[0150] Results

[0151] Figure 1 The correlation between methylation-sensitive fetal fraction estimates and SRY-based fetal fraction estimates for 24 samples is shown. When both SRY / SPIN4 and SRY / RPP30 calculations were included, the R for the positive correlation 2 was 0.88. Additionally, the R for the correlation between methylation-sensitive fetal fraction estimates and VeriSeq non-invasive prenatal test (NIPT) determinations 2 was 0.84 (Table 5).

[0152] Use with other fetal diagnostic assays

[0153] In the case of sufficient multiplexing, this fetal fraction quantification method can be used concurrently with other fetal cfDNA tests, including trisomy and microdeletion detection. In some embodiments, the assays can be performed in separate wells with the same sample because the method only employs a portion of the cfDNA obtained from the maternal sample. In some embodiments, if a multi-well test is required, MSRE digestion is performed within the droplets to determine the fetal fraction, which simplifies the workflow. In this case, the cfDNA eluate is added to each test well, and only the cfDNA used for fetal fraction determination will be digested due to the presence of MSRE in the fetal fraction test reaction mixture. In some embodiments, the assay can be performed in conjunction with diagnostic tests for single-gene disorders (such as, but not limited to, α-thalassemia or β-thalassemia, cystic fibrosis, or hemophilia A).

[0154] Example 2: Methylation-based highly multiplexed ddPCR assay and machine learning method for determining fetal cell-free DNA fraction in NIPT samples.

[0155] In this example, a methylation-based highly multiplexed ddPCR assay and a machine learning-based method are used to analyze the fetal cfDNA fraction in NIPT samples.

[0156] The fetal fraction (FF) is the proportion of fetal cfDNA in the blood of pregnant mothers. Estimation of the fetal fraction can be used, for example, to non-invasively assess fetal aneuploidy. Currently, next-generation sequencing (NGS) serves as the gold standard method for fetal fraction estimation. Methods include dissecting single nucleotide polymorphisms to analyze different genotypes between the fetus and the mother, measuring the proportion of Y chromosome cfDNA reads in male fetuses, and examining differences in methylation profiles. However, sequencing-based methods are not cost-effective. Droplet digital PCR (ddPCR) involves partitioning individual PCR reactions into microdroplets, allowing for high levels of sensitivity and accuracy, as well as cost reduction relative to NGS.

[0157] In Example 1, a methylation-based ddPCR assay was designed based on the different methylation patterns between maternal cfDNA and fetal cfDNA in NIPT samples to calculate the fetal fraction. This example describes a methylation-based highly multiplexed ddPCR assay to simultaneously quantify fetal and maternal cfDNA in the same ddPCR reaction with improved accuracy to quantify the fetal fraction in NIPT samples.

[0158] ddPCR assay design and machine learning method

[0159] The ddPCR assay uses 6 fluorescence channels (QX600 instrument, Bio-Rad Laboratories), with 6 - 10 targets per channel, targeting:

[0160] (i) Sites that are hypermethylated in fetal cfDNA and hypomethylated in maternal cfDNA to target fetal cfDNA after methylation-sensitive restriction enzyme digestion ("fetal");

[0161] (ii) Sites that are hypermethylated in maternal cfDNA and hypomethylated in fetal cfDNA to target maternal cfDNA after methylation-sensitive restriction enzyme digestion ("maternal");

[0162] (iii) Sites that are hypomethylated in both fetal and maternal cfDNA ("hypomethylated") to target the methylation-sensitive enzyme digestion efficiency together with "hypermethylated" as explained in (iv);

[0163] (iv) Sites hypermethylated in fetal and maternal cfDNA (“hypermethylated”), to be targeted for digestion by methylation-sensitive enzymes together with “hypomethylated”;

[0164] (v) For male fetuses, sites on the Y chromosome that do not contain CpG sites (i.e., methylation-insensitive regions) (“ChrY”); and

[0165] (vi) Control sites that do not contain CpG sites for performance control determination (“control”).

[0166] Estimation of fetal cfDNA fraction is performed using fetal / hypermethylated, maternal / hypermethylated, hypomethylated / hypermethylated, and chrY / control. The fetal / hypermethylated, maternal / hypermethylated, hypomethylated / hypermethylated, and chrY / control are calculated based on the λ ratios of “fetal” divided by “hypermethylated”, “maternal” divided by “hypermethylated”, “hypomethylated” divided by “hypermethylated”, and “chrY” divided by “control”.

[0167] Figure 3 and 4A An overview of the assay design is provided in -4B. Table 6 provides exemplary target sites for fetal fraction quantification that can be used in this ddPCR assay.

