Sequence conversion reaction using inversion ligation products

Abstract

Disclosed herein are methods, compositions, and kits related to the identification and / or quantification of target molecules using sequence conversion probes that may detect, using a single probe, a variety of targeted sequences. These methods and compositions may be particularly well suited for quantitative PCR and detection of very low copy signals by including one or more features for reducing digital PCR noise, including the use of inversion ligation products with an engineered circularizable signal specific region, a lock probe, and a cassette.

Description

SEQUENCE CONVERSION REACTION USING INVERSION LIGATIONPRODUCTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. provisional patent application no. 63 / 517,106, titled “SEQUENCE CONVERSION REACTION,” and filed on August 1, 2023, herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.FIELD

[0003] This disclosure relates to compositions, kits, and methods for detecting and quantifying molecular targets, and especially for detecting and quantifying DNA and RNA. The apparatuses and methods may find use in a variety of applications including prenatal testing, detection of pathogens and screening of cancer.BACKGROUND

[0004] There is a need for genetic assays that provide highly accurate and reproducible results. However, these highly reproducible results are difficult to achieve, when the amount of starting genetic material may be low in quantity and / or quality.

[0005] Non-invasive prenatal testing refers to testing the characteristics of a fetus from a pregnant female, from a tissue sample obtained from the pregnant female. Fetal cells and parts of fetal cells including fetal nucleic acid are present in the pregnant female’s bloodstream and the fetal tissue can provide a wealth of information. A blood sample can be easily and safely obtained from the pregnant female with no harm to the fetus. Advances in DNA amplification techniques such as polymerase chain reaction (PCR) and next generation sequencing (NGS) have led to substantial increases in our ability to perform prenatal testing and several prenatal tests are commercially available. Existing DNA amplification techniques, like dPCR, have various technical limitations such as nonspecific and biased amplification when trying to detect many different target sequences. These limitations can lead to results that cannot be interpreted. Next generation Sequences (NGS) techniquesprovide technical advantages but suffer from complex workflows associated with high cost and long turnaround time. Accordingly, there is a need for improved methods for performing these and other tests. In particular it would be desirable to enable dPCR for these tests due to low cost, fast turnaround time and relative ease of use especially compared to NGS.

[0006] The reliability of a quantitative measurement improves with the number of measurements (replicates). Often there is not enough material available to yield the number of measurements (copy number too low) needed to achieve the required accuracy and precision. For example, as mentioned before and as applicable to NIPT, the number of genomes (number of chromosome copies, number of allele copies) represented in 1 2 ml of plasma as cell-free DNA (cfDNA) is estimated to be approximately 1000 copies. This is (for example) not sufficient for NIPT by dPCR. Prior work has utilized hundreds of thousands of counts, using an alternative technology, to achieve the required performance.

[0007] Digital polymerase chain reaction (dPCR) is a biotechnological refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA or RNA. In dPCR the sample is separated into a large number of partitions (e.g. droplets, microfluidic chambers, etc.) and the reaction is carried out in each partition individually. This separation allows absolute quantification without the need for a standard curve. The main technical reason why dPCR is not commonly used in NIPT despite the listed theoretical advantages and years of development are problems with the fact that the copy number of cfDNA is too low to deliver the required statistical precision. Theoretically, one can perform multiplexed dPCR, amplifying many alleles on chromosomes of interest in order to address the inherent low copy number issue. However, primer-primer probe interactions in multiplexed dPCR causes unspecific and biased amplification, manifesting as a visible phenomenon called “rain”, a form of background noise in dPCR. This problem is in particular pronounced in challenging samples. Diagnostic tests require high robustness, and the assumption has to be that samples are challenging. PCR amplifies target exponentially and small differences in reaction efficiency lead to very different copy numbers at the end of the reaction (e.g. 40 plus cycles) even when the copy number at the beginning of the reaction was the same.

[0008] Even though digital PCR is a digital yes / no reaction, the data analysis ultimately depends on a positive reaction i.e. TaqMan signal above background. This in turn depends on having enough TaqMan probe cleaved for a partition to be considered positive, which depends on how much amplicon was made by the end of cycling. Having different PCR efficiencies in different partitions may result in different amounts of amplicon in differentpartitions and hence in some partitions less cut probe compared to others (lower signal). This may lead to “rain” and the difficulty to clearly distinguish positive from negative.

[0009] Furthermore, cfDNA fragments of typically less than 300 base pairs in length may be distributed into individual partitions, with hundreds or thousands of potential target sequences (measurands) from a given chromosome. In order to examine in a clinically relevant manner multiple (e.g., three or more) chromosomes the assay needs to be able to potentially amplify many e.g. 100 different allele sites (measurands) per chromosome. This means that, in this example, each partition may need enough of each of the 300 primer pairs (600 primers) and 300 different probes to support detection of each measurand. There has to be enough primer and probe in each partition to support the reaction of a single target measurand, while at the same time the unused primer and probes need to be present but not interfere with a single target measurand (unique sequence) or cause a false positive result. In practical terms such a reaction cannot be performed reliably. It would be beneficial to reduce the complexity of the reaction and provide reliable target detection and quantitation.

[0010] Previously described techniques to address the above-described problems have proven difficult to implement, and highly sensitive to background factors. Described herein are methods and compositions that may address these problems.SUMMARY OF THE DISCLOSURE

[0011] Described herein are methods, kits, and compositions that may address the limitations and needs described above. In particular, described herein are methods, including sequence conversion methods (e.g., sequence conversion assays or sequence conversion reactions) that are modified to improve signal while decreasing background significantly. In particular, these methods and compositions may include one or more of: the use of UNG cleanup, rolling circle amplification, and padlock primer extension (P-LEX). In particular, these methods and compositions (including kits) may use a synthetic circularizable signal specific region (SSR) that includes a target dU region (comprising one or more deoxyuridines), and circularization using a lock probe and a cassette sequence (e.g., complimentary to the lock probe) that may be ligated to the cut SSR to specifically circularize only the SSRs for those sequence conversion probes (SCPs) that bound to target. Once circularized, the entire mixture may be exposed to a Uracil-DNA glycosylase to form inverted cSSRs that may be detected by qPCR or dPCR when amplified. The process of circularizing using both a lock and a lock cassette then cutting (‘inverting’) the circularized surprisingly reduces the background ‘noise’ of these reactions by orders of magnitude, permitting them to detect even very low-copy sequences.

[0012] For example, described herein are methods, comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including one or more target-specific regions (TSRs), and a signal specific region (SSR), the SSR further comprising a forward primer region and a reverse primer region that are oriented away from each other and a dU region comprising one or more deoxyuridines between the forward and reverse primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSR to a lock probe and to a cassette sequence to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence between the 3’ hybridization site and the 5’ hybridization site to form a circular SSR; digesting the circular SSR with a Uracil-DNA glycosylase to form an inverted cSSR; amplifying a probe sequence on the inverted cSSR using a forward primer binding to the forward primer region and a reverse primer binding on the reverse primer region; and detecting the probe sequence.

[0013] In some cases it may be particularly beneficial where each SCP comprises two TSRs (e.g., at either end). Any of these methods may include the use of a lock probe that may have a flap region at a 3’ end of the lock probe that does not hybridize to the cSSR. The lock probe may be blocked to prevent extension; for example, the lock probe may have a phosphate at a 3' end of the lock probe to prevent extension by a DNA polymerase. The probe sequence may span a junction between the cSSR and the cassette sequence. Alternatively, the probe sequence may be a first engineered polynucleotide marker within the SSR. In some cases the amplifying the probe sequence comprises amplifying the probe sequence extending at least partially across the cassette sequence.

[0014] The lock probe may bind to the 3’ hybridization site comprising 1 or more base pairs on the 3’ end of the cSSR. The lock probe binding to the 3’ hybridization site may have a low melting temperature of less than 20 degrees C to prevent binding to the lock probe binding to the sequence conversion probe. Thus, the lock probe may be engineered to have a sequence with this melting temperature. In any of these methods and compositions, the forward primer region may be at least 4 base pairs from the 5’ end of the cSSR and at least 1 base pairs from the 3’ end of the cSSR.

[0015] In any of these methods and compositions, each SCP may comprise a pair of target-specific regions (TSRs), TSR1 and TSR2 positioned at either ends of the SSR, wherein hybridizing the plurality of different TSRs comprises hybridizing TSR1 and TSR2 to the one or more target measurand within the sample mixture, and wherein cutting the SCPs comprises cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs). In any of these methods and kits, detecting may comprise performing digital PCR.

[0016] Any of these methods may include combining a second plurality of SCPs with the sample mixture, wherein the second plurality of SCPs comprises a plurality of different TSRs configured to hybridize to different target measurands, and wherein each SCP of the second plurality of SCPs includes a same SSR that is different from the SSR of the first plurality of SCPs. The plurality of different TSRs of the first plurality of SCPs may be configured to hybridize to different target measurands are configured to hybridize to a first chromosome and the plurality of different TSRs of the second plurality of SCPs may be configured to hybridize to different target measurands are configured to hybridize to a second chromosome.

[0017] Cutting SCPs to release cSSRs may comprise nicking the SCPs without cutting the one or more target measurand. This may allow detection of the same measurand multiple times. The steps of hybridizing the plurality of TSRs to the one or more target measurand within the sample mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs may be repeated multiple times.

[0018] In any of these methods and compositions a cassette sequence may be a polynucleotide having a sequence that is complimentary to a region of the lock probe that is configured to span between the hybridized ends of the cSSR. The cassette sequence may also be a probe and may be equivalently referred to as a lock cassette or lock cassette probe.

[0019] Any appropriate Uracil-DNA glycosylase may be used (e.g., Uracil-N- glycosylase, UDG).

[0020] For example, a method may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including two target-specific regions (TSRs), and a circularizable signal specific region (SSR), wherein the circularizable SSR comprises one or more deoxyuridines between a forward primer region and a reverse primer region wherein the forward primer region is oriented away from the second primer region, the circularizable SSR further comprising a first engineered polynucleotide marker, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPsincludes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe and hybridizing a cassette sequence to the lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence to the 3’ end of the cSSR and to the 5’ end of the cSSR to form a circular SSR; cutting the circular SSR with a Uracil-DNA glycosylase to form an inverted cSSR; amplifying the first engineered polynucleotide marker from the inverted cSSR using a forward primer binding to the forward primer region and a reverse primer binding to the reverse primer region; and detecting the first engineered polynucleotide marker.

[0021] Also described herein are kits configured to perform any of these the methods. These kits may include any of these components in solution or dried (lyophilized), e.g., sequence conversion probes (SCPs), lock probes, cassette sequences (lock cassettes), etc.

[0022] Also described herein are methods of determining a fetal aneuploidy using any of the methods described herein. For example, a method may include: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP comprises: a first polynucleotide target-specific region (TSR1) and a second polynucleotide target-specific region (TSR2); a circularizable signal specific region (SSR) between TSR1 and TSR2, wherein the circularizable SSR comprises one or more deoxyuridines between a first primer region and a second primer region wherein the first primer region is oriented away from the second primer region, the circularizable SSR further comprising a first engineered polynucleotide marker, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands on a same chromosome, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the TSRls and TSR2s to target measurands in the mixture; cutting SCPs having TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe and hybridizing a cassette sequence to the lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence to the 3’ end of the cSSR and to the 5’ end of the cSSR to form a circular SSR; cutting the circular SSR with aUracil-DNA glycosylase to form an inverted cSSR; distributing the inverted cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain inverted cSSRs and some do not contain inverted cSSRs; amplifying the first engineered polynucleotide marker from the inverted cSSRs using the forward primer binding to the forward primer region and the reverse primer binding to the reverse primer region; detecting the first engineered polynucleotide marker; analyzing the engineered polynucleotide markers of the inverted cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

[0023] As mentioned, the methods described herein may include the use of a lock (padlock) probe or primer. For example, the methods described herein may include padlock primer extension. The use of padlock primer extension may include the use of a set of forward and reverse primers configured to bind only to the circularized signal specific region (SSR) after the SSR has been cut from the target specific region (TSR), and in which the binding sites for the forward and reverse primers are spaced apart from each other and from the site of circularization.

[0024] The methods and compositions described herein may improve sequence conversion methods that use a pair of target-specific regions flanking a sequence specific region. As used herein, a sequence conversion method may convert one (or more preferentially more) target regions, e.g., a target measurand or allele, into a signal specific region (SSR) that is engineered to include a polynucleotide marker region (“engineered polynucleotide marker”) and in some cases primer regions for copying and / or amplifying and / or detecting the engineered polynucleotide marker. A number of different target regions, which may share a common property, such as being part of a particular chromosome, gene, plasmid, genetic pathway, or the like, may share a common signal specific region, by linking the same signal specific region to these different target measurand regions (“target-specific regions” or TSR) in a subset of sequence conversion probes (SCPs). Multiple subsets of sequence conversion probes may then be combined. This may also allow reduction in the complexity and variability of the assay to provide substantial advantages in sensitivity, accuracy and precision.

[0025] In general, these methods include detection of a natural occurring nucleic acid sequence (and / or chromosome) by amplifying and detecting a synthetic sequence that is different from the original sequence and is not found in the target genome.

