Methods of measuring fetal fraction or maternal contamination and / or determining fetal genotype or fetal abnormality

Abstract

Disclosed herein are novel target genomic regions for performing prenatal testing, as well as corresponding primers and probes, and methods of performing prenatal testing, including fetal fraction estimation, aneuploidy assay, and 22q microdeletion assay, to obtain an aneuploidy score and a 22q microdeletion score, and, as appropriate, a fetal fraction adjusted aneuploidy score and a fetal fraction adjusted 22q microdeletion score. The methods can be particularly suitable for NIPT or IPT applications.

Description

Background Technology

[0001] Accurate diagnosis of chromosomal aneuploidy (such as trisomy of chromosomes 13, 18, and 21) requires accurate estimation of fetal fraction or maternal contamination in fetal DNA samples. This necessitates distinguishing fetal DNA from maternal DNA, even if their genetic sequences are highly similar.

[0002] Digital PCR (dPCR) testing is used in applications such as fetal fraction estimation and non-invasive prenatal testing (NIPT) for detecting chromosome copy number. These applications require highly accurate detection and counting of target sequences in cell-free DNA (cfDNA).

[0003] Aneuploidy detection typically requires numerous targets, often including multiple target sites on each target chromosome. This limits the use of target sequence-specific probe strategies, as amplification of multiple target sequences often leads to undesirable side effects, such as primer dimers and primer-probe interactions that affect detection performance. However, alternative probe strategies often produce poor signal intensity because the probe is not cleaved throughout the PCR cycle.

[0004] In recent years, multiplex dPCR methods have emerged, in which multiple target DNA sequences can be detected simultaneously in a single dPCR reaction. dPCR multiplexing requires the separation of dPCR data during the analysis stage, where the degree of separation can be discriminative and / or non-discriminative at the target level.

[0005] Existing multiplex dPCR methods known in the art include: probe fluorophore-based multiplexing, wherein one or more target sequence-specific probes contain fluorophores, each fluorophore having a unique excitation and emission spectrum; amplitude-based multiplexing, wherein the concentrations of dPCR amplification primers and dPCR probes vary according to a specific target sequence; and ratio-based multiplexing, wherein the ratio of two or more target sequence-specific probes labeled with fluorophores emitted in different color channels varies according to each specific target sequence.

[0006] In the context of dPCR applications of NIPT and / or IPT, the amount of DNA available is typically limited, necessitating highly accurate detection and counting of target sequences. For example, the average genomic equivalent copy number extracted from a tube of sample (e.g., a blood sample) may be around 3,000, of which approximately 4% to 15% may originate from the fetus. Therefore, in existing technologies, multiple target sites per chromosome are detected in a single assay, sometimes spanning more than one chromosome. However, amplification of multiple target sequences often leads to undesirable side effects. Furthermore, designing dPCR assays typically requires knowledge of the target cfDNA sequence; for example, in NIPT applications, the 5' and 3' ends of cfDNA within and between subjects often differ, which can affect the consistency of cfDNA copy number measurements and pose challenges to dPCR assay design, particularly for multiplex assays.

[0007] Therefore, given the current technology, there is an urgent need for a method for preparing and detecting target sequences in DNA that can reduce the number of sequence-specific primers and probes required for multiplex target amplification in dPCR. Such a method could be particularly beneficial for NIPT and / or IPT applications, which are not limited to dPCR and may include qPCR detection. Furthermore, there is a need for a probe that produces appropriate signal intensity in multiplex detection without generating undesirable side reactions.

[0008] The present invention aims to overcome one or more problems found in the prior art. Summary of the Invention

[0009] This disclosure generally relates to methods and systems for measuring fetal fractions or maternal contamination in samples obtained from pregnant human subjects and / or determining fetal genotypes or fetal abnormalities. This disclosure also relates to oligonucleotide probes activated by RNAseH2. This disclosure further relates to methods and systems for detecting oligonucleotides via polynucleotide amplification. In various aspects and embodiments, this disclosure provides methods for preparing and detecting target sequences in DNA that reduce the number of sequence-specific primers and probes required for amplifying multiplex targets in dPCR. Such methods are particularly advantageous for NIPT and / or IPT applications, which are not limited to dPCR and may include qPCR detection. This disclosure also provides probes that produce appropriate signal intensities in multiplex detection without generating undesirable side effects.

[0010] Advantageously, this disclosure provides methods, systems, and consumables, such as universal primers and probes, that allow the detection of a wide variety of oligonucleotides using a reduced number of different primers and probes compared to prior art methods and systems. Therefore, methods and systems for detection and diagnosis can be achieved through one or more of the following: reduced cost and complexity, ease of execution, increased efficiency, and / or improved reliability.

[0011] In an aspect of the invention, a method is provided for measuring fetal fraction or maternal contamination and / or determining fetal genotype or fetal abnormality in a sample obtained from a pregnant human subject, the method comprising: (a) providing a DNA sample; (b) incubating the DNA with at least one methylation-sensitive restriction endonuclease; (c) processing the DNA to generate a plurality of target single-stranded oligonucleotides; and (d) reverse transcribing the plurality of target single-stranded oligonucleotides using a plurality of reverse transcription primers to obtain a plurality of first double-stranded oligonucleotides, each reverse transcription primer comprising: a universal RNAseH2 probe sequence, and / or a universal forward primer sequence, wherein the plurality of reverse transcription primers comprises: at least one primer configured to anneal to one or more gene sites that are highly methylated in fetal DNA and lowly methylated in maternal DNA; and at least one primer configured to anneal to lowly methylated gene sites in fetal DNA. The primers are configured to anneal to one or more methylation sites in fetal DNA and maternal DNA that are highly methylated; and optionally, one or more of the following: at least one primer configured to anneal to one or more methylation-insensitive sites in fetal DNA and maternal DNA that are unlikely to exhibit aneuploidy; at least one primer configured to anneal to one or more sites in fetal DNA and maternal DNA that are both highly methylated; at least one primer configured to anneal to one or more sites in fetal DNA and maternal DNA that are both low methylated; (e) performing a template switching reaction on the plurality of first double-stranded oligonucleotides to generate a plurality of second double-stranded oligonucleotides using a plurality of TSOs, each TSO containing a universal reverse primer sequence; (f) amplifying the plurality of second double-stranded oligonucleotides to obtain a plurality of amplification products and a plurality of probe signals; and (g) detecting and quantifying the plurality of probe signals.

[0012] In an aspect of the invention, a method for determining a fetal genotype or fetal abnormality of a sample is provided, the method comprising: (a) providing a DNA sample; (b) processing the DNA to generate a plurality of target single-stranded oligonucleotides; (c) reverse transcribing the plurality of target single-stranded oligonucleotides using a plurality of reverse transcription primers to obtain a plurality of first double-stranded oligonucleotides, each reverse transcription primer comprising: a universal RNAseH2 probe sequence, and / or a universal forward primer sequence; (d) performing a template switching reaction on the plurality of first double-stranded oligonucleotides using a plurality of TSOs to generate a plurality of second double-stranded oligonucleotides, each TSO comprising a universal reverse primer sequence; (e) amplifying the plurality of second double-stranded oligonucleotides to obtain a plurality of amplification products and a plurality of probe signals; and (f) detecting and quantifying the plurality of probe signals.

[0013] In an aspect of the invention, a method for amplifying and detecting one or more oligonucleotides includes: (a) providing a plurality of target single-stranded oligonucleotides; (b) contacting at least one of the plurality of target single-stranded oligonucleotides with a first oligonucleotide, wherein the first oligonucleotide comprises: a 3' sequence configured to anneal to a sequence of the at least one target single-stranded oligonucleotide; a first universal oligonucleotide probe sequence corresponding to the annealed portion of the first universal oligonucleotide probe; and / or a first universal forward primer sequence corresponding to the annealed portion of a first universal forward amplification primer; and (c) generating at least one first double-stranded oligonucleotide by extending the first oligonucleotide to synthesize a complementary strand to the at least one target single-stranded oligonucleotide; and (d) contacting the at least one first double-stranded oligonucleotide with a second oligonucleotide, wherein the second oligonucleotide... The nucleotide comprises: a 3' sequence configured to anneal to a sequence toward or located at the 3' end of the complementary strand; a first universal reverse primer sequence corresponding to the annealed portion of the first universal reverse amplification primer; and (e) extension along the complementary strand of the at least one first double-stranded oligonucleotide to generate at least one second double-stranded oligonucleotide; and (f) providing: a first universal forward amplification primer; a first universal reverse amplification primer; and a first universal oligonucleotide probe and / or a first non-universal oligonucleotide probe configured to anneal to a sequence of the target single-stranded oligonucleotide; and (g) amplification reaction of the at least one second double-stranded oligonucleotide to generate a plurality of oligonucleotide amplification products and detection of a plurality of probe signals emitted by one or more universal oligonucleotide probes or one or more non-universal oligonucleotide probes, wherein the probe signals are emitted during the amplification reaction.

[0014] In an aspect of the invention, a method for amplifying and detecting one or more oligonucleotides includes: (a) providing a plurality of target single-stranded oligonucleotides; (b) contacting at least one of the plurality of target single-stranded oligonucleotides with a first oligonucleotide, wherein the first oligonucleotide comprises: a 3' sequence configured to anneal to a sequence of the at least one target single-stranded oligonucleotide; a first universal oligonucleotide probe sequence corresponding to the annealed portion of the first universal oligonucleotide probe; and / or a first universal forward primer sequence corresponding to the annealed portion of a first universal forward amplification primer; and (c) generating at least one first double-stranded oligonucleotide by extending the first oligonucleotide to synthesize a complementary strand to the at least one target single-stranded oligonucleotide; and (d) contacting the at least one first double-stranded oligonucleotide with a second oligonucleotide, wherein the second The oligonucleotide comprises: a 3' sequence configured to anneal to a sequence toward or located at the 3' end of the complementary strand; and a first universal reverse primer sequence corresponding to the annealed portion of the first universal reverse amplification primer; and (e) extending a second oligonucleotide along the complementary strand of the first double-stranded oligonucleotide to generate at least one second double-stranded oligonucleotide; and (f) providing: a first universal forward amplification primer, a first universal reverse amplification primer; and a first universal oligonucleotide probe and / or a first non-universal oligonucleotide probe configured to anneal to a sequence of the target single-stranded oligonucleotide; and (g) performing an amplification reaction on the at least one second double-stranded oligonucleotide to generate a plurality of oligonucleotide amplification products and detecting a plurality of probe signals emitted by one or more universal oligonucleotide probes or one or more non-universal oligonucleotide probes, wherein the probe signals are emitted during the amplification reaction.

[0015] In aspects and embodiments of the invention, the amplification reaction further includes: denaturing the at least one second double-stranded oligonucleotide to obtain: at least one antisense single-stranded oligonucleotide comprising a sequence of a complementary strand to the target single-stranded oligonucleotide, and at least one sense single-stranded oligonucleotide comprising the sequence of the target single-stranded oligonucleotide; annealing a first universal forward amplification primer to the at least one sense single-stranded oligonucleotide, annealing a first universal reverse amplification primer to the at least one antisense single-stranded oligonucleotide, annealing a first universal oligonucleotide probe to the at least one sense single-stranded oligonucleotide, or annealing a first non-universal oligonucleotide probe to the at least one sense single-stranded oligonucleotide, and quantifying the signals of the plurality of probes.

[0016] In an embodiment, amplifying the plurality of second double-stranded oligonucleotides further includes providing: an RNAseH2 enzyme, a plurality of universal forward amplification primers, a plurality of universal reverse amplification primers, a polymerase, and a plurality of universal RNAseH2-activated oligonucleotide probes (each comprising: a fluorophore, a quencher, a sequence configured to anneal to a universal RNAseH2 probe sequence, and at least one ribonucleotide separating the fluorophore and the quencher); or a plurality of non-universal RNAseH2-activated oligonucleotide probes (each comprising: a fluorophore, a quencher, a sequence configured to anneal to a target single-stranded oligonucleotide sequence, and at least one ribonucleotide separating the fluorophore and the quencher).

[0017] In an embodiment, amplifying the plurality of second double-stranded oligonucleotides or the amplification reaction further includes: denaturing the at least one second double-stranded oligonucleotide to obtain: at least one antisense single-stranded oligonucleotide containing a sequence of the complementary strand to the target single-stranded oligonucleotide, and at least one sense single-stranded oligonucleotide containing the sequence of the target single-stranded oligonucleotide; and annealing a first universal forward amplification primer to the at least one sense single-stranded oligonucleotide, annealing a first universal reverse amplification primer to the at least one antisense single-stranded oligonucleotide, annealing a first universal oligonucleotide probe to the at least one sense single-stranded oligonucleotide, or annealing a first non-universal oligonucleotide probe to the at least one sense single-stranded oligonucleotide.

[0018] In the implementation scheme, the sample may be obtained from any of the following sources: blood of a pregnant woman containing maternal and fetal DNA, plasma of a pregnant woman containing maternal and fetal DNA, serum of a pregnant woman containing maternal and fetal DNA, chorionic villi containing maternal and fetal DNA, amniotic fluid containing maternal and fetal DNA, or optionally fetal blood taken from the umbilical cord.

[0019] In the implementation scheme, the target single-stranded oligonucleotides are derived from the following sources: one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA; one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in both fetal and maternal DNA; one or more sites that are hypermethylated in both fetal and maternal DNA; and one or more sites that are hypomethylated in both fetal and maternal DNA; and are distinguishable from each other based on their respective probe signals.

[0020] In the implementation, the probe signals originate from the following sources: one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA; one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in fetal DNA and maternal DNA; one or more sites that are hypermethylated in both fetal DNA and maternal DNA; and one or more sites that are hypomethylated in both fetal DNA and maternal DNA; all of which are distinguishable from each other.

[0021] In the implementation scheme, two or more of the plurality of target single-stranded oligonucleotides are derived from at least two different chromosomes.

[0022] In an implementation scheme, at least one specific amplification product of the plurality of oligonucleotide amplification products derived from a specific chromosome is distinguishable from at least one other specific amplification product from a different specific chromosome, optionally wherein the plurality of distinguishable amplification products are at least about 10, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 150.

[0023] In an implementation, the method includes at least two groups of universal or non-universal RNAseH2 probes for targeting a plurality of different target chromosomes; for example, each of the groups of at least two universal or non-universal RNAseH2 probes includes up to 20, up to 12, up to 10, up to 8, up to 6, or up to 4 universal or non-universal RNAseH2 probes, optionally wherein at least one particular amplification product among the plurality of oligonucleotide amplification products is distinguishable from at least one other particular amplification product.

[0024] In the implementation, the at least one primer configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA, the at least one primer configured to anneal to one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA, the at least one primer configured to anneal to one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in fetal DNA and maternal DNA, the at least one primer configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA, and the at least one primer configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA, each comprises a different universal RNAseH2 probe sequence.

[0025] In the embodiments, the at least one primer configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA, the at least one primer configured to anneal to one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA, the at least one primer configured to anneal to one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in fetal DNA and maternal DNA, the at least one primer configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA, and the at least one primer configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA, comprises one or more different universal RNAseH2 probe sequences.

[0026] In one embodiment, the plurality of reverse transcription primers comprise at least three primers configured to anneal to one site that is hypermethylated in fetal DNA and hypomethylated in maternal DNA; and at least three primers configured to anneal to at least three sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA.

[0027] In an embodiment, the plurality of reverse transcription primers comprise at least one primer configured to anneal to at least three or 6-10 methylation-insensitive sites in fetal DNA and maternal DNA, at least one primer configured to anneal to at least three or 6-10 sites that are both hypermethylated in fetal DNA and maternal DNA, and at least one primer configured to anneal to at least three or 6-10 sites that are both hypomethylated in fetal DNA and maternal DNA.

[0028] In one embodiment, a site of the at least one primer that is unlikely to exhibit aneuploidy in fetal DNA and maternal DNA is configured to anneal with one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in fetal DNA and maternal DNA, the methylation-insensitive sites being located on the Y chromosome.

[0029] In one embodiment, the DNA is optionally divided into multiple independent volumes before being incubated with at least one methylation-sensitive restriction endonuclease; optionally, said independent volumes are droplets or micropores.

[0030] In one embodiment, the method is carried out in a reaction mixture, wherein the reaction mixture is in two or more partitions.

[0031] In an embodiment, the reaction mixture comprises: DNA extracted from a sample, the plurality of reverse transcription primers, the plurality of TSOs, the plurality of universal forward amplification primers, the plurality of universal reverse amplification primers, the plurality of universal RNAseH2 probes, or the plurality of non-universal RNAseH2 probes.

[0032] In an implementation, each of the plurality of universal or non-universal RNAseH2 probes forms a stem-loop and comprises, from 5' to 3': a first stem sequence, a loop sequence, and a second stem sequence, wherein the second stem sequence is the reverse complementary sequence of the first stem sequence, wherein the ribonucleotide is located in the loop sequence, and wherein the 5' universal sequence contains at least a portion of the loop sequence.

[0033] In the implementation scheme, the RNaseH2 enzyme is Thermococcus abyssalis ribonuclease HII (… Pyrococcus abyssi RNase H2 enzyme) or its mutants, *Firmianalyte* ribonuclease HII ( Pyrococcus furiosis RNase H2 enzyme) or its mutants, Thermococcus viride ribonuclease HII ( Pyrococcus horikoshii RNase H2 enzyme) or its mutants, thermococcal ribonuclease HII ( Thermococcus kodakarensis RNase H2 enzyme) or its mutants, or Thermococcus marinum ribonuclease HII ( Thermococcus litoralis RNase H2 enzyme or its mutants.

[0034] In the implementation scheme, the quantification of the plurality of probe signals further includes determining the corrected concentration of fetal DNA in the sample (fetal_FF, fetal_FF) and / or the corrected concentration of maternal DNA in the sample (maternal_FF, maternal_FF).

[0035] In the implementation plan, determining the corrected concentration of fetal DNA includes the following calculation steps (step 1a):

[0036] Where 'foetal_FF' represents the corrected fetal DNA concentration; [hyper] is the hypermethylated reference copy concentration derived from the signal in the RNAseH2 probe partition, wherein the RNAseH2 probe is configured to anneal with a universal RNAseH2 probe sequence of the reverse transcription primer, wherein the reverse transcription primer is configured to anneal with one or more sites that are hypermethylated in both fetal DNA and maternal DNA; [hypo] is the hypomethylated reference copy concentration derived from the signal in the RNAseH2 probe partition, wherein the RNAseH2 probe is configured to anneal with a universal RNAseH2 probe sequence of the reverse transcription primer. 2. Probe sequence annealing, wherein the reverse transcription primer is configured to anneal to one or more sites in fetal DNA and maternal DNA that are hypomethylated; [foetal] is the fetal DNA copy concentration based on RNAseH2 probe partitioning signals, wherein the RNAseH2 probe is configured to anneal to the universal RNAseH2 probe sequence of the reverse transcription primer, wherein the reverse transcription primer is configured to anneal to one or more sites in fetal DNA that are hypermethylated and in maternal DNA that are hypomethylated; 0.08 is a background correction factor derived from regression analysis of fetal methylation targets and chrY targets in male samples.

[0037] In the implementation plan, determining the corrected concentration of maternal DNA includes the following calculation steps (step 1b):

[0038] Where 'maternal_FF' represents the corrected maternal DNA concentration; [maternal] is the maternal DNA copy concentration derived from the signal in the RNAseH2 probe partition, wherein the RNAseH2 probe is configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, wherein the reverse transcription primer is configured to anneal with one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA; [hyper] is as defined above.

[0039] In the implementation scheme, the method further includes determining the average methylated fetal score based on the following calculation step (step 2) and performing customized background correction:

[0040] Where 'meth_FF' represents the average fetal methylation score; 'foetal_FF' and 'maternal_FF' are as defined above.

[0041] In an implementation, the method further includes determining a final fetal score (FF) value in the case of a male or female fetus (i.e., in the case of the presence or absence of chrY in the fetal score).

[0042] First, the proportion of chrY in the fetal fraction is determined based on the following calculation steps (step 3):

[0043] Where 'chrY_FF' is the proportion of the Y chromosome in the fetal fractional DNA sample; [chrY] is the concentration of chrY in the total DNA sample; and [control] is the total DNA concentration based on the methylation-insensitive amplification primer and probe set signal (in the partition).

[0044] In the next step, in the case of a male or female fetus, the final fetal score is calculated based on a threshold of 0.02 for the 'chrY_FF' value, according to the following calculation steps: For a male fetus, i.e., when chrY_FF is higher than the threshold of 0.02 (step 4a):

[0045] Or for female fetuses, i.e., when chrY_FF is 0.02 or less (step 4b): (If no chrY signal exceeding the threshold exists)

[0046] 'FF_calculated' is the final calculated fetal score; 'meth_FF' and 'chrY_FF' are defined above.

[0047] In an alternative embodiment, quantifying the plurality of probe signals further includes determining the corrected concentration of fetal DNA (FetCorr) in the sample and / or the corrected concentration of maternal DNA (MatCorr) in the sample, wherein determining the corrected concentration of fetal DNA includes the following calculation steps:

[0048] [Total] is the total copy number concentration derived from the RNAseH2 probe signal in the partition, wherein the probe is configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, which is configured to anneal with one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in fetal DNA and maternal DNA; [Hyper] is the hypermethylated reference copy number concentration derived from the RNAseH2 probe signal in the partition, wherein the probe is configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, which is configured to anneal with one or more sites that are hypermethylated in both fetal DNA and maternal DNA; [Hypo] [Fet] is the hypomethylation reference copy number concentration derived from the signal of the RNAseH2 probe in the partition, wherein the probe is configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, which is configured to anneal with one or more sites that are hypomethylated in both fetal DNA and maternal DNA; and [Fet] is the fetal DNA copy number concentration derived from the signal of the RNAseH2 probe in the partition, wherein the probe is configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, which is configured to anneal with one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; determining the corrected concentration of maternal DNA includes the following calculation steps:

[0049] [Mat] is the maternal DNA copy number concentration derived from the signal of the RNAseH2 probe in the partition, the probe being configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, the reverse transcription primer being configured to anneal with one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA.

[0050] In an implementation, the method further includes determining the fetal fraction (FF) in the sample, wherein determining the fetal fraction includes at least one of the following calculation steps (a)-(d): .

[0051] In an implementation scheme, the method further includes calculating an estimated fetal score based at least in part on the fetal score in the sample and the model.

[0052] In the implementation scheme, the model is a generalized additive model (GAM), a linear model, or a second-order polynomial model, at least in part based on a clinical fetal score dataset and a corresponding fetal score measurement dataset using next-generation sequencing (NGS).

[0053] In an implementation scheme, the method further includes determining the fetal fraction of a male fetus in a DNA sample, wherein determining the fetal fraction of a male fetus includes: or or ,

[0054] Wherein, [YChr] is the concentration of a Y chromosome-specific sequence derived from the signal of an RNAseH2 probe in a partition, the probe being configured to anneal with a universal RNAseH2 probe sequence of a reverse transcription primer, the reverse transcription primer being configured to anneal with one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in either fetal or maternal DNA, wherein the one or more methylation-insensitive sites are located on the Y chromosome.

[0055] In one embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA comprises one of SEQ ID NO: 13 to 20; in another embodiment, the sequence of the reverse transcription primer configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA comprises one of SEQ ID NO: 60 to 67; in another embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises one of SEQ ID NO: 21 to 27; in another embodiment, the sequence of the reverse transcription primer configured to anneal to one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises one of SEQ ID NO: 68 to 74; in yet another embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in either fetal DNA or maternal DNA comprises SEQ ID NO: One of SEQ ID NOs 39 to 47; in an embodiment, the sequence of the reverse transcription primer configured to anneal to one or more methylation-insensitive sites that are unlikely to exhibit aneuploidy in both fetal DNA and maternal DNA comprises one of SEQ ID NOs 86 to 94; in an embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA comprises one of SEQ ID NOs 28 to 34; in an embodiment, the sequence of the reverse transcription primer configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA comprises one of SEQ ID NOs 75 to 81; in an embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA comprises one of SEQ ID NOs 35 to 38, 58, and 59; in an embodiment, the sequence of the reverse transcription primer configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA comprises one of SEQ ID NOs 75 to 81. One of SEQ ID NO: 82 to 85, 105, 106; in an embodiment, at least one of the plurality of reverse transcription primers configured to anneal to one or more methylation-insensitive sites on the Y chromosome that are unlikely to exhibit aneuploidy in either fetal DNA or maternal DNA comprises one of SEQ ID NO: 48 to 57;In one embodiment, the sequence of the reverse transcription primer configured to anneal to one or more methylation-insensitive sites on the Y chromosome that are unlikely to exhibit aneuploidy in either fetal or maternal DNA comprises one of SEQ ID NO: 95 to 104; in another embodiment, the sequence of the universal reverse amplification primer comprises one of SEQ ID NO: 1 to 6.

[0056] In the implementation plan, the sampling is invasive. In some such implementation plans, the calculated fetal score instead includes measurements of maternal contamination.

[0057] In embodiments of the invention, the amplification reaction is dPCR, optionally using droplets for dPCR. In embodiments of the invention, the plurality of target single-stranded oligonucleotides are obtained from a plurality of parental oligonucleotides selected from any of the following: a plurality of denatured double-stranded or non-linear single-stranded parental oligonucleotides; and / or a plurality of parental oligonucleotides with 3' homopolymeric nucleic acid tails; and / or parental oligonucleotides of a desired length obtained by reducing their length through any of the following methods or combinations: digestion with at least one restriction endonuclease, thermal denaturation, fragmentase-mediated shearing, size selection, sonication; and / or a plurality of free oligonucleotides.

[0058] In embodiments of the present invention, the desired length of the plurality of target single-stranded oligonucleotides is up to about 25, up to about 50, up to about 100, up to about 200, up to about 300, up to about 400, up to about 500, up to about 600, up to about 700, up to about 800, up to about 900, up to about 1000, or up to about 1500 nucleotides, or between about 25 and 1500, between about 50 and 1000, between about 50 and 500, between about 75 and 300, or between about 100 and 200 nucleotides.

[0059] In an embodiment of the invention, the method can be carried out in a reaction mixture, wherein two or more reaction mixtures comprise two or more partitions containing the reaction mixture.

[0060] In embodiments of the present invention, the reaction mixture may comprise: the plurality of target single-stranded oligonucleotides, and at least one first oligonucleotide, and at least one second oligonucleotide, and at least one first universal forward amplification primer, and at least one first universal reverse amplification primer, and at least one universal oligonucleotide probe or at least one non-universal oligonucleotide probe.

[0061] In embodiments of the invention, the 3' end of the first oligonucleotide may be complementary to a sequence at or towards the 3' end of the target single-stranded oligonucleotide, optionally wherein said sequence comprises at least a terminal nucleotide at the 3' end of the target single-stranded oligonucleotide. In embodiments of the invention, the first oligonucleotide may comprise a 3' poly(A) sequence annealed to one or more 3' T nucleotides located at the 3' T-tail modified ends of a plurality of parent oligonucleotides, and the target single-stranded oligonucleotide is derived from said plurality of parent oligonucleotides. In embodiments of the invention, the strand complementary to the target oligonucleotide may be synthesized by a polymerase to generate a first double-stranded oligonucleotide, wherein said polymerase is a reverse transcriptase having terminal nucleotide transferase activity, wherein during reverse transcription, one or more 3' protruding terminal nucleotides are added to the 3' end of the complementary strand by the reverse transcriptase, optionally wherein during reverse transcription, one or more C nucleotides are added to the 3' end of the complementary strand by the reverse transcriptase. In embodiments of the invention, the second oligonucleotide may be a template-changing oligonucleotide (TSO) comprising one or more 3' nucleotides complementary to one or more protruding terminal nucleotides of the complementary strand. In an embodiment of the invention, the method may include adding one or more T nucleotides to the 3' end of the complementary strand of a first double-stranded oligonucleotide via T-tailing.

[0062] In an embodiment of the invention, at least one first double-stranded oligonucleotide may be denatured to produce: at least one single-stranded target oligonucleotide comprising the sequence of the target single-stranded oligonucleotide, and at least one single-stranded complementary strand comprising the sequence of a strand complementary to the target oligonucleotide.

[0063] In embodiments of the invention, the second oligonucleotide may comprise a poly(A) sequence configured to anneal with one or more T nucleotides added to the complementary strand via a T-tail. In embodiments of the invention, a first population of universal oligonucleotide probes may be configured to anneal with a second double-stranded oligonucleotide generated from a target single-stranded oligonucleotide derived from a specific chromosome, and a second population of universal oligonucleotide probes may be configured to anneal with a second double-stranded oligonucleotide generated from a target single-stranded oligonucleotide derived from a different specific chromosome.

[0064] In embodiments of the present invention, the method may include a population of at least two universal oligonucleotide probes for targeting multiple different chromosomes; for example, each of the populations of at least two universal or non-universal oligonucleotide probes may include up to 20, up to 12, up to 10, up to 8, up to 6, or up to 4 universal oligonucleotide probes, optionally wherein at least one specific amplification product among the plurality of oligonucleotide amplification products can be distinguished from at least one other specific amplification product.

[0065] In embodiments of the invention, all second double-stranded oligonucleotides can be amplified by a first universal reverse amplification primer. Advantageously, in embodiments of the invention, the concentration of the universal reverse amplification primer in the amplification reaction can be at least about 1.2 times, about 1.5 times, about 2 times, about 3 times, about 5 times, about 10 times, about 20 times, or about 50 times the concentration of the universal forward amplification primer.