[0168] To further improve the assessment of fetal fraction, machine learning linear regression, polynomial, and generalized additive models trained on clinical NIPT samples were developed. Due to the limitations of clinical NIPT samples, more than 70 samples were used to train these three models. The fetal / hypermethylated, maternal / hypermethylated, hypomethylated / hypermethylated, and chrY / control calculated from the highly multiplexed fetal fraction assay mentioned in Table 6 were also tested and used as input variables for these three models, while other metadata from clinical NIPT samples, such as gestational week, were used to improve the performance of the models.

[0169] Results

[0170] As Figures 5A - 5D and shown in Table 7, when using linear regression, polynomial, and generalized additive models, the mean percentage error (MAPE) and mean squared error (MSE) of ddPCR-based fetal fraction estimation are significantly reduced compared to previous analytical methods for NGS-based calculations.

[0171] For example, compared to the fetal fraction calculation using equations 1 - 11 (“FF_calculated value”), the fetal fraction estimation using the linear regression model trained on clinical samples (“LM”) has smaller errors, including smaller MAPE and MSE (see Figure 5Aand Table 7). Using NGS and linear models, the relationships between fetal / hypomethylated and maternal / hypomethylated variables and fetal fraction are as Figure 5B shown. To further improve the model, NGS was used to decipher the relationships between fetal / hypomethylated, maternal / hypomethylated, and fetal fraction using residual plots. Figure 5B The figures in show a non-linear relationship between fetal fraction and fetal / hypomethylated compared to the maternal / hypomethylated variable. Using the second-order term of fetal / hypomethylated in the polynomial model can improve performance metrics such as MAPE and MSE. For example, Figure 5C shows the fetal fraction estimated using the second-order polynomial model (“poly2LM”). Compared to previous methods, the second-order polynomial model has smaller errors, including smaller MAPE and MSE (see Figure 5A , 5C and Table 7).

[0172] Generalized additive models (GAMs) are generalized linear models that can model non-linear data with interpretability. Compared to previous methods, GAMs have smaller errors, including smaller MAPE and MSE (see Figure 5D and Table 7).

[0173] The ddPCR assays and fetal fraction calculation methods described in this example show great promise in improving the accuracy and reliability of ddPCR in fetal fraction estimation, which helps in ddPCR aneuploidy NIPT testing and makes it a useful tool for prenatal screening and diagnosis.

[0174] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes made in accordance with them will be contemplated by those skilled in the art and included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0175] Table 1. Examples of dPCR master mixes for in-partition digestion

[0176]

[0177]

[0178] Table 2. ddPCR MSRE digestion and thermal cycling protocol

[0179]

[0180] Table 3. Examples of dPCR multiplex assays and copy concentration readings

[0181]

[0182] Table 4. MSRE and reference target scenarios

[0183]

[0184]

[0185] Table 5. Correlation of methylation-sensitive digestion fetal fraction with orthogonal metrics.

[0186] Orthogonal metric Coefficient R2 SRY and SPIN4 (n = 3) 0.74 SRY and RPP30 (n = 3) 0.87 SRY and SPIN4 and RPP30 (n = 3) 0.88 VeriSeq NIPT 0.84

[0187] Table 6. Exemplary methylation-based fetal fraction ddPCR assay target regions as shown in human genome version 38

[0188]

[0189]

[0190] Table 7. Performance of fetal fraction estimation in different algorithms

[0191]

Claims

1. A method for estimating the fetal DNA fraction in a cfDNA sample obtained from a blood sample of a pregnant human subject, the method comprising a digital amplification reaction method, the digital amplification reaction method comprising: (a) partitioning an amplification reaction mixture comprising cfDNA from the cfDNA sample, amplification reagents, and a plurality of amplification sets into partitions, the plurality of amplification sets comprising primer and probe sets, wherein each amplification set comprises primers and probes for multiplex amplification and each amplification set generates an amplification product, and when a target is present, each amplification set comprises a different label distinguishable from the label of every other amplification set; and wherein the plurality of amplification sets comprises: (i) an amplification set targeting a site that is hypermethylated in fetal DNA and hypomethylated in maternal DNA ； and (ii) an amplification set targeting a site that is hypermethylated in maternal DNA and hypomethylated in fetal DNA; and optionally one or more of (iii), (iv), and (v): (iii) an amplification set targeting total cfDNA comprising a methylation-insensitive region from a chromosome that is unlikely to exhibit aneuploidy; (iv) an amplification set targeting a site that is hypermethylated in both fetal DNA and maternal DNA; (v) an amplification set targeting a site that is hypomethylated in both fetal DNA and maternal DNA; (b) incubating the cfDNA with a methylation-sensitive restriction enzyme (MSRE) mixture comprising at least one methylation-sensitive restriction enzyme that cuts unmethylated DNA; (c) amplifying the target nucleic acid sequences in the partitions, if present, to obtain amplification products; (d) detecting the signals of each different label from the amplification products in the partitions; and (e) quantifying the signals for each different label.