[0026] These assays may therefore allow the parallel processing of large amounts of genetic material with very low background. Furthermore, the methods and compositions (including kits) described herein may allow the sequence conversion probes to be used with either or both DNA and RNA including very small, previously difficult to assay, fragments of RNA and / or DNA. The sequence conversion methods described herein may, with great fidelity, convert target measurands (or multiple target measurands) to signal specific regions (e.g., engineered polynucleotide markers) that may be directly assayed, e.g., by binding to one or more signal probes or signal labels, and / or by genetic amplification and detection (e.g., digital PCR, etc.).

[0027] Also described herein are methods of amplifying the output (e.g., signal) from the SSRs by cycling (e.g., recycling) the use of the target measurand, and / or methods of amplifying the output by rolling amplification. Specifically, described herein are methods of the signal by making more signal specific region (cut and / or circularized signal specific region, cSSR) from the same amount of sequence conversion probe (SCP) and target measurand (e.g., sample material).

[0028] Any of the methods and apparatuses described herein may be configured to include Padlock Primer Extension (P-LEX). For example, described herein are methods, comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including one or more target-specific regions (TSRs), and a circularizable signal specific region (SSR), the circularizable SSR further comprising a first engineered polynucleotide marker including a forward primer region, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; replicating the circularization complex from the lock probe without ligating the ends of the cSSR; amplifying the replicated circularization complex using the forward primer binding to the forward primer region on thereplicated circularization complex; and detecting the first engineered polynucleotide marker from the replicated circularization complex.

[0029] The lock probe may be configured as a reverse primer relative to the cut SSR. The lock probe may bind to the 3’ hybridization site comprising 5 or more base pairs on the 3’ end of the cSSR. The lock probe binding to the 3’ hybridization site may have a low melting temperature, e.g., of less than 20 degrees C (or less than 18 degrees C, less than 16 degrees C, between 15-20 degrees C, etc.) to prevent binding to the lock probe binding to the sequence conversion probe.

[0030] In any of these examples, the forward primer region may be configured so that it does not overlap with either the 3’ hybridization site on the 3’ end of the cSSR and the 5’ hybridization site on the 5’ end of the cSSR for the lock probe. The forward primer region may be at least 5 base pairs from the 5’ end of the cSSR and at least 5 base pairs from the 3’ end of the cSSR.

[0031] In general, these methods may use circularization complexes without ligating the ends of the circularization complexes together (e.g., when using P-LEX as described herein). Omitting the use of ligase with P-LEX may also significantly reduce the background.

[0032] Alternatively or additionally, any of these methods may be configured to use uracil-N-glycosylase (UDG) to reduce the background of the assay. For example, any of these methods may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including one or more target-specific regions (TSRs), and a circularizable signal specific region (SSR), wherein the circularizable SSR comprises one or more deoxyuridines, the circularizable SSR further comprising a first engineered polynucleotide marker including a forward primer region, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; replicating the circularization complex by rolling circle amplification, wherein the amplified circularization complexes comprise thymidine in place of the one or more deoxyuridines from the cSSR; treating the circularized cSSRs with uracil-N-glycosylase (UDG) to remove uracil; and detecting the first engineered polynucleotide marker from the amplified circularization complex.

[0033] Any of the methods described herein may be configured to use both P-LEX and uracil-N-glycosylase (UDG) to remove background. Optionally these methods may or may not use rolling circle amplification as part of the method.

[0034] For example, described herein are method comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including one or more targetspecific regions (TSRs), and a circularizable signal specific region (SSR), wherein the circularizable SSR comprises one or more deoxyuridines, the circularizable SSR further comprising a first engineered polynucleotide marker including a forward primer region, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; replicating the circularization complex, wherein the amplified circularization complexes comprise thymidine in place of the one or more deoxyuridines from the cSSR; treating the circularized cSSRs with uracil-N-glycosylase (UDG) to remove uracil; and detecting the first engineered polynucleotide marker from the amplified circularization complex. Optionally rolling circle amplification may be used.

[0035] The forward primer region may be at least 5 base pairs from the 5’ end of the cSSR and at least 5 base pairs from the 3’ end of the cSSR. Alternatively or additionally, the forward primer region may not overlap with either the 3’ hybridization site on the 3’ end of the cSSR and the 5’ hybridization site on the 5’ end of the cSSR for the lock probe.

[0036] In some examples the circularized cSSR may include a gap filling region between the 5’ hybridization site on the 5’ end of the cSSR and the 3’ hybridization site on the 3’ end of the cSSR, wherein the gap filling region comprises a gap primer sequence. The gap filling region may be part of the lock probe.

[0037] The 3’ end of the lock probe may be blocked to prevent extension. The 3’ end of the lock probe may be blocked to prevent extension by an overhang flap region that does not hybridize to the circularized cSSR.

[0038] Each SCP may comprise a pair of target-specific regions (TSRs), TSR1 and TSR2 positioned at either ends of the SSR, wherein hybridizing the plurality of different TSRs comprises hybridizing TSR1 and TSR2 to the one or more target measurand within thesample mixture, and wherein cutting the SCPs comprises cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs).

[0039] Detecting may comprise detecting the first engineered polynucleotide marker by performing digital PCR using the gap primer sequence to amplify the first engineered polynucleotide markers. In some examples, detecting comprises detecting the first engineered polynucleotide marker by performing digital PCR.

[0040] In any of these methods, combining may further comprise combining a second plurality of SCPs with the sample mixture, wherein the second plurality of SCPs comprises a plurality of different TSRs configured to hybridize to different target measurands, and wherein each SCP of the second plurality of SCPs includes a same SSR that is different from the SSR of the first plurality of SCPs. The plurality of different TSRs of the first plurality of SCPs that are configured to hybridize to different target measurands are configured to hybridize to a first chromosome and the plurality of different TSRs of the second plurality of SCPs that are configured to hybridize to different target measurands are configured to hybridize to a second chromosome. Cutting SCPs to release cSSRs may comprise nicking the SCPs without cutting the one or more target measurand. Thus, any of these methods may include repeating the steps of hybridizing the plurality of TSRs to the one or more target measurand within the sample mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs.

[0041] Any of these methods may include removing un-cut SCP and non-circularized SSR before replicating the circularization complex by adding exonuclease.

[0042] Cutting the SCPs to release cut SSRs may comprise using BspQI to digest the hybridized plurality of different TSRs.

[0043] Replicating the circularization complex may comprise adding Phi29 and dTTPs.

[0044] In some examples a method may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including a first target-specific region (TSR1), a second target-specific region (TSR2) and a circularizable signal specific region (SSR) between TSR1 and TSR2, wherein the circularizable SSR comprises one or more deoxyuridines, the circularizable SSR further comprising a first engineered polynucleotide marker including a forward primer region, further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; amplifying by repeatedly: hybridizing the plurality of TSRls and the TSR2s to the one ormore target measurand within the sample mixture, and cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs) by nicking the SCPs without cutting the one or more target measurand, and hybridizing the cSSRs to a lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; replicating the circularization complex, wherein the amplified circularization complexes comprise thymidine in place of the one or more deoxyuridines from the cSSR; treating the circularized cSSRs with uracil-N-glycosylase (UDG) to remove uracil; and detecting the first engineered polynucleotide marker from the amplified circularization complex. Optionally rolling circle amplification may be used.

[0045] Also describe herein are methods of determining a fetal aneuploidy using any of these methods described herein. For example, a method of determining a fetal aneuploidy may include: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP includes: one or more polynucleotide target-specific regions (TSRs), a circularizable signal specific region (SSR), wherein the circularizable SSR comprises one or more deoxyuridines, the circularizable SSR comprising a first engineered polynucleotide marker including a forward primer region, further wherein the plurality of SCPs includes different pairs of TSRs; wherein each set of SCPs comprises the same SSR and a plurality of different TSRs configured to hybridize to different target measurands on a same chromosome; hybridizing the TSRs to target measurands in the mixture; cutting SCPs having TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; replicating the circularization complex, wherein the amplified circularization complexes comprise thymidine in place of the one or more deoxyuridines from the cSSR; treating the circularized cSSRs with uracil-N- glycosylase (UDG) to remove uracil; and analyzing the engineered polynucleotide markers of the cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number. Optionally, rolling circle amplification may be used.

[0046] Also described herein are kits configured to perform these methods

[0047] In general, the various improvements and features described herein may improve methods including a sequence conversion probe, including those described in U.S. Patent Application 18 / 001,575, filed June 10, 2021, titled “SEQUENCE CONVERSION REACTION” and PCT / US2022 / 052438, filed December 9, 2022, titled “SEQUENCE CONVERSION REACTION,” each of which is herein incorporated by reference in its entirety. For example, the features described herein (including the use of UNG cleanup, rolling circle amplification, and / or padlock primer extension (P-LEX)) may be used with any of the sequence conversion probes and reaction including them. For example, these features may be used with methods including: combining a plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a pair of polynucleotide target-specific region (TSR), an affinity tag, and a signal specific region (SSR), the SSR comprising a first engineered polynucleotide region (which may be referred to herein as a marker, and may be recognized by one or more specific primers), further wherein the plurality of SCPs include different pairs of TSRs; hybridizing the pairs of TSRs of the SCPs to the one or more target measurand; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs) without releasing SSRs of un-hybridized SCPs (and in some cases by nicking, so that the target measurand is not cut); isolating the cSSRs from un-hybridized SCPs and the hybridized TSRs; and detecting the first engineered polynucleotide marker from the cSSRs.

[0048] For example, these methods may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including a first target-specific region (TSR1), a second target-specific region (TSR2) and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of TSRls and the TSR2s to the one or more target measurand within the sample mixture; cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0049] In some examples, the method may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more targetmeasurand, wherein each SCP comprises a polynucleotide including a first target-specific region (TSR1), a second target-specific region (TSR2) and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; amplifying by repeatedly: hybridizing the plurality of TSRls and the TSR2s to the one or more target measurand within the sample mixture, and cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs) by nicking the SCPs without cutting the one or more target measurand, and hybridizing the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0050] In any of these methods, detecting may comprise detecting the first engineered polynucleotide marker by performing digital PCR using the circularized cSSRs (cirSSR) and one or more primers configured to hybridize to the one or more primer regions to amplify the first engineered polynucleotide markers.

[0051] Combining further comprises combining a second plurality of SCPs with the sample mixture, wherein the second plurality of SCPs comprises a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, and wherein each SCP of the second plurality of SCPs includes the same SSR that is different from the SSR of the first plurality of SCPs. The plurality of different TSRls and TSR2s of the first plurality of SCPs that are configured to hybridize to different target measurands may be configured to hybridize to a first chromosome and the plurality of different TSRls and TSR2s of the second plurality of SCPs that are configured to hybridize to different target measurands may be configured to hybridize to a second chromosome.

[0052] In any of these methods, cutting SCPs to release cSSRs may comprise nicking the SCPs without cutting the one or more target measurand. These methods may further include repeating the steps of hybridizing the plurality of TSRls and the TSR2s to the one or more target measurand within the sample mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs.

[0053] Any of these methods may include performing rolling amplification of the circularized cSSRs before detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0054] Either or both of TSR1 and TSR2 may comprise a Type IIS enzyme recognition site. For example, TSR1 and TSR2 may comprise different Type IIS enzyme recognition sites. In some examples TSR1 and TSR2 comprise a Type IIS enzyme recognition site and a Type II enzyme recognition site.

[0055] In general, TSR1 and TSR2 may be configured to hybridize to the target measurand at two regions that are spaced apart from each other by between about 1 and 50 bp (e.g., 0-50 bp, 5-50 bp, etc.).

[0056] The methods and compositions described herein may be adapted specifically for use in determining fetal aneuploidy. For example, described herein are methods of determining a fetal aneuploidy comprising: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP includes: a first polynucleotide target-specific region (TSR1), a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the plurality of SCPs includes different pairs of TSRs; wherein each set of SCPs comprises the same SSR and a plurality of different pairs of TSRs configured to hybridize to different target measurands on a same chromosome; hybridizing the pairs of TSRs to target measurands in the mixture; cutting SCPs having pairs of TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; distributing the circularized cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain cSSR and some do not contain cSSRs; analyzing the engineered polynucleotide markers of the cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

[0057] For example, a method of determining a fetal aneuploidy may include: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP includes: a first polynucleotidetarget-specific region (TSR1), a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the plurality of SCPs includes different pairs of TSRs; wherein each set of SCPs comprises the same SSR and a plurality of different pairs of TSRs configured to hybridize to different target measurands on a same chromosome; amplifying by repeatedly: hybridizing the pairs of TSRs to target measurands in the mixture, and cutting SCPs having pairs of TSRs that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs) by nicking the SCPs without cutting the one or more target measurand, and; hybridizing the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; distributing the circularized cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain cSSR and some do not contain cSSRs; analyzing the engineered polynucleotide markers of the cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

[0058] In any of these examples, a first set of the plurality of sets of SCPs may comprise a first plurality of different pairs of TSRs configured to hybridize to different target measurands on a first chromosome, and each SCP of the first set of the plurality of sets of SCPs may comprise a same first forward primer region and a same first reverse primer region indicating the first chromosome. A second set of the plurality of sets of SCPs may comprise a second plurality of different pairs of TSRs and the circularizable signal specific region may comprise a second signal specific region (SSR) wherein the second plurality of different pairs of TSRs are configured to hybridize to different target measurands on a second chromosome, and wherein each SCP of the second set of the plurality of sets of SCPs comprising a same second forward primer region and a same second reverse primer region may indicate the second chromosome, further the second forward primer region may be the same or different from the first forward primer region and the second reverse primer region may be the same or different from the second forward primer region.