[0066] In embodiments of the invention, two or more of the plurality of target single-stranded oligonucleotides may originate from at least two different chromosomes. In embodiments of the invention, at least one specific amplification product of the plurality of oligonucleotide amplification products derived from a specific chromosome may be distinguishable from at least one other specific amplification product from a different specific chromosome, optionally wherein the plurality of distinguishable amplification products are at least about 10, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 150. In embodiments of the invention, a first universal oligonucleotide probe may be configured to anneal to a complementary sequence to a universal forward primer sequence. In embodiments of the invention, the plurality of single-stranded oligonucleotides may originate from a cfDNA sample comprising maternal and fetal DNA from a pregnant human subject. In embodiments of the invention, the method includes quantifying the signals of the plurality of probes to estimate the fraction of fetal DNA in the cfDNA sample and / or to estimate the copy number of one or more chromosomes in the cfDNA sample.

[0067] In any aspect and embodiment of this disclosure, the sample / amplification mixture may be partitioned prior to the amplification reaction. In any aspect and embodiment, the partitioned sample or reaction mixture may contain two or more partitions. In embodiments, there may be about 2 to 1,000,000 partitions, about 50 to 1,000,000 partitions, about 100 to 100,000 partitions, or about 1,000 to 100,000 partitions, depending on the method of partitioning. For example, partitioning may be achieved by forming droplets of the sample or reaction mixture in immiscible liquids; or partitioning may be achieved by aliquoting multiple equal portions of the sample or reaction mixture into, for example, separate containers.

[0068] Within the scope of this disclosure, it is expressly intended that the invention encompasses any of the various aspects, embodiments, examples, and alternatives set forth in the foregoing paragraphs, clauses, claims, and / or the following description, particularly their respective features, and that any such features may be employed independently or in any combination. That is, unless such features are incompatible, all embodiments and / or features of any aspect or embodiment may be combined in any manner and / or combination. More specifically, it is specifically intended that any embodiment of any aspect may form an embodiment of any other aspect, and all such combinations are included within the scope of this disclosure. The applicant reserves the right to amend any originally filed claim or to file any new claim, including the right to amend any originally filed claim to subordinate to and / or incorporate any feature of any other claim, even if protection was not initially sought in this manner. Attached Figure Description

[0069] The nature and advantages of embodiments of the present invention can be better understood by referring to the following detailed description and accompanying drawings. Those skilled in the art will understand that variations and modifications can be made to the invention described in the drawings, in addition to those specifically depicted. It should be understood that the present invention includes all such variations and modifications. The scope of the invention is not limited to the specific drawings described herein, which are for illustrative purposes only.

[0070] Figure 1 : Fetal score estimation using methylation-sensitive enzyme digestion ddPCR and Y chromosome ddPCR. This figure illustrates the correlation between methylation-sensitive fetal score estimation and SRY-based fetal score assessment in the exemplary detections described in detail herein, including the Examples section.

[0071] Figure 2A Analysis of methylation patterns at maternal and fetal methylation sites, as well as at generally methylated or unmethylated sites (especially fetal hypermethylated sites).

[0072] Figure 2B Analysis of methylation patterns at maternal and fetal methylation sites, as well as at generally methylated or unmethylated sites (especially maternal hypermethylated sites).

[0073] Figure 2C Analysis of methylation patterns at maternal and fetal methylation sites, as well as at generally methylated or unmethylated sites (especially hypermethylated sites).

[0074] Figure 2D Analysis of methylation patterns at maternal and fetal methylation sites, as well as at generally methylated or unmethylated sites (especially hypomethylated sites).

[0075] Figure 3This illustrates how methylation-sensitive restriction endonucleases (MSREs) enable ddPCR to quantify fetal and maternal cfDNA based on differences in fetal and maternal cfDNA methylation. As shown, MSRE digestion is performed within a droplet, without interfering with the ddPCR workflow. Fetal and maternal cfDNA are simultaneously quantified in the same ddPCR reaction. 1) Quantification of hypermethylated fetal cfDNA after MSRE digestion of hypomethylated maternal cfDNA. 2) Quantification of maternal cfDNA after MSRE digestion of hypomethylated fetal cfDNA. 3) Quantification of total cfDNA from non-digested regions.

[0076] Figure 4A : A schematic diagram of multiplex ddPCR detection, specifically illustrating a common form of ddPCR detection.

[0077] Figure 4B : A schematic diagram of multiplex ddPCR detection, specifically illustrating the use of unique universal probes to combine multiple primer pairs targeting the same chromosome into a single fluorescent channel.

[0078] Figure 5A The illustration shows fetal score assessment using the linear model, polynomial model, and generalized additive model (GAM) developed in Example 2, specifically comparing fetal score assessment using the developed linear model (“LM”) and the previous method (“FF_calculated”) with fetal score assessment using next-generation sequencing (NGS).

[0079] Figure 5B A schematic diagram of fetal score assessment using the linear model, polynomial model, and generalized additive model (GAM) developed in Example 2 described herein is shown, specifically illustrating the fetal scores calculated with respect to the fetal / hypermethylation variable and the maternal / hypermethylation variable. The residual plots show a nonlinear relationship between the fetal / methylation variable (510) and the maternal / methylation variable (512).

[0080] Figure 5C The illustration shows fetal score assessment using the linear model, polynomial model and generalized additive model (GAM) developed in Example 2, specifically showing a comparison between fetal score assessment using a second-order polynomial model (“poly2LM”) and fetal score assessment using NGS.

[0081] Figure 5D: A schematic diagram of fetal score assessment using the linear model, polynomial model, and generalized additive model (GAM) developed in Example 2 described herein, specifically illustrating the GAM used for fetal score assessment and a comparison with fetal score assessment using NGS. MPAE, mean percentage error. MSE, mean squared error.

[0082] Figure 6 The working mechanism of molecular beacon fluorescent probes and the differences in cleavage between molecular beacons with RNA bases in their loop sequences and RNaseH2-activated fluorescent probes. (6A) During the first cycle of PCR, the 5' universal tail of the forward primer is incorporated into the amplified DNA template; (6B) In subsequent PCR cycles, a reverse strand of DNA containing the reverse complementary sequence of the universal tail incorporated in step A is synthesized. (6C) Standard molecular beacons (without RNA bases) bind to the reverse complementary sequence of the universal sequence located at the 3' end of the amplified DNA reverse strand. They bind to their targets in the annealing step and are released in the denaturation step. In the context of real-time PCR, the fluorescence signal can be monitored in real time during the annealing step of each PCR cycle. The fluorescence signal increases with the increase in the number of synthesized reverse DNA strands. In dPCR, the signal is read at the endpoint. To ensure maximum binding of the molecular beacons, a final denaturation step (i.e., 98°C for 10 min) can be added, followed by cooling to 4°C at a rate of 2.5°C / s. This causes all amplified DNA molecules and molecular beacons to be completely denatured and find their hybridization targets during the cooling step. Maximum fluorescence signal is generated during this step and remains stable at room temperature for readout. (6D) For molecular beacons containing at least one RNA base in their circular sequence, the molecular beacon binds to its target during the annealing step, and once the RNA:DNA hybrid structure is stable, RNaseH2 cleaves the RNA base of the molecular beacon. This produces a stronger fluorescence signal than binding of the molecular beacon itself alone. New molecular beacons with RNA bases can hybridize and be cleaved in each subsequent PCR cycle. F = fluorophore, Q = quencher, R = RNA base.

[0083] Figure 7Examples of RNaseH2-activated fluorescent probe design. (7A) The 5' universal tail of the forward primer contains the same sequence as the molecular beacon loop sequence. One base at the center of the molecular beacon loop changes from a DNA base to an RNA base. During the annealing step, the loop sequence of the molecular beacon binds to the inverse complementary sequence of the universal sequence, and the RNA base is cleaved by RNaseH2. (7B) The 5' universal tail of the forward primer may contain the same sequence as the loop sequence and a stem sequence of the molecular beacon. One base at the center of the molecular beacon loop changes from a DNA base to an RNA base. During the annealing step, the loop sequence and a stem sequence of the molecular beacon bind to the inverse complementary sequence of the universal sequence, and the RNA base is cleaved by RNaseH2. (7C) The 5' universal tail of the forward primer may contain the full-length sequence of the linear probe. One base at the center of the linear probe changes from a DNA base to an RNA base. During the annealing step, the full-length linear probe binds to the inverse complementary sequence of the universal sequence, and the RNA base is cleaved by RNaseH2. F = fluorophore, Q = quencher, R = RNA base.

[0084] Figure 8 Differences in fluorescence separation were tested using molecular beacon probes and linear probe configurations (with RNA bases) in the presence or absence of RNase H2.

[0085] Figure 9 The results of 60-fold detection were tested on the Bio-Rad QX600 droplet digital PCR system, comparing standard molecular beacons and RNaseH2 cleavable molecular beacons.

[0086] Figure 10 : A schematic diagram of a method for preparing and detecting target oligonucleotides involving template switching reactions.

[0087] Figure 11 Overview of exemplary multiplex configurations for the simultaneous preparation and detection of more than one target single-stranded oligonucleotides.

[0088] Figure 12 : A schematic diagram of a method for preparing and detecting target oligonucleotides involving template switching reactions and non-universal oligonucleotide probes.

[0089] Figure 13 : A schematic diagram of a method for preparing and detecting target oligonucleotides involving template switching reactions and T-tailing.

[0090] Figure 14 : A schematic diagram of a method for preparing and detecting target oligonucleotides containing parent oligonucleotides through restriction endonuclease digestion and template switching reactions.

[0091] Figure 15: A schematic diagram of a method for preparing and detecting target oligonucleotides involving an initial T-tailing reaction and a subsequent T-tailing reaction.

[0092] Figure 16 Schematic diagram of the 22q11.2 region and the most commonly used / established 22q11.2 microdeletion subtypes, where regions where breakage typically occurs are indicated in dark gray and labeled 'AA', 'A', 'B', 'C', 'D', and 'E'. Thick arrows represent deletion variants and regions targeted by an exemplary 6-fold ddPCR assay, where the corresponding fluorophores are assigned to probes at specific detection sites within each target region of the 22q11.2 region (as indicated by thin arrows).

[0093] Figure 17A Next-generation sequencing (NGS) coverage map shows the data obtained from sequencing the 22q11.2 region of three 22q11.2 microdeleted samples and one non-22q11.2 microdeleted sample.

[0094] Figure 17B The results obtained by genotyping three 22q11.2 microdeletion samples and one non-22q11.2 microdeletion sample using an exemplary 6-fold ddPCR 22q11.2 microdeletion detection.

[0095] Figure 18 : 2D amplitude plot, showing the results obtained from an exemplary six-fold 22q11.2 ddPCR assay used to distinguish 22q11.2 microdeletion variants.

[0096] Figure 19(A) shows the Z-score of the copy number ratio obtained by an exemplary 6-fold 22q11.2 ddPCR assay using a sample input of 5 ng, the sample comprising a formulated sample prepared by incorporating DNA from 'AA', 'A', and 'B' 22q11.2 microdeletion subtype samples into a DNA sample obtained from a non-22q11.2 microdeletion sample; (B) shows the Z-score of the copy number ratio obtained by an exemplary 6-fold 22q11.2 ddPCR assay using a sample input of 10 ng, the sample comprising a formulated sample prepared by incorporating DNA from 'AA', 'A', and 'B' 22q11.2 microdeletion subtype samples into a DNA sample obtained from a non-22q11.2 microdeletion sample; (C) shows the Z-score of the copy number ratio obtained by an exemplary 6-fold 22q11.2 ddPCR assay using a sample input of 5 ng. (A) shows the Z-score of the copy number ratio obtained by ddPCR detection, wherein the sample comprises a formulated sample prepared by incorporating DNA from 'A' to 'D' 22q11.2 microdeletion subtype samples into a DNA sample obtained from a non-22q11.2 microdeletion sample; and (D) shows the Z-score of the copy number ratio obtained by exemplary 6-fold 22q11.2 ddPCR detection using a sample input amount of 10 ng, wherein the sample comprises a formulated sample prepared by incorporating DNA from 'A' to 'D' 22q11.2 microdeletion subtype samples into a DNA sample obtained from a non-22q11.2 microdeletion sample. Detailed Implementation

[0097] All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, such as molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization.

[0098] Generally, the nomenclature used in this article and the laboratory procedures for cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization described below are well-known and commonly used in the field.

[0099] Unless otherwise stated, the present invention is practiced using conventional techniques of molecular biology and genetics, organic chemistry, nucleic acid chemistry, and hybridization, which are within the capabilities of those skilled in the art. Standard techniques are used for nucleic acid synthesis.

[0100] These techniques and procedures are generally performed according to conventional methods in the field (see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference).

[0101] Those skilled in the art will understand that, apart from the specific description, the invention described herein is open to variations and modifications. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps and features individually or collectively mentioned or pointed out in this specification, as well as any and all combinations of any two or more of the stated steps or features. The scope of the invention is not limited to the specific embodiments described herein, which are for illustrative purposes only.

[0102] As used herein, the term "comprising" means that any of the stated elements must be included, and other elements may be included optionally. "consistently of" means that any of the stated elements must be included, elements that substantially affect the essential and novel features of the listed elements are excluded, and other elements may be included optionally. "consisting of" means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. It should also be understood that the term "and / or" as used herein refers to and covers any and all possible combinations of one or more of the associated listed items.

[0103] As used herein, the terms “a,” “an,” or “the” can include not only aspects having one member but also aspects having more than one member. For example, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” can include plural references. Thus, for example, reference to “a bead” can include multiple such beads, and reference to “the sequence” can include reference to one or more sequences known to those skilled in the art, and so on.

[0104] the term:

[0105] As used herein, the term "oligonucleotide" is a macromolecule comprising at least two or more covalently linked nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond, such as a nucleic acid. Applicable oligonucleotides are as follows. Oligonucleotides can be natural or synthetic. For the purposes of this invention, such oligonucleotides can constitute nucleic acids. Typical nucleic acids include DNA and RNA. Oligonucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Oligonucleotides can be linear and single-stranded, and can be derived from linear or circular conformations, as well as single-stranded or double-stranded oligonucleotides, or can contain one or more double-stranded regions and one or more single-stranded regions. The terms "oligonucleotide" and "polynucleotide" are used interchangeably. Oligonucleotides are composed of nucleotides.

[0106] As used herein, the term "nucleotide" generally comprises a nucleobase, a sugar, and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically, adenine, guanine, thymine, uracil, and cytosine. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically comprise monophosphate, diphosphate, or triphosphate. The phosphate group may be attached to the 5' or 3' side of the nucleotide. For the purposes of this invention, the terms "nucleotide" and "base" are used interchangeably.

[0107] Oligonucleotides can comprise any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in any oligonucleotide of this invention can be modified, including one or more chemical modifications to the nucleotides. Modified nucleotides can appear alone or in combination with other nucleotide modifications of the same or different forms, and can be adjacent to or not adjacent to other nucleotide modifications of the same or different forms.

[0108] One or more nucleotides in any oligonucleotide of the present invention may be non-natural or synthetic nucleotides. Non-natural nucleotides may include analogs of natural nucleotides, as well as nucleotides modified on the base, sugar, and / or phosphate moieties (e.g., phosphate thioester backbones). The oligonucleotides of the present invention may also comprise any synthetic nucleic acid known in the art, including but not limited to peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers having nucleotide side chains. One or more nucleotides in the oligonucleotide may be modified with a label or tag. The oligonucleotide may comprise one or more spacer sequences. Modifications may also include 3' and 5' modifications, including but not limited to capping with a fluorophore (e.g., quantum dot) or another portion.

[0109] In many embodiments, the oligonucleotide will have fewer than 250 nucleotides, and in some embodiments, between 4 and 200, for example, 10 to 150 nucleotides.

[0110] Unless otherwise stated, a "template" or "target" refers to a polynucleotide sequence containing the polynucleotide to be amplified and which may be side-joined by one or a pair of primer hybridization sites. Therefore, a "target nucleic acid" may contain a target polynucleotide sequence adjacent to at least one primer hybridization site. In some cases, a "target nucleic acid" contains a target polynucleotide sequence side-joined by hybridization sites of both a "forward" primer and a "reverse" primer.

[0111] As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or higher-order aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those that provide additional charges, polarization, hydrogen bonds, electrostatic interactions, linker sites, and functional chemical groups to the bases of nucleic acid ligands or the entire nucleic acid ligand. Such modifications include, but are not limited to: peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., thiophosphates, methylphosphonates), 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications on exocyclic amines, substitution of 4-thiouridines, substitution of 5-bromo or 5-iodouracil; backbone modifications, methylation, unusual base pairing combinations, such as isobases, isocytosine, and isoguanine. Nucleic acids may also include non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, including, but not limited to, capping with a fluorophore (e.g., quantum dots) or another component.

[0112] As used herein, a "gene" is a segment of nucleic acid (usually DNA) that encodes the sequence information required to produce a polypeptide or ribonucleic acid gene product. It comprises regions before and after the coding region (5' UTR and 3' UTR) and spacer sequences (introns) between the individual coding regions (exons). Conveniently, the term also includes control sequences necessary for gene expression (e.g., enhancers, silencers, promoters, terminators, etc.), which may be adjacent to or far from the associated coding sequence, as well as the coding and / or transcribed regions that encode the gene product.

[0113] The term "NIPT" refers to non-invasive prenatal testing, which involves determining the risk of a fetus being born with a chromosomal abnormality (such as trisomy 21) by detecting fetal cfDNA present in a sample (usually a blood sample) from the pregnant woman.

[0114] The term "copy number variation" (CNV) refers to the variable number of copies of a specific DNA segment between different individual genomes or in different cell or tissue types within an individual genome. Variant regions are of variable length, typically defined as segments longer than 1,000 base pairs but less than 5 megabases. CNVs include both extra copies of the sequence and deletions of genetic material, which can arise from duplication, deletion, or other structural changes (e.g., recombination). Such regions may contain one or more genes or may not contain any groups. CNVs can be detected by qPCR or dPCR, where the copy number of a specific genomic region can be expressed as the number of molecules or partitions containing target oligonucleotides originating from that specific genomic region.

[0115] As used herein, the term "expression" refers to the process by which information encoded in a nucleotide sequence that may contain a gene or pseudogene is transcribed to produce a functional gene product. The expression of a given gene can be detected using techniques such as qPCR or dPCR, where gene expression can be represented by the number of molecules or regions detected that contain target oligonucleotides derived from the transcription of a given RNA through a gene or pseudogene.

[0116] For the purposes of this invention, the term "amplification reaction" refers to any in vitro method used to amplify copies of a target nucleic acid sequence in a linear or exponential manner. Components of an amplification reaction include, but are not limited to, primers, oligonucleotide templates, polymerases, and nucleotides. Amplification reaction methods include, but are not limited to, dual-primer methods, such as polymerase chain reaction (PCR); ligase methods, such as DNA ligase chain reaction (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990)) (LCR); QBeta, RNA replicase, and RNA transcription-based amplification reactions (e.g., amplification involving RNA polymerization of T7, T3, or 5P6 primers), such as transcription amplification systems (TAS), nucleic acid sequence-based amplification (NASBA), and autonomous sequence replication (3SR); isothermal amplification reactions (e.g., single-primer isothermal amplification (SPIA)); and other methods known to those skilled in the art.

[0117] As used herein, the term "polymerase chain reaction" or "PCR" refers to a method in which a specific segment or subsequence of target double-stranded DNA is amplified geometrically. PCR is well known to those skilled in the art; see, for example, U.S. Patents 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., 1990. Exemplary PCR reaction conditions typically involve two or three cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step, followed by a separate extension step.

[0118] In this embodiment, the amplification reaction may comprise a digital PCR (dPCR) reaction, such as a droplet digital PCR (ddPCR) reaction. Methods for performing PCR in droplets are described, for example, in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of which are incorporated herein by reference. In this embodiment, the QX200, QX600, or QX One droplet digital PCR (ddPCR) system (Bio-Rad) may be used. In this embodiment, the amplification reaction may comprise a qPCR reaction.

[0119] "Amplification" refers to the step of placing a solution under conditions sufficient to allow polynucleotide amplification, provided all components of the reaction are intact. Components of an amplification reaction include, for example, primers, polynucleotide templates, polymerases, nucleotides, etc. The term "amplification" typically refers to an "exponential" increase in the number of target nucleic acids. However, as used herein, "amplification" can also refer to a linear increase in the number of selected target sequences of nucleic acids, as obtained through cyclic sequencing or linear amplification. In some implementations, "amplification" refers to PCR amplification using first and second amplification primers.

[0120] The reaction characteristics of the amplification reaction, such as the amplification rate, or the reaction output, such as the number of detectable molecules, can be detected and measured. These amplification methods may include detection reagents or detectable tags, which can be detected using any of a variety of detection devices. Exemplary detection methods include optical detection (e.g., fluorescence or chemiluminescence) and other methods known to those skilled in the art. As a non-limiting example, a detection device equipped with a module that generates excitation light that can be absorbed by the fluorophore and a module that detects the light emitted by the fluorophore can be used to detect fluorescent tags. In embodiments, the amplification reaction may be qPCR or dPCR.

[0121] A “primer” refers to an oligonucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as the starting point for nucleic acid synthesis. Primers can have various lengths. In embodiments, primers are typically less than 60 nucleotides in length, for example, between about 12 and 35 nucleotides. In embodiments, primers are typically less than 100 nucleotides in length, for example, between 18 and 55 nucleotides. The length and sequence of primers used for PCR can be designed based on principles known to those skilled in the art, see, for example, Innis et al. (ed.), 1990. Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers may include one or more modified or non-natural nucleotide bases. In some cases, primers are labeled. In some instances, primers may also contain nucleic acid sequences that do not involve hybridization with the amplified target, such as sequences that hybridize with another labeled oligonucleotide, sequences that hybridize with a capture oligonucleotide, or tag sequences such as barcodes.

[0122] An oligonucleotide or a portion thereof is annealed with another oligonucleotide in a suitable annealing buffer at a specific temperature in a manner that minimizes nonspecific annealing. For the purposes of this invention, the terms "annealing" and "hybridization" are used interchangeably. In embodiments, a primer or a portion thereof hybridizes with a sequence shared by a set of target oligonucleotides. In embodiments, the specific temperature at which specific hybridization occurs is room temperature. In embodiments, the specific temperature at which specific hybridization occurs is above room temperature. In embodiments, the specific temperature at which specific hybridization occurs is at least about 37°C, about 40°C, about 42°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C, for example, about 50°C to about 70°C, or about 55°C to about 65°C. The specific temperature at which specific hybridization occurs may be about 5°C lower than the calculated melting temperature of the primer.

[0123] The term "fully complementary sequence" refers to two hybrid sequences that share 100% sequence complementarity. The term "partially complementary sequence" refers to two hybrid sequences that share less than 100% sequence complementarity, including one or more mismatches between the two annealed nucleotide sequences.

[0124] In the implementation scheme, if at least 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 are present, for example, at least about 15, then the primer or a portion thereof can hybridize with the primer binding site. Alternatively, if fewer than 1, 2, 3, 4, 5, or 6 complementary mismatches are present on at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 consecutive complementary nucleotides, then the primer or a portion thereof can hybridize with the primer binding site.

[0125] "Polymerase" refers to an enzyme that performs template-guided synthesis of oligonucleotides (such as DNA). The term encompasses full-length polypeptides and domains possessing polymerase activity. DNA polymerases are well known to those skilled in the art. The DNA polymerase used in the compositions and methods disclosed herein can be any polymerase capable of replicating DNA molecules. The DNA polymerase can be a thermostable polymerase. Thermostable DNA polymerases are known in the art and are commercially available.

[0126] Thermostable polymerases have been isolated from various thermophilic bacteria, such as *Thermophyton aquatilis* (…). Thermus aquaticus Taq), Vibrio fibrillosa ( Pyrococcus furiosus Pfu), Warwick's fireball ( Pyrococcus woesei Pwo), thermophilic steatobacterium ( Bacillus sterothermophilus Bst), acidophilic thermosulfuric leaf fungus ( Sulfolobus acidocaldarius Sac), sulfur mineral sulfide fungi ( Sulfolobus solfataricus Sso), cryptotrophic thermococci ( Pyrodictium occultum Poc), Thermophilic cocci ( Pyrodictium abyssi Pab) and thermoautotrophic methanobacteria ( Methanobacterium thermoautotrophicum , Mth), and other species.

[0127] The DNA polymerase may be Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENTTm, DEEPVENT Tm, or their active mutants, variants, or derivatives, as known in the art. In various aspects and embodiments, the DNA polymerase may be Taq DNA polymerase. In other aspects and embodiments, the DNA polymerase may be a high-fidelity DNA polymerase (e.g., iProofrm high-fidelity DNA polymerase, Phusion® high-fidelity DNA polymerase, Q5 high-fidelity DNA polymerase, Platinum® Taq high-fidelity DNA polymerase, Accura® high-fidelity polymerase).

[0128] In some implementations, the DNA polymerase may be a fast-start or hot-start polymerase (e.g., FastStart). TM Taq DNA polymerase, FastStart TM High-fidelity DNA polymerase or iTaq TM DNA polymerase.

[0129] Polymerases also include a class called "reverse transcriptases," which are typically used to synthesize complementary strands of template RNA oligonucleotides, but can also synthesize complementary strands of DNA or cDNA oligonucleotides (see Gerard GF, and D'Alessio JM, Chapter 6 (73-93), Methods in Molecular Biology, Vol.16: Enzymes of Molecular Biology, edited by MM Burell 1993 Humana Press Inc., Totoa, NJ). Reverse transcriptases are well known to those skilled in the art. In embodiments, a suitable reverse transcriptase used in the methods, systems, and compositions disclosed herein can be any polymerase capable of synthesizing complementary strands. In embodiments, the reverse transcriptase exhibits terminal nucleotide transferase activity, which includes the non-template-dependent addition of one or more terminal nucleotides to the 3' end of an oligonucleotide sequence synthesized by reverse transcription that is complementary to the template sequence.

[0130] The term "extending along the sequence of..." refers to the synthesis of a sequence complementary to the target template sequence, wherein the complementary sequence is synthesized along the sequence of the target template sequence. In an embodiment, extending the complementary strand of the at least one first double-stranded oligonucleotide along the second oligonucleotide includes synthesizing a sequence complementary to the second oligonucleotide, thereby generating at least one second double-stranded oligonucleotide longer than the first double-stranded oligonucleotide. In an embodiment, extending the second oligonucleotide along the complementary strand of the first double-stranded oligonucleotide includes synthesizing a sequence complementary to the complementary strand of the first double-stranded oligonucleotide, thereby generating the second double-stranded oligonucleotide.

[0131] As used herein, the term "oligonucleotide probe" or "probe" refers to an oligonucleotide conjugated to a portion that can emit a detectable signal under desired conditions. For example, the probe portion may alter the signal state depending on whether the probe anneals with a target nucleic acid. In embodiments, as described herein, the probe comprises a nucleic acid that alters the signal state depending on whether the probe anneals with a target nucleic acid. In some embodiments, the probe nucleic acid comprises a blocked 3' end that prevents polymerase from extending the probe during annealing.

[0132] During amplification reactions such as PCR, oligonucleotide probes can be further extended, as described in this article. Suitable probes include fluorescent probes, such as TaqMan. TMProbes. These probes may require hydrolysis by the 5' to 3' exonuclease activity of DNA polymerase to separate the fluorescent portion of the probe from the quenching portion in the probe structure, thereby allowing a fluorescent signal to be generated during amplification. Any suitable probe can be used. For example, suitable fluorescent probes include molecular beacons, which contain stem-loop structures that increase the specificity of the probe binding to its target. During the annealing step of the amplification reaction, the loop portion of the molecular beacon probe can anneal with the target sequence, thereby denaturing the stem and separating the fluorescent portion from the quenching portion, resulting in detectable fluorescence from the fluorescent portion.

[0133] Fluoresceins or fluorescent agents can include a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and their derivatives. Fluoresceins and quenchers have been reported in the literature and are therefore known to those skilled in the art, and many fluorophores and quenchers are readily available from commercial suppliers in the biotechnology industry. References for fluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, DL, J. of ChemicalPhysics 21: 836-850 (1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40: 83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47:819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal. Chem. 67: 1197-1203 (1995), these references are incorporated herein by reference in their entirety. Non-limiting examples of fluorophores include cyanine, luciferin (e.g., 5'-carboxyluciferin (FAM), Oregon Green, and Alexa 488), HEX, rhodamine (e.g., N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)), eosin, coumarin, pyrene, tetrapyrrole, arylmethane, oxazine, polymeric dots, and quantum dots. In embodiments, the probe may include a blocked 3' end, preventing polymerase extension annealing.

[0134] The term "cell-free DNA sample" or "cfDNA sample" refers to a nucleic acid sample containing extracellular DNA obtained from any cell-free biological fluid, such as whole blood, urine, saliva, or other biological fluid treated to remove cells. In a typical embodiment, the cfDNA used for analysis is obtained from whole blood treated to remove cells, such as plasma or serum samples. Therefore, as used herein, the term "cfDNA" refers to DNA that can be recovered from the noncellular components of bodily fluids, such as blood.

[0135] Methylation status refers to the presence or absence of methyl groups at a specific DNA sequence. In some embodiments, DNA methylation refers to the presence or absence of methylcytosine at one or more CpG dinucleotides in a DNA sequence. The term "methylation status" or "methylation condition" in relation to CpG dinucleotide methylation refers 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 status at one or more specific methylation sites in a DNA sequence includes "unmethylated," "fully methylated," and "hemimethylated." For the purposes of this application, the term "hypermethylated" refers to a region where, for a specific subset of samples (e.g., fetal DNA or maternal DNA), the average frequency of methylation is greater than 80% as determined by methylation-based sequencing. "Hypomethylated" refers to a region where, for a specific subset of samples (e.g., fetal DNA or maternal DNA), the average frequency of methylation is less than 20% as determined by methylation-based sequencing.

[0136] Methylation-sensitive restriction endonucleases (MSREs) are enzymes that cut DNA at specific unmethylated cytosine residues, but do not cut at the recognition sequence when the cytosine residues are methylated.