2. The method according to claim 1, wherein the plurality of amplification sets comprises (iii) an amplification set targeting total cfDNA comprising a methylation-insensitive region from a chromosome that is unlikely to exhibit aneuploidy.

3. The method according to claim 1 or 2, wherein each of the amplification sets of (i) and (ii) comprises primers and probes for targeting at least three sites.

4. The method according to claim 1 or 2, wherein each of the amplification sets of (i)-(v) comprises primers and probes for targeting at least three sites or 6-10 sites.

5. The method according to any one of claims 1 to 4, further comprising an amplification set targeting a methylation-insensitive region of the Y chromosome.

6. The method according to any one of claims 1 to 5, further comprising an amplification set targeting a site that is hypomethylated in both fetal and maternal cfDNA; and / or an amplification set targeting a site that is hypermethylated in both fetal and maternal cfDNA.

7. The method according to any one of claims 1 to 6, wherein the amplification reaction mixture further comprises a control target fully methylated synthetic DNA sequence and / or a fully unmethylated form of the same synthetic DNA sequence.

8. The method according to any one of claims 1 to 7, wherein the digital amplification reaction method is a digital PCR method.

9. The method according to claim 8, wherein the digital PCR method is a droplet digital PCR method.

10. The method according to any one of claims 1 to 9, wherein the amplification reaction mixture comprises the MSRE mixture, and the incubation is carried out after the partitioning and before the amplification.

11. The method according to any one of claims 1 to 9, wherein step (b) is carried out before the partitioning and the digested cfDNA is added to the amplification reaction mixture.

12. The method according to any one of claims 1 to 11, wherein the MSRE mixture comprises at least two, at least three or at least four methylation-sensitive restriction enzymes; and / or wherein the MSRE mixture comprises restriction enzymes selected from HhaI, HpaII, AciI, HpyCH4IV and BsaHI.

13. The method according to claim 12, wherein the MSRE mixture comprises at least two, three or four of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV and BsaHI.

14. The method according to claim 12 or claim 13, wherein the MSRE mixture comprises at least HhaI and HpyCH4IV.

15. The method according to any one of claims 1 to 14, wherein the cfDNA sample is obtained from plasma or serum.

16. The method according to any one of claims 1 to 15, wherein each label is a fluorescent label.

17. The method according to claim 16, wherein the probe is a molecular beacon probe comprising a fluorescent label.

18. The method according to any one of claims 1 to 15, wherein each probe is an oligonucleotide hybridized to a complementary oligonucleotide, the complementary oligonucleotide comprising a label that provides a detectable signal.

19. The method according to any one of the preceding claims, further comprising determining a normalized copy concentration of each said target based on the number of targets (N i ) in the amplification set.

20. The method according to any one of the preceding claims, further comprising determining a corrected concentration of fetal cfDNA in the cfDNA sample (fetal 校正 ) and / or a corrected concentration of maternal cfDNA in the cfDNA sample (maternal 校正 ), wherein determining the corrected concentration of fetal cfDNA comprises calculating: wherein [Total] is the total cfDNA copy concentration, which is based on the signals in the partitions obtained from the amplification set targeting total cfDNA, the total cfDNA comprising methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy; [Hypermethylation] is the hypermethylation reference copy concentration, which is based on the signals in the partitions obtained from the amplification set targeting sites that are hypermethylated in fetal DNA and maternal DNA; [Hypomethylation] is the hypomethylation reference copy concentration, which is based on the signals in the partitions obtained from the amplification set targeting sites that are hypomethylated in fetal DNA and maternal DNA; and [Fetal] is the fetal cfDNA copy concentration, which is based on the signals in the partitions obtained from the amplification set targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and determining the corrected concentration of maternal cfDNA comprises calculating: wherein [Maternal] is the maternal cfDNA copy concentration, which is based on the signals in the partitions obtained from the amplification set targeting sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA.

21. The method according to any one of the preceding claims, further comprising determining a fetal fraction (FF) in the cfDNA sample, wherein determining the fetal fraction comprises at least one of the following calculations (a)-(d):

22. The method according to claim 21, further comprising: estimating a fetal fraction at least in part based on the fetal fraction in the cfDNA sample and a model calculation.