[0059] As mentioned, in any of these methods cutting SCPs to release cSSRs may comprise nicking the SCPs without cutting the one or more target measurand, and the methodmay include repeating the steps of hybridizing the pairs of TSRs to the target measurands in the mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs. Alternatively or additionally the method may include performing rolling amplification of the circularized cSSRs.

[0060] Either or both of TSR1 and TSR2 may comprise a Type IIS enzyme recognition site. In some examples TSR1 and TSR2 comprise different Type IIS enzyme recognition sites. In some examples TSR1 and TSR2 comprise a Type IIS enzyme recognition site and a Type II enzyme recognition site. TSR1 and TSR2 may be configured to hybridize to the target measurand at two regions that are spaced apart from each other by between about 1 and 50 bp.

[0061] Also described herein are kits configured and adapted specifically to perform these methods, including (but not limited to) the methods of determining fetal aneuploidy.

[0062] Also described herein are sequence conversion probes (SCPs) that include: a first target-specific region (TSR1) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR1 has at least 80% identity to a first single region of one chromosome within a human genome, wherein the TSR1 includes a restriction site for a first type Ils restriction enzyme having a cut or nicking site that is at or near a first end of the TSR1, further wherein the TSR1 has a GC content of greater than 50%; a second targetspecific region (TSR2) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR2 has at least 80% identity to a second single region of the one chromosome within the human genome and is within between 5 and 50 base pairs of the first single region, wherein the TSR2 includes a restriction site for a second type Ils restriction enzyme or a type II restriction enzyme having a cut or nicking site that is at or near a second end of the TSR2, further wherein the TSR2 has a GC content of greater than 50%, wherein the first type Ils restriction enzyme is different from the second type Ils restriction enzyme or the type II restriction enzyme; and a sequence-specific region (SSR) having a polynucleotide sequence extending greater than 40 bp, wherein the polynucleotide sequence of the SSR sequence does not occur in the human genome, further wherein the SSR has a CG content of greater than 50%, wherein the TSR1 is joined to the SSR at the first end of the SSR and the TSR 2 is joined to the SSR at the second end of the SSR, further wherein the restriction site is not part of a hairpin structure in the SCP.

[0063] These SCPs may be part of a kit. For example, described herein are kits comprising a plurality of sequence conversion probes (SCP), wherein each SCP of the plurality of SCPs comprises: a first plurality of target-specific regions (TSRls) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of each of the TSRlshas at least 80% identity to a first region of a first chromosome within a human genome, wherein the TSRls each include a restriction site for a first type Ils restriction enzyme having a cut or nicking site that is at or near a first end of the TSR1, further wherein each of the TSRls has a GC content of greater than 50%; a second plurality of target-specific regions (TSR2s) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of each of the TSR2s has at least 80% identity to a second region of the first chromosome within the human genome that is within between 5 and 50 base pairs of one of the TSRls, wherein the TSR2s include a restriction site for a second type Ils restriction enzyme or a type II restriction enzyme having a cut or nicking site that is at or near a second end of the TSR2, further wherein the TSR2 has a GC content of greater than 50%, wherein the first type Ils restriction enzyme is different from the second type Ils restriction enzyme or the type II restriction enzyme; and a sequence-specific region (SSR) common to each of the SCPs of the plurality of SCPs having a polynucleotide sequence extending greater than 40 bp, wherein the polynucleotide sequence of the SSR sequence does not occur in the human genome, further wherein the SSR has a CG content of greater than 50%, wherein the TSR1 is joined to the SSR at the first end of the SSR and the TSR 2 is joined to the SSR at the second end of the SSR, further wherein the restriction site is not part of a hairpin structure in the SCP.

[0064] Any of these kits may include a lock probe having a polynucleotide sequence configured to circularize the SSR.

[0065] The kit may include a second plurality of SCPs, wherein each SCP of the second plurality of SCPs comprises a pair of target-specific regions (TSRs) that are configured to bind to a second chromosome, further wherein each SCP of the second plurality of SCPs comprises a second sequence-specific region (SSR) common to each of the SCPs of the second plurality of SCPs. The second SSR may have a polynucleotide sequence extending greater than 40 bp and does not occur in the human genome. In some examples the second SSR has a CG content of greater than 50%.

[0066] In general, the signal specific region (SSR) (e.g., the engineered polynucleotide marker) may be designed to be approximately the same size, GC content, etc. between different signal specific regions so that they will be amplified with approximately the same frequency and / or efficiency, to prevent bias in the detection and / or measurements.

[0067] In any of the methods described herein, the cut SSRs (cSSRs) may be isolated from the un-cut (e.g., full length SCPs). For example, isolating the cSSRs may comprise using the affinity tag to separate the un-hybridized SCPs and the hybridized TSRs from the cSSRs.

[0068] In general, the sample mixture may be a biological sample or extracted from a biological sample. For example, the sample mixture may be blood or an extract of blood or any other bodily fluid and / or tissue. For example, as described herein in some variations the sample mixture is a blood sample taken from a pregnant woman that includes both maternal and fetal genetic material.

[0069] In some variations the TSR may be between the affinity tag and the SSR on each SCP. As mentioned, any appropriate target measurand may be used. For example, the one or more target measurand may be a DNA or an RNA. For example, the one or more target measurand may be an mRNA, a micro-RNA, an rRNA, an snRNA, or an RNAi, snoRNA, a guide RNA, a ribonuclease, a Y RNA, a telomerase RNA component, an antisense RNA (aRNA), a CRISPR RNA, a long noncoding RNA, a Piwi-interacting RNA, a small interfering RNA, a short hairpin RNA, a trans-acting siRNA, a repeat associated siRNA, an enhancer RNA, viral RNA, satellite RNA, genomic DNA, cfDNA, circulating tumor DNA, cell free fetal DNA (cffDNA), cell free maternal DNA, single stranded DNA (ssDNA), etc.

[0070] The target-specific region (TSR) of the sequence conversion probe (SCP) may be any appropriate length. For example, the TSR may be between about 13 and 100 bp long, e.g., between about 13-50 bp, between about 13-45 bp, between about 13-40 bp, between about 13-35 bp long, between about 13-30 bp long, etc. (e.g., between about 14-80 bp, between about 15-80 bp, etc.). In some variations, the TSR may be configured to hybridize to the one or more target measurand with 3 or fewer mismatches (e.g., with 2 or fewer mismatches, with 1 or fewer mismatches, etc.). The TSR is typically selected so that it represents a region of the genome that is unique to greater than 80%, meaning that the targetspecific region does not share 80% or more identity with any other region in the genome of the target organism (e.g., the human genome). The TSR is selected as described herein so that it will hybridize to only a single (target) region of the genome. The methods and compositions described herein may, however, allow for individual variations (e.g., polymorphisms) that may be present between individuals while still only hybridizing to the specified (unique) target region. For example, the methods described herein may allow hybridizing of the region with 80% or greater identity. Because the TSR is selected to be a region that is unique in the target genome up to 80% identity (allowing some variation / polymorphisms), the TSR may be used to identify target regions that are 80% or more (e.g., 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 95% or more, etc.) identity.

[0071] The sequence conversion probe (SCP) may include any appropriate affinity tag.The affinity tag may be on the target-specific region, e.g., opposite from the signal specificregion (SSR) of the SCP. In some variations the affinity tag is on the distal end of the TSR (e.g., opposite from the SSR, which may be either a 5’ or 3’ end). Although many of the examples described herein include biotin as the affinity tag (which may bind to a streptavidin group), any other affinity tag that may allow specific and secure binding between the affinity tag and the affinity partner may be used. For example, the affinity tag may be one or more of: a peptide tag or epitope tag or polynucleotide tag. Non-limiting examples of affinity tags may include: Albumin-binding protein (ABP), Alkaline Phosphatase (AP), AU1 epitope, AU5 epitope, Bacteriophage T7 epitope (T7-tag), Bacteriophage V5 epitope (V5-tag), Biotincarboxy carrier protein (BCCP), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), Chloramphenicol Acetyl Transferase (CAT), Cellulose binding domain (CBP), Chitin binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase (DHFR), E2 epitope, FLAG epitope, Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glutathione S-transferase (GST), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag (HAT), Horseradish Peroxidase (HRP), HSV epitope, Ketosteroid isomerase (KSI), KT3 epitope, LacZ, Luciferase, Maltose-binding protein (MBP), Myc epitope, NusA, PDZ domain, PDZ ligand, Polyarginine (Arg-tag), Polyaspartate (Asp-tag), Polycysteine (Cys-tag), Polyhistidine (His-tag), Polyphenylalanine (Phe-tag), Profinity eXact, Protein C, SI -tag, S-tag, Streptavadin-binding peptide (SBP), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep-tag, Streptavadin, Small Ubiquitin-like Modifier (SUMO), Tandem Affinity Purification (TAP), T7 epitope, Thioredoxin (Trx), TrpE, Ubiquitin, Universal, VSV-G, etc.

[0072] As mentioned, any of these methods may include cutting the SCPs that are hybridized to the one or more target measurands with a nuclease. Any appropriate nuclease may be used. For example, the nuclease may be one or more of: a flap endonuclease / 5’ nuclease, a double-strand nuclease, an RNA specific nuclease, a CAS. In some variations, the nuclease may comprise one of: an RNase H, a duplex-specific nuclease (DSN) (e.g. Evrogen cat# EA 001), a double-stranded DNase (dsDNase), and a restriction endonuclease. For example, the nuclease may be RNase H2. The one or more target measurand may be an RNA, and the first engineered polynucleotide marker may comprise a DNA. In general, the cut signal specific region (cSSR) may be isolated from the non-cut SCP (including non-cut SCP’s own SSR) by binding the affinity tag to a substrate to remove the un-hybridized SCPs and the hybridized TSRs. In some variations the substrate is a solid phase substrate, such as, e.g., a magnetic bead or microwell titer plate.

[0073] Any of the sequence conversion probes may include one or more predetermined cutting or cleavage regions, e.g., between the SSR and the TSR (and / or part of the TSR). Insome variations, particularly those using a RNase H2, for example, the SCP may include one or more ribose configured to serve as a cleavage site for cutting the SCPs having TSRs that are hybridized to the one or more target measurands.

[0074] In any of these methods, detecting the first engineered polynucleotide marker from the cSSRs may include amplifying the first engineered polynucleotide marker using one or more primers specific to the SSR.

[0075] As mentioned, the cut signal specific region may be detected specifically and effectively by one or more techniques described herein. For example, the SSR (and particularly the engineered polynucleotide marker) may be detected by amplifying the first engineered polynucleotide marker using a primer configured to hybridize to the first forward primer region and a primer configured to hybridize to the first reverse primer region. For example, detecting may include detecting the first engineered polynucleotide marker from the cSSRs by amplifying the first engineered polynucleotide marker using primers configured to hybridize to the different first forward primer regions and primers configured to hybridize to the first reverse primer regions. Alternatively or additionally, detecting the first engineered polynucleotide marker may include hybridizing the cSSR to a labeled signal probe and detecting a signal from the labeled signal probe. As described in greater detail herein, in some variations, the SSRs described herein may include only one primer region (and not a pair); for example, isothermal amplification and RCA with one primer may be used.

[0076] Detecting the first engineered polynucleotide marker may include hybridizing the cSSR to a labeled signal probe and to a capture probe and detecting a signal from the labeled signal probe.

[0077] As mentioned, also described herein are kits to perform any of the methods described herein. For example a kit may include a first plurality of sequence conversion probes (SCPs) directed against a plurality of target measurands, wherein each SCP comprises a pair of polynucleotide target-specific regions (TSRs) configured to hybridize with a target measurand of the plurality of target measurands, wherein the TSRs flank a signal specific region (SSR), the SSR comprising a first engineered polynucleotide marker; a pair of nuclease configured to cut or nick SCPs having TSRs that are hybridized to target measurand; and a substrate comprising a binding partner that specifically binds to the affinity tag.

[0078] In some variations a kit includes: a first plurality of sequence conversion probes (SCPs) directed against a plurality of target measurands, wherein each SCP comprises a pair of polynucleotide target-specific regions (TSRs) configured to hybridize with one target measurand of the plurality of target measurands, wherein the TSRs flank a signal specific region (SSR), the SSR comprising a first engineered polynucleotide marker; a nucleaseconfigured to cut SCPs having TSRs that are hybridized to target measurand; and a substrate comprising a binding partner that specifically binds to the affinity tag.