[0137] As used herein, a "methylation-sensitive" genomic region refers to genomic DNA that can be methylated, such as at CpG sequences, such that the site cannot be cleaved by methylation-sensitive restriction endonucleases in the methylated state, but can be cleaved in the absence of methylation. As used herein, "cleavable" means that at least 50% of the DNA is cleaved by methylation-sensitive restriction endonucleases when the recognition sequence is not methylated, compared to when the recognition sequence is methylated. Therefore, the detection of amplification products from cfDNA containing methylation sites after MSRE digestion indicates that the cfDNA is methylated at that site.

[0138] The term "partitioning" or "being partitioned" refers to separating a sample into multiple parts or "partitions". Partitioning is usually physical, such that the contents of one partition do not mix with, or are substantially not mixed with, the contents of adjacent partitions.

[0139] The partitions can be solid or fluid. In some embodiments, the partitions are solid partitions, such as microchannels or micropores. In some embodiments, the partitions are fluid partitions, such as droplets. In some embodiments, the fluid partitions (e.g., droplets) are mixtures of immiscible fluids (e.g., water and oil). In some embodiments, the fluid partitions (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil). Exemplary pore arrays and pore descriptions can be found, for example, in U.S. Patent Nos. 9,103,754 and 10,391,493. Pore arrays (nanopores, micropores, collections of pores) can be used to capture solid supports, optionally at known, addressable locations. Thus, pore arrays can be configured to facilitate bead capture in the form of a single solid support or optionally in small groups of solid supports. Exemplary micropore arrays and methods for delivering beads to and analyzing them are described, for example, in PCT / US2021 / 034152.

[0140] The partition can be a fluid, such as a droplet. In an embodiment, the fluid partition, such as a droplet, can contain a mixture of immiscible fluids, such as water and oil. In an embodiment, the droplet can contain an emulsion composition. In an embodiment, the droplet can be an aqueous droplet surrounded by an immiscible carrier fluid (e.g., oil). In an embodiment, the droplet can be an oily droplet surrounded by an immiscible carrier fluid (e.g., an aqueous solution). In an embodiment, the droplets can be relatively stable and have minimal aggregation between two or more droplets. In an embodiment, less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the droplets generated from the sample aggregate with other droplets. The emulsion can also have limited flocculation, which is the process by which the dispersed phase detaches from the suspension in the form of aggregates. Methods for emulsion formation are described, for example, in published patent applications WO 2011 / 109546 and WO 2012 / 061444, each of which is incorporated herein by reference in its entirety.

[0141] Methods and compositions for delivering reagents to one or more partitions include microfluidic methods known in the art; droplet or microcapsule coalescence, aggregation, fusion, rupture, or degradation (e.g., as described in US 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO2010 / 151,776); and combinations thereof, each of which is incorporated herein by reference in its entirety. In embodiments, the components of the reaction mixture can be combined by the methods described above, particularly if the method is performed in a stepwise manner as previously described.

[0142] In embodiments where the phase is divided into droplets, the droplets can be formed by flowing an oil phase through an aqueous sample containing the sample and reaction components. The oil phase may or may not contain a fluorinated base oil, which may be further stabilized by combination with a fluorinated surfactant (e.g., a perfluoropolyether). In embodiments, the base oil comprises one or more of HFE 7500, FC-40, FC-43, FC-70, or another common fluorinated oil. In embodiments, the oil phase comprises anionic fluorinated surfactants. In embodiments, the anionic fluorinated surfactant is an ammonium salt of Krytox (Krytox-AS), an ammonium salt of Krytox FSH, or a morpholine derivative of Krytox FSH. Krytox-AS may be present at concentrations of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w / w). In embodiments, the concentration of Krytox-AS is about 1.8%. In the embodiments, the concentration of Krytox-AS is approximately 1.62%. The morpholine derivatives of Krytox FSH may be present at concentrations of approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w / w). In the embodiments, the concentration of the morpholine derivatives of Krytox FSH is approximately 1.8%. In the embodiments, the concentration of the morpholine derivatives of Krytox FSH is approximately 1.62%. In the embodiments, the oil phase further comprises additives for adjusting oil properties, such as vapor pressure, viscosity, or surface tension. Non-limiting examples include perfluorooctanol and 1H,1H,2H,2H-perfluorodecanol. In the embodiments, 1H,1H,2H,2H-perfluorodecyl alcohol is added at concentrations of about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0% (w / w). In the embodiments, 1H,1H,2H,2H-perfluorodecyl alcohol is added at a concentration of about 0.18% (w / w). In an embodiment, the emulsion is formulated to produce highly monodisperse droplets with a liquid-like interfacial film, which can be converted into microcapsules with a solid-like interfacial film by heating; such microcapsules can serve as bioreactors, capable of retaining their contents throughout the incubation period (see, for example, U.S. Patent No. 10,378,048, which is incorporated herein by reference in its entirety). The conversion to microcapsule form can occur upon heating. For example, such conversion can occur at temperatures greater than about 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 95°C.During heating, a fluid or mineral oil coating can be used to prevent evaporation. Excess continuous phase oil may or may not be removed before heating. The biocompatible capsules resist agglomeration and / or flocculation over a wide range of thermal and mechanical processing conditions. After conversion, the microcapsules can be stored at approximately -70°C, -20°C, 0°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0143] Microcapsule / microdroplet partitions according to any aspect and embodiment of this disclosure are resistant to aggregation, especially at high temperatures. Therefore, the capsules can be incubated at very high densities (e.g., the number of partitions per unit volume). In embodiments, greater than 100,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 5,000,000, or 10,000,000 partitions can be incubated per mL. In some embodiments, sample-probe incubation occurs in a single well, e.g., in the well of a microtiter plate, without mixing between partitions. The microcapsules may also contain other components required for incubation. In the implementation plan, samples are divided into at least 500 partitions, at least 1,000 partitions, at least 2,000 partitions, at least 3,000 partitions, at least 4,000 partitions, at least 5,000 partitions, at least 6,000 partitions, at least 7,000 partitions, at least 8,000 partitions, at least 10,000 partitions, at least 15,000 partitions, at least 20,000 partitions, and at least 30,000 partitions. Partitions, at least 40,000 partitions, at least 50,000 partitions, at least 60,000 partitions, at least 70,000 partitions, at least 80,000 partitions, at least 90,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 300,000 partitions, at least 400,000 partitions, at least 500,000 partitions, at least 600,000 partitions, up to At least 700,000 partitions, at least 800,000 partitions, at least 900,000 partitions, at least 1,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000 partitions, at least 20,000,000 partitions, at least 30,000 partitions. 000 partitions, at least 40,000,000 partitions, at least 50,000,000 partitions, at least 60,000,000 partitions, at least 70,000,000 partitions, at least 80,000,000 partitions, at least 90,000,000 partitions, at least 100,000,000 partitions, at least 150,000,000 partitions, or at least 200,000,000 partitions.

[0144] In the embodiments, the generated droplets are substantially uniform in shape and / or size. For example, in the embodiments, the droplets are substantially uniform in average diameter. In the embodiments, the average diameter of the generated droplets is approximately 0.001 micrometers, approximately 0.005 micrometers, approximately 0.01 micrometers, approximately 0.05 micrometers, approximately 0.1 micrometers, approximately 0.5 micrometers, approximately 1 micrometer, approximately 5 micrometers, approximately 10 micrometers, approximately 20 micrometers, approximately 30 micrometers, approximately 40 micrometers, approximately 50 micrometers, approximately 60 micrometers, approximately 70 micrometers, approximately 80 micrometers, approximately 90 micrometers, approximately 100 micrometers, approximately 150 micrometers, approximately 200 micrometers, approximately 300 micrometers, approximately 400 micrometers, approximately 500 micrometers, approximately 600 micrometers, approximately 700 micrometers, approximately 800 micrometers, approximately 900 micrometers, or approximately 1000 micrometers. In the embodiments, the average diameter of the generated droplets is less than about 1000 micrometers, less than about 900 micrometers, less than about 800 micrometers, less than about 700 micrometers, less than about 600 micrometers, less than about 500 micrometers, less than about 400 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 50 micrometers, or less than about 25 micrometers. In the embodiments, the generated droplets are non-uniform in shape and / or size.

[0145] In the implementation plan, the generated droplets are substantially uniform in volume. For example, in the implementation scheme, the average volume of the generated droplets is approximately 0.001 nL, approximately 0.005 nL, approximately 0.01 nL, approximately 0.02 nL, approximately 0.03 nL, approximately 0.04 nL, approximately 0.05 nL, approximately 0.06 nL, approximately 0.07 nL, approximately 0.08 nL, approximately 0.09 nL, approximately 0.1 nL, approximately 0.2 nL, approximately 0.3 nL, approximately 0.4 nL, approximately 0.5 nL, approximately 0.6 nL, approximately 0.7 nL, approximately 0.8 nL, approximately 0.9 nL, approximately 1 nL, approximately 1.5 nL, approximately 2 nL, approximately 2.5 nL, approximately 3 nL, approximately 3.5 nL, approximately 4 nL, approximately 4.5 nL, approximately 5 nL, approximately 5.5 nL, approximately 6 nL, approximately 6.5 nL, approximately 7 nL, and approximately 7.5 nL. The droplet volumes are approximately nL, 8 nL, 8.5 nL, 9 nL, 9.5 nL, 10 nL, 11 nL, 12 nL, 13 nL, 14 nL, 15 nL, 16 nL, 17 nL, 18 nL, 19 nL, 20 nL, 25 nL, 30 nL, 35 nL, 40 nL, 45 nL, or 50 nL. In one embodiment, the average droplet volume is approximately 50 picoliters to approximately 2 nanoliters. In another embodiment, the average droplet volume is approximately 0.5 nanoliters to approximately 50 nanoliters. In yet another embodiment, the average droplet volume is approximately 0.5 nanoliters to approximately 2 nanoliters.

[0146] In embodiments where the partitions are solid, the partitions can be microchannels or micropores, wherein an array of pores (i.e., one or more groups of nanopores, micropores, or pores) can be used to capture a solid support, such as beads, optionally at an addressable, known location. Thus, the pore array can be configured to facilitate bead capture in the form of a single solid support or optionally in the form of a small group of solid supports. Exemplary pore arrays and pore descriptions can be found, for example, in U.S. Patent Nos. 9,103,754 and 10,391,493, which are incorporated herein by reference in their entirety. Exemplary micropore arrays and methods for delivering beads to and analyzing them are described, for example, in PCT / US2021 / 034152, which is incorporated herein by reference in its entirety. The reaction mixture partitions can be picoliter pores, nanopores, or micropores. A partition containing the reaction mixture can be a picoliter, nanoliter, or microliter reaction chamber, such as a picoliter, nanoliter, or microliter capsule. In embodiments, the reaction mixture partitions can be picoliter, nanoliter, or microliter channels.

[0147] For the purposes of this invention, the term "reaction mixture" refers to an aqueous solution containing reagents and oligonucleotides for carrying out any of the methods described herein. The term "amplification reaction mixture" refers to a reaction mixture configured to amplify a target nucleic acid.

[0148] The reaction mixture may contain, but is not limited to, enzymes, aqueous buffers, salts, and nucleoside triphosphates. The reaction mixture may contain nucleotides. The nucleotides used in the methods described herein can be any nucleotide used for nucleic acid polymerization. Nucleotides can be naturally occurring, modified, derived, or artificial. In embodiments, the nucleotides will generally be unlabeled. In embodiments, the reaction mixture may contain one or more buffers or salts to optimize reaction efficiency and specificity. A wide variety of buffers and salt solutions, as well as modified buffers, are known in the art. For example, in embodiments, the buffer is TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, or CAPS. In embodiments, the salt is potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, or lithium acetate. In embodiments, the reaction mixture may contain a salt (e.g., potassium chloride) at a concentration of about 10 mM to about 100 mM.

[0149] The reaction mixture may contain one or more stabilizers to optimize reaction efficiency and specificity. Stabilizers used in the methods described herein include, but are not limited to: polyols (glycerol, threitol, etc.), polyethers including cyclic polyethers, polyethylene glycol, organic or inorganic salts (e.g., ammonium sulfate, sodium sulfate, sodium molybdate, sodium tungstate, organic sulfonates, etc.), sugars, polyols, amino acids, peptides or carboxylic acids, quenchers and / or scavengers such as mannitol, glycerol, reduced glutathione, superoxide dismutase, bovine serum albumin (BSA) or gelatin, spermidine, dithiothreitol (or mercaptoethanol), and / or detergents such as TRITON® X-100 [octylphenol (ethylene glycol ether)], THESIT® [polyoxyethylene 9 lauryl ether (Polidocanol C12 E9)], TWEEN® (polyoxyethylene sorbitan monolaurate 20, NP40), and BRIJ-35 (polyoxyethylene 23 lauryl ether).

[0150] Once formed, the reaction mixture can be placed under conditions that allow the production of suitable target single-stranded oligonucleotides, such as a denaturation step. The reaction mixture can be placed under primer annealing conditions, such as a first or second oligonucleotide, and extended using one or more primers annealed to the target oligonucleotide. The reaction mixture can be placed under primer extension conditions, which can be, but are not limited to, conditions suitable for PCR, allowing the primers to anneal to the target oligonucleotide or a prepared oligonucleotide (if present) and extended by polymerase in a template-specific manner; this process can be repeated one or more cycles. The oligonucleotide probe signal can be monitored at the endpoint or in real-time (i.e., continuously or per cycle).

[0151] The terms “forward” and “reverse” are arbitrary designations used to indicate the annealing of the forward and reverse primers with the opposite strands of the double-stranded DNA molecule, and when annealed, their 3' ends face each other, so that an amplicon is formed under PCR conditions.

[0152] Used to estimate the fetal fraction in cell-free DNA from maternal samples or DNA from fetus or maternal samples. method

[0153] Accurate diagnosis of chromosomal aneuploidy (e.g., trisomy of chromosomes 13, 18, and 21) requires accurate estimation of the fetal fraction of cell-free DNA (cfDNA) in maternal plasma. cfDNA levels in maternal whole blood are low, ranging from 0–100 ng / mL. While the median fetal fraction of cfDNA is approximately 11% in early pregnancy, it may be less than 4% in early pregnancy (when non-invasive prenatal testing (NIPT) is recommended) (Wang et al., Prenat. Diagn. 33:662–666, 2013). Furthermore, despite high genetic sequence similarity, it is essential to differentiate fetal cfDNA from maternal cfDNA.

[0154] In one aspect, this disclosure provides a digital PCR (dPCR) method for quantifying fetal fractional cfDNA in maternal plasma using methylation-sensitive restriction endonuclease (MSRE) digestion. In some embodiments, this disclosure provides a method for estimating the fetal DNA fraction in cfDNA samples obtained from blood samples of pregnant human subjects.

[0155] The disclosed method can be used in conjunction with both non-invasive and invasive samples; for example, samples from amniocentesis or chorionic villus sampling (CVS).

[0156] In some embodiments, the method includes a digital amplification reaction method comprising: (a) dispensing (e.g., assigning to) partitions of an amplification reaction mixture containing cfDNA from a cfDNA sample, amplification reagents, and a plurality of amplification sets, each amplification set containing primer and probe sets, wherein each amplification set contains primers and probes for multiplex amplification, and each amplification set generates an amplification product containing a uniquely labeled marker distinguishable from the markers of each of the other amplification sets, wherein the plurality comprises: (i) amplification sets targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and (ii) amplification sets targeting sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA; and optionally (iii), (iv), and (v) One or more of the following: (iii) an amplification group targeting total cfDNA, said total cfDNA comprising methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy; (iv) an amplification group targeting sites that are hypermethylated in both fetal and maternal DNA; (v) an amplification group targeting sites that are hypomethylated in both fetal and maternal DNA; (b) incubating the cfDNA with a mixture of methylation-sensitive restriction endonucleases (MSREs) comprising at least one methylation-sensitive restriction endonuclease that cleaves unmethylated (e.g., hypomethylated) DNA; (c) amplifying the target nucleic acid sequence in the partition, if present, to obtain amplification products; (d) detecting the signal from each unique marker of the amplification products in the partition; and (e) quantifying the signal from each unique marker. In some embodiments, the plurality of amplification groups comprises (iii) an amplification group targeting total cfDNA, said total cfDNA comprising methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy. In some embodiments, each amplification group of (i) and (ii) comprises primers and probes targeting at least three sites. In some embodiments, each amplification group in (i)-(v) contains primers and probes targeting at least three sites or between 6 and 10 sites. In some embodiments, the method further includes using amplification groups targeting methylation-insensitive regions of the Y chromosome. In other embodiments, the method may further include using amplification groups targeting sites that are hypomethylated in both fetal and maternal cfDNA; and / or amplification groups targeting sites that are hypermethylated in both fetal and maternal cfDNA. In some embodiments, the amplification reaction mixture also contains a control target of a fully methylated synthetic DNA sequence and / or a fully unmethylated version of the same synthetic DNA sequence. In some embodiments, the digital amplification reaction method is a digital PCR method, such as droplet digital PCR. In some embodiments, the amplification reaction mixture in the partitions contains an MSRE mixture, and incubation occurs after partitioning and before amplification.In some embodiments, step (b) is performed prior to partitioning, and the enzyme-digested cfDNA is added to the amplification reaction mixture. In some embodiments, the MSRE mixture contains at least two, at least three, or at least four methylation-sensitive restriction endonucleases; and / or the MSRE mixture contains a restriction endonuclease selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture contains at least two or three of the restriction endonucleases HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture contains 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 containing a fluorescent label. In some embodiments, each probe is an oligonucleotide that hybridizes to a complementary oligonucleotide containing a label providing a detectable signal. In some embodiments, incubation of the cfDNA with the MSRE mixture occurs within partitions. In some embodiments, incubation of the cfDNA with the MSRE mixture occurs in the whole solution prior to partitioning (a). In some embodiments, the method further includes determining the normalized copy concentration of each target, for example based on the number of targets (N) in the amplified group. i ).

[0157] In an implementation scheme, the method further includes determining the corrected concentration (foetal_FF) of fetal DNA in the sample and / or the corrected concentration (maternal_FF) of maternal DNA in the sample. In an implementation scheme, determining the corrected concentration of fetal DNA includes the following calculation steps (step 1a):

[0158] Wherein 'foetal_FF' represents the corrected concentration of fetal DNA; [hyper] is the hypermethylation reference copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA; [hypo] is the hypomethylation reference copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA; and [foetal] is the fetal DNA copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA.

[0159] In the implementation plan, determining the corrected concentration of maternal DNA includes the following calculation steps (step 1b):

[0160] Where 'maternal_FF' represents the corrected concentration of maternal DNA; where [maternal] is the maternal DNA copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA; and [hyper] is as defined above.

[0161] In the implementation scheme, the method further includes determining the average fetal methylation score and performing a custom background correction based on the calculation (step 2):

[0162] Where 'meth_FF' represents the average fetal methylation score; 'foetal_FF' and 'maternal_FF' are defined as above.

[0163] In an implementation, the method further includes determining a final fetal score (FF) value in the case of a male or female fetus (i.e., in the case of the presence or absence of chrY in the fetal score).

[0164] First, the proportion of chrY in the fetal fraction is determined based on calculations (step 3):

[0165] Where 'chrY_FF' is the proportion of the Y chromosome in the fetal fractional DNA sample; [chrY] is the concentration of chrY in the total DNA sample; and [control] is the concentration of DNA in the sample.

[0166] In the next step, based on a threshold of 0.02 for the 'chrY_FF' value, the final fetal score is calculated for male or female fetuses. For male fetuses, i.e., when chrY_FF is higher than the threshold of 0.02, the calculation is based on the following steps (step 4a):

[0167] Or for female fetuses, i.e., when chrY_FF is 0.02 or less (step 4b): (If no chrY signal exceeding the threshold exists)

[0168] 'FF_calculated' is the final calculated fetal score; 'meth_FF' and 'chrY_FF' are defined as above.

[0169] In an alternative embodiment, the method may include determining the corrected concentration (FetCorr) of fetal cfDNA in the cfDNA sample and / or the corrected concentration (MatCorr) of maternal cfDNA in the cfDNA sample using alternative methods, wherein determining the corrected concentration of fetal cfDNA includes the following calculation steps:

[0170] Wherein, [Total] is the total cfDNA copy concentration based on the amplified region signal, the amplified group targeting total cfDNA containing methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy; [Hyper] is the hypermethylated reference copy concentration based on the amplified region signal, the amplified group targeting sites that are hypermethylated in both fetal and maternal DNA; [Hypo] is the methylated reference copy concentration based on the amplified region signal, the amplified group targeting sites that are hypomethylated in both fetal and maternal DNA; and [Fet] is the fetal cfDNA copy concentration based on the amplified region signal, the amplified group targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA. Determining the corrected concentration of maternal cfDNA includes the following calculation steps:

[0171] Wherein [Mat] is the maternal cfDNA copy concentration based on the amplified region signal, the amplified region targeting sites that are 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 calculation steps (a)-(d): or .

[0172] In some embodiments, the method further includes calculating an estimated fetal score based at least in part on the fetal score 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, based at least in part on a clinical fetal score dataset and a corresponding fetal score measurement dataset using next-generation sequencing (NGS).

[0173] 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 a male fetus includes: or or ,

[0174] [YChr] is the concentration of Y chromosome-specific sequences based on the amplified group region signal, which targets the Y chromosome methylation-insensitive region.

[0175] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA are one or more sites within the following range: .

[0176] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are one or more sites within the following range: .

[0177] In some embodiments of the methods or kits described herein, the sites that are hypermethylated in both fetal DNA and maternal DNA are one or more sites within the following range: .

[0178] In some embodiments of the methods or kits described herein, the sites that are hypomethylated in both fetal DNA and maternal DNA are one or more sites within the following range:

[0179] In some embodiments of the methods or kits described herein, methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy are one or more sites within the following range: .

[0180] In some embodiments of the methods or kits described herein, the methylation-insensitive regions of the Y chromosome lie within one or more of the following ranges: .

[0181] In another aspect, this disclosure provides a digital amplification kit for estimating the fraction of fetal DNA in cfDNA samples obtained from plasma or serum samples of pregnant human subjects, the kit comprising: (a) An amplification reaction mixture comprising amplification reagents and a plurality of amplification sets, each amplification set comprising primer and probe sets, wherein each amplification set contains a unique marker distinguishable from the markers of each other set, and each set contains primers and probes for multiplex amplification, and wherein the plurality of amplification sets comprises: (i) Amplomes targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and (ii) an amplification group targeting sites that are highly methylated in maternal DNA and lowly methylated in fetal DNA; and optionally one or more of (iii), (iv) and (v); (iii) Amplification of total cfDNA, which contains methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy; (iv) Amplification of sites that are highly methylated in both fetal and maternal DNA; (v) Amplification sets targeting sites that are hypomethylated in both fetal and maternal DNA. In some embodiments, each amplification set in (i) and (ii) contains primers and probes targeting at least three sites.

[0182] In some embodiments, the kit further comprises an amplified genome targeting methylation-insensitive regions of the Y chromosome. In some embodiments, the kit further comprises a fully methylated synthetic DNA sequence and / or a fully unmethylated version 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 to a complementary oligonucleotide containing a label that provides a detectable signal. In some embodiments, the kit further comprises a mixture of methylation-sensitive restriction endonucleases (MSREs) containing at least one MSRE that cleaves unmethylated DNA. In some embodiments, the MSRE mixture contains at least two, at least three, or at least four methylation-sensitive restriction endonucleases; and / or wherein the MSRE mixture contains a restriction endonuclease selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the kit contains a methylation-sensitive restriction endonuclease (MSRE) mixture comprising at least two, three, or more of the restriction endonucleases HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture comprises at least HhaI and HpyCH4IV. In some embodiments, the kit further comprises a methylation-sensitive restriction endonuclease (MSRE) mixture comprising at least two, three, or more of the restriction endonucleases HhaI, HpaII, AciI, HpyCH4IV, and BsaHI.

[0183] In various aspects and implementation schemes, datasets were created based on fetal scores calculated using the disclosed methods and corresponding fetal scores measured using next-generation sequencing (NGS) on different NIPT clinical samples from human subjects. Several models were developed that consider not only the calculated fetal scores and corresponding NGS measurements, but also parameters such as gestational age and Y chromosome calculations.

[0184] This disclosure provides a method for estimating the fetal DNA fraction in a cfDNA sample obtained from a cell-free biological sample from the mother. As described herein, the method includes a digital amplification reaction comprising evaluating cfDNA obtained from a pregnant subject (typically a human subject) to determine the methylation status of differentially methylated sites in fetal cfDNA and maternal cfDNA using a methylation-sensitive restriction endonuclease. Specifically, the method includes: analyzing the methylation status of one or more sites in fetal and maternal cfDNA that are highly methylated in fetal DNA and lowly methylated in maternal DNA; and detecting the methylation status of one or more sites that are highly methylated in maternal DNA and lowly methylated in fetal DNA. In some embodiments, the method further includes amplifying sites targeting total cfDNA containing methylation-insensitive genomic regions from chromosomes unlikely to exhibit aneuploidy, providing the ability to quantify the concentration of total DNA in the sample (i.e., both fetal and maternal). In some embodiments, the method further includes detecting methylation-insensitive regions of the Y chromosome as needed, for example, for determining fetal sex. In some embodiments, the method further includes assessing sites that are hypomethylated in both fetal and maternal cfDNA and / or assessing sites that are hypermethylated in both fetal and maternal cfDNA as internal controls for MSRE digestion. Assessing the levels of these various chromosomal regions with different methylation profiles provides the ability to quantify the fraction of fetal cfDNA derived from maternal samples. Estimation of the fetal fraction is important for quality control in non-invasive prenatal testing and for predicting fetal aneuploidy.

[0185] In some embodiments, the fetal score calculated / determined according to the methods of this disclosure can be used in methods for assessing fetal aneuploidy (e.g., trisomy, such as chromosomes 13, 18, or 21), chromosomal deletions (e.g., microdeletions, such as microdeletions in chromosome 22), or other genomic alterations (e.g., gene mutations associated with diseases such as α- or β-thalassemia, cystic fibrosis, sickle cell anemia, or hemophilia A). For example, the fetal score calculated / determined according to the methods of this disclosure can be used for quality control in such methods.

[0186] Components

[0187] In this disclosure, cell-free DNA obtained from pregnant subjects is evaluated to determine the amount of fetal cfDNA in maternal blood, i.e., the fraction of fetal cfDNA in maternal blood. cfDNA samples from pregnant subjects are digested with one or more methylation-sensitive restriction endonucleases, followed by amplification of multiple target sites with different methylation profiles in both fetal and maternal DNA. The fraction of fetal cfDNA can be calculated based on the level of differentially methylated DNA.

[0188] CfDNA

[0189] The cell-free DNA used in this invention is obtained from cell-free biological fluid samples (typically blood samples). Therefore, in a typical embodiment, the sample is a plasma or serum sample. Isolation of cfDNA can be achieved using a variety of different methods, such as by using 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 (kits) and ThermoFisher (e.g., MagMax) TM (Reagent kits, etc.). The disclosed methods can be used in conjunction with both non-invasive and invasive samples; for example, samples from amniocentesis or CVS.

[0190] Methylation-sensitive restriction endonucleases

[0191] cfDNA is digested with one or more methylation-sensitive restriction endonucleases (MSREs). These enzymes cleave unmethylated regions of DNA but not methylated regions. Therefore, amplification products from highly methylated regions of DNA will be more abundant than those from low-methylated regions.

[0192] In some embodiments, one restriction endonuclease is used. In alternative embodiments, two MSREs are used. In other embodiments, at least three MSREs are used. In still other embodiments, at least four MSREs are used. Exemplary restriction endonucleases 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 used in the analysis.

[0193] In some embodiments, the reaction mixture is digested whole with one or more MSREs before being partitioned into partitions (described in detail below). However, in a preferred embodiment, the cfDNA is added to the dPCR reaction mixture along with PCR reagents for the target amplification site and one or more restriction endonucleases. Restriction endonuclease digestion can then be performed in the partitions, but before amplification.

[0194] In addition, after overall MSRE digestion, the target in the cfDNA elution buffer can be pre-amplified, for example, to reduce the degree of detection multiplexing required to obtain sufficient sensitivity and accuracy.

[0195] Amplification target

[0196] Determining the fetal score typically involves multiplex amplification of each targeted hypomethylated or hypermethylated site in the genome being evaluated. Therefore, in some implementations, for each class of DNA that may be used in the detection, at least two sites, or at least three sites, or at least four sites or more are targeted: hypermethylated sites in fetal cfDNA, hypomethylated sites in fetal cfDNA, hypermethylated sites in maternal cfDNA, hypomethylated sites in maternal cfDNA, sites from methylation-insensitive regions of chromosomes unlikely to exhibit aneuploidy, sites from methylation-insensitive regions of the Y chromosome, sites hypomethylated in both fetal and maternal cfDNA, and sites hypermethylated in both fetal and maternal cfDNA.

[0197] Differential methylation sites between maternal and fetal cfDNA have been described (see, for example, Ionnides, Mol. Genet. Genomic Med 8:e1094, 2020; Hatte et al., PLOS ONE DOI: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 ONEJuly 31, 2015, pp. 1-12; DoI:10.1371 / journal.pone.0128918. Differential methylation sites can also be identified. For example, methylation-based sequencing can be used to identify hypermethylated and hypomethylated sequences from collections of fetal, maternal, and non-pregnant samples. Exemplary target sites are listed in Table 6A, and one, some, or all of the target sites listed in Table 6A can be used, optionally in combination with other target sites not listed in Table 6A. Table 6B shows exemplary primer and probe sets for detecting each target site in Table 6A. Two alternative reverse primer sequences are provided for each target site; the reverse primers of SEQ ID NO: 154 to 200 include an optional 6-nucleotide (ATGACT) 5' end sequence as an extension to increase the annealing temperature of the primer in the second and all subsequent amplification rounds.