23. The method according to claim 22, wherein the model is a generalized additive model (GAM), a linear model, or a second-order polynomial model that is at least in part based on a set of clinical fetal fraction data and a corresponding set of fetal fraction measurements using next-generation sequencing (NGS).

24. The method according to any one of claims 5 to 20, further comprising determining a fetal fraction of a male fetus in the cfDNA sample, wherein determining the fetal fraction of the male fetus comprises: where [YChr] is the concentration of the Y chromosome-specific sequence, based on signals in partitions obtained from an amplification set targeting a methylation-insensitive region of the Y chromosome.

25. The method according to any one of claims 1 to 24, wherein the sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA are one or more or all of the sites within:

26. The method according to any one of claims 1 to 25, wherein the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are one or more or all of the sites within: 。 27. The method according to any one of claims 1 to 26, wherein the sites that are hypermethylated in both fetal DNA and maternal DNA are one or more or all of the sites within: 。 28. The method according to any one of claims 1 to 27, wherein the sites that are hypomethylated in both fetal DNA and maternal DNA are one or more or all of the sites within: 。 29. The method according to any one of claims 1 to 28, wherein the methylation-insensitive regions of chromosomes that are less likely to exhibit aneuploidy are one or more or all of the sites within:

30. The method according to claim 5, wherein the methylation-insensitive region of the Y chromosome is within one or more or all of:

31. A digital amplification kit for estimating a fetal DNA fraction in a cfDNA sample obtained from a plasma or serum sample from a pregnant human subject, the kit comprising: (a) an amplification reaction mixture comprising amplification reagents and a plurality of amplification sets, the plurality of amplification sets comprising primer and probe sets, wherein each amplification set comprises a distinct label distinguishable from the labels of each of the other sets, and each set comprises primers and probes for multiplex amplification, and wherein the plurality of amplification sets comprises: (i) an amplification set targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and (ii) An amplification set targeting sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA; and optionally one or more of (iii), (iv), and (v); (iii) An amplification set targeting total cfDNA containing methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy; (iv) An amplification set targeting sites that are hypermethylated in both fetal DNA and maternal DNA; (v) An amplification set targeting sites that are hypomethylated in both fetal DNA and maternal DNA.

32. The kit according to claim 31, wherein each of the amplification sets of (i) and (ii) comprises primers and probes for targeting at least three sites.

33. The kit according to any one of claims 31 to 32, further comprising an amplification set targeting a methylation-insensitive region of the Y chromosome.

34. The kit according to any one of claims 31 to 33, further comprising a fully methylated synthetic sequence of DNA and / or a fully unmethylated form of the same synthetic sequence.

35. The kit according to any one of claims 31 to 34, wherein each label is a fluorescent label.

36. The kit according to claim 35, wherein the probe is a molecular beacon probe comprising a fluorescent label.

37. The kit according to any one of claims 31 to 34, wherein each probe is an oligonucleotide hybridizing with a complementary oligonucleotide comprising a label that provides a detectable signal.

38. The kit according to any one of claims 31 to 37, further comprising a methylation-sensitive restriction enzyme (MSRE) mixture comprising at least one MSRE that cuts unmethylated DNA.

39. The kit according to claim 38, wherein the MSRE mixture comprises at least two, at least three, or at least four methylation-sensitive restriction enzymes; and / or wherein the MSRE mixture comprises restriction enzymes selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI.

40. The kit according to any one of claims 31 to 37, wherein the MSRE mixture comprises at least two, three, or more of the restriction enzymes HhaI, HpaII, AciI, HpyCH4IV, and BsaHI.

41. The kit according to claim 39 or 40, wherein the MSRE mixture comprises at least HhaI and HpyCH4IV.

42. The kit according to any one of claims 31 to 40, wherein the sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA are sites within one or more or all of the following:

43. The kit according to any one of claims 31 to 40, wherein the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are sites within one or more or all of the following: 。 44. The kit according to any one of claims 31 to 40, wherein the sites hypermethylated in fetal DNA and maternal DNA are sites within one or more or all of the following: 。 45. The kit according to any one of claims 31 to 40, wherein the sites hypomethylated in fetal DNA and maternal DNA are sites within one or more or all of the following:

46. The kit according to any one of claims 31 to 40, wherein the methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy are sites within one or more or all of the following:

47. The kit according to claim 33, wherein the methylation-insensitive region of the Y chromosome is within one or more or all of the following:

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