[0079] For example, a kit may include: a first plurality of sequence conversion probes (SCPs) directed against a first plurality of target measurands, wherein each SCP comprises: a pair of polynucleotide target-specific region (TSR) configured to hybridize with one target measurand of the first plurality of target measurands, and wherein the TSRs flank a signal specific region (SSR), the SSR comprising a first engineered polynucleotide marker between a first forward primer region and a first reverse primer region; one or more nuclease configured to cut or nick the SCPs having TSRs that are hybridized to target measurand; a first forward primer configured to hybridize to the first forward primer region and a first reverse primer configured to hybridize to the first reverse primer region; and a substrate comprising a binding partner that specifically binds to the affinity tag.

[0080] The plurality of SCPs may comprise a plurality of different first forward primer regions and first reverse primer regions, further wherein there are substantially fewer different first forward primer regions and first reverse primer regions than there are different TSRs. The plurality of SCPs may comprise a same first forward primer region and first reverse primer region.

[0081] Any of these kits may include a first forward primer configured to hybridize to the first forward primer region and a first reverse primer configured to hybridize to the first reverse primer region. In some variations, the kit may include one or more additional pluralities of sequence conversion probes (SCPs) directed against one or more additional pluralities of target measurands that are different from the first plurality of target measurands and different from each other.

[0082] In some variations of the kit, the SCPs comprise at least one ribose configured to serve as a cutting site for the nuclease. Any of these kits may include a capture probe configured to hybridize to the engineered polynucleotide marker and a capture probe configured to hybridize to the engineered polynucleotide marker or a complex of the capture probe and the engineered polynucleotide marker. Any of these kits may include a nucleic acid amplification master mix.

[0083] Also described herein are applications of the sequence conversion methods described. For example, described herein are methods of nucleic acid amplification. For example, a method may include: combining a plurality of sets of sequence conversion probes (SCPs) with a sample mixture including a plurality of target measurands, wherein each set of SCPs comprises a plurality of SCPs, wherein each SCP includes: a pair of polynucleotide target-specific regions (TSRs), and a signal specific region (SSR) comprising an engineeredpolynucleotide marker and one or more primer regions, wherein each set of SCPs comprises a plurality of different TSRs configured to hybridize to different target measurands and the same SSR; hybridizing the TSRs of the SCPs of the plurality of sets of SCPs to the target measurands; nicking SCPs having TSRs that are hybridized to target measurands to release cut SSRs (cSSRs) without releasing SSRs of un-hybridized SCPs or cutting the measurand; circularizing the cSSRs to functionally isolate them from un-hybridized SCPs and the hybridized TSRs; and performing digital PCR using the cSSRs and primers configured to hybridize to the one or more primer regions of the cSSRs to amplify the engineered polynucleotide markers.

[0084] In some variations the assays described herein may include or may be part of a method such as a digital PCR method. For example, a method of digital PCR may include: combining a mixture of genetic material with a mixture of sequence conversion probes (SCPs), wherein the SCPs each include a pair of target-specific regions (TSRs) configured to hybridize to one of a plurality of target measurands in the genetic material, and a signal specific region (SSR) comprising one of a set of engineered polynucleotide markers that indicates on which chromosome the target measurand that hybridizes to the TSR is located; hybridizing the TSRs of the mixture of SCPs to any target measurands in the genetic material; cutting or nicking the SCPs having hybridized TSRs to release cut signal specific regions (cSSRs) from the hybridized SCPs; optionally removing or destroying any SCPs that were not cut; and performing digital PCR using the pool of cSSRs and one or more primers specific to the cSSR that amplify the engineered polynucleotide markers of the cSSRs. Any of these methods may also include removing or destroying material / fetal genetic material that is bound to the TSR. Thus later steps of the method may proceed without the material / fetal genetic material.

[0085] The method may include performing digital PCR using the pool of cSSRs and the one or more primers comprises performing digital PCR using the cSSRs and a single pair of primers specific to all of the cSSRs. Any of these methods may include performing digital PCR using the pool of cSSRs and the one or more primers comprises performing digital PCR using a plurality of primers, wherein the total number of primers is less than a total number of target measurands.

[0086] The mixture of genetic material may comprise a mixture of maternal and fetal genetic material from a maternal blood sample.

[0087] As mentioned, cutting may comprise cutting with a nuclease (e.g., a flap endonuclease / 5’ nuclease, a double-strand nuclease, an RNA specific nuclease, a CAS, etc.) and in particular nicking endonucleases.

[0088] In general, the engineered polynucleotide markers described herein may be noncognate with the genetic material.

[0089] Performing digital PCR may comprise distributing the pool of cSSRs (and in some examples, circularized SSRs) into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain cSSRs and some do not contain cSSRs. These methods may include analyzing the engineered polynucleotide markers of the cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid chromosome in the reaction samples. The method may include determining the presence or absence of an aneuploidy by comparing said first number with said second number, wherein a differential between the first number and the second number indicates the presence of polyploidy and / or aneuploidy.

[0090] The mixture of maternal and fetal genetic material may be combined with the plurality of SCPs and may include combining the mixture of maternal and fetal genetic material with a second plurality of SCPs, wherein the second plurality of SCPs comprises a second plurality of different TSRs pairs configured to hybridize to different target measurands from the maternal and fetal genetic material, and each SCP of the second plurality of SCPs comprises the one or more second primer regions and one of a second plurality of different engineered polynucleotide markers. The first plurality of different engineered polynucleotide markers may indicate a first chromosome of the maternal and fetal genetic material and the second plurality of different engineered polynucleotide markers indicate a second chromosome of the maternal and fetal genetic material. The first plurality of different engineered polynucleotide markers may indicate different chromosomes of the maternal and fetal genetic material.

[0091] The engineered polynucleotide markers may each indicate a chromosome of the maternal and fetal genetic material. The plurality of SCPs may include a plurality of different TSRs configured to hybridize to target measurands on different chromosomes from the maternal and fetal genetic material, and the first plurality of different engineered polynucleotide markers may indicate which chromosome of the maternal and fetal genetic material a TSR corresponds to.

[0092] In general, one or more of the engineered polynucleotide markers, and the one or more second primer regions may be non-cognate with the maternal and fetal genetic material. The plurality of SCPs may comprise a plurality of different TSRs configured to hybridize todifferent target measurands on the same chromosome from the maternal and fetal genetic material.

[0093] The plurality of SCPs may comprise a plurality of different TSRs configured to hybridize to different target measurands on different chromosomes from the maternal and fetal genetic material. Distributing the cSSRs (and in some cases circularized SSRs) into the plurality of reaction samples may comprise distributing the cSSRs at low volume and / or high dilution whereby more engineered polynucleotide markers are detected in samples containing a trisomic or increased copy number of target measurand.

[0094] The mixture of maternal and fetal genetic material may be taken from less than 4 mL of blood.

[0095] The methods and compositions described herein may indicating the number of copies (or partial copies) of the one or more chromosomes being examined (e.g., chromosome 13, 18 and 21); for example, one or each one can be aneuploid (and / or polyploid). For example, 2 out of 3 may be euploid, 1 out of three may be euploid, and / or all of them or none of them may be euploid. Thus, the method described herein generally do not require the presence of a predefined euploid reference, and these methods may take the ratio and whichever one is “more” is aneuploid.

[0096] In any of the methods described herein, the assay may examine more than two chromosome (e.g., 13, 18 and 21, and in some variations, Y, plus X).

[0097] Any of the kits described herein may be used or adapted for use with a digital PCR method. For example, a kit that may be used for digital PCR as described herein may include, for example: a mixture of sequence conversion probes (SCPs), wherein the SCPs each include a pair of target-specific region (TSR) configured to hybridize to one of a plurality of target measurands in from a plurality of human chromosomes, a signal specific region (SSR) comprising one of a set of engineered polynucleotide markers that indicate on which chromosome the target measurand that hybridizes to the TSR is located, wherein the engineered polynucleotide marker is flanked by a forward primer region and reverse primer region; a nuclease configured to cut or nick SCPs having TSRs that are hybridized to target measurand; and a first forward primer configured to hybridize to the first forward primer region and a first reverse primer (in some variations) configured to hybridize to the first reverse primer region. The kit may include a set of probes configured to uniquely identify individual engineered polynucleotide markers.

[0098] Another variation of a method as described herein includes the use of one or more sequence conversion probes configured as inversion (e.g., padlock) probes. For example, described herein are methods comprising: combining a plurality of sets of circularizingsequence conversion probes (cSCPs) with a sample mixture including a plurality of target measurands, wherein each set of cSCPs comprises a plurality of cSCPs, wherein each cSCP includes: a first end comprising a sense probe configured to hybridize to a first target-specific region, a second end comprising an antisense probe configured to hybridize to a second target-specific region, and a signal specific region (SSR) comprising an engineered polynucleotide marker a forward primer region and a reverse primer region, wherein each set of cSCPs comprises the same SSR and a plurality of different first and second target-specific regions configured to hybridize to different target measurands; hybridizing the cSCPs of the plurality of sets of cSCPs to the target measurands to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSCPs of the circularization complexes; and performing digital PCR using circularized cSCPs and primers configured to hybridize to the forward primer region and the reverse primer region (e.g., in some variations, if needed) of the cSSRs to amplify the engineered polynucleotide markers.

[0099] For example, a method may include: combining a plurality of subsets of circularizing sequence conversion probes (cSCPs) with a sample mixture including a plurality of target measurands to form a combined mixture, wherein each subset of cSCPs comprises a plurality of cSCPs each having a same signal specific region (SSR) including an engineered polynucleotide marker and a pair of target-specific regions (TSRs) configured to hybridize to a different target measurand; hybridizing cSCPs of the plurality of subsets of cSCPs to the target measurands to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSCPs of the circularization complexes; contacting un-circularized cSCPs in the combined mixture with exonuclease to cut the un-circularized cSCPs; and performing digital PCR using the circularized cSCPs and primers configured to hybridize to one or more primer region of the SSR to amplify the engineered polynucleotide markers.

[0100] A sequence conversion probe configured as an inversion probe method may be configured as a method of determining a fetal aneuploidy.

[0101] As mentioned, the engineered polynucleotide marker may be flanked by the forward and reverse primer regions, further comprising isolating the circularized cSCPs from linear cSCPs using an exonuclease. The number of primers may be much less than the number of target measurands. The sample mixture may include a mixture of maternal and fetal genetic material from a maternal blood sample. Performing digital PCR may include distributing the pool of cSCPs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain cSCPs and some do not contain cSCPs. Themethod may further include analyzing the engineered polynucleotide markers of the cSCPs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid chromosome in the reaction samples.

[0102] Any of these methods may include determining the presence or absence of an aneuploidy by comparing said first number with said second number, wherein a differential between the first number and the second number indicates the presence of aneuploidy. The circularizing agent may be, for example, a ligase.

[0103] In general, the one or more target measurand may be a DNA or an RNA. The methods described herein may use or be adapted for use with isothermal amplification techniques (e g. NASBA, LAMP, NEAR, WGA, SDA, HD A, RD A, CPT etc ).

[0104] Also described herein are sequence conversion probes (SCPs) and methods of engineering them. For example, a sequence conversion probe (SCP), may include: a targetspecific region (TSR) extending between 15 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR is at least 80% identity to only a single region of one chromosome within a human genome, wherein the TSR includes a restriction site for a type Ils restriction enzyme having a cut site that is at or near a first end of the TSR, further wherein the TSR has a GC content of greater than 50%; and a sequence-specific region (SSR) having a polynucleotide sequence extending greater than 40 bp (e.g., greater than 45 bp, greater than 50 bp, greater than 55 bp, between 40 and 100 bp, between 40-90 bp, between 40-80 bp, between 50-100 bp, between 50-90 bp, between 50-80 bp, etc.), wherein the polynucleotide sequence of the SSR sequence does not occur in the human genome, further wherein the SSR has a CG content of greater than 50%, wherein the TSR is joined to the SSR at the first end, further wherein the restriction site is not part of a hairpin structure in the SCP. The SSR may include a forward primer region and a reverse primer region. For example, the SSR may include a forward primer region and a reverse primer region and an abasic region between the forward primer region and the reverse primer region.

[0105] The SCP may have a predicted secondary structure having an absolute minimum free energy (MFE) of less than 50 kcal / mol (e.g., less than 45, less than 40, less than 35, less than 30, etc.) kcal / mol.

[0106] The cut site may be for a type Ils restriction enzyme and may be at the 5’ end of the first TSR (e.g., TSR1). The first and second TSRs may have a melting temperature (Tm) of greater than 55 degrees C. The first and second TSRs may have a GC content of greater than 50%. The type Ils restriction enzyme may be one of: Bsal and BspQI. In some examplesthe type Ils restriction enzyme is one of: Bsal, BspQI, BbsI-HF, BtsCI, Btsl-v2, BtsIMutl, Faul, BsrDI, BsrI, BsmBI, BsmFI, or Bsml.