[0198] Sites originating from chromosomes unlikely to exhibit aneuploidy can originate from autosomes other than chromosomes 21, 13, or 18. In some embodiments, these sites originate from chromosome 3. Methylation-insensitive regions refer to areas on chromosomes that do not contain CpG sites and are therefore unlikely to be methylated in any cell, and lack the recognition sequence of the MSRE used in the described method, meaning that even if these sites are not methylated, they will not be cleaved by MSRE.

[0199] Sites originating from the methylation-insensitive region of the Y chromosome refer to methylation-insensitive sequences unique to the Y chromosome, making their detection indicate the presence of the Y chromosome, i.e., a male fetus.

[0200] For the purposes of this application, differentially methylated sites are those where the methylation patterns between fetal and maternal DNA show a statistically significant difference (p<0.015) according to a two-sample Kolmogorov-Smirnov test. Furthermore, for the selection of methylation sites in fetal cfDNA, sites with an average methylation frequency greater than 80% in fetal cfDNA (i.e., hypermethylation) and an average methylation frequency less than 20% in maternal DNA (i.e., hypomethylation) were selected. Similarly, for the selection of methylation sites in maternal cfDNA, sites with an average methylation frequency greater than 80% in maternal cfDNA (i.e., hypermethylation) and an average methylation frequency less than 20% in fetal DNA (i.e., hypomethylation) were selected.

[0201] For the selection of hypermethylation target sites as reference sites, sites with an average methylation frequency greater than 80% in both fetal cfDNA and maternal cfDNA were selected.

[0202] For the selection of hypomethylation target sites as reference sites, sites with an average methylation frequency of less than 20% in both fetal cfDNA and maternal cfDNA were selected.

[0203] As an illustration, based on the detection of differences in fetal and maternal methylation, the results of such selections for fetal hypermethylation, maternal hypermethylation, reference hypermethylation, and reference hypomethylation sites are provided respectively in... Figures 2A to 2D In some implementations, the target sites in Table 6A are detected according to the methods described herein.

[0204] Primers and probes

[0205] Primer and probe sequences used to detect amplified products of a desired target can be designed based on known principles. The amplified products are detected using detectable labels. Those skilled in the art will understand that many labeling configurations exist for detecting amplified products. In some embodiments, oligonucleotides are labeled with detectable reagents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, etc.

[0206] 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 (e.g., a fluorescent label). In some embodiments, complementary oligonucleotides that hybridize with non-target regions of the primers or probes are labeled with a detectable label (e.g., a fluorescent label).

[0207] In some implementations, the probe is a TAQMAN. TM probe, SCORPION TM probe, ECLIPSE TM Probes, molecular beacon probes, double-stranded probes, double hybridization probes, or double quenching probes.

[0208] In some embodiments, the oligonucleotide (e.g., primer or probe) is labeled with a detectable tag (e.g., fluorescent label). In some embodiments, the reagent is a fluorophore. A large number of fluorophores are available, including those from commercial suppliers. Non-limiting examples of fluorophores include cyanine (e.g., Cy3, Cy5), indolecarbonyl cyanine (e.g., Quasar® 570, Quasar® 670, and Quasar® 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-carboxyfluorescein (VIC), 6-carboxy-4'-,5'-dichloro-2'-,7'-dimethoxyfluorescein (JOE), 4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein (HEX), 4,7,2',7'-tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-5'-fluoro-7',8'-benzo-1,4-dichloro-6-carboxyfluorescein (NED), Oregon Green and Alexa 488), rhodamine (e.g., N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) and 5- and 6-carboxy-X-rhodamine (ROX)), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC)), Atto dye, eosin, coumarin, pyrene, tetrapyrrole, arylmethane, and oxazine. In some embodiments, the dye is selected from Cy3, Cy5, Cy4.4, ROX, Atto, FAM, HEX, JOE, QUASAR, rhodamine, TAMRA, TET, Texas Red, TYE, and VIC.

[0209] In some embodiments, the detection of the amplified product is performed using a reporter-quencher pair. The reporter-quencher pair can be selected from succinate dyes, including fluorescein and rhodamine dyes. Many suitable forms of these compounds are commercially available, having substituents on the phenyl group, which can serve as binding sites or as binding functional groups for attachment to oligonucleotides. Another class of fluorescent compounds used as reporter groups are naphthylamines, having an amino group at the α or β position. Such naphthylamine compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-phenylamino-8-naphthylsulfonate, and 2-p-tolylamino-6-naphthylsulfonate. Other dyes include 3-phenyl-7-isocyanate coumarin; acridines such as acridine 9-isothiocyanate; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazole; stilbenes; pyrene, etc.

[0210] Suitable examples of quenchers may be selected from 6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo)benzoic acid (DABYL), tetramethylrhodamine (TAMRA), BHQ-OTM, BHQ-1™, BHQ-2™, and BHQ-3™, each available from Biosearch Technologies, Inc., Novato, California; Qy7™, QSY-9™, QSY-21™, and QSY-35™, each available from Molecular Probes, Inc.; and ZEN from IntegratedDNA Technologies. TM and TAO TM Dual-quenching probes. Fluorescent and dark quenchers and their associated optical properties (from which exemplary reporter-quencher pairs can be selected) are listed and described, for example, in RW Sabnis, HANDBOOK OF FLUORESCENT DYES AND PROBES, John Wiley and Sons, New Jersey, 2015.

[0211] Primers can be designed taking into account the recognition sequences of one or more MSREs used in the reaction. Therefore, for example, primers and target regions to be amplified can be selected to avoid the presence of one or more MSRE recognition sequences in the primers and / or in the amplicon generated during the amplification reaction.

[0212] In some implementations, multiple amplicones can be detected using probes of the same color. For example, in some implementations, 2-20 amplicones, such as 6-10, are detected using a single-color probe. In some implementations, multiple color signals are used. For example, in some implementations, a first color signal is used to detect regions that are highly methylated in fetal cfDNA compared to maternal cfDNA, a second color signal is used to detect regions that are highly methylated in maternal cfDNA compared to fetal cfDNA, and optionally, a third color can be used to detect Y chromosome-specific sequences (if present). Additional colors can be used to detect regions that are highly methylated in both fetal and maternal cfDNA, regions that are lowly methylated in both fetal and maternal cfDNA, or as controls to confirm that the detection was performed as expected.

[0213] Other amplification reaction components

[0214] The reagent mixture may also contain other reagents, such as amplification reagents, including one or more of, for example, buffers, salts, nucleotides, stabilizers, primers, polymerases, or nuclease-free water. In some embodiments, additives, such as tetramethylammonium chloride (TMAC), DMSO, DTT, or betaine, may be used to improve amplification specificity or yield.

[0215] partition

[0216] Dispensing the reaction mixture (e.g., cfDNA, digital amplification reaction components, and methylation-sensitive restriction endonuclease) into multiple 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 microplates, capillaries, oil emulsions, and miniaturized chamber arrays for partitioning. In some embodiments, the partitions are droplets. Methods for generating such droplets include droplet or microcapsule coalescence, aggregation, fusion, rupture, or degradation (e.g., as described in US 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151,776); and combinations thereof. Therefore, for example, in a method of partitioning into droplets, droplets can be formed as an emulsion with an immiscible fluid (e.g., oil), such that the overall solution forms droplets containing reaction mixture reagents (including cfDNA template). Methods of emulsion formation are described, for example, in patent applications WO 2011 / 109546 and WO 2012 / 061444.

[0217] In some embodiments, the amplification reaction is a droplet digital PCR reaction. Methods for performing PCR in droplets are described, for example, in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of which are incorporated herein by reference. In some embodiments, the QX600 droplet digital PCR (ddPCR) system (Bio-Rad) is used.

[0218] In some implementations, any of a variety of detection 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 detection device equipped with a module that generates excitation light that can be absorbed by the fluorophore and a module that detects the light emitted by the fluorophore can be used to detect fluorescent labels.

[0219] In some embodiments, the detector also includes processing capabilities for handling partitioned samples (e.g., droplets), wherein a single partitioned sample enters the detector, is detected, and then exits the detector. In some embodiments, the partitioned samples (e.g., droplets) can be detected sequentially as the partitioned samples flow. In some embodiments, the partitioned samples (e.g., droplets) are arranged on a surface, and the detector moves relative to the surface, detecting the signal at each location containing a single partition. Examples of detectors are provided in WO 2010 / 036352, the contents of which are incorporated herein by reference. In some embodiments, detectable markers in the partitioned samples can be detected sequentially without causing the partitioned samples to flow (e.g., using a chamber slide).

[0220] 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 background signal subtraction, target sequence and / or reference sequence allocation, and data quantification. The host computer can be used to display, store, retrieve, or calculate fetal fractions in samples; store, retrieve, or calculate raw data from nucleic acid testing; or display, store, retrieve, or calculate any sample or source information useful in the method.

[0221] A host computer can be configured with many different hardware components and can be manufactured in many sizes and styles (e.g., desktop PC, laptop, tablet PC, handheld computer, server, workstation, mainframe). It may include standard components such as a monitor, keyboard, disk drive, CD and / or DVD drive, etc. When the host computer is connected to a network, the connection can be provided through any suitable transmission medium (e.g., wired, optical, and / or wireless media) and any suitable communication protocol (e.g., TCP / IP); the host computer may include suitable network hardware (e.g., modem, Ethernet card, WiFi card). The host computer can run any of a variety of operating systems, including UNIX, Linux, Microsoft Windows, macOS, or any other operating system.

[0222] The computer code used to implement various aspects of this invention may 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 a host computer or compiled to be executed on a host computer. The code may also be written or distributed in low-level languages ​​such as assembly language or machine language.

[0223] Scripts or programs incorporating various features of this invention can be encoded on a variety of computer-readable media for storage and / or transmission. Examples of suitable media include magnetic disks or magnetic tapes, optical storage media such as optical discs (CDs) or DVDs (Digital Universal Optical Discs), flash memory, and carrier signals suitable for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet.

[0224] Calculation of fetal fraction

[0225] Data processing can be used to generate fetal score calculations, optionally, and fetal sex determination (when needed), using the copy concentrations of detected fetal-specific or maternal-specific target sites. Table 3 illustrates the various copy concentrations provided on six exemplary fluorescence channels using six exemplary detection sets. As described herein, copy concentrations from one or more fetal-specific or maternal-specific target sites and various control target sites can be determined in partitions of 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, said target sites being cleaved with one or more MSREs and detected, for example, by probes in digital detection. The signals of one or more target sites can be multiplexed such that, for example, probes detecting different fetal hypermethylated target sites have probes of the same color, and the total number of partitions with that color signal divided by the number of targets indicates the fetal copy concentration.

[0226] In some implementations, each copy concentration (e.g., each of six copy concentrations) is calculated by dividing by the number of detections in the associated multiplex detection before any downstream calculations. N i Normalization is performed. The number of partitions that are positive for a specific signal (e.g., a detectable wavelength or "color") can represent amplicons from multiple targets (each detected by a probe of the same color). In these implementations, N i This represents the number of multiple targets detected in the amplified group (and, for example, detected using probes with the same color label). In one illustrative embodiment, the average corrected concentration of fetal and maternal cfDNA can be obtained by interpolation within high-methylation and low-methylation reference corrections, as shown in the equations of steps 1a and 1b. (Step 1a) (Step 1b)

[0227] Wherein 'foetal_FF' represents the corrected concentration of fetal DNA; [hyper] is the hypermethylation reference copy concentration based on the amplified component region signal, wherein the amplified component targets sites that are hypermethylated in both fetal and maternal DNA; [hypo] is the methylation reference copy concentration based on the amplified component region signal, wherein the amplified component targets sites that are hypomethylated in both fetal and maternal DNA; and [foetal] is the fetal cfDNA copy concentration based on the amplified component region signal, wherein the amplified component targets sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; wherein 'maternal_FF' represents the corrected concentration of maternal DNA; [maternal] is the maternal cfDNA copy concentration based on the amplified component region signal, wherein the amplified component targets sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA.

[0228] The proportion of the Y chromosome (chrY) in the fetal fraction can be determined and used to accurately determine the fetal fraction based on fetal sex. For example, the proportion of the Y chromosome in the fetal fraction ('chrY_FF') can be determined based on the following calculation (step 3):

[0229] Where [chrY] is the concentration of chrY in the total DNA sample; and [control] is the total concentration of DNA.

[0230] For example, a male fetus can be identified when the 'chrY_FF' value is higher than the threshold of 0.02. Subsequently, a fetal score can be determined based on the calculations in steps 4a (for male fetuses) and 4b (for female fetuses), as follows: (Step 4a) (Step 4b)

[0231] Where 'FF_calculated' is the final calculated fetal score; and 'meth_FF' and 'chrY_FF' are defined as above.

[0232] In another alternative implementation, the average corrected concentration of fetal and maternal cfDNA can be obtained by interpolation within high-methylation and low-methylation reference corrections, as shown in Equations 1 and 2.

[0233] Wherein, [Total] is the total cfDNA copy concentration based on the amplified region signal, the amplified group targeting total cfDNA, the total cfDNA containing methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy; [Hyper] is the hypermethylated reference copy concentration based on the amplified region signal, the amplified group targeting sites that are hypermethylated in both fetal and maternal DNA; [Hypo] is the methylated reference copy concentration based on the amplified region signal, the amplified group targeting sites that are hypomethylated in both fetal and maternal DNA; [Fet] is the fetal cfDNA copy concentration based on the amplified region signal, the amplified group targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and [Mat] is the maternal cfDNA copy concentration based on the amplified region signal, the amplified group targeting sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA.

[0234] Furthermore, several relationships exist between independently measured metrics. Therefore, the relationships expressed in Equation 3-5 can be used as measures of data quality.

[0235] [YChr] is the concentration of Y chromosome-specific sequences based on the amplified group region signal, which targets the Y chromosome methylation-insensitive region.

[0236] Male fetuses can be identified by confirming that the Y chromosome copy concentration is non-zero and satisfies the relationship shown in Equation 5. Furthermore, in the case of Y chromosome aneuploidy, the coefficient will be 1 instead of 2.

[0237] In the case of female or male fetuses, fetal scores can be calculated using, for example, the four methods shown in Equations 6-9, with the average being taken for a more accurate value. Furthermore, if the calculated fetal score fails to converge to the average, it indicates a problem with data quality.

[0238] In the case of male fetuses, the calculation of two other fetal fractions (Equations 10 and 11) becomes possible by using data including the concentration of Y chromosome copies.

[0239] Fetal fraction estimation model

[0240] To improve the accuracy of fetal score calculation, a model was developed that takes the calculated fetal score as input and outputs an adjusted fetal score. The goal of this model is to minimize the average error between fetal scores calculated from clinical samples and fetal scores measured using next-generation sequencing (NGS) on the same clinical samples. In one example, the model aims to reduce the mean absolute percentage error, or mean squared error.

[0241] For example, suppose x1, x2, x3, …, xN represent the calculated fetal scores, and z1, z2, z3, …, zN are the corresponding NGS measurements, for example, z1 corresponds to x1, z2 corresponds to x2, and so on. Further, suppose the estimation model is represented as a function f(), which takes the calculated values ​​x1, x2, …, xN as input and calculates estimated fetal scores y1, y2, y3, …, yN for each calculated score, for example, y1 = f(x1), y2 = f(x2), …, yN = f(xN). The error of each estimate 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 is, for example, (e1) 2 + e2 2 + … + eN 2 ) / N, is minimized. In another example, f() can be designed to minimize the mean absolute percentage error (MAPE), such as (|e1| / z1 + |e2| / z2 + … + |eN| / zN) / N, where |.| is the absolute value operation.

[0242] In one example, once the model is designed using clinical or training data, it can be used to estimate the fetal score based on the calculated fetal score. In another example, the model can be dynamic and can be updated using additional training data.

[0243] In one example, the function f( f(x) is a linear function. For example, f(x) = ax + b. In another example, random noise can be included in the model, such as f(x) = ax + b + n, where n is a random variable with a predefined distribution.

[0244] Figure 5AThis paper illustrates the effect of applying a linear model on the calculated fetal score and its impact on MSE and MAPE. In section 501, the calculated fetal score is plotted against the corresponding NGS fetal score. The calculated fetal score data showed a MAPE of 28% and an MSE of 6.9 × 10⁻⁶. -4 In 503, the same data from 501 are represented using box plots. In 502, a linear model is applied to the calculated fetal scores to obtain the estimated fetal scores depicted. As shown in the figure, the estimates are in better agreement with the corresponding NGS values, which is also validated by the reduced MSE and MAPE values. Using the linear model, MAPE is reduced to 19% and MSE to 2.8 × 10⁻⁶. -4 In 504, the same data as in 502 are represented using box plots. Comparing the box plots in 504 and 503 reveals smaller variations in the data, and the estimated fetal score is closer to the measured NGS value than the calculated fetal score.

[0245] In one example, the function f() can be a polynomial. For example, f(x) = a0 + a1x + a2x 2 + a3 x 3 +… + aM x M M determines the order of the polynomial. For example, for a second-order polynomial, M=2 and f(x) = a0 + a1x + a2x 2 Using optimization criteria, such as minimizing MSE or MAPE, and applying the model to clinical data, coefficients a0, a1, and a2 can be calculated.

[0246] Figure 5B Residual plots were drawn. In plot 510, the calculated fetal score is plotted against the foetal / hyper variable. In plot 512, compared to the foetal / hyper residual plot, the calculated fetal score is plotted against the maternal / hyper variable. This plot does not show a linear relationship between foetal / hyper and the calculated fetal score.

[0247] Figure 5C This section explains the effect of applying a linear model on the calculated fetal scores and its impact on MSE and MAPE. A second-order polynomial model was developed using the same calculated fetal score data as in section 501. The second-order polynomial model (poly2LM) was applied to the calculated fetal scores to obtain the depicted estimated fetal scores. Figure 5C As shown, the estimated values ​​are more consistent with the corresponding NGS values ​​along a line, which is also verified by the reduced MSE and MAPE values. Using a second-order polynomial model, MAPE is reduced to 17% and MSE to 2.5 × 10⁻⁶. -4.

[0248] In one example, a generalized additive model (GAM) can be used to estimate the fetal score based on the calculated fetal score. GAM can be used to model nonlinearities using additive models. For example, the calculated fetal score may have different relationships with the corresponding NGS values ​​at different NGS values. A piecewise model can be developed that defines different estimation functions. For example, the NGS range can be divided into K regions, such as K = 20, 30, or 40, and each region can be estimated using splines, such as 2nd, 3rd, 4th, or different orders of splines. Furthermore, GAM can use other parameters as variables in the model, such as gestational age, maternal weight, etc.

[0249] Figure 5D This section illustrates the effect of applying GAM on the calculated fetal score and its impact on MSE and MAPE. The same calculated fetal score data as in 501 are used. GAM is applied to the calculated fetal score to obtain the depicted estimated fetal score. Figure 5D As shown, the estimated values ​​are more consistent with the corresponding NGS values ​​along a line, which is also verified by the reduced MSE and MAPE values. Using GAM, MAPE is reduced to 13% and MSE to 1.5 × 10⁻⁶. -4 .

[0250] Reagent test kit

[0251] In another aspect, this disclosure provides a digital amplification kit for estimating the fraction of fetal DNA in a cfDNA sample obtained from a blood sample (such as a plasma or serum sample) of a pregnant subject (e.g., a human). The kit may include any components described herein with respect to the method. In some embodiments, the kit comprises an amplification reaction mixture containing amplification reagents and a plurality of amplification groups, each amplification group containing sets of primers and probes, wherein each amplification group contains a unique marker distinguishable from the markers of each other group, and each group contains primers and probes for multiplex amplification, and wherein the plurality of amplification groups comprises (i) amplification groups targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and / or (ii) amplification groups targeting sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA. In some embodiments, the kit also comprises amplification groups targeting total cfDNA comprising methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy. In some embodiments, the kit comprises an amplification set targeting sites that are hypermethylated in both fetal and maternal DNA; and / or an amplification set targeting sites that are hypomethylated in both fetal and maternal DNA. In some embodiments, the kit also comprises an amplification set targeting methylation-insensitive regions of the Y chromosome. In some embodiments, as described elsewhere herein, one or more sites are selected from Table 6A. In some embodiments, the kit as described in this paragraph comprises a fully methylated synthetic DNA sequence and / or a fully unmethylated version of the same synthetic sequence. In some embodiments, the probe used for detection is a molecular beacon probe, such as a fluorescently labeled molecular beacon probe.

[0252] In some implementations, each amplification group of (i) and (ii) contains primers and probes that target at least two sites, at least three sites or more sites (e.g., 1-20 sites, or 6-10 sites).

[0253] In some embodiments, each label in the amplification group is a fluorescent label. In some embodiments, one or more primers or probes contain regions that do not hybridize with the target amplification site but are complementary to an oligonucleotide that provides a detectable signal.

[0254] In some embodiments, the kit comprising one or more amplification groups as described above further comprises at least one methylation-sensitive restriction endonuclease that cleaves unmethylated DNA. In some embodiments, the kit comprises one or more restriction endonucleases selected from HhaI, HpaII, AciI, HpyCH4IV, or BsaHI. In some embodiments, the methylation-sensitive restriction endonuclease (MSRE) mixture comprises at least one, two, at least three, or at least four methylation-sensitive restriction endonucleases selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the kit comprises a methylation-sensitive restriction endonuclease (MSRE) mixture containing at least two, three, or more restriction endonucleases selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI. In some embodiments, the MSRE mixture contains at least HhaI and HpyCH4IV.

[0255] In some implementations, the reaction mixture is lyophilized.

[0256] In some embodiments, the kit comprises a standard ddPCR kit, a primer and probe set for fetal fraction detection as described herein, and at least one MSRE. In some embodiments, such kits also comprise at least one PCR enhancer, such as TMAC and / or salt.

[0257] In some implementations, the kit comprises a standard ddPCR kit, a set of primers and probes for fetal fraction detection as described herein, at least one MSRE, and a stabilizer (e.g., trehalose, potassium glutamate, ammonium sulfate) (lyophilized together in a mixture).

[0258] In some implementations, the kit comprises a standard ddPCR kit, a set of primers and probes for fetal fraction detection as described herein; a stabilizing reagent (lyophilized together); and at least one MSRE, which may or may not be lyophilized.

[0259] RNaseH2 activated universal fluorescent probe

[0260] Various probe formats have been used to detect amplification products in whole or in specific regions (e.g., dPCR or droplet dPCR). The most commonly used fluorescent probe in qPCR (quantitative PCR) and dPCR (digital PCR) is TaqMan. TMProbes, which require hydrolysis by the 5' to 3' exonuclease activity of DNA polymerase to produce a fluorescent signal during each amplification signal. Another type of fluorescent probe is the molecular beacon, which has a stem-loop structure, increasing the specificity of the probe binding to its target. A disadvantage of molecular beacons is that the fluorescent signal they produce is not as strong as that of hydrolyzed probes because the probe is not cleaved throughout the PCR cycle. In the context of dPCR for reading the endpoint fluorescent signal, the molecular beacon hybridizes to its target in the final step, which involves denaturing the amplification product (e.g., 98°C for 10 min) followed by cooling to 4°C. The default configuration for quantifying target DNA with fluorescently hydrolyzed probes is to use primer pairs and fluorescent probes specific to each target amplicon. In the case of multiplex detection, this means that each target amplicon requires a three-component set: a forward primer, a reverse primer, and a fluorescent probe.

[0261] In some embodiments, a method for detecting target nucleic acids in a sample is provided. In some embodiments, the method includes: (a) forming a reaction mixture comprising: sample nucleic acids; a plurality of forward primers containing a 3' target-specific forward sequence; a plurality of reverse primers containing a 3' target-specific reverse sequence, wherein the forward or reverse primers further contain a 5' universal sequence; probe nucleic acids comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the 5' universal sequence, or both, and (iv) at least one ribonucleotide having a separation between the fluorophore and the quencher; a DNA polymerase; and an RNaseH2 enzyme. (b) Annealing the forward primer with the target nucleic acid in the sample nucleic acid and extending the forward primer with polymerase using the target nucleic acid as a template to form a first-strand extension product; (c) Annealing the reverse primer with the first-strand extension product and extending the reverse primer with polymerase using the first-strand extension product as a template to form a second-strand extension product, wherein if the reverse primer contains a 5' universal sequence, the first-strand extension product contains the reverse complementary sequence of the 5' universal sequence, and if the forward primer contains a 5' universal sequence, the second-strand extension product contains the reverse complementary sequence of the 5' universal sequence; and (d) Annealing the probe nucleic acid with the reverse complementary sequence of the 5' universal terminal sequence, and RNase H2 enzyme cleaving the annealed probe at the ribonucleotide, thereby separating the quencher from the fluorophore to generate a detectable signal indicating the presence of the target nucleic acid.

[0262] In some implementations, (a)-(d) are carried out in multiple partitions, wherein the target nucleic acid is distributed among multiple partitions such that at least some partitions do not contain the target nucleic acid.

[0263] In some implementations, the sample nucleic acid is cell-free DNA. In some implementations, the sample nucleic acid is derived from the pregnant woman and contains both maternal DNA and fetal DNA.

[0264] In some implementations, the partitions are droplets. In some implementations, the partitions are micropores. In some implementations, the micropores are sealed by caps, air gaps, or oil layers.

[0265] In some implementations, the detectable signal is monitored in real time. In some implementations, the detectable signal is monitored at the end of the method.

[0266] In some embodiments, the forward primer contains a 5' universal sequence, and the concentration of the reverse primer is higher than that of the forward primer.

[0267] In some embodiments, the forward primer contains a 5' universal sequence, and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid.

[0268] In some embodiments, the probe nucleic acid is a linear probe. In some embodiments, the linear probe comprises 80-100% of a 5' universal sequence. In some embodiments, the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, or 30) nucleotides annealed to the inverse complementary sequence of the 5' universal sequence. In some embodiments, the probe is non-universal and binds to a target sequence present in the target nucleic acid or oligonucleotide.

[0269] In some embodiments, the forward primer contains a 5' universal sequence, and the inverse complementary sequence of the 5' universal sequence on the linear probe and the second strand extension product forms a double strand, which has a higher melting temperature than the double strand formed by the 5' universal sequence of the forward primer and its inverse complementary sequence. In some embodiments, the reverse primer contains a 5' universal sequence, and the inverse complementary sequence of the 5' universal sequence on the linear probe and the first strand extension product forms a double strand, which has a higher melting temperature than the double strand formed by the 5' universal sequence of the reverse primer and its inverse complementary sequence.

[0270] In some embodiments, the probe nucleic acid forms a stem-loop and comprises, from 5' to 3': a first stem sequence, a loop sequence, and a second stem sequence, the second stem sequence being the reverse complementary sequence of the first stem sequence, wherein the ribonucleotide is located within the loop sequence, and wherein the 5' universal sequence comprises at least a portion (or all) of the loop sequence. In some embodiments, the 5' universal sequence also comprises at least a portion (or all) of the second stem sequence. In some embodiments, the first stem sequence and the second stem sequence are each 4-10 nucleotides in length (e.g., 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the loop sequence is between 5-50 nucleotides in length (e.g., 10-40 or 17-32). In some embodiments, the 5' universal sequence comprises the entire loop sequence, the entire second stem sequence, or the entire loop sequence and the entire second stem sequence.

[0271] In some embodiments, the probe nucleic acid forms a stem-loop and comprises, from 5' to 3': a first stem sequence, a loop sequence, and a second stem sequence, the second stem sequence being the inverse complementary sequence of the first stem sequence, wherein a ribonucleotide is located within the loop sequence, and wherein the target sequence of the target nucleic acid or oligonucleotide comprises at least a portion (or all) of the loop sequence. In some embodiments, the target sequence of the target nucleic acid or oligonucleotide also comprises at least a portion (or all) of the second stem sequence. In some embodiments, the first stem sequence and the second stem sequence are each 4-10 nucleotides in length (e.g., 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the loop sequence is between 5-50 nucleotides in length (e.g., 10-40 or 17-32). In some embodiments, the target sequence of the target nucleic acid or oligonucleotide comprises the entire loop sequence, the entire second stem sequence, or the entire loop sequence and the entire second stem sequence.

[0272] In some implementations, the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers to allow amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

[0273] In some embodiments, the RNaseH2 enzyme is Thermococcus abyssalis ribonuclease HII (H2O). Pyrococcus abyssi RNase H2 enzyme) or its mutants, *Firmianalyte* ribonuclease HII ( Pyrococcus furiosis RNase H2 enzyme) or its mutants, Thermococcus viride ribonuclease HII ( Pyrococcus horikoshiiRNase H2 enzyme) or its mutants, thermococcal ribonuclease HII ( Thermococcus kodakarensis RNase H2 enzyme) or its mutants, or Thermococcus marinum ribonuclease HII ( Thermococcus litoralis RNase H2 enzyme or its mutants.

[0274] In some embodiments, the reaction mixture comprises: (a) a first set of forward and reverse primers, wherein the first set targets a first plurality of different target nucleic acids, and the forward or reverse primers of the first set comprise a first 5' universal sequence; and a first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the first 5' universal sequence, or both; and (b) a second set of forward and reverse primers, wherein the second set targets a second plurality of different target nucleic acids, and the forward or reverse primers of the second set comprise a first 5' universal sequence. The primers contain a second 5' universal sequence different from the first 5' universal sequence, and a second set of probe nucleic acids containing (i) a second fluorophore, (ii) a second quencher, and (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the second 5' universal sequence, or both, such that a first amplicon from the first set of forward and reverse primers and having the first 5' universal sequence can be distinguished from a second amplicon from the second set of forward and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0275] In some implementations, the DNA polymerase lacks 5'-3' exonuclease activity.