[0107] For example, a sequence conversion probe may include: a pair of target-specific regions (TSRs) extending between 15 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR is at least 80% identity to only a single region of one chromosome within a human genome, wherein the TSRs may each include a restriction site for a type Ils restriction enzyme (e.g., two different type IIS restriction enzymes on TSR1 and TSR2, or a type IIS and a type II restriction enzyme) having cut sites that are at or near an end of each of the TSRs; and a sequence-specific region (SSR) extending greater than 40 bp (e.g., greater than 45 bp, greater than 50 bp, greater than 55 bp, between 40 and 100 bp, between 40-90 bp, between 40-80 bp, between 50-100 bp, between 50-90 bp, between 50-80 bp, etc.), wherein a polynucleotide sequence of the SSR sequence does not occur in the human genome and will not anneal to human chromosomal polynucleotides. The SSR may include a primer, such as (but not limited to) a forward primer region and a reverse primer region and an abasic region between the forward primer region and the reverse primer region, wherein the TSR is joined to the SSR at the first end, further wherein the cognate restriction site is not part of a hairpin structure in the SCP and the SCP has a GC content that is greater than 50%. In some cases, e.g., where rolling amplification is used, a single primer may be used, or two parts of a primer that are joined into a single primer when circularized, are used.

[0108] For example, described herein are sequence conversion probes (SCPs) having: a pair of target-specific regions (TSR) extending between 15 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSRs are at least 80% identity to only a single region of one chromosome within a human genome; and a sequence-specific region (SSR) comprising a sequence of SEQ ID NO. 1, wherein the TSRs flank the SSR.

[0109] For example, a sequence conversion probe (SCP) may include: a target-specific region (TSR) extending between 15 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR is at least 80% identity to only a single region of one chromosome within a human genome; and a sequence-specific region (SSR) comprising: a polynucleotide of SEQ ID NO. 10 coupled to an abasic region that is coupled to a polynucleotide of SEQ ID NO. 11, wherein the TSR is joined to the SSR.

[0110] Also described herein are kits including any of the SCPs, and preferentially multiple types of SCPS that all have the same SSR. For example, a kit may include: a first sequence conversion probes (SCP) comprising: a first target-specific region (TSR) extending between 13 and 50 base pairs (bp) (e.g., between 14 and 50 bp, between 15 and 50 bp, etc.), wherein a polynucleotide sequence of the first TSR is at least 80% identity (at least 81%, atleast 82%, at least 83%, at least 84%, at least 85% identity, etc.) to only a single region of one chromosome within a human genome, wherein the first TSR includes a restriction site for a type Ils restriction enzyme having a cut site that is at or near a first end of the first TSR, further wherein the first TSR has a GC content of greater than 50%; and a first sequencespecific region (SSR) having a polynucleotide sequence extending greater than 40 bp (e.g., greater than 45 bp, greater than 50 bp, greater than 55 bp, between 40 and 100 bp, between 40-90 bp, between 40-80 bp, between 50-100 bp, between 50-90 bp, between 50-80 bp, etc.), wherein the polynucleotide sequence of the first SSR sequence does not occur in the human genome, further wherein the first SSR has a CG content of greater than 50%, wherein the first TSR is joined to the first SSR at the first end, further wherein the restriction site is not part of a hairpin structure in the first SCP; and a second SCP comprising: a second TSR extending between 15 and 50 base pairs (bp), wherein a polynucleotide sequence of the second TSR is at least 80% identity to only a single region of one chromosome within the human genome, wherein the second TSR includes the restriction site for the type Ils restriction enzyme having the cut site that is at or near a first end of the second TSR, further wherein the second TSR has a GC content of greater than 50%; and a second SSR having the same polynucleotide sequence as the first SSR, wherein the second TSR is joined to the second SSR at the first end, further wherein the restriction site is not part of a hairpin structure in the second SCP.

[0111] Any of these kits may also include primers (e.g., one or more of: a forward primer, a reverse primer, a link probe, etc.).

[0112] For example, described herein are methods, comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including a pair of targetspecific regions (TSRs) and a circularizable signal specific region (SSR), the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the first plurality of SCPs comprise a plurality of different pairs of TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the pairs of TSRs to the one or more target measurand within the sample mixture; nicking or cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs) without releasing SSRs of un-hybridized SCPs; and hybridizing each of the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0113] All or portions of the methods and compositions (including kits) described herein may be used with and / or may improve the methods and compositions described in International Patent Application No. PCT / US2021 / 036766, filed June 10, 2021, and titled “SEQUENCE CONVERSION REACTION,” (which claims priority to U.S. provisional patent application no. 63 / 037,575, titled “SEQUENCE CONVERSION REACTION,” and filed on June 10, 2020, and to U.S. provisional patent application no. 63 / 166,955, titled “SEQUENCE CONVERSION REACTION,” filed on March 26, 2021), and is herein incorporated by reference in its entirety.

[0114] Also described herein are sequence conversion probes (SCPs) that include a pair of target specific regions (TSRs) that flank a signal specific region (SSR) in which both TSRs hybridize to adjacent regions of a target polynucleotide, e.g., of a chromosome. These SCPs may be referred to herein as tandem TSR SCPs and may be used in any of the methods and compositions (e.g., kits) described herein, which may result in significantly lower background for any assays performed. As used herein, tandem TSRs may be arranged on either side (flanking) an SSR. Tandem TSRs may be configured to bind to adjacent (immediately adjacent or separated by a small gap, e.g., between 1-50 bp, 2-50 bp, 5-50 bp, etc.) regions of the target measurand (e.g., polynucleotide).

[0115] For example, described herein are sequence conversion probe (SCP), the SCP comprising: a first target-specific region (TSR1) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR1 has at least 80% identity to only a first single region of one chromosome within a human genome, wherein the TSR1 includes a restriction site for a first type Ils restriction enzyme having a cut site that is at or near a first end of the TSR1, further wherein the TSR1 has a GC content of greater than 50%; a second target-specific region (TSR2) extending between 14 and 50 base pairs (bp), wherein a polynucleotide sequence of the TSR2 has at least 80% identity to only a second single region of the one chromosome within a human genome and is within between 10 and 50 base pairs of the first single region, wherein the TSR2 includes a restriction site for a second type Ils restriction enzyme having a cut site that is at or near a second end of the TSR2, further wherein the TSR2 has a GC content of greater than 50%; and a sequence-specific region (SSR) having a polynucleotide sequence extending greater than 40 bp, wherein the polynucleotide sequence of the SSR sequence does not occur in the human genome, further wherein the SSR has a CG content of greater than 50%, wherein the TSR1 is joined to the SSR at the first end of the TSR1 and the TSR 2 is joined to the SSR at the second end of the TSR2, further wherein the cognate restriction site is not part of a hairpin structure in the SCP.

[0116] For example, a method may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including a first target-specific region (TSR1), a second target-specific region (TSR2) and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of TSRls and the TSR2s to the one or more target measurand within the sample mixture; cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs); and hybridizing each of the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0117] In some examples method may include: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including a first target-specific region (TSR1), a second target-specific region (TSR2) and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of TSRls and TSR2s to the one or more target measurand within the sample mixture; cutting SCPs having TSRls and TSR2s that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); and hybridizing each of the cSSRs to a lock probe having a first sequence to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker by performing digital PCR using the circularized cSSRs (cirSSR) and one or more primers configured to hybridize to the one or more primer regions to amplify the first engineered polynucleotide markers.

[0118] In some examples a method may include: combining a plurality of sets of sequence conversion probes (SCPs) with a sample mixture including a plurality of target measurands, wherein each set of SCPs comprises a plurality of SCPs, wherein each SCP includes: a first polynucleotide target-specific region (TSR1), a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the plurality of SCPs include different pairs of TSRs; wherein each set of SCPs comprises the same SSR, and a plurality of different pairs of TSRs, each configured to hybridize to a different target measurand; hybridizing the pairs of TSRs to target measurands in the sample mixture; cutting SCPs having pairs of TSRs that are hybridized to target measurands to release cut SSRs (cSSRs); hybridizing each of the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; and detecting the first engineered polynucleotide marker from the circularized cSSRs.

[0119] Any of these methods may include the additional steps of components of the procedures described above when using an SCP that is not a tandem TSR SCP.

[0120] Also described herein are methods of determining a fetal aneuploidy using a tandem TSR SCP. For example, the method may include: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP includes: a first polynucleotide target-specific region (TSR1), a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs and a circularizable signal specific region (SSR) between TSR1 and TSR2, the circularizable SSR comprising a first engineered polynucleotide marker including one or more primer regions, further wherein the plurality of SCPs includes different pairs of TSRs; wherein each set of SCPs comprises the same SSR and a plurality of different pairs of TSRs configured to hybridize to different target measurands; hybridizing the pairs of TSRs to target measurands in the mixture; cutting SCPs having pairs of TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); hybridizing each of the cSSRs to a lock probe to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; distributing the circularized cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain cSSR and some do not contain cSSRs; analyzing the engineered polynucleotide markers of the cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples;and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

[0121] A method of determining a fetal aneuploidy may comprise: combining a mixture of maternal and fetal genetic material with a plurality of sequence conversion probes (SCPs), wherein each SCP comprises a first polynucleotide target-specific region (TSR1), a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs and a signal specific region (SSR) between TSR1 and TSR2, wherein the pair of TSRs is configured to hybridize to a target measurand from the maternal and fetal genetic material, and wherein the SSR comprises an engineered polynucleotide marker including one or more primer regions, further wherein the plurality of SCPs comprises a plurality of different pairs of TSRs configured to hybridize to different target measurands from the maternal and fetal genetic material, and each SCP of the plurality of SCPs comprises one of a first plurality of different engineered polynucleotide markers; hybridizing the pairs of TSRs of the plurality of SCPs to the target measurands; cutting SCPs from the plurality of SCPs having pairs of TSRs that are hybridized to the target measurands (in some cases without leaving an overhang, or optionally, leaving an overhand), to release cut SSRs (cSSRs); hybridizing each of the cSSRs to a lock probe having a first polynucleotide sequence, to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; distributing the circularized cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain circularized cSSRs and some do not contain circularized cSSRs; analyzing the engineered polynucleotide markers of the circularized cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; determining the presence or absence of a fetal aneuploidy by identifying a differential between the first number and the second number.

[0122] A method of determining a fetal aneuploidy may include: combining a mixture of maternal and fetal genetic material with a plurality of sequence conversion probes (SCPs) comprising a first subset of SCPs and a second subset of SCPs, wherein each of the SCPs in the plurality of SCPs comprises: a first plurality of pairs of TSRs including a first polynucleotide target-specific region (TSR1) and a second polynucleotide target-specific region (TSR2), the TSR1 and TSR2 forming a pair of TSRs, and a signal specific region (SSR) between each TSR1 and TSR2, wherein the pair of TSRs is configured to hybridize to a target measurand from the maternal and fetal genetic material, and wherein the SSRcomprises an engineered polynucleotide marker having a forward primer region and a reverse primer region, further wherein the first subset of SCPs comprises a first plurality of different pairs of TSRs configured to hybridize to different target measurands on a first chromosome, and each SCP of the first subset of SCPs comprises a same first forward primer region and a same first reverse primer region indicating the first chromosome, further wherein the second subset of SCPs comprises a second plurality of TSRs including a third polynucleotide targetspecific region (TSR3), a fourth polynucleotide target-specific region (TSR4), the TSR3 and TSR4 forming a second pair of TSRs and a signal specific region (SSR) between TSR3 and TSR4, wherein the second plurality of TSRs are configured to hybridize to different target measurands on a second chromosome, and each SCP of the second subset of SCPs comprises a same second forward primer region and a same second reverse primer region indicating the second chromosome, wherein the second forward primer region is the same or different from the first forward primer region and the second reverse primer region is the same or different from the second forward primer region; hybridizing the first pairs of TSRs and the second pairs of TSRs of the plurality of SCPs to the target measurands; cutting SCPs from the plurality of SCPs having first pairs of TSRs and second pairs of TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); isolating the cSSRs; hybridizing each of the cSSRs to a lock probe having a first polynucleotide sequence, to produce circularization complexes; contacting the circularization complexes with a circularizing agent to circularize the cSSRs; distributing the circularized cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain circularized cSSRs and some do not contain circularized cSSRs; analyzing the engineered polynucleotide markers of the circularized cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

[0123] A method may include: combining a plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including first target-specific region (TSR1), a second targetspecific region (TSR2), and a signal specific region (SSR) between TSR1 and TSR2, the SSR comprising a first engineered polynucleotide marker, further wherein the plurality of SCPs include different TSRls and TSR2s; hybridizing the TSRls and TSR2s of the SCPs to the one or more target measurand; cutting SCPs having TSRls and TSR2s that are hybridized tothe one or more target measurands to release cut SSRs (cSSRs); and detecting the first engineered polynucleotide marker from the cSSRs.

[0124] Any of these methods may include digital PCR using tandem TSR SCPs. For example, a method of digital PCR may include: combining a mixture of genetic material with a mixture of sequence conversion probes (SCPs), wherein the SCPs each include a fist targetspecific region (TSR1), a second target-specific region (TSR2), forming a pair of TSRs that are configured to hybridize to one of a plurality of target measurands in the genetic material, and a signal specific region (SSR) comprising one of a set of engineered polynucleotide markers that each indicates a subset of the target measurands, wherein the SSR is between TSR1 and TSR2; hybridizing the TSRs of the mixture of SCPs to target measurands in the genetic material; cutting the SCPs having hybridized pairs of TSRs to release cut signal specific regions (cSSRs) from the hybridized SCPs; removing any SCPs that were not cut using an affinity marker adjacent to the TSRs of the SCPs to form a pool of cSSRs; and performing digital PCR using the pool of cSSRs and one or more primers specific to the cSSR that amplify the engineered polynucleotide markers of the cSSRs.