[0276] A reaction mixture is also provided. In some embodiments, the reaction mixture comprises: a plurality of forward primers containing a 3' target-specific forward sequence; a plurality of reverse primers containing a 3' target-specific reverse sequence, wherein the forward or reverse primers further contain a 5' universal sequence; a probe nucleic acid comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the 5' universal sequence, or both, and (iv) a ribonucleotide having at least one separating the fluorophore and the quencher; a DNA polymerase; and an RNaseH2 enzyme.

[0277] In some embodiments, the reaction mixture further comprises sample nucleic acid. In some embodiments, the sample nucleic acid is cell-free DNA. In some embodiments, the sample nucleic acid is derived from the pregnant woman and contains both maternal and fetal DNA.

[0278] In some embodiments, the reaction mixture is located in multiple partitions, with the target nucleic acid distributed among the partitions such that at least a portion of the partitions does not contain the target nucleic acid. In some embodiments, the partitions are droplets. In some embodiments, the partitions are micropores. In some embodiments, the micropores are sealed by caps, air gaps, or oil layers.

[0279] In some embodiments, the forward primer contains a 5' universal sequence, and the concentration of the reverse primer is higher than that of the forward primer.

[0280] In some embodiments, the forward primer contains a 5' universal sequence, and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid.

[0281] In some embodiments, the probe nucleic acid is a linear probe. In some embodiments, the linear probe comprises 80-100% of the 5' universal sequence. In some embodiments, the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, or 30) nucleotides annealed to the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide. In some embodiments, the linear probe comprises 80-100% of the target sequence of the target nucleic acid or oligonucleotide. In some embodiments, the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, or 30) nucleotides annealed to the target sequence of the target nucleic acid or oligonucleotide.

[0282] In some embodiments, the probe nucleic acid forms a stem-loop and comprises, from 5' to 3': a first stem sequence, a loop sequence, and a second stem sequence, the second stem sequence being the reverse complementary sequence of the first stem sequence, wherein the ribonucleotide is located within the loop sequence, and wherein the 5' universal sequence comprises at least a portion of the loop sequence. In some embodiments, the 5' universal sequence further comprises at least a portion of the second stem sequence. In some embodiments, the first stem sequence and the second stem sequence are each 4-10 nucleotides in length (e.g., 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the loop sequence is between 5-50 nucleotides in length (e.g., 10-40 or 17-32 nucleotides). In some embodiments, the 5' universal sequence comprises the entire loop sequence, the entire second stem sequence, or the entire loop sequence and the entire second stem sequence.

[0283] In some implementations, the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers to allow amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

[0284] In some embodiments, the RNaseH2 enzyme is Thermococcus abyssalis ribonuclease HII (H2O). Pyrococcus abyssi RNase H2 enzyme) or its mutants, *Firmianalyte* ribonuclease HII ( Pyrococcus furiosis RNase H2 enzyme) or its mutants, Thermococcus viride ribonuclease HII ( Pyrococcus horikoshii RNase H2 enzyme) or its mutants, thermococcal ribonuclease HII ( Thermococcus kodakarensis RNase H2 enzyme) or its mutants, or Thermococcus marinum ribonuclease HII ( Thermococcus litoralis RNase H2 enzyme or its mutants.

[0285] In some embodiments, the reaction mixture comprises: (a) a first set of forward and reverse primers, wherein the first set targets a first plurality of different target nucleic acids, and the forward or reverse primers of the first set comprise a first 5' universal sequence; and a first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the first 5' universal sequence, or both; and (b) a second set of forward and reverse primers, wherein the second set targets a second plurality of different target nucleic acids, and the forward or reverse primers of the second set comprise a first 5' universal sequence. The primers contain a second 5' universal sequence different from the first 5' universal sequence, and a second set of probe nucleic acids containing (i) a second fluorophore, (ii) a second quencher, and (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the second 5' universal sequence, or both, such that a first amplicon from the first set of forward and reverse primers and having the first 5' universal sequence can be distinguished from a second amplicon from the second set of forward and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0286] In some implementations, the DNA polymerase lacks 5'-3' exonuclease activity.

[0287] A mixture is also provided comprising: (a) a first set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, the reverse primers comprising a 3' target-specific reverse sequence, wherein the forward or reverse primers of the first set further comprise a first 5' universal sequence, wherein the first set targets a first plurality of different target nucleic acids, and a first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the first 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; and (b) a second set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, the reverse primers comprising a 3' target-specific reverse sequence, wherein the first set of forward or reverse primers further comprises a first 5' universal sequence, wherein the first set targets a first plurality of different target nucleic acids, and a first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the first 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; and (b) a second set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, the reverse primers comprising a first fluorophore, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; and (v) a second set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, the reverse primers comprising a first fluorophore, and (v) at least The primers contain a 3' target-specific reverse sequence, wherein the forward or reverse primers of the second set further contain a second 5' universal sequence, wherein the second set targets a second plurality of different target nucleic acids, and a second set of probe nucleic acids, which contains (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the second 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher, such that a first amplicon from the first set of forward and reverse primers and having the first 5' universal sequence can be distinguished from a second amplicon from the second set of forward and reverse primers and having the second 5' universal sequence, based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0288] In some embodiments, the mixture also contains RNaseH2 enzyme.

[0289] Methods for detecting target nucleic acids using probes containing ribonucleotides are provided. When the probe binds to a universal sequence of the target nucleic acid introduced via a primer, the probe is cleaved by an RNaseH2 enzyme. For example, the probes described herein may have a fluorophore and a quencher portion separated by ribonucleotides, such that the signal from the fluorophore in the intact probe is relatively quenched relative to the signal when the probe is cleaved at the ribonucleotides by the RNaseH2 enzyme. The inventors have found that such probes are particularly useful in digital amplification detection and will also be used for real-time bulk amplification detection. Some advantages of these methods include better separation of signal (target) from background (no target) and the ability to use DNA polymerases lacking 5'-3' exonuclease activity. The methods described herein can also be used in various multiplex forms, allowing the detection of a large number of different amplicons as needed. Other advantages will become apparent from the remainder of this disclosure.

[0290] These methods may involve forming a reaction mixture containing sample nucleic acids, forward and reverse primers, probes as described herein, DNA polymerase, and RNase H2 enzyme. Each of these will be discussed in turn, followed by their usage.

[0291] The target nucleic acid is any DNA sequence (e.g., dsDNA, cDNA) to be detected from the sample. The biological sample can be obtained from any organism, such as animals, plants, fungi, pathogens (e.g., bacteria or viruses), or any other organism. In some embodiments, the biological sample is derived from animals, such as mammals (e.g., humans or non-human primates, cattle, horses, pigs, sheep, cats, dogs, mice, or rats), birds (e.g., chickens), or fish. The biological sample can be any tissue or bodily fluid obtained from an organism, such as blood, blood aliquots or blood products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum or saliva, tissues (e.g., kidney, lung, liver, heart, brain, nerve tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, such as primary cultures, explants and transformed cells, stem cells, feces, urine, etc. In some embodiments, the sample is a cell-free nucleic acid sample. In some implementations, the cell-free DNA sample comes from a pregnant woman (e.g., a cell-free sample containing maternal and fetal DNA from maternal blood), or from a person who has, has had, or is suspected of having cancer, or an organ transplant recipient.

[0292] The disclosed method can be used with samples obtained non-invasively or invasively (e.g., amniocentesis or CVS samples).

[0293] The reaction mixture will contain multiple forward primers, each containing one or more 3' target-specific forward sequences. The terms "forward" and "reverse" are arbitrary designations, referring to the forward and reverse primers annealing to the opposite strand of a double-stranded DNA molecule, and upon annealing, their 3' ends facing each other, thus forming an amplicon under PCR conditions. The 3' target-specific forward sequences can vary in composition and length and can be designed to specifically amplify a specific target nucleic acid in different nucleic acid mixtures. In some embodiments, the 3' target-specific forward sequence is between 10 and 30 nucleotides in length, annealed to or adjacent to the target nucleic acid; other lengths may be used in other embodiments.

[0294] Multiple forward primers will comprise a number of primer copies to achieve the desired amplification. In multiplex reactions, the multiple forward primers will further comprise multiple different forward primers with different 3' target-specific forward sequences, allowing amplification of different targets in the same reaction. As a simple example, the reaction may have copies of a first forward primer (with a first 3' target-specific forward sequence) and a second forward primer (with a second (different from the first) 3' target-specific forward sequence), allowing amplification of the first and second targets present in the sample (if present). As discussed in more detail below, the number of different forward primers can be high, for example, at least 2, 5, 10, 20, 30, 40, 50, 70, 100, 120 or more, such as 2-200, in the same reaction. As will be discussed below, in some embodiments, a universal sequence will be located on the forward primer, and in this case, at the 5' end of the forward primer.

[0295] In some implementations, multiple reverse primers are provided. For example, in some implementations, for each forward primer with a unique 3' target-specific forward sequence, there will be a reverse primer with a unique 3' target-specific reverse sequence, such that the forward and reverse primers amplify a specific target nucleic acid together. Similar to the forward primers, in the multiplex option, reverse primers with different 3' target-specific reverse sequences can be used in the reaction mixture.

[0296] The forward or reverse primers will also contain a 5' universal sequence, but in some embodiments not both are included, and in some embodiments neither is included. The 5' universal sequence is a sequence common to a set of primers, but it is not annealed to the target sequence in the initial amplification round (it is not target-specific). See, for example, Figure 6 The 5' universal sequence can have any desired length, as long as it is long enough to allow the probe to anneal during the reaction, as described below. In some embodiments, for example, the 5' universal sequence is between 10 and 50 nucleotides long, such as 15-40, 15-35, or 20-35 nucleotides long. At least a portion (in some embodiments, all) of the 5' universal primer and at least a portion of the probe sequence (including the probe's ribonucleotides) will have the same sequence as each other, such that the reverse complementary sequence of the probe (including the ribonucleotides) and the universal sequence will anneal, thereby allowing the RNaseH2 enzyme to cleave at the ribonucleotides, as further explained below.

[0297] In embodiments providing forward and reverse primer pairs, it may be advantageous to include a higher quantity (e.g., a higher concentration) of primers lacking a 5' universal sequence compared to primers having a 5' universal sequence. For example, in embodiments where the forward primer has a 5' universal sequence, in some cases, the concentration of the reverse primer will be higher (e.g., at least 1.5, 2, 3, 5, or 10 times higher) than the concentration of the forward primer. Similarly, in embodiments where the reverse primer has a 5' universal sequence, in some cases, the concentration of the forward primer will be higher (e.g., at least 1.5, 2, 3, 5, or 10 times higher) than the concentration of the reverse primer. For example, in some embodiments, the concentration of the primer lacking the 5' universal sequence is 500-2,000 nM (e.g., 900 or 1,000 nM), while the primer with the 5' universal sequence is provided at a concentration of 50-200 nM (e.g., 90 or 100 nM). This is useful for generating an excess of strands with the reverse complementary sequence of the 5' universal sequence, which are detected by the probe.

[0298] In some multiple implementations, some primers may have different 5' universal sequences, allowing the use of different "color" probes in the same reaction. For example, in some implementations, a first set of primers (forward or reverse primers) has a first 5' universal sequence, and a second set of primers (forward or reverse primers) has a second 5' universal sequence. In these implementations, the reaction mixture has a first probe and a second probe, the sequence of the first probe being partially or entirely identical to the first 5' universal sequence, and the sequence of the second probe being partially or entirely identical to the second 5' universal sequence. If the first and second probes have different fluorophores or quenchers, such that the emitted signals have different wavelengths (colors), then the signals of the first and second probes can be distinguished. While the above example is illustrated with two 5' universal sequences and associated probes, more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, etc.) 5' universal sequences and associated probes can be used, where each probe can be distinguished from the other probes by its signal wavelength.

[0299] Furthermore, in some embodiments, each set of primers having the same 5' universal sequence may include primers having more than one different 3' target-specific forward (or reverse, depending on which primer) sequence. Thus, for example, a first set of forward primers may include 2, 5, 10, or 20 or more different 3' target-specific forward sequences, each having a first 5' universal sequence and thus detectable by a first probe; a second set of forward primers may include 2, 5, 10, or 20 or more different 3' target-specific forward sequences, each having a second 5' universal sequence and thus detectable by a second probe. When all of these are in a single reaction mixture, this allows for the monitoring of a very large number of different amplifications. For example, the inventors have used six distinguishable probes, each set of probes detecting 20 different amplification reactions having its own 5' universal sequence, thereby allowing for the monitoring of 120 different reactions in a digital format.

[0300] Probe nucleic acids utilize energy transfer between two parts, such as a donor fluorophore and an acceptor part separated by ribonucleotides. In some embodiments, the probe nucleic acid comprises a fluorophore part separated by ribonucleotides and a quencher part (i.e., an acceptor that absorbs energy released by the donor fluorophore but does not emit light itself), such that upon ribonucleotide cleavage, the two parts are separated, thereby generating a detectable signal. The fluorophore and quencher may be located at the ends of the probe, or one or both may be attached to internal nucleotides within the probe. In some embodiments, proximity of the quencher to the fluorophore in the intact probe results in quenching of the fluorescence signal, while the cleaved probe results in an increased detectable signal due to attenuation or elimination of quenching. In some embodiments using two or more different probes, the different probes will have different fluorophores or quenchers, or both, such that the different probes emit signals at different wavelengths, which can be distinguished from each other by the sensor. Because some quenchers can quench a wide range of wavelengths, in some embodiments, the same quencher is used for two or more different probes, each probe having a different fluorophore emitting at different wavelengths.

[0301] Fluorescent agents can include a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and their derivatives. Numerous fluorophores and quenchers have been reported in the literature and are therefore known to those skilled in the art, and many are readily available from commercial suppliers in the biotechnology industry. Literature sources for fluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, DL, J. of Chemical Physics 21: 836-850 (1953); Hochstrasser et al., Biophysical Chemistry45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem .,40: 83-114 (1971); Stryer, L. Ann. Rev. Biochem. , 47: 819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal. Chem. 67: 1197-1203 (1995). Non-limiting examples of fluorophores include cyanine, fluorescein (e.g., 5'-carboxyfluorescein (FAM), Oregon Green, and Alexa 488), HEX, rhodamine (e.g., N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)), eosin, coumarin, pyrene, tetrapyrrole, arylmethane, oxazine, polymeric dots, and quantum dots. In some embodiments, the fluorophore is selected from HEX, FAM, Cy5, Cy5.5, ROX, Atto 590, Alexa 405, Pacific Blue, ABY, and Texas Red. Exemplary quenchers may include, but are not limited to, Iowa Black® FQ, Iowa Black® RQ, Black Hole Quencher®-1, Black Hole Quencher®-2, DABCYL, and ZEN internal quenchers.

[0302] The nucleotide sequence of the probe (including the ribonucleotide positions) will contain at least 25%, and more preferably at least 30, 40, 50, 60, 70, 80, 90, or 100% of the 5' universal sequence. For example, in some embodiments, the probe may contain 10-50, e.g., 15-30 nucleotides identical to the 5' universal sequence. At the alignment position, the ribonucleotide will contain the same nucleotide as the 5' universal sequence (but in ribonucleotide form), unless that position in the 5' universal sequence is thymine, in which case the ribonucleotide is its RNA equivalent, i.e., uracil. The nucleotide sequence will consist of non-ribonucleotides other than one or more ribonucleotides that separate the fluorophore and quencher portions. The non-ribonucleotides may be, for example, deoxyribonucleotides (e.g., forming DNA) or analogues thereof. In some embodiments, the probe may comprise one or more non-natural nucleotides or analogues thereof (e.g., peptide nucleic acids (PNAs) or locked nucleic acids (LNAs)) or other nucleotides that have a higher affinity for the reverse complement of the 5' universal sequence compared to primers (composed of DNA) having a 5' universal sequence. The enhanced affinity (higher melting temperature (Tm) of the probe for the reverse complement of the 5' universal sequence compared to the affinity of primers having a 5' universal sequence for the reverse complement of the 5' universal sequence can be used to further improve signal-to-noise ratio discrimination.

[0303] The nucleotide sequence of the probe (including the ribonucleotide position) will contain at least 25% of the target sequence of the target nucleic acid or oligonucleotide, and in embodiments, at least 30, 40, 50, 60, 70, 80, 90, or 100%. For example, in some embodiments, the probe may contain 10-50, e.g., 15-30 consecutive nucleotides identical to the target sequence of the target nucleic acid or oligonucleotide. At the alignment position, the ribonucleotide will contain the same nucleotide as the target sequence of the target nucleic acid or oligonucleotide (but in ribonucleotide form), unless that position in the target sequence of the target nucleic acid or oligonucleotide is thymine, in which case the ribonucleotide is its RNA equivalent, i.e., uracil. The nucleotide sequence may consist of non-ribonucleotides other than one or more ribonucleotides that separate the fluorophore and quencher portions. Non-ribonucleotides may be, for example, deoxyribonucleotides (e.g., forming DNA) or analogues thereof. In some embodiments, the probe may comprise one or more non-natural nucleotides or analogues thereof (e.g., peptide nucleic acids (PNAs) or locked nucleic acids (LNAs)) or other nucleotides that have a higher affinity for the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide compared to the affinity of a primer (composed of DNA) for the target sequence of the target nucleic acid or oligonucleotide. The enhanced affinity (higher melting temperature (Tm)) of the probe for the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide compared to the affinity of a primer for the target sequence of the target nucleic acid or oligonucleotide can be used to further improve signal-to-noise ratio discrimination.

[0304] As described above, one or more ribonucleotides are positioned between the fluorophore and quencher in the probe, such that when the RNaseH2 enzyme cleaves the ribonucleotide, the fluorophore and quencher diffuse into each other to generate a detectable signal. Furthermore, the RNaseH2 enzyme cleaves the ribonucleotide annealed to its deoxyribonucleotide counterpart against a background of two annealed strands (in this example, the inverse complementary sequence of the probe and the 5' universal sequence). The RNaseH2 enzyme cleaves more efficiently if multiple (e.g., 2, 3, 4, 5, 6, or more) annealed nucleotides are present on either side of the ribonucleotide. Therefore, in some embodiments, the ribonucleotide is positioned in the middle segment of the probe, or at least at a location more than 2, 3, 4, 5, 6, or more nucleotides away from either end of the probe.

[0305] Optionally, the probe may be modified at its 3' end to prevent polymerase from extending it. In some embodiments, a fluorophore or quencher is attached to the 3' end of the probe, which prevents polymerase from extending the probe. In other embodiments, the 3' end of the probe may be modified to block extension. Non-restrictive modifications to the 3' end may include, for example, 3' reverse dT, a 3' C3 spacer sequence, 3' amino, or 3' phosphorylation.

[0306] Various probe forms can be used. In some embodiments, the probe is a linear probe, meaning that the sequence within the probe does not form a stem-loop structure or otherwise anneal itself. In other embodiments, the probe may be in stem-loop form, with the ribonucleotide located in the loop portion. See, for example, Figure 7 The probe concentration can be selected to avoid background while generating a detectable signal. For example, in some embodiments, the probe is provided at a concentration of 100-1000 nM, such as 500 nM. In some embodiments, the probe concentration is lower than the concentration of the restriction primer (i.e., the primer with the 5' universal sequence), and in some embodiments, it is 2-10 times the concentration of the restriction primer.

[0307] Linear probes can have any desired length. Exemplary probe nucleic acids can have, for example, at least 10 (e.g., at least 15, 20, 25, 30) consecutive or non-consecutive nucleotides that are anticomplementary to the 5' universal sequence. In some embodiments, the linear probe will contain at least 80%, 90%, or 100% identical sequences to the 5' universal probe sequence, thereby improving the probe's ability to compete for the anticomplementary sequence of the universal 5' sequence compared to primers having the 5' universal sequence.

[0308] Linear probes can have any desired length. Exemplary probe nucleic acids can have, for example, at least 10 (e.g., at least 15, 20, 25, 30) consecutive or discontinuous nucleotides that are anticomplementary to the target sequence of the target nucleic acid or oligonucleotide. In some embodiments, the linear probe will contain at least 80%, 90%, or 100% identical sequences to the target sequence of the target nucleic acid or oligonucleotide, thereby improving the probe's ability to compete for the anticomplementary sequence of the universal 5' sequence compared to primers containing the target sequence of the target nucleic acid or oligonucleotide.

[0309] The stem-loop probe will have a first stem sequence, a loop sequence, and a second stem sequence, the second stem sequence being the inverse complementary sequence of the first stem sequence, such that the two stem sequences self-anneal to form a stem-loop. At least a portion of the loop sequence containing ribonucleotides will have a sequence identical to the 5' universal sequence. In some embodiments, at least a portion of the second stem sequence is also identical to a sequence in the 5' universal sequence. In some embodiments, the loop sequence or the second stem sequence, or both, are identical to adjacent sequences in the 5' universal sequence; see, for example, Figure 7 .

[0310] The stem-loop probe will have a first stem sequence, a loop sequence, and a second stem sequence, the second stem sequence being the inverse complementary sequence of the first stem sequence, such that the two stem sequences self-anneal to form a stem-loop. At least a portion of the loop sequence containing ribonucleotides will have a sequence identical to the target sequence of the target nucleic acid or oligonucleotide. In some embodiments, at least a portion of the second stem sequence is also identical to a sequence in the target sequence of the target nucleic acid or oligonucleotide. In some embodiments, the loop sequence or the second stem sequence, or both, are identical to adjacent sequences in the target sequence of the target nucleic acid or oligonucleotide.

[0311] The lengths of the stem and loop sequences can be varied as needed. In some embodiments, the first and second stem sequences are each 4-10 nucleotides long (e.g., 4, 5, 6, 7, 8, 9, or 10), although other lengths are also possible. In some embodiments, the loop sequence is between 5-50 nucleotides long (e.g., 10-40 or 17-32), although other lengths are also possible.

[0312] The reaction mixture may contain a DNA polymerase, which extends at least one primer in a template-dependent manner. In some embodiments, the DNA polymerase is a thermostable polymerase. Thermostable polymerases have been isolated from a variety of thermophilic bacteria, such as *Thermophilus aquaticus* (…). Thermus aquaticus Taq), Vibrio fibrillosa ( Pyrococcus furiosus Pfu), Warwickia fireball ( Pyrococcus woesei Pwo), thermophilic steatobacterium ( Bacillus sterothermophilus Bst), acidophilic thermosulfuric leaf fungus ( Sulfolobus acidocaldarius Sac), sulfur mineral sulfide fungi ( Sulfolobus solfataricus , Sso), cryptotrophic thermococci ( Pyrodictium occultum Poc), Thermophilic cocci (Poc), Abyssal thermophilic cocci ( Pyrodictium abyssi Pab) and thermoautotrophic methanobacterium ( 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 its active mutants, variants, or derivatives. 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). TMHigh-fidelity DNA polymerases, including Phusion® High-fidelity DNA polymerase, Q5 High-fidelity DNA polymerase, Platinum® Taq High-fidelity DNA polymerase, and Accura® High-fidelity polymerase. In some implementations, the DNA polymerase is a fast-start polymerase (e.g., FastStart). TM Taq DNA polymerase or FastStart TM (High-fidelity DNA polymerase). In some embodiments, the polymerase lacks 5'-3' exonuclease activity, for example, as found in B family polymerases.

[0313] As described above, methods and compositions can involve the activity of RNase H2 enzymes. RNase H2 enzymes cleave ribonucleotides in the RNA / DNA double helix. Various RNase H2 enzymes are known and can be used in the methods described herein. Exemplary RNase H2 enzymes have been described, such as those from *Cyclocarya abyssica* (…). Pyrococcus abyssi ), Vibrio parahaemolyticus ( Pyrococcus furiosus ), Thermococcus viride ( Pyrococcus horikoshii ), Kodakara thermococcus ( Thermococcus kodakarensis ) and coastal thermococcus ( Thermococcus litoralis In addition, a number of mutant enzymes have been developed from these natural enzymes, for example, as described in WO2018 / 031625, which is incorporated by reference.

[0314] The method described herein involves forming a reaction mixture using some or all of the components described above, as well as reagents necessary for primer extension (e.g., amplification).

[0315] The amplification reaction mixture will also contain nucleotides. The nucleotides used in the methods described herein can be any nucleotide used for nucleic acid polymerization. Nucleotides can be naturally occurring, unusual, modified, derived, or artificial. In the embodiments described herein, the nucleotides will typically be unlabeled.

[0316] In some embodiments, the amplification reaction mixture contains one or more buffers or salts. Various buffers and salt solutions, as well as modified buffers, are known in the art. For example, in some embodiments, the buffer is TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, or CAPS. In some embodiments, the salt is potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, or lithium acetate. In some embodiments, the amplification reaction mixture contains a salt (e.g., potassium chloride) at a concentration of about 10 mM to about 100 mM.

[0317] In some embodiments, the amplification reaction mixture contains one or more stabilizers. Stabilizers used in the methods described herein include, but are not limited to, polyols (glycerol, threitol, etc.), polyethers including cyclic polyethers, polyethylene glycol, organic or inorganic salts (e.g., ammonium sulfate, sodium sulfate, sodium molybdate, sodium tungstate, organic sulfonates, etc.), sugars, polyols, amino acids, peptides or carboxylic acids, quenchers and / or scavengers (e.g., mannitol, glycerol, reduced glutathione, superoxide dismutase, bovine serum albumin (BSA) or gelatin, spermidine, dithiothreitol (or mercaptoethanol)), and / or detergents such as TRITON® X-100 [octylphenol (ethylene glycol ether)], THESIT® [polyoxyethylene 9 lauryl ether (Polidocanol C 12 E9), TWEEN® (polyoxyethylene sorbitan monolaurate 20, NP40) and BRIJ®-35 (polyoxyethylene 23 lauryl ether).

[0318] Once formed, the reaction mixture is placed under conditions that allow for primer extension using one or more primers that anneal to the target nucleic acid to be detected. The reaction mixture is placed under primer extension conditions, which can be, but are not limited to, PCR conditions, allowing the primers to anneal to the target nucleic acid (if present) and be extended by the polymerase in a template-specific manner (i.e., target nucleic acid). The reaction can be performed monolithically or in partitions, and the signal can be monitored at the endpoint or in real time, for example, continuously or in each cycle.

[0319] In embodiments involving forward and reverse primers, when the target nucleic acid is present, the forward primer is extended to form a first strand, and then the first strand is used as a template to extend the reverse primer to form a second strand complementary to the first strand. Depending on whether the 5' universal sequence is on the forward or reverse primer, the first or second strand will each contain a 5' universal sequence, and therefore the second or first strand will each contain the reverse complementary sequence of the 5' universal sequence. Since the probe nucleic acid and RNase H2 enzyme are also in the reaction mixture, as the reverse complementary sequence of the 5' universal sequence is generated, the probe will anneal to the reverse complementary sequence of the 5' universal sequence, and the annealed probe will be cleaved at the ribonucleotide by the RNase H2 enzyme, separating the fluorophore and quencher to produce a detectable fluorescent signal. The amount of this signal can indicate the amount or at least the presence of the target nucleic acid in the overall reaction, or, if the reaction is in partitions, the number of partitions with a signal above a threshold will be proportional to the amount of target nucleic acid in the sample. Typically, the amount of sample nucleic acid and the number of partitions are selected according to a Poisson distribution such that at least some partitions are empty.

[0320] In embodiments involving forward and reverse primers, when the target nucleic acid is present, the forward primer is extended to form a first strand, and then the first strand is used as a template to extend the reverse primer to form a second strand complementary to the first strand. Depending on whether the target sequence of the target nucleic acid or oligonucleotide is on the forward or reverse primer, the first or second strand will contain the target sequence of the target nucleic acid or oligonucleotide chain, respectively, and therefore the second or first strand will contain the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide, respectively. In embodiments, since the probe nucleic acid and RNase H2 enzyme are also in the reaction mixture, as the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide is generated, the probe will anneal to the reverse complementary sequence of the target sequence of the target nucleic acid or oligonucleotide, and the annealed probe will be cleaved at the ribonucleotide by the RNase H2 enzyme, separating the fluorophore and quencher to produce a detectable fluorescent signal. The amount of this signal can indicate the amount or at least presence of the target nucleic acid in the overall reaction, or, if the reaction is in partitions, the number of partitions with signals above a threshold will be proportional to the amount of target nucleic acid in the sample. Typically, based on the Poisson distribution, the amount of nucleic acid in the sample and the number of partitions are chosen such that at least some partitions are empty.

[0321] Methods and compositions for partitioning samples are described, for example, in published patent applications WO 2010 / 036,352, US 2010 / 0173,394, US 2011 / 0092,373 and US 2011 / 0092,376, the contents of which are incorporated herein by reference in their entirety. Multiple mixture partitions may be multiple emulsion droplets or multiple micropores, etc.

[0322] In some embodiments, sample nucleic acids can be partitioned into multiple mixture partitions, and then one or more amplification primers, probes, enzymes, oligonucleotides, or combinations thereof can be introduced into the multiple mixture partitions. Methods and compositions for delivering reagents to one or more mixture partitions include microfluidic methods known in the art; the merging, aggregation, fusion, rupture, or degradation of droplets or microcapsules (e.g., as described in US 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151,776); and combinations thereof.

[0323] In some embodiments, the amplification reaction is a droplet digital PCR reaction. Methods for performing PCR in droplets are described, for example, in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of which are incorporated herein by reference. In some embodiments, the QX200, QX600, or QX One droplet digital PCR (ddPCR) system (Bio-Rad) is used.

[0324] In some implementations, any of a variety of detection 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 detection device equipped with a module that generates excitation light that can be absorbed by the fluorophore and a module that detects the light emitted by the fluorophore can be used to detect fluorescent labels.

[0325] In some embodiments, the detector further includes processing capabilities for handling partitioned samples (e.g., droplets), wherein a single partitioned sample enters the detector, is detected, and then exits the detector. In some embodiments, the partitioned samples (e.g., droplets) can be detected sequentially as the partitioned samples flow. In some embodiments, the partitioned samples (e.g., droplets) are arranged on a surface, and the detector moves relative to the surface, detecting a signal at each location containing a single partition. Examples of detectors are provided in WO 2010 / 036352, the contents of which are incorporated herein by reference. In some embodiments, detectable markers in the partitioned samples can be detected sequentially without causing the partitioned samples to flow (e.g., using a chamber slide).

[0326] 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.

[0327] Kits and mixtures for carrying out the methods are also provided. Kits may consist, for example, of plastic containers containing mixtures as described herein, optionally accompanied by instructions for use. For example, in some embodiments, forward primers or mixtures of forward and reverse primers as described herein are provided, which can be mixed by the end user with RNase H2 enzyme, probes, polymerases, and / or other reagents as described herein.

[0328] In some embodiments, the mixture comprises at least a first forward primer and a reverse primer, the first forward primer comprising a 3' target-specific forward sequence and the reverse primer comprising a 3' target-specific reverse sequence, wherein the forward or reverse primer further comprises a first 5' universal sequence, and a probe nucleic acid comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher. In some embodiments, the mixture comprises a first set of forward and reverse primers as described above, each forward or reverse primer having the same 5' universal sequence but having different (e.g., at least 2, 3, 4, 5, 8, 10, 15, 20 or more) 3' target-specific forward sequences and 3' target-specific reverse sequences, such that at least 2, 3, 4, 5, 8, 10, 15, 20 or more different target nucleic acids can be amplified by the primers and detected by the probe nucleic acid.

[0329] In some implementations, the mixture may contain at least two different individually detectable probes and two sets of primers: a first set of primers (forward and reverse) containing a first 5' universal sequence whose reverse complementary sequence is detectable by the first probe; and a second set of primers (forward and reverse) containing a second 5' universal sequence whose reverse complementary sequence is detectable by the second probe. For each set of primers, one of the forward or reverse primers contains the 5' universal sequence, but not both. Any one or both sets of primers may contain multiple different primers with different 3' target-specific sequences, allowing amplification of different target nucleic acids within the same set of primers.

[0330] For example, in some embodiments, the mixture comprises: (a) a first set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, the reverse primers comprising a 3' target-specific reverse sequence, wherein the forward or reverse primers of the first set further comprise a first 5' universal sequence, wherein the first set targets a first plurality of different target nucleic acids, and a first set of probe nucleic acids, the first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the first 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; and (b) a second set of forward and reverse primers, the forward primers comprising a 3' target-specific forward sequence, The reverse primer contains a 3' target-specific reverse sequence, wherein the forward or reverse primer of the second set further contains a second 5' universal sequence, wherein the second set targets a second plurality of different target nucleic acids, and a second set of probe nucleic acids, the second set of probe nucleic acids containing (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%), or at least 10 (e.g., at least 15, 20, 25 or more) nucleotides of the second 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher, such that a first amplicon from the first set of forward and reverse primers and having a first 5' universal sequence can be distinguished from a second amplicon from the second set of forward and reverse primers and having a second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0331] The mixture may further contain third, fourth, fifth, sixth or more groups of primers and probes, as described above, but targeting different target nucleic acids and having probes that detect different universal sequences as described herein, allowing for a higher degree of multiplexing.

[0332] Preparation method of oligonucleotides for simultaneous detection by amplification

[0333] Digital PCR (dPCR) testing is used in applications such as fetal fraction estimation and non-invasive prenatal testing (NIPT) for chromosome copy number detection, which requires highly accurate detection and counting of target sequences in cell-free DNA (cfDNA). In recent years, multiplex dPCR methods have emerged, in which multiple target DNA sequences can be detected simultaneously in a single dPCR reaction. dPCR multiplexing requires the separation of dPCR data during the analysis stage, where the degree of separation can be discriminative and / or non-discriminative at the target level.

[0334] Currently known multiplex dPCR methods in this field include: probe fluorophore-based multiplexing, wherein one or more target sequence-specific probes contain fluorophores, and each fluorophore has a unique excitation and emission spectrum; amplitude-based multiplexing, wherein the concentrations of dPCR amplification primers and dPCR probes vary based on a specific target sequence; and ratio-based multiplexing, wherein the ratio of two or more target sequence-specific probes labeled with fluorophores emitted in different color channels varies for each specific target sequence.

[0335] In the context of dPCR NIPT applications, the amount of cfDNA available is typically limited, necessitating highly accurate detection and counting of target sequences. For example, approximately 3,000 genomic equivalent copies can be extracted on average from a single sample (e.g., a blood sample), of which about 4% to 15% may originate from the fetus. Therefore, in existing technologies, multiple target sites per chromosome are detected in a single assay, and in some cases, across more than one chromosome. However, amplification of multiple target sequences often leads to undesirable side effects, such as primer dimers and primer-probe interactions, which can affect assay performance. Furthermore, knowledge of the target cfDNA sequence is often required to design dPCR assays, and for NIPT applications, for example, the 5' and 3' ends of cfDNA often vary within and between subjects, which can affect the consistency of cfDNA copy number measurements and pose challenges to dPCR assay design, particularly multiplex assays.

[0336] This article discloses a novel method for preparing oligonucleotides for simultaneous detection via multiplex amplification reactions, comprising adding an oligonucleotide to a target single-stranded oligonucleotide, wherein the oligonucleotide comprises a sequence configured to anneal with a universal amplification primer during the amplification reaction, and optionally comprises a universal probe sequence configured to anneal with a universal oligonucleotide probe during the amplification reaction. The correspondingly prepared oligonucleotides are suitable for amplification using universal primers and detection using universal probes. This method may be particularly suitable for designing and executing amplification reactions (e.g., dPCR, such as droplet dPCR), and / or for higher-order multiplexing in qPCR.

[0337] In aspects and embodiments of the method, the method includes preparing a single-stranded oligonucleotide for detection by amplification, wherein the preparation includes adding a universal sequence to a target single-stranded oligonucleotide. The oligonucleotide prepared according to this disclosure is suitable for subsequent amplification using primers that target the universal sequence added to the target single-stranded oligonucleotide, rather than specifically targeting the sequence of the target single-stranded oligonucleotide. The oligonucleotide prepared according to this disclosure can be detected using an oligonucleotide probe that can anneal to the universal sequence added to the prepared oligonucleotide, and / or can anneal to the sequence of the target single-stranded oligonucleotide, and emit a detectable signal during any suitable amplification reaction (e.g., PCR). The signal derived from the detection of the prepared oligonucleotide via probe-mediated amplification can be appropriately processed to calculate the abundance of the target oligonucleotide in a sample, wherein the target single-stranded oligonucleotide can be derived from a target nucleotide sequence, such as, but not limited to, one or more specific chromosomal sites, or RNA derived from a specific gene.

[0338] The method is applicable to both digital amplification detection and real-time on-the-spot amplification detection. The aspects and embodiments of this disclosure address various technical problems that those skilled in the art may encounter when implementing prior art methods and systems, such as eliminating or at least reducing undesirable primer dimers and / or primer-probe interactions. Furthermore, the aspects and embodiments of this disclosure are advantageous in enabling the design of primers to target the variable 5' and 3' ends of cfDNA in the amplification reaction, and can reduce the number of primers and probes required during such amplification reactions. Therefore, the beneficial technical effects of the claimed invention may include one or more of the following: reducing the formation of primer dimers and / or primer-probe interactions, reducing the risk of inconsistent primer-target binding due to variability at the 5' and 3' ends of the target cfDNA, reducing subsequent amplification bias, and increasing the probability of amplifying the target cfDNA. Therefore, the claimable invention is advantageous in that it improves the sensitivity and consistency of multiplex amplification reactions including detection steps, thereby achieving unprecedented high-order multiplexing levels. Advantageously, in aspects and embodiments, the methods and systems of this disclosure can reduce primer and probe design constraints associated with short oligonucleotide sequences (such as miRNAs), for example, by adding universal sequences corresponding to the annealing portions of universal amplification primers and probes, which anneal during the amplification reaction. Furthermore, the addition of universal sequences is not limited by the sequence at the 3' or 5' end of the target sequence or by knowledge of that sequence, which provides greater advantages for short target oligonucleotides. Additionally, the addition of universal sequences can include a reaction in which the 3' or 5' end sequence does not introduce design limitations, which is particularly beneficial for short target oligonucleotides with a limited number of sequences available for design.

[0339] Suitable target oligonucleotides

[0340] The term "target single-stranded oligonucleotide" refers to any single-stranded oligonucleotide sequence derived from a sample suitable for the method described. In embodiments, the target single-stranded oligonucleotide may be derived from single-stranded oligonucleotides, denatured nonlinear single-stranded oligonucleotides, or denatured double-stranded oligonucleotides, and may be linear or cyclic oligonucleotides. In embodiments, the target single-stranded oligonucleotide may consist of DNA, cDNA, or RNA, wherein the RNA may contain a poly(a)tail, such as mRNA, and / or may be ncRNA, such as microRNA (miRNA), small interfering RNA (siRNA), or piwi-interacting RNA (piRNA).

[0341] The target single-stranded oligonucleotide can be of any length, where the length corresponds to the number of nucleotides constituting the oligonucleotide. In embodiments, the target single-stranded oligonucleotide is approximately 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, or about 1500 nucleotides long. In embodiments, the target single-stranded oligonucleotide is between about 25 and 1500, about 50 and 500, about 50 and 300, or about 100 and 200 nucleotides long.

[0342] The term "multiple target single-stranded oligonucleotides" refers to two or more target single-stranded oligonucleotides, wherein the two or more specific target single-stranded oligonucleotides may contain different sequences, and / or wherein the two or more target single-stranded oligonucleotides may originate from different genomic sites. In embodiments, two or more target single-stranded oligonucleotides in the multiple target single-stranded oligonucleotides may originate from a single chromosome, or may originate from at least two different chromosomes. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more target single-stranded oligonucleotides may originate from a single chromosome. In embodiments, the multiple target single-stranded oligonucleotides comprise about 2 to 500, about 3 to 300, about 5 to 200, about 10 to 150, about 20 to 120, or about 25 to 100 unique target single-stranded oligonucleotides.

[0343] In the implementation scheme, multiple target single-stranded oligonucleotides are obtained from multiple parental oligonucleotides. These parental oligonucleotides may be selected from, but are not limited to: multiple denatured double-stranded or non-linear single-stranded parental oligonucleotides; and / or multiple 3'-terminal homo-polynucleic acid-tailed parental oligonucleotides; and / or multiple parental oligonucleotides processed to reduce their length to obtain multiple target single-stranded oligonucleotides of the desired length.

[0344] In the implementation scheme, processing the plurality of parental oligonucleotides to obtain a plurality of target single-stranded oligonucleotides of desired length may include, but is not limited to, any one or any combination of the following methods: digestion with at least one restriction endonuclease, thermal denaturation, fragment enzyme-mediated shearing, size selection and / or sonication.

[0345] In the implementation scheme, at least one restriction endonuclease may be selected from, but is not limited to, any of the following restriction endonucleases: Acc65I, AcclI, AflII, AluI. ,ApaI,AvrII,BamHI-HF,BglII,BsoBI,BssSI-v2,CviAII,CviQI , DpnII, EaeI, EcoRI-HF, FatI, HaeIII HindIII-HF , HinfI, HpaI, HphI, Hpy188I, Hpy188III, HpyCH4V, KpnI-HF, MfeI-HF, MscI, MseI , NciI, NcoI-HF, NlaIII, RsaI, SacI-HF, SphI-HF, TfiI and / or XhoI.

[0346] In one embodiment, the method may include dephosphorylating the 3' ends of a plurality of parental oligonucleotides, optionally wherein the dephosphorylation is performed by shrimp alkaline phosphatase. In a further embodiment, the method may include adding a nucleotide to the 3' ends of a plurality of parental oligonucleotides by an enzyme having terminal transferase activity, optionally wherein the nucleotide is thymine (T), and the addition is performed by a T-tailing enzyme (e.g., terminal deoxynucleotidyl transferase).

[0347] In the implementation scheme, the length of the target single-stranded oligonucleotides in the plurality of target single-stranded oligonucleotides is up to about 25, up to about 50, up to about 100, up to about 200, up to about 300, up to about 400, up to about 500, up to about 600, up to about 700, up to about 800, up to about 900, up to about 1000, or up to about 1500 nucleotides.

[0348] In the implementation scheme, the length of the target single-stranded oligonucleotides in the plurality of target single-stranded oligonucleotides is between about 25 to 1500, about 50 to 1000, about 50 to 500, about 75 to 300, or about 100 to 200 nucleotides.

[0349] The term "fragmentase" refers to an enzyme-based reagent that cuts DNA to produce fragments of a desired size based on treatment time. In an embodiment, the fragmentase may be NEBNext® dsDNA Fragmentase®.

[0350] In the implementation scheme, the multiple target single-stranded oligonucleotides may include linear and / or cyclic oligonucleotides.

[0351] Suitable samples

[0352] The methods and systems disclosed herein can be performed on any suitable oligonucleotide derived from a sample in which the target oligonucleotide sequence to be detected is present. The disclosed methods and systems can be performed on one or more suitable oligonucleotides derived from a sample known to contain or suspected of containing one or more target oligonucleotides. Alternatively, the invention can be performed on one or more suitable oligonucleotides derived from a sample to confirm the presence of one or more unknown target oligonucleotides in the sample.

[0353] The oligonucleotides can be derived from biological samples. The methods and systems disclosed herein can be performed on oligonucleotides derived from samples obtained or extracted from any organism. The organism or microorganism can be prokaryotic or eukaryotic and generally belongs to one of five kingdoms: Plantae, Animalia, Fungi, Prokaryotes, and Protists. The methods and systems disclosed herein can also be performed on oligonucleotides derived from samples obtained or extracted from any virus.

[0354] The sample is typically a fluid sample. Alternatively, if the sample source is solid or semi-solid, it can be subsequently processed to provide a fluid sample. Examples of such samples are feces, skin, tissue, hair, bone, and muscle. The sample may contain bodily fluids from a patient. Samples may be selected, for example, from urine, blood, plasma, serum, lymph, saliva, interstitial fluid, tears, mucus, or amniotic fluid. Typically, the sample source is human, but it may also come from another animal, particularly a mammal such as a mouse model organism, or from commercially raised animals such as horses, cattle, sheep, or pigs; or it may come from pets such as cats or dogs.

[0355] The sample can be a non-biological sample. A non-biological sample can be a fluid sample. Examples of non-biological samples include surgical fluids, water (such as drinking water, seawater, or river water), and industrial samples (such as laboratory testing reagents, or samples obtained from the synthesis of polymer reagents).

[0356] In the implementation, one or more target single-stranded oligonucleotides are derived from blood samples, wherein the samples may contain plasma and / or serum, and wherein the samples are derived from pregnant human female subjects, particularly wherein the samples contain maternal and fetal cfDNA. The disclosed method is applicable to both non-invasive and invasive samples, such as samples from amniocentesis or CVS.

[0357] Preparation of oligonucleotides for amplification reactions

[0358] The term "first oligonucleotide" in this disclosure refers to an oligonucleotide comprising: a 3' sequence configured to anneal with the sequence of at least one of a plurality of target single-stranded oligonucleotides; a first universal oligonucleotide probe sequence corresponding to the annealed portion of a first universal oligonucleotide probe, wherein the first universal oligonucleotide probe sequence of the first oligonucleotide is configured to anneal with the complementary sequence of the first universal oligonucleotide probe; and / or a first universal forward primer sequence corresponding to the annealed portion of a first universal forward amplification primer, wherein the first universal forward primer sequence of the first oligonucleotide is configured to anneal with the complementary sequence of the first universal forward amplification primer.

[0359] In the implementation, the 3' sequence configured to anneal with the sequence of at least one target single-stranded oligonucleotide can be configured to anneal with the sequence of a gene, PCR product, or RNA molecule, or in an alternative implementation, it can be configured to anneal with a nucleotide homopolymer of parent oligonucleotides added to a plurality of parent oligonucleotides.

[0360] The term "universal oligonucleotide probe" refers to an oligonucleotide probe configured to anneal to a complementary sequence to a first oligonucleotide universal oligonucleotide probe sequence. In an embodiment, the first universal oligonucleotide probe refers to one or more universal oligonucleotide probes sharing the same sequence configured to anneal to a complementary sequence to the first universal oligonucleotide probe sequence. In an embodiment, the second universal oligonucleotide probe refers to one or more universal oligonucleotide probes sharing the same sequence different from the first universal oligonucleotide probe, and the second universal oligonucleotide probe is configured to anneal to a second universal oligonucleotide probe sequence.

[0361] The term "universal RNaseH2 probe" refers to an oligonucleotide probe, specifically a universal oligonucleotide probe, configured to anneal to the complementary sequence of a first oligonucleotide's universal oligonucleotide probe sequence. This probe includes a fluorophore and a quencher portion separated by ribonucleotides, such that the signal from the fluorophore in the intact probe is relatively quenched relative to the signal when the probe is cleaved at the ribonucleotide by the RNaseH2 enzyme. In an embodiment, the first universal RNaseH2 probe refers to one or more universal RNaseH2 probes sharing the same sequence configured to anneal to the complementary sequence of the first universal RNaseH2 probe sequence. In an embodiment, the second universal RNaseH2 probe refers to one or more universal RNaseH2 probes sharing the same sequence different from the first universal RNaseH2 probe, and the second universal RNaseH2 probe is configured to anneal to a second universal RNaseH2 probe sequence.

[0362] In the implementation scheme, the term "non-universal oligonucleotide probe" refers to an oligonucleotide probe, specifically a non-universal oligonucleotide probe, configured to anneal to the sequence of a sense single-stranded oligonucleotide generated by denaturing at least one second double-stranded oligonucleotide. In other words, the non-universal oligonucleotide probe is complementary to and annealed to the sequence of the target single-stranded oligonucleotide.

[0363] In the implementation, the term "non-universal RNaseH2 probe" refers to an oligonucleotide probe comprising a fluorophore and a quencher portion separated by ribonucleotides, such that the signal from the fluorophore in the intact probe is relatively quenched relative to the signal when the probe is cleaved at the ribonucleotide by the RNaseH2 enzyme. This probe is configured to anneal to the sequence of a sense single-stranded oligonucleotide generated by denaturing at least one second double-stranded oligonucleotide. In other words, the non-universal RNaseH2 probe is complementary to and annealed to the sequence of the target single-stranded oligonucleotide.

[0364] The term "universal oligonucleotide probe sequence" refers to a sequence present in a universal oligonucleotide probe and that may also be present in a first oligonucleotide. In an embodiment, the first universal oligonucleotide probe sequence comprises a sequence different from the second universal oligonucleotide probe sequence, wherein the first universal oligonucleotide probe sequence and the second universal oligonucleotide probe sequence may be present in the same or different oligonucleotides, such as the first oligonucleotide.

[0365] The term "universal RNaseH2 probe sequence" refers to a sequence present in a universal RNaseH2 probe and that may be present in a first oligonucleotide. In an embodiment, the first universal RNaseH2 probe sequence comprises a sequence different from the second universal RNaseH2 probe sequence, wherein the first universal RNaseH2 probe sequence and the second universal RNaseH2 probe sequence may be present in the same or different oligonucleotides, such as the first oligonucleotide.

[0366] In the context of the first oligonucleotide, the term "universal forward primer sequence" refers to a sequence present in both the universal forward primer sequence of the first oligonucleotide and the first universal forward amplification primer. In an embodiment, the first universal forward primer sequence of the first oligonucleotide comprises a sequence different from the second universal forward primer sequence of the first oligonucleotide, wherein the second universal forward primer sequence of the first oligonucleotide is configured to anneal with a complementary sequence of the second universal forward amplification primer. In an embodiment, one or more first oligonucleotides may comprise a first universal forward primer sequence and optionally a second universal forward primer sequence.

[0367] The term "universal forward amplification primer" refers to an oligonucleotide primer configured to anneal to a complementary sequence to the universal forward primer sequence of a first oligonucleotide. In an embodiment, the first universal forward amplification primer refers to one or more universal forward amplification primers sharing the same sequence configured to anneal to a complementary sequence to the first universal forward primer sequence. In an embodiment, the second universal forward amplification primer refers to one or more universal forward amplification primers sharing the same sequence, which differs from the first universal forward amplification primer, and configured to anneal to the second universal forward primer sequence.

[0368] The term "first double-stranded oligonucleotide" refers to a double-stranded oligonucleotide produced by contacting a target single-stranded oligonucleotide with a first oligonucleotide and by extending the first oligonucleotide along the sequence of the target single-stranded oligonucleotide to synthesize the complementary strand of the target single-stranded oligonucleotide.

[0369] In one embodiment, the first double-stranded oligonucleotide comprises a target strand containing the sequence of a target single-stranded oligonucleotide; and a complementary strand containing a sequence complementary to the target single-stranded oligonucleotide sequence and the sequence of the first oligonucleotide.

[0370] In the implementation scheme, the same first oligonucleotide may be provided for contacting more than one specific target single-chain oligonucleotide among a plurality of target single-chain oligonucleotides, or the same first oligonucleotide may be provided for contacting one target single-chain oligonucleotide among a plurality of target single-chain oligonucleotides.

[0371] The term "second oligonucleotide" refers to an oligonucleotide comprising: a 3' sequence configured to anneal to the 3' end of a sequence of the complementary strand of a first double-stranded oligonucleotide; and a first universal reverse primer sequence corresponding to the annealed portion of a first universal reverse amplification primer, wherein the first universal reverse primer sequence of the second oligonucleotide is configured to anneal to the complementary sequence of the first universal reverse amplification primer. In embodiments, the second oligonucleotide may comprise a first universal oligonucleotide probe sequence.

[0372] The term "universal reverse primer sequence" refers to a sequence present in both the second oligonucleotide and the first universal reverse amplification primer. In an embodiment, the first universal reverse primer sequence of the second oligonucleotide comprises a sequence different from the second universal reverse primer sequence of the second oligonucleotide, wherein the second universal reverse primer sequence of the second oligonucleotide is configured to anneal to a complementary sequence of the second universal reverse amplification primer. In an embodiment, one or more second oligonucleotides may comprise the first universal reverse primer sequence.

[0373] The term "universal reverse amplification primer" refers to an oligonucleotide primer configured to anneal to a complementary sequence to the universal reverse primer sequence of a second oligonucleotide. In an embodiment, a first universal reverse amplification primer refers to one or more universal reverse amplification primers sharing the same sequence, configured to anneal to a complementary sequence to the first universal reverse primer sequence. In an embodiment, a second universal reverse amplification primer refers to one or more universal reverse amplification primers sharing the same sequence, which differs from the first universal reverse amplification primer and is configured to anneal to a complementary sequence to the second universal reverse primer sequence.

[0374] In embodiments, for example, during a reverse transcription reaction, one or more C nucleotides (optionally three) are added to the 3' end of the complementary strand by reverse transcriptase. The universal reverse amplification primer may contain one or more G nucleotides configured to anneal with said one or more C nucleotides. In embodiments, for example, when the parental single-stranded oligonucleotide has been digested with at least one restriction endonuclease, the universal reverse amplification primer may contain a portion of the recognition and cleavage site sequence of a specific restriction endonuclease, such as the sequence of the target oligonucleotide resulting from cleavage with the restriction endonuclease. In embodiments, the universal reverse amplification primer may be configured to anneal with the sequence of the restriction endonuclease site, which can advantageously reduce the generation of nonspecific amplification products that may be generated by TSO annealing and extension to one or more C nucleotide homopolymers.

[0375] The term "second double-stranded oligonucleotide" refers to an oligonucleotide that is primarily double-stranded but may contain single-stranded nucleotide regions, such as universal primer sequences.

[0376] In this invention, the second double-stranded oligonucleotide is produced by contacting a first double-stranded oligonucleotide with at least one second oligonucleotide and synthesizing the second double-stranded oligonucleotide.

[0377] In aspects and implementations, the second double-stranded oligonucleotide comprises: a target strand comprising: the sequence of the target single-stranded oligonucleotide and the sequence of the second oligonucleotide; and a complementary strand comprising: a sequence complementary to the target single-stranded oligonucleotide sequence and the sequence of the first oligonucleotide.

[0378] In the implementation scheme, the complementary strand of the second double-stranded oligonucleotide may also contain a sequence complementary to the second oligonucleotide.

[0379] In the implementation scheme, the target strand of the second double-stranded oligonucleotide may also contain a sequence complementary to the first oligonucleotide.

[0380] In one embodiment, one or more first oligonucleotides may comprise a first universal forward primer sequence. In another embodiment, the first universal forward primer sequence of the first oligonucleotide comprises a sequence different from a second universal forward primer sequence of the first oligonucleotide, wherein the second universal forward primer sequence corresponds to the annealing portion of a second universal forward amplification primer, and wherein the second universal forward primer sequence of the first oligonucleotide is configured to anneal to a complementary sequence of the second universal forward amplification primer.

[0381] In one embodiment, one or more second oligonucleotides may comprise a first universal reverse primer sequence. In another embodiment, the first universal reverse primer sequence of the second oligonucleotide comprises a sequence different from the second universal reverse primer sequence of the second oligonucleotide, wherein the second universal reverse primer sequence corresponds to the annealing portion of the second universal reverse amplification primer, and wherein the second universal reverse primer sequence of the second oligonucleotide is configured to anneal to a complementary sequence of the second universal reverse amplification primer.

[0382] In the implementation scheme, the same second oligonucleotide can be provided for contacting more than one specific first double-stranded oligonucleotide, or the same second oligonucleotide can be provided for contacting all first double-stranded oligonucleotides, wherein the second oligonucleotide can be TSO.

[0383] In an implementation scheme, the first universal forward primer sequence may be located at or toward the 5' end of the first oligonucleotide, and / or the first universal reverse primer sequence may be located at or toward the 5' end of the second oligonucleotide as defined in the present invention.

[0384] In an embodiment, the 3' end of the first oligonucleotide may be fully or partially complementary to the sequence at or toward the 3' end of the target single-stranded oligonucleotide. In an embodiment, the partially complementary sequence of at least one first oligonucleotide to at least one target single-stranded oligonucleotide contains one or more base pair mismatches. In an embodiment, the number of base pair mismatches may be 1, 2, 3, 4, 5, or 6.

[0385] In an embodiment, the first oligonucleotide may anneal to a sequence at the 3' end of the target single-stranded oligonucleotide, wherein the sequence comprises at least the terminal nucleotide at the 3' end of the target single-stranded oligonucleotide, or may anneal to a sequence located at or near the 3' end of the target single-stranded oligonucleotide. In an embodiment, the first oligonucleotide may anneal to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides located at or near the 3' end of the target single-stranded oligonucleotide.

[0386] In embodiments of the present invention, the first double-stranded oligonucleotide may comprise a double-stranded nucleotide region and a single-stranded nucleotide region, wherein the single-stranded nucleotide region may comprise a sequence corresponding to the first oligonucleotide. In embodiments, this sequence may comprise part or all of a universal forward primer sequence of the first oligonucleotide, and in embodiments, it may also comprise part or all of a universal oligonucleotide probe sequence of the first oligonucleotide.

[0387] In an embodiment, the 3' end of at least one second oligonucleotide may be fully or partially complementary to the 3' end of, or toward, the complementary strand of, the target single-stranded oligonucleotide. In an embodiment, the partially complementary sequence of at least one second oligonucleotide with (at least one) first double-stranded oligonucleotide contains one or more base pair mismatches. In an embodiment, the number of base pair mismatches may be 1, 2, 3, 4, 5, or 6, wherein the base pair mismatches may be sequential or discontinuous.

[0388] In an embodiment, the second oligonucleotide may be annealed to a sequence at the 3' end of the complementary strand of the target single-stranded oligonucleotide synthesis, wherein the sequence comprises at least a terminal nucleotide at the 3' end of the complementary strand, or may be annealed to a sequence located at or near the 3' end of the complementary strand. In an embodiment, the second oligonucleotide may be annealed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides located at or near the 3' end of the complementary strand of the target single-stranded oligonucleotide synthesis.

[0389] In the implementation scheme, the second double-stranded oligonucleotide is synthesized by extending the complementary strand of the first double-stranded oligonucleotide along the second oligonucleotide; that is, by extending the complementary strand along the prominent sequence of the second oligonucleotide.

[0390] In other embodiments, the second double-stranded oligonucleotide is synthesized by extending the second oligonucleotide along the complementary strand of the first double-stranded oligonucleotide, wherein the synthesized oligonucleotide sequence contains a sequence complementary to the complementary strand.

[0391] In one embodiment, the second double-stranded oligonucleotide may comprise a double-stranded nucleotide region and a single-stranded nucleotide region, wherein the single-stranded nucleotide region may comprise the sequence of the first oligonucleotide, wherein the sequence may comprise part or all of the universal forward primer sequence of the first oligonucleotide, and in another embodiment, may also comprise part or all of the universal oligonucleotide probe sequence of the first oligonucleotide. In another embodiment, the single-stranded nucleotide region of the second double-stranded oligonucleotide may comprise the sequence of the second oligonucleotide, wherein the sequence may comprise part or all of the universal reverse amplification primer of the second oligonucleotide, and in another embodiment, may also comprise part or all of the universal oligonucleotide probe sequence of the second oligonucleotide.

[0392] In an implementation, for example, when the target single-stranded oligonucleotide contains a polyA tail, such as a target single-stranded oligonucleotide containing mRNA, the first oligonucleotide may contain a poly(T) sequence.