[0125] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0115] FIG. 1 A illustrates an example of a sequence conversion method using a sequence conversion probe (SCP) including a signal specific region (SSR) that may be circularized by a lock probe and a ligase (forming a circularized SSR or cirSSR). In FIG. 1 A, the assay includes a plurality of sequence conversion probes that, after a target sequence region (TSR) of the SCP hybridizes to a target region (e.g., of measurand), may be cut by a restriction enzyme to release the signal specific region that may be circularized by a lock probe and a ligase to form cirSSR. Linear DNA (including hybridized DNA) may be digested, and remaining cirSSR may be amplified and / or detected. This may be part of a digital PCR quantification method, e.g., to detect chromosome ploidy.

[0116] FIG. IB illustrates in more detail the circularization and amplification steps shown in FIG. 1 A.

[0117] FIG. 1C shows another example, similar to FIG. IB, in which the cut SSR is configured as a circularizable SSR that may be circularized by a lock probe in the presence of ligase. In FIG. 1C, the SSR does not necessarily include a separate first and second hybridization region (e.g., Hybl, Hyb2), but the lock probe may be configured to hybridize to the ends of the SSR where the ends may include the probe region and a primer region (e.g., forward primer).

[0118] FIG. ID illustrates one example of a workflow that may be used for the procedures described herein, including but not limited to the procedure of FIG. 1A (e.g., as part of a method for detecting chromosomal ploidy).

[0119] FIG. IE illustrates an example of a workflow that may be used for some of the methods described herein.

[0120] FIGS. 2A-2B illustrate examples of a variation of a sequence conversion method configured as a primer-driven assay. In this example, a first primer-coupled target-specific probe is configured to hybridize to a target measurand and be cut, e.g., by a nuclease, to release a sequence conversion primer (SC primer) that is then used to selectively extend a sequence conversion template (SCT). Extended SCT (the “copy of the SCT” or cSCT) may then be separated from un-extended SCT, and the extended SCT, or a signal specific region (SSR) on the copy, may be detected. In FIG. 2A the SC primer is combined with an affinity tagged sequence conversion template (SCT) and extended to make a copy of the sequence conversion template. In FIG. 2B the SC primer may be affinity tagged and, when hybridized to a circularized (“Cyclic template”) sequence conversion template (SCT) to form a copy of the sequence conversion template (cSCT). The cSCT may be separated from the affinity tagged SCT (and from the original genetic material) and used for quantification, e.g., by direct quantification or by amplification (e.g., by digital PCR, etc.).

[0121] FIG. 3 A illustrates an example of an SCP formed as described herein, including aSSR (having an abasic region between a revere primer region and a forward primer region), and a TSR including a BspQI restriction site.

[0122] FIG. 3B illustrates an example of an SCP as described herein, similar to that shown in FIG.3A, and including a 5' SSR probe orientation.

[0123] FIG. 4A-4D, which is similar to FIG. IB, schematically illustrates one example of an SSR lock probe (FIG. 4A) that may be circularized using a lock probe (FIGS. 4B, 4C) and used to quantify a genetic signal (FIG. 4D), as described herein.

[0124] FIG. 5A illustrates an example of a first portion of a sequence conversion method as described herein. In FIG. 5 A, a sequence conversion probe (SCP) for converting a target polynucleotide (target measurand) into an engineered polynucleotide combined with a samplemixture to be examined for a target measurand (e.g., shown as genomic DNA in FIG. 5 A). SSR is released (as cSSR) only from those SCPs that hybridized to target measurand by digestion of hybridized SCPs.

[0125] FIG. 5B schematically illustrates conversion of the cSSR from FIG. 5 A into a sequence (which does not include any genomic, e.g., target measurand, sequence) that may be detected using nucleic acid amplification and / or by hybridization. In FIG. 5B an SSR extension probe is selectively annealed to the 3’ end of the cSSR by ligation and the resulting final amplicon (cSSR+SSR extension probe) is a linear polynucleotide product that may be detected to indicate the presence and / or amount of target measurand in the sample.

[0126] FIG. 5C schematically illustrates another example of conversion of the cSSR from FIG. 5 A into a sequence (which does not include any genomic, e.g., target measurand, sequence) that may be detected using nucleic acid amplification and / or by hybridization. In FIG. 5C an SSR extension probe is added to the 3’ end of the cSSR and the entire product (of cSSR+SSR extension probe) is circularized. The resulting circular SSR (cirSSR) may be detected to indicate the presence and / or amount of target measurand in the sample.

[0127] FIG. 6A shows an example a sequence conversion probe with a 5’ TSR as described herein.

[0128] FIG. 6B illustrates a mixture of potential synthesized products from a chemically synthesized SCP which may potentially lead to background.

[0129] FIG. 7A illustrates one example of an SCP having tandem TSR regions (“tandem TSR SCP”).

[0130] FIG. 7B shows an example of a chemical byproduct when forming a tandem TSR SCP.

[0131] FIG. 8 schematically illustrates hybridization of a tandem TSR SCP as described herein.

[0132] FIG. 9 schematically illustrates one example of an SCP having a pair of tandem TSRs each with recognition sites for nicking restriction enzymes.

[0133] FIG. 10 schematically illustrates cycling (e.g., recycling) to amplify the production of cut SSRs (cSSRs) when using tandem TSRs by reusing the target measurand.

[0134] FIG. 11 is a graph illustrating the effect of cycling ) to amplify the production of cut SSRs (cSSRs) when using tandem TSRs, for different concentrations of measurand.

[0135] FIG. 12 is a graph showing the increase in production of cSSR when cycling using different concentrations of target measurand.

[0136] FIG. 13 is a graph showing the increased sensitivity for target measurand using cycling to amplify the production of cut SSRs (cSSRs) when using tandem TSRs.

[0137] FIG. 14 graphically illustrates the use of SCPs with tandem TSRs and cycling across different types of target measurand.

[0138] FIG. 15 is a graph showing the effective use of cycling with tandem TSRs across different sources of measurand for different examples of SCPs.

[0139] FIG. 16 schematically illustrates one example of rolling circle amplification which may be used in any stage of the methods described herein after the formation of the cSSR and circularization of the cSSRs.

[0140] FIGS. 17A and 17B illustrate examples of a step of hybridizing a lock probe that is blocked to prevent extension which may be part of any of the methods and compositions (e.g., kits) described herein. In FIG. 17A the lock probe is blocked by the phosphate at the 3’ end. In FIG. 17B the lock probe is blocked by the inclusion of a 3’ overhang (as well as a phosphate at the 3’ end).

[0141] FIG. 17C illustrates an example of a step of hybridizing a lock probe and a lock cassette sequence (e.g., probe cassette) and ligating the cassette into a cut SSR.

[0142] FIG. 17D is a table showing that cassette ligation (as shown in FIG. 17C) may increase ligation specificity in the SCP reactions described herein and dramatically reduce background.

[0143] FIG. 18A schematically illustrates an example of a sequence conversion method using a sequence conversion probe (SCP) including a signal specific region (SSR) including one or more deoxyuridines; the SSR may be circularized by a lock probe and a ligase as described herein.

[0144] FIG. 18B schematically illustrates an example of a portion of a sequence conversion method as described herein in which one or more deoxyuridines (dU) may be used to cut a circularized and ligated cSSR including a cassette region to form an inverted cSSR.

[0145] FIG. 19 illustrates a gap filling lock probe design comprising a primer sequence in the gap filling region that may increase ligation specificity as described herein.

[0146] FIG. 20 schematically illustrates an example of one variation of a method of using a sequence conversion probe to amplify a signal from a target.

[0147] FIG. 21 schematically illustrates an example of one variation of a method of using a sequence conversion probe to amplify a signal from a target using rolling circle amplification and UNG / UDG cleanup.

[0148] FIG. 22 schematically illustrates an example of one variation of a method of using a sequence conversion probe to amplify a signal from a target using a padlock primer extension as described herein.

[0149] FIGS. 23A and 23B show additional detail to the method of using padlock primer extension.

[0150] FIG. 24A illustrates one example of a sequence specific region including forward and reverse primers for padlock primer extension.

[0151] FIG. 24B illustrates a modified sequence specific region including forward and reverse primers for padlock primer extension.

[0152] FIGS. 25A-25D illustrate examples of assays using different variations of forward and reverse primers for padlock primer extension.

[0153] FIG. 26 illustrates a method combining a number of the components described herein for performing a highly sensitive and low-noise sequence conversion assay to detect one or more target sequences, including the use of a lock probe, lock cassette and dU-cutting to form an inverted cSSR (which may be referred to as “dU ligation inversion”).

[0154] FIGS. 27A-27B illustrate the surprising effectiveness of dU ligation inversion. FIG. 27A shows a graph output of a background assay and FIGS. 27B is a table showing the results.

[0155] FIGS. 28A-28B illustrate the use of dU ligation inversion using in a digital PCR assay under different conditions.

[0156] FIG. 29 is a comparison of SCPs having one TSR versus two TSRs, showing an advantage in some cases for two-TSR SCPs.DETAILED DESCRIPTION

[0157] Provided herein are compositions, kits, and methods for the detection and quantitation of genetic markers, including in particular the quantification of chromosomes, including detection of trisomy. The methods and compositions described herein can simplify and improve the detection and quantitation of genetic analytes in bacterial, human or species, including pathogens, as well as detection of chromosomes, genes, etc. These genetic markers may be referred to as measurands, and the methods and compositions described herein may provide methods for converting a plurality of measurands into a smaller number or even a single unified measurand. For example, the disclosure herein provides methods for converting a plurality of different nucleic acid targets for one or more alleles present on a chromosome into a single measurand. This approach may simplify assays using nucleic acid amplification and hybridization quantification techniques, such as digital counting methods, including (but not limited to) digital PCR. It may also improve the accuracy of those methods. The current state of the art for performing non-invasive prenatal testing (NIPT) by PCR requires many unique primers and / or probes as each unique measurand requires twounique primers and may require a unique probe as well. The methods and compositions disclosed herein simplifies the chemistry to a few or even as little as one primer pair (2 primers) and 3 unique probes, for example, when using NIPT for detecting trisomy 13, 18 and 21. This approach is broadly applicable to hybridization quantification methods.

[0158] For example, this disclosure may significantly simplify the design for NIPT dPCR assays, may offer flexibility to easily select target measurand sequences including shorter sequences, may reduce the complexity of an assay for NIPT to a few or even a single primer pair for all chromosomes and may only require a single probe per chromosome, while allowing enough alleles on each chromosome to be targeted / tested to meet the statistically required number of replicates to achieve needed clinical performance.

[0159] The methods and compositions (including kits) described herein may require only two primers and three probes to allow digital PCR analysis, resulting in a clean reaction.Starting with maternal and fetal DNA extracted from 1 2 mL of plasma to achieve 100 000 counts per chromosome 13, 18 and 21 it is estimated with conventional methods 600 primers and 300 probes are needed, for 200 000 counts 1200 primers and 600 probes.

[0160] These simplifications increase the robustness of the assay and allow targeting a high number of sequences without having to worry about primer / primer / probe interaction causing an unspecific reaction.Definitions:

[0161] As used herein: accuracy refers to closeness of a measurement to a specific or correct value. Analyte refers to a substance of interest in an analysis, such as a nucleic acid of a specific chromosome, a specific virus, a specific bacterium, etc.

[0162] Analyte specific SSR refers to a common signal specific region (SSR) sequence for a defined analyte (e.g. a specific chromosome, a specific virus, a specific bacterium, etc.). The "analyte specific SSR" is connected on sequence conversion probe (SCPs) with different "measurand specific TSR". These sequence conversion probe (SCP) detect a common analyte by detecting a plurality of analyte specific measurands (e.g. different alleles on chromosomes or pathogens).

[0163] Aneuploidy refers to the state where the wrong number of chromosomes (e.g., the wrong number of full chromosomes or the wrong number of chromosome segments, such as the presence of deletions or duplications of a chromosome segment) is present in a cell. In the case of a somatic human cell it may refer to the case where a cell does not contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. In the case of a human gamete, it may refer to the case where a cell does not contain one of each of the 23 chromosomes. In the case of a single chromosome type, it may refer to the case where more or less than twohomologous, but non-identical chromosome copies are present, or where there are two chromosome copies present that originate from the same parent. In some embodiments, the deletion of a chromosome segment is a microdeletion.

[0164] In the Figures, “B” refers to an affinity tag, including (but not limited to) biotin or another affinity tag. The affinity tag may allow removal of unwanted moieties such as following hybridization and cutting steps.

[0165] Chromosome refers to either a full chromosome, or a segment or section of a chromosome.

[0166] CRISPR-Cas refers to a system for controlled or programmable genome engineering. CRISPR refers to clustered regularly interspaced short palindromic repeats. Cas is an endonuclease that cuts DNA at a location specified by a specific RNA (may be referred to as a guide RNA).

[0167] cirSSR is a circularized exonuclease resistant signal specific region (SSR) derived from a cSSR.

[0168] cSSR is a signal specific region (SSR) of a sequence conversion probe (SCP) after cutting such as by e.g., a nuclease.

[0169] cTSR is a target-specific region (TSR) portion of the sequence conversion probe (SCP) after cutting by e.g., a nuclease.

[0170] Digital PCR (dPCR) is an alternate method to conventional qRT-PCR for absolute quantification and detection of nucleic acid molecules. dPCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions; some of these reactions contain the target nucleic acid molecule (positive) while others do not (negative) and amplifying the sample. A single starting molecule of DNA or cDNA can be amplified a million-fold or more. During amplification, dye-labeled probes are used to detect sequencespecific targets. When no target sequence is present, no signal accumulates.Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without the need for standards or endogenous controls.

[0171] Duplex refers to DNA:DNA double strand or DNA:RNA double strand.

[0172] Lock Probe refers to an oligonucleotide which, in the presence of ligase, facilitates formation of a cirSIR.

[0173] The term “kit” (or “kit of parts”) refers to an article of manufacture including one or more containers and, optionally, a data carrier. The one or more containers may be filled with one or more of the reagents described herein. Additional containers may be included in the kit that contain, e.g., diluents, buffers and further reagents such as dNTPs. The datacarrier may be a non-electronic data carrier, e.g., a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronic data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronic data carrier. The access code may allow the access to a database, e.g., an internet database, a centralized, or a decentralized database. The data carrier may include instructions for the use of the kit in the methods described herein. The data carrier may include a cut-off value or reference level. In case that the data carrier includes an access code which allows the access to a database, the threshold value or reference level is deposited in this database. In addition, the data carrier may comprise information or instructions on how to carry out the methods in this disclosure.

[0174] As used herein, a measurand refers to a nucleic acid representing one of many possible sequence alleles of (or in) an analyte. Thus, a target measurand refers to a portion of a genetic material (e.g., chromosome, DNA, RNA, etc.) that is being identified and / or measured.

[0175] Measurand specific TSR refers to a target-specific region (TSR) specific for a measurand. Different "measurand specific TSRs" can be linked to a common "analyte specific SSR".

[0176] NIPT refers to non-invasive prenatal testing and in some cases may refer to trisomy-based analysis on cell-free DNA in maternal blood or plasma.

[0177] Nucleic acid or nucleic acid molecule refers to DNA and / or RNA.

[0178] Nuclease refers to an enzyme that degrades nucleic acids by breaking bonds between nucleotides in the nucleic acids. Nucleases can act on DNA (DNase) and / or RNA (RNase). A duplex specific nuclease (DSN) selectively cuts DNA in a nucleic acid duplex (DNA / DNA) or heteroduplex (e.g., RNA / DNA). A duplex specific nuclease may leave one strand or part of one strand intact. Many nucleases are commercially available and include, but are not limited to: Restriction enzymes, 5 ’-3’ nucleases, RNase H, RNase H2, RNase HII, RNase V, duplex-specific nuclease (DSN), lambda exonuclease, T7 exonuclease, exonuclease III, RecJf, exonuclease I, exonuclease T, exonuclease V, BAL-31 nuclease, mung bean nuclease, DNase I, micrococcal nuclease, T7 endonuclease I, T5 exonuclease, Cas9 and any combination thereof. Information regarding nucleases and their substrates is found, e.g., in Mishra, Nawin C; (Nucleases: Molecular Biology and Applications, Wiley- Interscience (ISBN: 978-0-471-39461-7). Additional nucleases of interest include but are not limited to: RNase VI (e.g., for cutting dsRNA), oligoribonucleases, (e.g., for cutting oligonucleotides), exoribonuclease II (e.g., for cutting mature miRNAs).

[0179] Oligonucleotide refers to a single-stranded multimer of nucleotides from 2 to about 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are under 10 to 50 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides or "RNA oligonucleotides") or deoxyribonucleotide monomers (i.e., may be oligodeoxyribonucleotides or "DNA oligonucleotides") or chemical linker. Oligonucleotides may be 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotides in length, for example.

[0180] Padlock Probe refers to an oligonucleotide composed of two target complementary segments connected by a linker that may carry detectable functions. The two ends of the linear oligonucleotide probes are brought in juxtaposition by hybridization to a target sequence (e.g. a Lock Probe). This juxtaposition allows the two probe segments to be covalently joined by the action of a DNA ligase. In some examples described herein, a padlock probe may be a specific type of inversion probe in which there is no gap between the target complementary regions at the ends of the probe (e.g., the target complementary regions span the entire target region upon hybridization, leaving no gaps). In general, an inversion probe may include no gaps (as in a padlock probe) or may include a gap (e.g., a gap of 1 bp, a gap of 2 bp, a gap of 3 bp, etc.). Inversion probes having larger gaps (e.g., > 2-5 bp, >2 bp, >3 bp, >4 bp, >5 bp, etc.) may be referred to as connector inversion probes.

[0181] Primer refers to a single DNA molecule (a DNA oligomer) or a collection of DNA molecules (DNA oligomers) in which the DNA molecules are identical, or nearly so, and where the primer contains a region that is designed to hybridize to a targeted locus (e.g. a targeted polymorphic locus or a nonpolymorphic locus) and may contain a priming sequence designed to allow PCR amplification.

[0182] Primer orientation refers to the direction in which the elongation of the primer in DNA synthesis occurs.

[0183] R refers to one or more Ribonucleotides or other nucleotide analog or a recognition site for a Restriction Enzyme; they may allow site-specific cutting upon hybridization to a nucleic acid target.

[0184] SCA refers to sequence conversion method.

[0185] SCORE refers to a sequence conversion reaction. A sequence conversion reaction “converts” a nucleic acid into a different form (sometimes referred to as a “proxy”).

[0186] SCP refers to sequence conversion probe.

[0187] SSR refers to a signal specific region.

[0188] Sequence refers to a DNA sequence or a genetic sequence. It may refer to the primary, physical structure of the DNA molecule or strand in an individual. It may refer to the sequence of nucleotides found in that DNA molecule, or the complementary strand to the DNA molecule. It may refer to the information contained in the DNA molecule as its representation in silico.

[0189] Subject relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, e.g., an animal such as a rodent, a rabbit, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In some embodiments, the subject is a pregnant human female.

[0190] A substrate refers to a substance on which an enzyme acts. For example, T21, T18, T13 refer to trisomy of chromosomes 21, 18, 13, respectively.

[0191] TSR refers to target-specific region. A unique target-specific region represents a unique measurand. (e.g. a specific allele sequence)

[0192] An analyte can be quantified by a sum of measurements, e.g. the analyte human chromosome 21 includes potentially thousands of measurands represented by thousands of detectable sequences across chromosome 21. Chromosome 21 has a length of ~48 Mbases. This theoretically represents 480,000 unique measurands of an average length of 100 bases.

[0193] This method uses, in some embodiments "analyte specific SSRs," e.g. chromosome specific SSR, linked to "measurand specific TSR" (e.g., specific to a unique sequence allele on the chromosome).Sequence Conversion method

[0194] A sequence conversion method (e.g., a method of performing a sequence conversion reaction) may detect a target of interest by converting the target of interest from a target measurand into a different form, e.g., an engineered “signal specific region” (SSR) that may act as a proxy for the target measurand, and then detecting the signal specific region (proxy). In some examples, the target measurand may be, for example, part of a chromosome allele. A sequence conversion probe (SCP) may have at least two different functional domains: a first, target-specific region (TSR) is configured to hybridize to the measurand of a target, and the second, the signal specific region (SSR) is engineered to be readily amplifiable and / or detectable.

[0195] The target-specific region of the sequence conversion probe (SCP) may hybridize to a target measurand (e.g., a portion of a chromosome of interest) under appropriate annealing conditions to form a target-sequence conversion probe complex (target-SCP). The target-sequence conversion probe complex (target-SCP) may have a double stranded region where the target measurand and target-specific region (TSR) of the sequence conversionprobe are hybridized and a single stranded region, the signal specific region (SSR). When complexed with a target measurand, the sequence conversion probe (SCP) may include a nuclease recognition site and exposing the target-sequence conversion probe complex (target- SCP) to the nuclease results in cutting of the sequence conversion probe (SCP) at the nuclease recognition site and forming, in some examples, a double stranded region where the target and target-specific region (TSR) of the sequence conversion probe is hybridized and a separate cleaved off single stranded region, the signal specific region. Although the target measurand is shown as a polynucleotide fragment, it may be any size, and may be part of a longer polynucleotide chain. The target measurand may be any appropriate polynucleotide, and the SCP may be adapted for use with a particular type of target (e.g., RNA, DNA, length of oligonucleotide etc.).

[0196] In some examples the affinity tag may be a biotin (B) and its binding partner, streptavidin (SA) may be coupled to a solid substrate (e.g., magnetic beads or microtiterplate). The sequence conversion probe (SCP) may be labeled with an affinity tag to aid in removal of unwanted forms of the sequence conversion probe; in particular, they may be labeled on the target-specific region. The streptavidin on the magnetic beads will bind to biotin affinity tag on the nucleic acid. The biotin-labeled nucleic acid may be the singlestranded uncleaved SCP or the target-specific region that is cleaved from the sequence conversion probe (which may be hybridized to the target measurand. Thus, uncleaved sequence conversion probe (SCP) (and in this variation, target DNA) may be removed from the reaction. A magnet field can be used to separate the streptavidin coated magnetic beads and bound biotinylated DNA from the signal specific region. For example, the cleaved signal specific regions can be removed from the magnetic beads (or vice versa), such as by using magnets or a spin column to separate out the magnetic beads. The solution can be analyzed for the presence of the signal specific region to determine the presence or absence of target measurand in the original sample. Any appropriate detection method may be used. For example, amplification and detection or hybridization and detection may be used.Amplification and detection may be performed using a nucleotide amplification technique, including but not limited to digital PCR. Amplification may use one or more primers, such as in some variations a forward primer (primer a) and a reverse primer (primer b). In some variations this amplification may be quantified by use of a florescent marker, such as the Taqman assay including a probe with a fluorophore (R) and a quencher (Q). Other amplification detection techniques may be used.

[0197] Direct visualization of the cleaved SSR (cSSR) may also be used. For example, hybridization and detection may use a capture probe attached or made to be attached to asolid phase and configured to hybridize to the signal specific region. Alternatively or additionally, a signal probe that is configured to bind (e.g., hybridize) the SSR (or the combination of the SSR and capture probe) may be used, as shown. The signal probe may include a signal label, such as a fluorescent label. The capture probe and / or signal probe may hybridize by annealing to the SSR, and / or they may specifically bind by other molecular interaction. For example, the probe(s) may be antibody probes (or portions of antibodies, such as Fab fragments, etc.), aptamer probes, or the like.

[0198] The SSR may be engineered to have a predetermined length, or range of lengths, polynucleotide content (e.g., GC ratio, etc.), and / or sequence. In particular in variations in which multiple different SSRs are used, either to indicate different specific target measurands (individual measurands) or to identify groups of target measurands (e.g., grouped by gene, by chromosome, by genetic pathway, by pathogen, mutations indicative of cancer, etc.), the SSRs may be distinct from each other, but may be engineered to have similar functional properties, such as melting temperature, primer regions, length, etc. This may help prevent bias that may otherwise occur during detection (e.g., amplification and / or binding).

[0199] Any of these assays may include the use of multiple SCPs and / or subsets of SCPs. Each subset of SCP may include the same SSR and different TSRs, as will be described in more detail below. In some variations the SSR that is released (cut or cleaved) from the SCP may be unlabeled or not directly labeled. In other variations the SSR may be directly labeled. A target measurand may be converted into an engineered signal specific region (SSR), and then may be detected.

[0200] Any of the methods and compositions (e.g., kits) described herein may be used to provide input into a procedure including digital PCR to provide quantitative data.

[0201] The methods and compositions described herein may also be used to detect very short target measurand sequences.

[0202] In general, the target-specific region may be designed so that binding to the target measurand is optimal. For example, the TSR may be configured to bind to at least a 5 mer and can be directed to the coding (gene) portions of the genome. The sequences may be restricted to contain at least half GC (so 3 to 6 for 6 mers). When identifying good target measurand regions, regions that are repeated (e.g., multiple copy regions) may be preferred, because they may increase the overall signal or maybe avoided if e.g. accurate chromosome counting is desired.

[0203] The use of a sequence conversion assay in which the sequence conversion probe is configured to be an inversion probe (e.g., a padlock probe) may be particularly advantageous. As described above, in some variations very strict tolerances may be necessary, particularlyin variations adapted for use with NIPT, as described herein. Sequence conversion assays that include a circularizing SSR may address this need, particularly where all non-circularized DNA (including hybridized polynucleotides and un-hybridized polynucleotides) may be digested and removed (e.g., by the use of a nuclease, such as a DNase, exonuclease, etc.). This approach may not necessarily rely on the affinity marker (e.g., SA-biotin) SCP cleanup, and in some variations may not include an affinity marker on the SCP. Optionally, the affinity marker may be included.