[0393] The term "extending...along...sequence" refers to the synthesis of a sequence complementary to a target template sequence, wherein the complementary sequence is synthesized along the sequence of the target template sequence. In an embodiment, extending the complementary strand of at least one first double-stranded oligonucleotide along a second oligonucleotide includes synthesizing a sequence complementary to the second oligonucleotide, thereby producing at least one second double-stranded oligonucleotide longer than the first double-stranded oligonucleotide. In an embodiment, extending the second oligonucleotide along the complementary strand of the first double-stranded oligonucleotide includes synthesizing a sequence complementary to the complementary strand of the first double-stranded oligonucleotide, thereby producing the second double-stranded oligonucleotide.

[0394] In one embodiment, the complementary strand of the target oligonucleotide can be synthesized via reverse transcription to generate a first double-stranded oligonucleotide. The reverse transcription reaction may involve a reverse transcriptase with terminal C-transferase activity, such as MuMLV. In such embodiments, during reverse transcription, one or more C-nucleotides can be added to the 3' end of the complementary strand via reverse transcriptase. In another embodiment, the second oligonucleotide may be a template-transfer oligonucleotide (TSO).

[0395] For the purposes of this invention, the term "template-transfer oligonucleotide" (TSO) refers to an oligonucleotide comprising one or more nucleotides configured to anneal with a protruding nucleotide sequence of the complementary strand of a first double-stranded oligonucleotide. In embodiments, the protruding nucleotide sequence is synthesized via a reverse transcription reaction comprising a reverse transcriptase (RT) having terminal nucleotide transferase activity (e.g., MuMLV). In this manner, one or more nucleotides can be added to the 3' end of the synthetic complementary strand of the target single-stranded oligonucleotide during the reverse transcription reaction. Not wishing to be bound by any particular theory, the annealing of the TSO with the protruding one or more nucleotides allows the RT to extend the sequence of the complementary strand of the first double-stranded oligonucleotide by synthesizing a sequence complementary to the TSO.

[0396] In one embodiment, the second oligonucleotide is TSO, the 3' end sequence of which is configured to anneal to or near the 3' end of the complementary strand of the first double-stranded oligonucleotide, and the 3' end sequence is at least partially complementary to a protruding nucleotide added to the complementary strand by a reverse transcriptase (RT) having terminal nucleotide transferase activity during the synthesis of the first double-stranded oligonucleotide. In another embodiment, a universal reverse amplification primer anneals to the complementary sequence of the template-converting oligonucleotide.

[0397] In an embodiment, the sequence in the TSO that is at least partially complementary to (e.g., added by reverse transcriptase) one or more protruding nucleotides may contain one or more 3' G bases; typically three 3' G bases. In an embodiment, the 3' G bases may be modified, or may be RNA or LNA bases. In an embodiment, the sequence in the TSO that is at least partially complementary to (e.g., added by reverse transcriptase) one or more protruding nucleotides contains rGrGrG at the 3' end, which is annealed to the protruding C nucleotide.

[0398] In one embodiment, the TSO comprises a 5' biotinylate moiety and / or one or more 5' base-free sites. In another embodiment, the TSO comprises a closed 3' end, wherein the 3' end may be closed using a C3 spacer sequence.

[0399] In one embodiment, synthesizing the second double-stranded oligonucleotide involves annealing one or more 3' G bases of TSO to one or more terminal 3' C bases of the complementary strand of the first double-stranded oligonucleotide, and using a polymerase to extend the complementary strand to synthesize a sequence complementary to TSO.

[0400] In an embodiment where the second oligonucleotide is TSO and the universal reverse amplification primer is configured to anneal to the complementary sequence of TSO, the universal reverse amplification primer may comprise any one of SEQ ID NO: 1 to 6.

[0401] In one embodiment, the 3' ends of multiple target single-stranded oligonucleotides may be dephosphorylated, wherein dephosphorylation is performed by shrimp alkaline phosphatase. In another embodiment, one or more T nucleotides may be added to the 3' end of the target single-stranded oligonucleotide via T-tailing, wherein T-tailing is performed by a T-tailing enzyme (e.g., but not limited to terminal deoxynucleotidyl transferase). In yet another embodiment, the first oligonucleotide may contain a poly(A) sequence that anneals to one or more 3' T nucleotides of the target single-stranded oligonucleotide.

[0402] In one embodiment, one or more T nucleotides are added to the 3' end of the complementary strand of the first double-stranded oligonucleotide via a T-tail. In another embodiment, at least one first double-stranded oligonucleotide is denatured to produce at least one single-stranded target oligonucleotide containing a target single-stranded oligonucleotide sequence, and at least one single-stranded complementary strand containing a sequence of a strand complementary to the target single-stranded oligonucleotide. In yet another embodiment, one or more T nucleotides are added to the 3' end of the complementary strand via a T-tail.

[0403] In one embodiment, the second oligonucleotide comprises a poly(A) sequence configured to anneal with one or more T nucleotides added via a T-tail to the complementary strand of the first double-stranded oligonucleotide or the (denatured) single-stranded complementary strand. In embodiments where the second oligonucleotide comprises a poly(A) sequence, the universal reverse amplification primer may comprise any one of SEQ ID NO: 7 to 12.

[0404] Oligonucleotide probes

[0405] Probes suitable for the method include fluorescent probes, such as TaqMan. TM Probes. Such probes may require hydrolysis by the 5' to 3' exonuclease activity of a DNA polymerase to separate the fluorescent portion from the quenching portion in the probe structure, thereby allowing a fluorescent signal to be generated during amplification. Any suitable probe can be used. For example, suitable fluorescent probes include molecular beacons, which contain a stem-loop structure that increases the probe's specificity for binding to its target. During the annealing step of the amplification reaction, the loop portion of the molecular beacon probe can anneal with the target sequence, thereby denaturing the stem and separating the fluorescent portion from the quenching portion, resulting in detectable fluorescence from the fluorescent portion. Further suitable fluorescent probes include RNaseH2-activated probes (e.g., disclosed in U.S. Application No. 18 / 370,566, which is incorporated herein by reference) that contain a fluorophore and a quencher portion separated by ribonucleotides, such that the signal from the fluorophore in the intact probe is relatively quenched relative to the signal when the probe is cleaved at the ribonucleotides by the RNaseH2 enzyme.

[0406] In embodiments, at least one of the one or more universal oligonucleotide probes may be a linear probe, and / or at least one of the one or more oligonucleotide probes may form a stem-loop. In embodiments, the stem-loop may be 5' to 3' comprising: a first stem sequence, a loop sequence, and a second stem sequence, wherein the second stem sequence is the reverse complementary sequence of the first stem sequence. In embodiments, the stem-loop sequence of the universal oligonucleotide probe may be annealed to the complementary sequence of the first oligonucleotide probe sequence as defined in this invention, and / or may be annealed to the complementary sequence of the universal forward primer sequence of the first oligonucleotide.

[0407] In embodiments, at least one of the one or more non-universal oligonucleotide probes may be a linear probe, and / or at least one of the one or more oligonucleotide probes may form a stem-loop as described above. In embodiments, the stem-loop sequence of the non-universal oligonucleotide probe may anneal to the strand of a second double-stranded oligonucleotide that substantially contains the target single-stranded oligonucleotide sequence, and / or to a sense single-stranded oligonucleotide (containing the target single-stranded oligonucleotide sequence). In embodiments, the stem-loop sequence of the non-universal oligonucleotide probe may anneal to the complementary strand of the second double-stranded oligonucleotide (i.e., the strand of the second double-stranded oligonucleotide that is (substantially) complementary to the target strand sequence), and / or to an antisense (or complementary) single-stranded oligonucleotide (containing the sequence of the strand complementary to the target single-stranded oligonucleotide, which is (substantially) complementary to the target strand sequence).

[0408] In one implementation, the oligonucleotide probe is a universal RNaseH2 probe. In another implementation, the oligonucleotide probe is a non-universal RNaseH2 probe.

[0409] In an embodiment, at least one of one or more universal oligonucleotide probes is annealed with a first oligonucleotide according to the present disclosure, and / or at least one of one or more oligonucleotide probes is annealed with a second oligonucleotide according to the present disclosure.

[0410] In the implementation, the term "first universal oligonucleotide probe" refers to one or more universal oligonucleotide probes sharing the same oligonucleotide sequence, configured to anneal to a complementary sequence of the first universal oligonucleotide probe sequence of a first oligonucleotide or a second oligonucleotide, or to a sense single-stranded oligonucleotide generated by denaturation of a second double-stranded oligonucleotide. In the implementation, the first universal oligonucleotide probe is configured to anneal to a complementary sequence of the first universal oligonucleotide probe sequence in a second double-stranded oligonucleotide, wherein the first universal oligonucleotide probe sequence does not correspond to the sequence of the target single-stranded oligonucleotide.

[0411] The term "second universal oligonucleotide probe" refers to one or more universal oligonucleotide probes that share the same sequence, which is different from the first universal oligonucleotide probe, and is configured to anneal a sequence complementary to the sequence of the second universal oligonucleotide probe and the first or second oligonucleotide.

[0412] In the implementation, the term "first non-universal oligonucleotide probe" refers to one or more non-universal oligonucleotide probes sharing the same oligonucleotide sequence configured to anneal with the sequence of a sense single-stranded oligonucleotide generated by denaturation of at least one second double-stranded oligonucleotide. In other words, the first non-universal oligonucleotide probe is complementary to and annealed with the sequence of the target single-stranded oligonucleotide.

[0413] The term “second non-universal oligonucleotide probe” refers to one or more non-universal oligonucleotide probes that share the same sequence, which is different from the first non-universal oligonucleotide probe, and are configured to anneal with a different sequence of a sense single-stranded oligonucleotide generated by denaturation of at least one second double-stranded oligonucleotide.

[0414] In the implementation, the term "first non-universal RNaseH2 probe" refers to one or more non-universal RNaseH2 probes sharing the same oligonucleotide sequence configured to anneal to the sequence of a sense single-stranded oligonucleotide generated by denaturation of at least one second double-stranded oligonucleotide. In other words, the first non-universal RNaseH2 probe is complementary to and annealed to the sequence of the target single-stranded oligonucleotide.

[0415] The term “second non-universal RNaseH2 probe” refers to one or more non-universal RNaseH2 probes that share the same sequence, which is different from the first non-universal RNaseH2 probe, and are configured to anneal with a different sequence of a sense single-stranded oligonucleotide generated by denaturation of at least one second double-stranded oligonucleotide.

[0416] In the implementation, the term "first fluorophore" refers to one or more fluorophores that are identical to each other in a universal or non-universal oligonucleotide probe. The term "second fluorophore" refers to one or more fluorophores that are identical to each other but different from the first fluorophore in a universal or non-universal oligonucleotide probe. For example, the first and second fluorophores may have different excitation and emission spectra. In the implementation, it is desirable that the selection of the fluorophore can be based on a specific target single-stranded oligonucleotide sequence. In the implementation, the first and second universal or non-universal oligonucleotide probes may contain the same first fluorophore, or they may contain the first and second fluorophores (which are different).

[0417] In the implementation scheme, the term "first group of universal oligonucleotide probes" refers to one or more universal oligonucleotide probes containing a first fluorophore, wherein the first group may include first, second, third, fourth, fifth or more universal oligonucleotide probes, each with a different sequence. The term "second group of universal oligonucleotide probes" refers to one or more universal oligonucleotide probes containing a second fluorophore, wherein the second group may include first, second, third, fourth, fifth or more universal oligonucleotide probes, each with a different sequence.

[0418] In one embodiment, the first and second groups of the universal oligonucleotide probes are configured to anneal with different single-stranded oligonucleotides. In another embodiment, the first and second groups of the universal oligonucleotide probes are configured to anneal with sequences of target single-stranded oligonucleotides derived from a specific chromosome, and the second group of the universal oligonucleotide probes is configured to anneal with sequences of target single-stranded oligonucleotides derived from a different specific chromosome.

[0419] In the implementation scheme, the universal oligonucleotide probes can be mutually exclusive among different groups of universal oligonucleotide probes; for example, the first and second groups of universal oligonucleotide probes can have a first universal oligonucleotide probe and a third universal oligonucleotide probe, and the second group of universal oligonucleotide probes can have a second universal oligonucleotide probe and a fourth universal oligonucleotide probe.

[0420] In an implementation, the method includes a group of at least two universal oligonucleotide probes for targeting a corresponding plurality (e.g., at least two) different chromosomes; for example, the group of at least two universal oligonucleotide probes may include up to 20, up to 12, up to 10, up to 8, up to 6, or up to 4 different universal oligonucleotide probes for targeting a corresponding number of different chromosomes.

[0421] In the embodiments, the term "first group of non-universal oligonucleotide probes" refers to one or more non-universal oligonucleotide probes containing a first fluorophore, wherein the first group may include first, second, third, fourth, fifth, or more non-universal oligonucleotide probes, each comprising a different nucleotide sequence and therefore including a different target binding sequence. The term "second group of non-universal oligonucleotide probes" refers to one or more non-universal oligonucleotide probes containing a second fluorophore, wherein the second group may include first, second, third, fourth, fifth, or more non-universal oligonucleotide probes, each comprising a different nucleotide sequence and therefore including a different target binding sequence. In the embodiments, the first and second groups of non-universal oligonucleotide probes may be configured to anneal with different single-stranded oligonucleotides. In the embodiments, the first group of non-universal oligonucleotide probes is configured to anneal with a target single-stranded oligonucleotide sequence derived from a specific chromosome, and the second group of non-universal oligonucleotide probes is configured to anneal with a target single-stranded oligonucleotide sequence derived from a different specific chromosome.

[0422] In embodiments, the non-universal oligonucleotide probes can be mutually exclusive among different groups of non-universal oligonucleotide probes; for example, a first group of non-universal oligonucleotide probes has a first non-universal oligonucleotide probe and a third non-universal oligonucleotide probe, and a second group of non-universal oligonucleotide probes has a second non-universal oligonucleotide probe and a fourth non-universal oligonucleotide probe. In embodiments, the method includes at least two groups of non-universal oligonucleotide probes for targeting corresponding plurality of different chromosomes; for example, the at least two groups of non-universal oligonucleotide probes include up to 20, up to 12, up to 10, up to 8, up to 6, or up to 4 different groups of non-universal oligonucleotide probes for targeting a corresponding number of different chromosomes.

[0423] In one embodiment, the universal oligonucleotide probe anneals to a complementary sequence of the universal oligonucleotide probe sequence located at or toward the 5' end of the first oligonucleotide. In another embodiment, the universal oligonucleotide probe anneals to a complementary sequence of the universal oligonucleotide probe sequence located at or toward the 5' end of the second oligonucleotide. In yet another embodiment, the template-changing oligonucleotide may contain the universal oligonucleotide probe sequence. In yet another embodiment, the universal oligonucleotide probe anneals to a sequence complementary to the universal oligonucleotide probe sequence of the template-changing oligonucleotide.

[0424] In one embodiment, the first universal oligonucleotide probe may be configured to anneal to a sequence complementary to the universal forward primer sequence of the first oligonucleotide. In another embodiment, the first universal oligonucleotide probe may be configured to anneal to a sequence complementary to the universal reverse primer sequence of the second oligonucleotide.

[0425] reaction mixture

[0426] In the embodiments, the reaction mixture may contain a plurality of target single-stranded oligonucleotides, at least one first oligonucleotide, at least one second oligonucleotide, at least one first universal forward amplification primer, at least one first universal reverse amplification primer; and at least one universal oligonucleotide probe and / or at least one non-universal oligonucleotide probe.

[0427] In embodiments, the preparation of the oligonucleotide and the amplification of the second double-stranded oligonucleotide can be performed stepwise. For example, a first step may include the preparation of the oligonucleotide, comprising generating at least one second double-stranded oligonucleotide from a plurality of target single-stranded oligonucleotides, and a second step may include an amplification reaction of at least one second double-stranded oligonucleotide. In embodiments where the method is performed stepwise, the reaction mixture may comprise a plurality of target single-stranded oligonucleotides, at least one first oligonucleotide, and at least one second oligonucleotide. In embodiments where the method is performed stepwise, the reaction mixture may further comprise at least one second double-stranded oligonucleotide, at least one first universal forward amplification primer, at least one first universal reverse amplification primer, at least one universal oligonucleotide probe, and / or at least one non-universal oligonucleotide probe; wherein at least one first universal forward amplification primer, at least one first universal reverse amplification primer, and at least one universal oligonucleotide probe and / or at least one non-universal oligonucleotide probe are provided after the generation of at least one second double-stranded oligonucleotide. In embodiments, the method may be performed as a one-pot reaction.

[0428] Once formed, the reaction mixture can be placed under conditions that allow for the generation of suitable target single-stranded oligonucleotides, such as a denaturation step. The reaction mixture can be placed under conditions for primer annealing and extension (e.g., a first or second oligonucleotide) using one or more primers annealed to the target oligonucleotide to be detected. The reaction mixture can be placed under primer extension conditions, which can be, but are not limited to, conditions suitable for PCR, allowing the primers to anneal to the target oligonucleotide or prepared oligonucleotides (if present) and be extended by polymerase in a template-specific manner; this process can be repeated one or more cycles. The oligonucleotide probe signal during the amplification reaction can be monitored at the endpoint or in real-time (i.e., continuously or per cycle).

[0429] Partitioning and amplification of the reaction mixture

[0430] In an embodiment, the method may be carried out as a single reaction mixture in a holistic manner, or it may be carried out in two or more reaction mixtures, wherein the two or more reaction mixtures may contain two or more partitions comprising the reaction mixture.

[0431] In this embodiment, the generation of one or more second double-stranded oligonucleotides suitable for the amplification reaction and the amplification reaction itself both occur in the same partition. In this embodiment, the partition may contain a reaction mixture, thus constituting a partition containing a reaction mixture, or it may contain only a portion of the reaction mixture required to perform the method, but not all of the necessary reagents and components, or it may be empty. In this embodiment, the generation of at least one second double-stranded oligonucleotide may be carried out monolithically or in a single reaction, followed by partitioning the at least one second double-stranded oligonucleotide and the reaction mixture, wherein the amplification reaction is carried out in the partition.

[0432] Amplification reaction and detection of amplification products and probe signals

[0433] In embodiments, multiple oligonucleotide amplification products are generated from a group of second double-stranded oligonucleotides, wherein a first group of second double-stranded oligonucleotides may be generated from multiple different target single-stranded oligonucleotides, for example, derived from 2 or more, 5 or more, 10 or more (e.g., at least 20) different genomic targets on a chromosome. In embodiments, a second group of second double-stranded oligonucleotides may be generated from multiple different target single-stranded oligonucleotides, for example, derived from 2 or more, 5 or more, 10 or more (e.g., at least 20) different genomic targets on a chromosome different from the first group of second double-stranded oligonucleotides. Therefore, in aspects and embodiments of this disclosure, a group of second double-stranded oligonucleotides may comprise at least one first subgroup of second double-stranded oligonucleotides (having the same sequence, i.e., synthesized from target single-stranded oligonucleotides derived from the same genomic target) and a second subgroup of second double-stranded oligonucleotides (having the same sequence, but the sequence differs from the sequence of the first subgroup of second double-stranded oligonucleotides, wherein the second subgroup of second double-stranded oligonucleotides originates from target single-stranded oligonucleotides corresponding to different genomic targets, which are identical for all second double-stranded oligonucleotides in the second subgroup).

[0434] It is understood that a plurality of second double-stranded oligonucleotides may comprise one or more, such as a group of 1 to 23 second double-stranded oligonucleotides. It is understood that a plurality of second double-stranded oligonucleotides may comprise two or more, such as a subgroup of about 2 to about 150, about 2 to about 100, about 2 to about 50, or about 2 to about 25 second double-stranded oligonucleotides.

[0435] In the implementation scheme, the second double-stranded oligonucleotide group may contain 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, or 20 or more second double-stranded oligonucleotide subgroups.

[0436] In the implementation scheme, an amplification product containing at least one target single-stranded oligonucleotide sequence from a first chromosome can be detected by a first signal among multiple probe signals, and an amplification product containing at least one target single-stranded oligonucleotide sequence from a second chromosome can be detected by a second signal among multiple probe signals.

[0437] In the implementation scheme, a first signal among the multiple probe signals and amplification products can be distinguished from a second signal among the multiple probe signals. In the implementation scheme, the first signal among the multiple probe signals may originate from a target single-stranded oligonucleotide derived from a specific chromosome, while the second signal among the multiple probe signals may originate from target single-stranded oligonucleotides derived from different chromosomes.

[0438] In the implementation scheme, a first, second, third, fourth, fifth, or more signals from a plurality of probe signals and amplification products may be generated. In the implementation scheme, the plurality of probe signals and amplification products may comprise at least about 10, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 150 of the plurality of probe signals and amplification products.

[0439] In one embodiment, a first universal forward amplification primer is provided for amplifying one or at least one subgroup of the second double-stranded oligonucleotides. In another embodiment, all second double-stranded oligonucleotides in a subgroup can be amplified using the first universal forward amplification primer.

[0440] In one embodiment, a first universal reverse amplification primer is provided for amplifying one or at least one subgroup of the second double-stranded oligonucleotides. In another embodiment, all second double-stranded oligonucleotides in a subgroup can be amplified using the first universal reverse amplification primer.

[0441] In an embodiment, at least one specific amplification product derived from a first subgroup of the second double-stranded oligonucleotide can be distinguished from at least one other amplification product derived from a second subgroup of the second double-stranded oligonucleotide. In an embodiment, at least one specific amplification product among a plurality of oligonucleotide amplification products can be distinguished from at least one other specific amplification product, wherein at least one specific amplification product derived from a specific chromosome among the plurality of oligonucleotide amplification products can be distinguished from at least one other specific amplification product derived from a different specific chromosome. In an embodiment, the number of distinguishable amplification products can be at least about 10, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 150.

[0442] In this implementation, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 different amplification products (obtained from the amplification of the corresponding number of second double-stranded oligonucleotide subsets) are simultaneously detected. In this implementation, the universal oligonucleotide probe is a molecular beacon probe. In this implementation, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 different amplification products are simultaneously detected. In this implementation, the non-universal oligonucleotide probe is a TaqMan probe.

[0443] In embodiments, any suitable detection device can be used to detect the detection reagent or detectable label, such as an oligonucleotide probe, in the partition. Exemplary detection methods include optical detection (e.g., fluorescence or chemiluminescence). As a non-limiting example, a detection device equipped with a module that generates excitation light that can be absorbed by the fluorophore and a module that detects the light emitted by the fluorophore can be used to detect fluorescent labels. In embodiments, the detector further includes processing capability for handling partition samples (e.g., droplets), wherein a single partition sample enters the detector, is detected, and then exits the detector. In embodiments, the partition samples (e.g., droplets) can be detected sequentially as the partition samples flow. In embodiments, the partition samples (e.g., droplets) are arranged on a surface, and the detector moves relative to the surface, detecting a signal at each location containing a single partition. Examples of detectors are provided, for example, in WO 2010 / 036352, the contents of which are incorporated herein by reference. In embodiments, detectable labels in the partition samples can be detected sequentially without causing the partition samples to flow (e.g., using a chamber slide). After acquiring the fluorescence detection data, a general-purpose computer system (e.g., a “host computer”) can be used to store and process the data.

[0444] In an embodiment, as one or more copies of a sense single-stranded oligonucleotide (derived from the denaturation of one or more second double-stranded oligonucleotides) are generated during the amplification reaction, a universal or non-universal oligonucleotide probe is annealed with the sense single-stranded oligonucleotide. In an embodiment, the annealed universal or non-universal oligonucleotide probe may be cleaved by an enzyme, wherein cleavage results in the separation of the fluorophore from the quencher, or wherein annealing of the universal or non-universal oligonucleotide probe results in the separation of the fluorophore from the quencher, thereby generating a detectable fluorescent signal. In an embodiment, the enzyme may be, but is not limited to, a DNA polymerase, such as Taq polymerase. In an embodiment, when the probe is an RNase H2-activated probe according to U.S. Application No. 18 / 370,566, the RNase H2 enzyme may cleave the probe at the ribonucleotide sequence of the RNase H2-activated probe. The amount of this signal may indicate the quantity of the target nucleic acid in the overall reaction or at least its presence, or, if the reaction is performed in partitions, the number of partitions with a signal above a selected threshold detection value may be proportional to the quantity of the target single-stranded oligonucleotide or the second double-stranded oligonucleotide derived from the specific target single-stranded oligonucleotide in the sample. In the implementation scheme, the amount of target oligonucleotides and the number of partitions are selected such that at least some partitions are empty, as determined by a Poisson distribution.

[0445] In embodiments, the concentration of the universal reverse amplification primer can be higher than the concentration of the universal forward amplification primer. In embodiments, the concentration of the universal reverse amplification primer can be at least about 1.2, 1.5, 2, 3, 5, 10, 20, or 50 times higher than the concentration of the universal forward amplification primer. For example, in embodiments, the concentration of the universal forward amplification primer can be between about 50 and 200 nM (e.g., about 90 or 100 nM), while the universal reverse amplification primer is provided at a concentration between about 500 and 2000 nM (e.g., about 900 or 1000 nM). In aspects and embodiments, such a ratio may be advantageous for generating an excess of the denatured second double-stranded oligonucleotide sense single-stranded oligonucleotide. The amplified sense single-stranded oligonucleotide comprises the complementary sequence of the universal oligonucleotide probe sequence and / or the complementary sequence of the universal forward primer sequence (the universal oligonucleotide probe can anneal with either or both during the amplification reaction), and the sequence of the target single-stranded oligonucleotide (the non-universal oligonucleotide probe can anneal with this sequence during the amplification reaction), thereby enabling the detection of the prepared oligonucleotide.

[0446] In embodiments, multiplexing of the reaction may include adding first and second oligonucleotides to the target single-stranded oligonucleotide with different universal oligonucleotide probe sequences, thereby allowing the use of different oligonucleotide probes containing different colored fluorophores in the same reaction. In embodiments, multiplexing of the reaction may include adding a first oligonucleotide to the target single-stranded oligonucleotide with a different universal forward primer sequence, thereby allowing the use of multiple different universal forward amplification primers in the same reaction, such as first, second, or more universal forward amplification primers. In embodiments, multiplexing of the reaction may include adding a second oligonucleotide to the target single-stranded oligonucleotide with a different universal reverse primer sequence, thereby allowing the use of multiple different universal reverse amplification primers in the same reaction, such as first, second, or more universal reverse amplification primers. In these ways, a large number of different amplification reactions can be performed and monitored, for example, in a single reaction mixture and amplification reaction. In embodiments, two or more, three or more, four or more, five or more, or six or more (e.g., six) distinguishable probes may be used. In such implementations, each distinguishable probe sequence can anneal with up to 2, 5, 10, or 20 (e.g., 20) different target second double-stranded oligonucleotides during the amplification reaction, wherein each group of, for example, 20 different target second double-stranded oligonucleotides is amplified using a single universal forward amplification primer. For example, when using 6 distinguishable probes, and each distinguishable probe sequence is capable of annealing with 20 different target second double-stranded oligonucleotides, 120 target second double-stranded oligonucleotides can be amplified and detected using only a single universal reverse amplification primer (in combination with an appropriate universal forward amplification primer).

[0447] In embodiments where multiple single-stranded oligonucleotides are derived from cfDNA samples containing maternal and fetal DNA from pregnant human subjects, the methods and systems of this disclosure may include estimating the fraction of fetal DNA in the cfDNA sample. In embodiments, the methods and systems may include estimating the copy number of one or more chromosomes, such as, but not limited to, any of the following chromosomes or any combination thereof: chromosome 21, chromosome 18, chromosome 13, the long arm of chromosome 22 (chromosome 22q), the X chromosome, and the Y chromosome.

[0448] In the implementation, the methods and systems may include estimating the copy number of one or more chromosomes or loci associated with a disease, such as, but not limited to, any of the following diseases or any combination thereof: Down syndrome, Edwards syndrome, and Patau syndrome.

[0449] The disclosed method can be used for both non-invasive and invasive samples (e.g., samples from amniocentesis or CVS). In one embodiment, the sample can be obtained via an invasive method. In another embodiment, the sample can be obtained via a non-invasive method.

[0450] In the implementation scheme, the sample may contain an average of 3,000 genomic equivalent copies extracted from one tube of blood, of which approximately 4% to approximately 15% may be derived from the fetus.

[0451] Target genomic regions and corresponding primers and probes for performing prenatal testing

[0452] This article discloses target genomic regions for performing prenatal testing, along with corresponding primers and probes. These primers and probes are suitable for simultaneously amplifying and detecting multiple genetic markers on different chromosomes, such as assessing chromosomal copy number variations in fetal DNA.

[0453] Aneuploidy, defined as an abnormality in the number of chromosomes, is a major cause of developmental disorders and pregnancy complications. Common aneuploidies include trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). Prenatal testing for these chromosomal abnormalities is crucial for informing parents of potential outcomes and enabling them to make timely medical decisions.

[0454] Conventional methods for detecting aneuploidy, such as karyotype analysis or fluorescence in situ hybridization (FISH), while effective, require invasive sampling methods, such as amniocentesis or chorionic villus sampling (CVS). Advances in non-invasive prenatal testing (NIPT) now enable the detection of cell-free fetal DNA (cfDNA) from maternal blood using PCR-based techniques.

[0455] As disclosed herein, the term "PCR-based detection" refers to any molecular diagnostic technique that includes or is substantially composed of the use of polymerase chain reaction (PCR), including but not limited to: dPCR, droplet dPCR, and qPCR.

[0456] In some embodiments, the amplification reaction is a droplet digital PCR reaction. Methods for performing PCR in droplets are described, for example, in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of which are incorporated herein by reference. In some embodiments, the QX200, QX600, or QX One droplet digital PCR (ddPCR) system (Bio-Rad) is used.

[0457] It is worth noting that singleton PCR-based assays, which detect only one chromosomal target at a time, are generally insufficient for detecting aneuploidy due to their limited scope and inability to simultaneously detect abnormalities. Multiplex PCR-based assays offer the ability to detect multiple chromosomal targets in a single reaction, enabling the detection of multiple chromosomal regions (e.g., sites on chromosomes 21, 18, 13, X, and Y) in a single reaction. This capability is crucial for simultaneously screening for multiple aneuploidies, increasing diagnostic rates while minimizing the number of required reactions, thus providing a more comprehensive and efficient method for prenatal aneuploidy detection. Therefore, by combining multiple primer-probe sets in a single reaction, multiplex assays reduce reagent usage, manpower, and time compared to singleton assays that run individually for each chromosome. This makes the assay more cost-effective and scalable for high-throughput clinical environments.

[0458] Multiplex PCR testing also exhibits relatively high sensitivity and specificity by utilizing highly specific primers and probes to amplify target regions uniquely associated with the target chromosome. This minimizes the risk of false positives or false negatives, especially in cases of low abundance of cfDNA in maternal plasma. Furthermore, given that all target sites are amplified simultaneously, multiplex testing advantageously requires only a relatively small amount of cfDNA for detection. This is advantageous for situations with low concentrations of fetal DNA in maternal plasma and for the high sensitivity required to accurately detect aneuploidy, deletions, or other genetic abnormalities. Therefore, multiplex testing is a powerful tool for screening pregnancies at risk of aneuploidy without invasive procedures.

[0459] In general, throughout this disclosure, detection based on non-invasive DNA / oligonucleotide samples (e.g., non-invasive prenatal testing (NIPT)) can be multiplexed to increase signal strength and reliability. For example, such detections may contain up to 60, 50, 30, or 20 primer and probe sets per target oligonucleotide / chromosome. In embodiments, NIPT detections may contain 2 to 60, 3 to 50, 4 to 40, 5 to 30, or 6 to 20 primer and probe sets per target oligonucleotide / chromosome. Specifically, NIPT detections may contain 6 to 18, 6 to 16, 6 to 14, or 6 to 12 primer and probe sets per target oligonucleotide / chromosome, for example, 6, 7, 8, 9, 10, 11, 12, 13, or 14 primer and probe sets per target oligonucleotide / chromosome.

[0460] In general, throughout this disclosure, assays based on invasive DNA / oligonucleotide samples (e.g., invasive prenatal testing (IPT) assays) may or may not be multiplexed. For example, such assays may contain up to 20 primer and probe sets per target oligonucleotide / chromosome; for example, up to 18, 16, 14, 12, 10, 8, or 6 primer and probe sets. In embodiments, IPT assays may contain 1 to 20, 2 to 18, 3 to 16, 4 to 14, or 5 to 12 primer and probe sets per target oligonucleotide / chromosome; particularly 6, 7, 8, 9, 10, 11, or 12 primer and probe sets per target oligonucleotide / chromosome.

[0461] Multiple designs also allow for the inclusion of reference sites in unaffected chromosomes or stable genomic regions. These internal controls ensure the quality and validity of the assay, addressing potential variability in sample quality, DNA extraction, and amplification efficiency.

[0462] Furthermore, multiplex PCR-based assays provide a flexible platform for expanded assay combinations, where assays can be easily adapted to include additional chromosomal targets (such as microdeletions or duplications) or biomarkers for other conditions, offering customizable solutions for comprehensive prenatal genetic testing.

[0463] It is worth noting that cfDNA derived from the mother and fetus is highly fragmented, with an average size of approximately 150–200 base pairs. This fragmentation may pose challenges to certain genomic assays, such as reducing target availability or resulting in low amplification efficiency.

[0464] The disclosed multiplex detection regions, primers, and probes overcome this technical challenge by integrating multiple primer sets targeting multiple regions. This increases the probability of amplifying at least one intact target region, even if some fragments are degraded or missing. Appropriately, multiple regions of each chromosome can be detected to ensure robust detection. For example, for trisomy 21, several different regions of chromosome 21 can be targeted. If one region is missing due to fragmentation, another intact fragment can still be amplified, thus maintaining the reliability of the detection. Therefore, the multiplex form ensures that the small amount of cfDNA in a sample that can be amplified (and / or detected) is used efficiently, as a single reaction amplifies multiple sites simultaneously, reducing the need for relatively large DNA inputs or additional reactions, which would be challenging for fragmented cfDNA.

[0465] Furthermore, the use of primers designed for amplifying very short DNA regions (e.g., between approximately 60 and 100 bp, or between approximately 65 and 75 bp) ensures compatibility with the fragmented nature of cfDNA. Shorter amplicons are more likely to be present intact in the cfDNA library, thus allowing for efficient amplification despite the small size of the cfDNA fragments.

[0466] In the implementation scheme, prenatal testing using the PCR-based methods disclosed herein includes chromosomal aneuploidy testing.

[0467] In the implementation scheme, prenatal testing using the PCR-based methods disclosed herein includes 22q microdeletion detection, appropriately 22q11.2 microdeletion detection.

[0468] In the implementation scheme, prenatal testing using the PCR-based methods disclosed herein includes chromosomal aneuploidy detection and 22q microdeletion detection, appropriately 22q11.2 microdeletion detection.

[0469] Those skilled in the art will understand that identifying a set of genomic regions to target in multiplex prenatal testing, and generating high-performance primers and probes for multiplex prenatal testing, is a complex task, both individually and collectively, as it involves the interaction of multiple technical, biological, and practical factors.

[0470] Prenatal testing typically relies on circulating cell-free fetal DNA (cfDNA) in maternal blood, which exists at low concentrations (approximately 10-15% of total cfDNA). Therefore, identifying genomic regions in cfDNA that reliably represent the fetus while minimizing maternal DNA interference is challenging, although the signal-to-noise ratio of the assay may vary depending on the selected region. Similarly, primers suitable for such assays must reliably amplify fetal-specific regions without substantial interference from the relatively abundant maternal DNA.

[0471] Furthermore, as those skilled in the art will understand, many genomic regions exhibit population-specific variation. A region selected for one population may not provide reliable results in another population due to factors such as allele frequency, SNP density, the presence of polymorphisms or repetitive sequences (e.g., with variable length or composition), high / variable GC content, or nonspecific background binding of primers to non-target genomic regions (which have higher similarity to target genomic regions in different populations). This can affect primer binding efficiency and specificity. Therefore, identifying target genomic regions and primers that consistently perform well across different populations is challenging and often requires extensive data analysis.

[0472] Primers used for prenatal testing should have appropriate sensitivity and reliability to detect subtle changes such as aneuploidy, duplication, or deletion, requiring consistent amplification across the entire genome and robust differentiation of copy number variations (CNVs). Appropriate target genomic regions should provide consistent coverage during sequencing or other downstream processes. Uneven amplification or sequencing biases (e.g., GC bias) can impair the detection of abnormalities.

[0473] The selected region should also be highly informative (e.g., targeting a specific genomic region, such as on a specific chromosome suspected of aneuploidy, on a chromosome arm, or in a specific region of a chromosome arm), while amplification or sequencing in multiplex detection is technically feasible. Some regions may be biologically desirable, but as mentioned above, they are difficult to target due to high GC content, repetitive sequences, or structural complexity.

[0474] Primers must specifically bind to the target genomic region without amplifying homologous or off-target sequences. This is particularly challenging in multiplex assays because multiple primers interact simultaneously, increasing the likelihood of primer dimer formation and mispriming. Furthermore, interactions such as secondary structure formation can degrade detection performance. Additionally, in multiplex assays, primers for different regions are configured to exhibit similar amplification efficiencies to ensure balanced amplification, especially considering that multiplex assays are designed to work robustly across various sample types. Over-amplification of one region may mask results for other regions and potentially reduce or eliminate the validity of the results.

[0475] Computational tools are typically used to predict and minimize these interactions and ensure compliance with clinical performance standards, such as sensitivity for detecting low-frequency fetal DNA chromosomal variations and high specificity to avoid false positives. However, those skilled in the art will understand that these computational predictions are not always accurate reflections of biological systems, as the interaction of biological, chemical, and technical factors means there is no direct method to predict which primer combinations will perform well in multiplex detection. Even with advanced computational tools, selecting regions that consistently perform well in multiplex detection requires iterative empirical testing.

[0476] In fact, many theoretically suitable regions, primers, and probes may fail due to unexpected problems such as secondary structure or amplification artifacts. It is worth noting that there is no direct or predictable solution to this optimization problem, as improving one factor (e.g., informatics) may impair another (e.g., primer interactions). Therefore, determining suitable target genomic regions, primers, and probes requires iterative testing, validation, and optimization of the primer set.

[0477] The inventors have discovered for the first time that the disclosed target genomic regions are suitable for performing multiplex prenatal testing, and that the disclosed primers and probes targeting any such target genomic regions for performing multiplex prenatal testing should advantageously exhibit high sensitivity (detection of very few false positive events) and discriminative ability (indicated by clear separation between positive wells / droplets and negative wells / droplets).

[0478] Furthermore, the disclosed target genomic regions, primers, and probes are suitable for performing multiplex prenatal assays in droplets, which presents additional challenges compared to bulk PCR reactions. Droplets are much smaller than bulk reactions, typically in the nanoliter or picoliter range. This significantly limits the total amount of template DNA, primers, and other reagents available in each droplet, increasing the risk of random effects such as uneven amplification or target loss. Moreover, achieving the correct primer concentrations across multiple primers in multiplex assays is more challenging due to the volume constraints and the possibility of uneven primer distribution among droplets, potentially leading to preferential amplification of some targets and inhibition of others. Additionally, each droplet encapsulates a small and potentially uneven subset of the DNA template, so not all droplets may contain all target sequences, resulting in incomplete amplification or target loss. Furthermore, random partitioning becomes a more significant problem when template concentrations are low, as some droplets may be completely lacking certain target sequences. In multiplex PCR in droplets, primer dimer formation and competition for reagents (e.g., DNA polymerase, dNTPs) can be more pronounced due to the confined environment, potentially hindering the simultaneous amplification of multiple targets. Furthermore, detecting multiplex PCR results in droplets after amplification is more complex. Each droplet needs to be detected individually, typically requiring advanced imaging systems and detection procedures for multiple readouts.

[0479] The disclosed target genomic regions, primers, and probes were determined after extensive research by the inventors, including initial selection based on differential methylation profiles and initial design (including multiple rounds of adjustment of the Tm of all multiplex primers and the annealing temperature of the amplification reaction to reduce nonspecific amplification caused by incorrect primer annealing, and / or to achieve effective annealing temperatures for all primers to ensure uniform amplification).

[0480] Following the initial selection and design are several rounds of iterative validation (including amplification and sequencing of the resulting regions), reduction selection (to remove regions that produce false positives, exhibit poor separation, exhibit off-target effects, and / or form undesirable primer products (e.g., primer dimers)), and further validation (e.g., testing for new primer-target or primer-primer interactions that arise from new reduction selections of region / primer / probe combinations).

[0481] The coordinates corresponding to these target areas are shown in Table 14 below. Table 14. Target regions: Numbered based on the '+' strand; the forward primer binds to one of the '+' or '-' strands; the reverse primer binds to the other of the '+' or '-' strands.

[0482] In one aspect of the invention, a primer set for detecting chromosomal abnormalities in PCR-based prenatal testing is disclosed, comprising:

[0483] At least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on chromosome 13, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on chromosome 13, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0484] And at least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on chromosome 18, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on chromosome 18, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0485] And at least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on chromosome 21, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on chromosome 21, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0486] And at least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on the long arm of chromosome 22, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on the long arm of chromosome 22, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0487] And at least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on the X chromosome, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on the X chromosome, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0488] And at least a first forward primer configured to anneal to at least 12 nucleotides of a first forward primer region on the Y chromosome, and at least a first reverse primer configured to anneal to at least 12 nucleotides of a first reverse primer region on the Y chromosome, wherein the first forward primer region and the reverse primer region are selected from one or more or all of the following: ;

[0489] In the implementation, the first forward primer is configured to anneal with at least 13 nucleotides of the first forward primer region, such as at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides.

[0490] In one embodiment, the first reverse primer is configured to anneal with at least 13 nucleotides of the first reverse primer region, such as at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides.

[0491] In the implementation, the first forward primer is configured to anneal with at least 13 consecutive nucleotides of the first forward primer region, such as at least 14 consecutive nucleotides, at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, or at least 21 consecutive nucleotides.

[0492] In one embodiment, the first reverse primer is configured to anneal with at least 13 consecutive nucleotides of the first reverse primer region, such as at least 14 consecutive nucleotides, at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, or at least 21 consecutive nucleotides.

[0493] In an implementation, the at least first forward primer configured to anneal with the first forward primer region of chromosome 13 may contain only the first forward primer, or may contain first and second forward primers; first, second and third forward primers; or first, second, third and fourth forward primers.

[0494] In an implementation, the at least first reverse primer configured to anneal with the first reverse primer region of chromosome 13 may contain only the first reverse primer, or may contain first and second reverse primers; first, second and third reverse primers; or first, second, third and fourth reverse primers.

[0495] In an implementation, the at least first forward primer configured to anneal with the first forward primer region of chromosome 18 may contain only the first forward primer, or may contain first and second forward primers; first, second and third forward primers; or first, second, third and fourth forward primers.

[0496] In an implementation, the at least first reverse primer configured to anneal with the first reverse primer region of chromosome 18 may contain only the first reverse primer, or may contain first and second reverse primers; first, second and third reverse primers; or first, second, third and fourth reverse primers.

[0497] In an implementation, the at least first forward primer configured to anneal with the first forward primer region of chromosome 21 may contain only the first forward primer, or may contain first and second forward primers; first, second and third forward primers; or first, second, third and fourth forward primers.

[0498] In an implementation, the at least first reverse primer configured to anneal with the first reverse primer region of chromosome 21 may contain only the first reverse primer, or may contain first and second reverse primers; first, second and third reverse primers; or first, second, third and fourth reverse primers.

[0499] In an implementation, the at least first forward primer configured to anneal with the first forward primer region of the long arm of chromosome 22 may contain only the first forward primer, or may contain first and second forward primers; first, ...

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, including: (a) The amplification reaction mixture is divided into multiple partitions, the amplification reaction mixture comprising cfDNA from the cfDNA sample, amplification reagents, and multiple amplification sets comprising primer and probe sets, wherein each amplification set contains primers and probes for multiplex amplification, and each amplification set generates an amplification product containing a uniquely labeled marker distinguishable from the markers of each of the other amplification sets when the target is present; and wherein the multiple amplification sets comprise: (i) Amplomes targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and (ii) an amplified group targeting sites that are highly methylated in maternal DNA and lowly methylated in fetal DNA, and optionally one or more of (iii), (iv) and (v): (iii) Targeting the amplified group containing total cfDNA from methylation-insensitive regions of chromosomes that are unlikely to exhibit aneuploidy; (iv) Amplification of sites that are highly methylated in both fetal and maternal DNA; (v) Amplification of sites that are hypomethylated in both fetal and maternal DNA; (b) Incubate cfDNA with a mixture of methylation-sensitive restriction endonucleases (MSREs) containing at least one methylation-sensitive restriction endonuclease that cleaves unmethylated DNA; (c) If present, amplify the target nucleic acid sequence in the partition to obtain the amplification product; (d) Detecting the signal from each unique marker of the amplification product in the partition; and (e) Quantify the signal for each unique marker.

2. The method of claim 1, wherein (iii) the amplification group containing total cfDNA from methylation-insensitive regions of chromosomes unlikely to exhibit aneuploidy is included in the plurality of amplification groups.

3. The method according to claim 1 or 2, wherein each amplification group 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 amplification group (i) to (v) comprises primers and probes for targeting at least three sites or between 6 and 10 sites.

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

6. The method according to any one of claims 1 to 5, further comprising an amplification group targeting sites that are hypomethylated in both fetal and maternal cfDNA; and / or an amplification group targeting sites that are 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 of a fully methylated synthetic DNA sequence and / or a fully unmethylated version 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 occurs after the partitioning and before the amplification.

11. The method according to any one of claims 1 to 9, wherein step (b) is performed before the partitioning, and the enzyme-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 endonucleases; and / or wherein the MSRE mixture comprises a restriction endonuclease selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI.

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

14. The method of claim 12 or 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 marker is a fluorescent marker.

17. The method of claim 16, wherein the probe is a molecular beacon probe containing a fluorescent label.

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

19. The method according to any one of claims 1 to 18, comprising determining the normalized copy concentration of each target based on the number (Ni) of targets in the amplicon.

20. The method according to any one of claims 1 to 19, further comprising determining the corrected concentration (Fet_FF) of fetal cfDNA in the cfDNA sample and / or the corrected concentration (Maternal_FF) of maternal cfDNA in the cfDNA sample, wherein determining the corrected concentration of fetal cfDNA comprises the following calculation steps: Wherein [hyper] is a hypermethylation reference copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypermethylated in both fetal DNA and maternal DNA; [hypo] is a hypomethylation reference copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypomethylated in both fetal DNA and maternal DNA; and [foetal] is a fetal DNA copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and / or Determining the corrected concentration of maternal cfDNA involves the following calculation steps: Wherein [maternal] is the maternal DNA copy concentration based on the probe signal, the probe being configured to anneal to one or more sites that are hypomethylated in fetal DNA but hypermethylated in maternal DNA; and [hyper] is as defined above.

21. The method of claim 20, further comprising determining a fetal fraction (FF) in the cfDNA sample based on whether the sample is associated with a male or female fetus, wherein determining the fetal fraction includes: Determining the average fetal methylation score (meth_FF) involves the following calculation steps: The proportion of chrY in the fetal fraction is determined based on the following calculation steps: Where 'chrY_FF' is the proportion of the Y chromosome in the fetal fractional DNA sample; [chrY] is the concentration of chrY in the total DNA sample; and [control] is the total concentration of sample DNA based on probe signals, the probe being configured to anneal to one or more sites containing methylation-insensitive regions from chromosomes unlikely to exhibit aneuploidy; and wherein: (i) When chrY_FF is higher than 0.02, the fetus is determined to be male, and the calculation of the fetal score includes the following calculation steps: or (ii) When chrY_FF is 0.02 or lower, the fetus is determined to be female, and the fetal score is calculated using the following steps: 'FF_calculated' is the final calculated fetal score; and 'meth_FF' and 'chrY_FF' are defined as above.

22. The method according to claim 20 or 21, further comprising: The estimated fetal score is calculated at least in part based on the fetal score and model in the cfDNA sample.

23. The method of claim 22, wherein the model is at least in part based on a generalized additive model (GAM), a linear model, or a second-order polynomial model of a clinical fetal score dataset and a corresponding fetal score measurement dataset using next-generation sequencing (NGS).

24. The method according to any one of claims 1 to 23, wherein the sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA are sites located in one or more of the following regions: 。 25. The method according to any one of claims 1 to 24, wherein the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are sites located in one or more of the following regions: 。 26. The method according to any one of claims 1 to 25, wherein the sites that are hypermethylated in both fetal DNA and maternal DNA are sites located in one or more of the following regions: 。 27. The method according to any one of claims 1 to 26, wherein the sites hypomethylated in both fetal DNA and maternal DNA are sites located in one or more of the following regions: 。 28. The method according to any one of claims 1 to 27, wherein the methylation-insensitive region from a chromosome unlikely to exhibit aneuploidy is a site located within one or more of the following regions: 。 29. The method according to any one of claims 1 to 28, wherein the methylation-insensitive region of the Y chromosome is located in one or more of the following regions: 。 30. A DNA amplification kit for estimating the fraction of fetal DNA in cfDNA samples obtained from plasma or serum samples of pregnant human subjects, the kit comprising: (a) An amplification reaction mixture comprising amplification reagents and a plurality of amplification sets, each amplification set comprising primer and probe sets, wherein each amplification set contains a unique marker distinguishable from the markers of each other set, and each set contains primers and probes for multiplex amplification, and wherein the plurality of amplification sets comprises: (i) Amplomes targeting sites that are hypermethylated in fetal DNA and hypomethylated in maternal DNA; and (ii) an amplification group targeting sites that are highly methylated in maternal DNA and lowly methylated in fetal DNA; and optionally one or more of (iii), (iv) and (v); (iii) Amplification of total cfDNA, which contains methylation-insensitive regions from chromosomes that are unlikely to exhibit aneuploidy; (iv) Amplification of sites that are highly methylated in both fetal and maternal DNA; (v) Amplification of sites that are hypomethylated in both fetal and maternal DNA.

31. The kit of claim 30, wherein each amplification group of (i) and (ii) contains primers and probes for targeting at least three sites.

32. The kit according to claim 30 or 31 further comprises an amplicon targeting a methylation-insensitive region of the Y chromosome.

33. The kit according to any one of claims 30 to 32 further comprises a fully methylated synthetic DNA sequence and / or a fully unmethylated version of the same synthetic sequence.

34. The kit according to any one of claims 30 to 33, wherein each label is a fluorescent label.

35. The kit of claim 34, wherein the probe is a molecular beacon probe containing a fluorescent label.

36. The kit according to any one of claims 30 to 35, wherein each probe is an oligonucleotide that hybridizes with a complementary oligonucleotide, the complementary oligonucleotide comprising a label that provides a detectable signal.

37. The kit according to any one of claims 30 to 36 further comprises a mixture of methylation-sensitive restriction endonucleases (MSREs), said MSRE mixture containing at least one MSRE that cleaves unmethylated DNA.

38. The kit of claim 37, wherein the MSRE mixture comprises at least two, at least three, or at least four methylation-sensitive restriction endonucleases; and / or wherein the MSRE mixture comprises a restriction endonuclease selected from HhaI, HpaII, AciI, HpyCH4IV, and BsaHI.

39. The kit according to any one of claims 1 to 38, wherein the MSRE mixture comprises at least two, three or more of the restriction endonucleases HhaI, HpaII, AciI, HpyCH4IV and BsaHI.

40. The kit according to claim 38 or 39, wherein the MSRE mixture comprises at least HhaI and HpyCH4IV.

41. The kit according to any one of claims 30 to 40, wherein the sites of high methylation in fetal DNA and low methylation in maternal DNA are sites located in one or more of the following regions: 。 42. The kit according to any one of claims 30 to 41, wherein the sites that are hypermethylated in maternal DNA and hypomethylated in fetal DNA are sites located in one or more of the following regions: 。 43. The kit according to any one of claims 30 to 42, wherein the sites of high methylation in both fetal DNA and maternal DNA are sites located in one or more of the following regions: 。 44. The kit according to any one of claims 30 to 43, wherein the sites hypomethylated in both fetal DNA and maternal DNA are sites located in one or more of the following regions: 。 45. The kit according to any one of claims 30 to 44, wherein the methylation-insensitive region from a chromosome unlikely to exhibit aneuploidy is a site located within one or more of the following regions: 。 46. ​​The kit according to any one of claims 30 to 45, wherein the methylation-insensitive region of the Y chromosome is located in one or more of the following regions: 。 47. The method of any one of claims 1 to 29, or the kit of any one of claims 30 to 46, wherein at least one forward primer targeting the amplification group of sites that are highly methylated in fetal DNA and lowly methylated in maternal DNA comprises the sequence of one of SEQ ID NO: 13 to 20.

48. The method according to any one of claims 1 to 29 and 47, or the kit according to any one of claims 30 to 47, wherein the sequence of at least one forward primer targeting a site of amplification that is hypermethylated in fetal DNA and hypomethylated in maternal DNA comprises one of SEQ ID NO: 60 to 67.

49. The method according to any one of claims 1 to 29, 47 and 48, or the kit according to any one of claims 30 to 48, wherein at least one forward primer targeting the amplification group of sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises the sequence of one of SEQ ID NO: 21 to 27.

50. The method according to any one of claims 1 to 29 and 47 to 49, or the kit according to any one of claims 30 to 49, wherein the sequence of at least one forward primer targeting the amplification group of sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises one of SEQ ID NO: 68 to 74.

51. The method according to any one of claims 1 to 29 and 47 to 50, or the kit according to any one of claims 30 to 50, wherein at least one forward primer targeting amplification sites in fetal DNA and maternal DNA that are methylation-insensitive and unlikely to exhibit aneuploidy comprises a sequence of one of SEQ ID NO: 41 to 49.

52. The method according to any one of claims 1 to 29 and 47 to 51, or the kit according to any one of claims 30 to 51, wherein the sequence of at least one forward primer targeting a site in the amplification group that is methylation-insensitive and unlikely to exhibit aneuploidy in fetal DNA and maternal DNA comprises one of SEQ ID NO: 88 to 96.

53. The method according to any one of claims 1 to 29 and 47 to 52, or the kit according to any one of claims 30 to 52, wherein at least one forward primer targeting the amplification group of sites that are hypermethylated in both fetal DNA and maternal DNA comprises the sequence of one of SEQ ID NO: 28 to 34.

54. The method according to any one of claims 1 to 29 and 47 to 53, or the kit according to any one of claims 30 to 53, wherein the sequence of at least one forward primer targeting a site that is hypermethylated in both fetal DNA and maternal DNA comprises one of SEQ ID NO: 75 to 81.

55. The method according to any one of claims 1 to 29 and 47 to 54, or the kit according to any one of claims 30 to 54, wherein at least one forward primer targeting the amplification group of sites that are hypomethylated in both fetal DNA and maternal DNA comprises the sequence of one of SEQ ID NO: 35 to 40.

56. The method according to any one of claims 1 to 29 and 47 to 55, or the kit according to any one of claims 30 to 55, wherein the sequence of at least one forward primer targeting a site of hypomethylation in both fetal DNA and maternal DNA comprises one of SEQ ID NO: 82 to 87.

57. The method of any one of claims 1 to 29 and 47 to 56, or the kit of any one of claims 30 to 56, wherein at least one forward primer targeting a site on the Y chromosome that is methylation insensitive and unlikely to exhibit aneuploidy in fetal DNA and maternal DNA comprises a sequence of one of SEQ ID NO: 50 to 59.

58. The method according to any one of claims 1 to 29 and 47 to 57, or the kit according to any one of claims 30 to 57, wherein the sequence of at least one forward primer targeting a site on the Y chromosome that is methylation insensitive and unlikely to exhibit aneuploidy in fetal DNA and maternal DNA comprises one of SEQ ID NO: 97 to 106.

59. The method according to any one of claims 1 to 29 and 47 to 58, or the kit according to any one of claims 30 to 58, wherein at least one reverse primer targeting the amplification group of sites that are highly methylated in fetal DNA and lowly methylated in maternal DNA comprises the sequence of one of SEQ ID NO: 154 to 161.

60. The method according to any one of claims 1 to 29 and 47 to 59, or the kit according to any one of claims 30 to 59, wherein the sequence of at least one reverse primer targeting a site of amplification that is hypermethylated in fetal DNA and hypomethylated in maternal DNA comprises one of SEQ ID NO: 107 to 114.

61. The method according to any one of claims 1 to 29 and 47 to 60, or the kit according to any one of claims 30 to 60, wherein at least one reverse primer targeting the amplification group of sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises the sequence of one of SEQ ID NO: 162 to 168.

62. The method according to any one of claims 1 to 29 and 47 to 61, or the kit according to any one of claims 30 to 61, wherein the sequence of at least one reverse primer targeting the amplification group of sites that are hypomethylated in fetal DNA and hypermethylated in maternal DNA comprises one of SEQ ID NO: 115 to 121.

63. The method of any one of claims 1 to 29 and 47 to 62, or the kit of any one of claims 30 to 62, wherein at least one reverse primer targeting amplification sites in fetal DNA and maternal DNA that are methylation-insensitive and unlikely to exhibit aneuploidy comprises a sequence of one of SEQ IDs 182 to 190.

64. The method of any one of claims 1 to 29 and 47 to 63, or the kit of any one of claims 30 to 63, wherein the sequence of at least one reverse primer targeting amplification sites in fetal DNA and maternal DNA that are methylation-insensitive and unlikely to exhibit aneuploidy comprises one of SEQ ID NO: 135 to 143.

65. The method according to any one of claims 1 to 29 and 47 to 64, or the kit according to any one of claims 30 to 64, wherein at least one reverse primer of the amplification group targeting sites that are hypermethylated in both fetal DNA and maternal DNA comprises the sequence of one of SEQ ID NO: 169 to 175.

66. The method according to any one of claims 1 to 29 and 47 to 65, or the kit according to any one of claims 30 to 65, wherein the sequence of at least one reverse primer targeting a site of hypermethylation in both fetal DNA and maternal DNA comprises one of SEQ ID NO: 122 to 128.

67. The method of any one of claims 1 to 29 and 47 to 66, or the kit of any one of claims 30 to 66, wherein at least one reverse primer of the amplification group targeting sites that are hypomethylated in both fetal DNA and maternal DNA comprises a sequence of one of SEQ ID NO: 176 to 181.

68. The method according to any one of claims 1 to 29 and 47 to 67, or the kit according to any one of claims 30 to 67, wherein the sequence of at least one reverse primer targeting a site of hypomethylation in both fetal DNA and maternal DNA comprises one of SEQ ID NO: 129 to 134.

69. The method of any one of claims 1 to 29 and 47 to 68, or the kit of any one of claims 30 to 68, wherein at least one reverse primer targeting the amplification group of sites on the Y chromosome that are methylation-insensitive and unlikely to exhibit aneuploidy in fetal DNA and maternal DNA comprises a sequence of one of SEQ ID 191 to 200.

70. The method of any one of claims 1 to 29 and 47 to 69, or the kit of any one of claims 30 to 69, wherein the sequence of at least one reverse primer targeting a site on the Y chromosome that is methylation-insensitive and unlikely to exhibit aneuploidy in fetal DNA and maternal DNA comprises one of SEQ ID NO: 144 to 153.

71. The method according to any one of claims 1 to 70, wherein the partition is a droplet.

72. The method according to any one of claims 1 to 70, wherein the partition is a micropore.

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