[0204] As described above, in some variations, the SSR may be engineered so that the SSR only becomes PCR-detectable after being separated from the SCP and circularized, e.g., by including primers oriented away from each other rather than towards each other (FIG. IB). This may further ensure that the final signal is only generated by the released SSR (e.g., cut SSR) that has been circularized. In general, as described above, the SSR may include an engineered polynucleotide marker; different SCPs may include the same SSR, providing a reductionist assay in which multiple target-specific regions (e.g., all shaping a higher-order relationship, such as a same chromosome, same gene, same allele, etc.) may be coupled to the same SSR output. In case where the SSR only becomes PCR-detectable after being separated from the SCP and circularized the exonuclease cleanup step becomes optional because the linear (uncircularized) SSR cannot be amplified and generate a signal.

[0205] FIG. 1 A illustrates one example of a method of implementing such an embodiment. In FIG. 1 A, the cut SSR forms a protected single stranded DNA circle (closed circle inversion probe) from the released SSR. This circularized SSR (cirSSR) is resistant to exonuclease treatment, allowing subsequent degradation of the intact linear SCP through exonucleases, as shown in FIG. 1 A. This reaction is highly specific because the SCP cannot be circularized; the SSR may be circularized when it is released from the SCP.

[0206] For example, in FIG. 1 A, the SCP includes an SSR and TSR region (which may be oriented with either the TSR at the 3’ end or at the 5’ end, as described above. Optionally the TSR may be coupled to (or may include) an affinity marker; in some variations, no affinity marker is included. If included, the affinity marker may be used to bind to a solid phase (e.g., Streptavidin-coated magnetic beads) as described above in reference to FIG. IB. The SCP may also include a sequence specific region (SSR) that is configured to be circularized after being released by cutting with a nuclease, as shown in FIG. 1 A. In this example, when the cut SSR is hybridized to a lock probe and combined with ligase, the cut SSR circularizes to form a circularized SSR (cirSSR). To circularize the linear cSSR it may be hybridized to a specific lock probe, allowing ligase-mediated circularization after the cSSR was released by restriction enzyme activity from the SCP upon hybridization to itsDNA target. The lock probe includes regions that recognized the 5’ end and the 3’ end of the cut SSR (with one end complementary to the end of the cut SSR in an inverted orientation and the opposite end complementary to the other end of the cut SSR in an un-inverted orientation, so that the cut SSR is hybridized in a circular manner). After ligation, one or more nuclease (e.g., exonuclease) may optionally be added to digest linear DNA, including intact SCP. The circularized SSR will not be cut by one or more enzymes that digest linear DNA and may be used to remove the uncut SCP as well as the target DNA (measurand DNA) and TSR. The circular cSSR is then amplified in the downstream PCR or dPCR without unspecific signal contribution by the undigested SCP. Only the circular SSR may be detected, e.g., using primers regions in the SSR that are oriented so that they may only be amplified when the SSR is circularized, resulting in a detectable probe, as shown in FIG. 1 A. As mentioned above, an optional affinity tag (i.e. biotin) may be used for one or more additional cleanup steps in addition to the circularization and exonuclease digestion. It may be beneficial to engineer into the SSR between the primer sites a restriction site allowing linearization of the cirSSR with a restriction enzyme before start of PCR.

[0207] In general, the use of a nuclease (e.g., an endonuclease, such as a DNase) in the method shown in FIG. 1 A is optional (indicated by the dashed arrow). Because the direction of the priming regions on the SSR are only correctly oriented once the SSR has been cut and circularized, the amplification and / or detection may be sufficiently specific even in the presence of the un-circularized SSR (including full or truncated SCP).

[0208] FIG. IB shows a more detailed example of the circularization of a cut SSR from a sequence conversion probe including an inversion probe as described herein. In FIG. IB, the cut SSR is engineered to include a 5’ hybridization end region (first hybridization region, Hybl) and a 3’ hybridization end region (second hybridization region, Hyb2) as well as a forward primer region and a second primer region and a probe region. Optionally, the first hybridization region (Hybl) may comprise part of the forward primer binding region and the second hybridization region (Hyb2) may comprise part of the probe binding region, as shown in FIG 1C. This optional design to use part of the forward primer and / or probe binding region in the hybridization regions results in an SSR of reduced length compared to an SSR with separate hybridization and primer binding regions. This optional design is possible due to the fully synthetic and engineered nature of the SSR. The engineered probe region may be a synthetic (e.g., engineered, non-naturally occurring region) that may be used for detection of the cut SSR, as described herein. In FIG. IB, the cut SSR may include the forward primer region (for hybridizing to a first, forward, primer) and a reverse primer region (for hybridizing to a second, reverse, primer). In the cut SSR, the forward and reverse primersmay be arranged in tandem. These regions may be oriented so that once circularized, following hybridization to a lock probe, as shown in FIG. IB, and ligated to circularize the cut SSR, the forward and reverse primers may be oriented towards each other, allowing replication (e.g., amplification) of the region of the cut SSR including the probe. In FIG. IB, this is shown by the final amplicon, which includes the forward primer region at the 5’ end and the reverse primer region at the 3’ end, with the probe between the forward and reverse primer. In some variations the probe may include or incorporate one or both of Hybl and Hyb2. Thus, in this example, the padlock structure of the SSR only becomes PCR detectable after being separated from the SCP and circularized. In the ligated, circularized inversion probe shown in FIG. IB (which in some variations may be padlock probe), the star shown in the circularized SSR represents a ligated nick, which may allow a PCR primer to amplify across the ligated junction. In some variations, the probe may be configured to include a TaqMan probe binding region. As described, to further improve specificity, primer regions (for hybridizing to primers) and probe regions may be arranged on the SSR in such a manner that they only become PCR amplifiable after circularization, forming a padlock probe. The padlock arrangement may depend on cut SSR circularization and may create a specific cSSR signal even in the presence of SCP. This system may be highly specific, because the primers that hybridize to the forward and reverse primer regions in the SSR may point in the wrong direction (e.g., away from each other) when the cut SSR is in the linear form, but may point in the right direction (e.g., towards each other) in the circularized cut SSR configuration. In FIGS. 1 A-1C, the inversion and / or padlock probes are configured to hybridize to sequences that are not found in the genomic target. Instead, the hybridization sequences are derived from a synthetic target.

[0209] FIG. ID illustrates one example of a method (e.g., workflow) as described herein. This method may be used with a circular cSSR as described. In this example, the method incorporates the general method described in FIG. 1 A, in which the sequence conversion probe is configured to be an inversion probe; in particular, in this variation the SCP includes SSR that is configured to be circularized by a lock probe. The lock probe may be generic to all of the cut SSRs (e.g., the 3’ and 5’ end regions of the SSRs having different engineered identification (probe) regions may be the same); alternatively, different cut SSRs may have different lock probes that may be used (e.g., as part of a pool of lock probes).

[0210] In FIG. ID, the workflow may begin by adding a mixture including the target measurand to the chamber (e.g., well or tube) 2101; the mixture may contain, for example, a mixture of material and fetal genomic material (e.g., as from a maternal blood sample). This material may be an extract of the fetal and maternal DNA (e.g., a 50 pL portion of theextracted DNA in FIG. ID). The SCP material may be added to the mixture including the measurand 2102. The SCP material may be, for example, a portion of a mixture of SCPs that include a plurality of different target-specific regions (TSRs), where the different TSRs are divided into two or more subsets sharing the same SSR, as described above. For example, in FIG. ID, 20 pL of the SCP mixture may be added to the mixture and heated to melt the DNA (e.g., heated to approximately 95°C), then cooled (e.g., to approximately 50°C) to allow hybridization of the TSRs to the measurand in the mixture 2102. Following hybridization, a nuclease (e.g., a restriction enzyme) may be added to the mixture 2103, to cut any SCP that has hybridized to a measurand. For example, 10 pL of restriction enzyme may be added and incubated at 1 hour (e.g., at 50°C) followed by heat-inactivation of the restriction enzyme (20 minutes at 80°C); this may release the SSR by cutting the SCP. The lock probe(s) and ligase may then be added (together or sequentially) to circularize the SSR 2104. For example, in FIG. ID, 20 pL of the ligase and lock probe(s) may be added and incubated for 2 hours at 50°C. Following circularization, in some variations the linear DNA in the mixture may be digested 2105, e.g., by (optionally) adding a nuclease (e.g., 10 pL of exonuclease) and incubating (e.g., 1 hour at 37°C, followed by heat inactivation at, e.g., 80°C for 20 minutes). A sample of the resulting mixture may then be taken 2106 for detection and / or amplification (e.g., a 5-10 pL sample of the 100 to 110 pL supernatant may be used in one or more dPCR procedure as described herein). Note that as the material is added to the well or tube as shown in FIG. ID, the mixture may optionally be mixed (agitated, vortexed, etc.).

[0211] This workflow, which in this example does not depend on a biotin streptavidin clean-up, may be highly specific for cut SSR amplification and may prevent all or most of the background that may otherwise arise from un-cut SCP, when using an exonuclease digestion destroying the un-cut SCP.

[0212] Another example of a workflow for use with a sequence conversion method as described herein, similar to that of FIG. ID, is shown in FIG. 21. As in FIG. ID, in FIG. IE the amounts (shown in pL) are exemplary only; other volumes may be used. Similarly, the temperatures and times are examples only and may be modified. In FIG. IE the sequence conversion probe is configured to be cut to release a cut SSR (e.g., a cS SR and / or a short cSSR). The resulting cSSR may be kept linear, or it may be circularized. In some examples an additional primer, such as an SSR extension probe may be ligated to an end (e.g., the 3’ end) using a lock probe.

[0213] In FIG. IE, the workflow may begin by adding a mixture to be tested to identify the presence and / or amount of a target measurand to a chamber (e.g., well or tube); the mixture may contain, for example, a mixture of material and fetal genomic material (e.g., asfrom a maternal blood sample). This material may be an extract of the fetal and maternal DNA (e.g., a 50 pL portion of the extracted DNA in FIG. IE). The SCP material may be added to the mixture potentially including the measurand. The SCP material may be, for example, a portion of a mixture of SCPs that include a plurality of different target-specific regions (TSRs), where the different TSRs are divided into two or more subsets sharing the same SSR, as described above. For example, in FIG. IE, 10 pL of an SCP mixture may be added to the sample mixture and heated to melt the DNA (e.g., heated to approximately 95°C), then cooled (e.g., to approximately 50°C) to allow hybridization of the TSRs to any measurand in the mixture. Following hybridization, a nuclease (e.g., a restriction enzyme such as a type Ils restriction enzyme) may be added to the mixture, to cut SCP that has hybridized to a measurand, releasing a cut SSR (cSSR). For example, 10 pL of restriction enzyme may be added and incubated at 1 hour (e.g., at 50°C) followed by heat-inactivation of the restriction enzyme (20 minutes at 80°C). Optionally, the mixture with cSSRs (e.g., short cSSRs) may be cleaned by adding streptavidin magnetic beads, removing uncleaved SCP and cut TSR by separating the streptavidin magnetic beads from the mixture including the cSSRs.

[0214] In some examples, as described in greater detail below, lock probe(s), SSR extension probe, and ligase may then be added (together or sequentially) to ligate the SSR extension probe to an end of the short cSSR. In some examples this may result in circular cSSR (cirSSR); alternatively the cSSR (“long cSSR” comprising the short cSSR and SSR extension probe) may be left linear. For example, in FIG. IE, 10 pL of ligase and lock probe(s) may be added and incubated for 2 hours at 50°C. In some examples (e.g., when circularized) linear DNA in the mixture may be digested, e.g., by (optionally) adding a nuclease (e.g., 10 pL of exonuclease) and incubating (e.g., 1 hour at 37°C, followed by heat inactivation at, e.g., 80°C for 20 minutes). A sample of the resulting mixture may then be taken for detection and / or amplification (e.g., a 70-80 pL supernatant sample may be used in one or more dPCR procedure as described herein). Note that as the material is added to the well or tube as shown in FIG. IE, the mixture may optionally be mixed (agitated, vortexed, etc.). This workflow may be highly specific for cut SSR amplification and may prevent all or most of the background that may otherwise arise from un-cut SCP.

[0215] Another variation of a sequence conversion method is shown in FIGS. 2A and 2B and includes a primer driven sequence conversion method. In the primer-driven variation of the sequence conversion method described herein the primer-coupled target-specific probe (PCTSP) may be used to initially hybridize with the target and release a primer (a sequence conversion primer or SC Primer) when a target measurand is present. The PCTSP once hybridized to a target measurand is cleaved by a nuclease (e.g., exonuclease) to release theSC Primer. The SC primer may then prime extension of a sequence conversion template (SCT) that includes a signal specific region (SSR), forming a copy of the sequence conversion template (cSCT). The SCT includes an affinity tag (shown as biotin in FIG. 2A), or in some variations may be bound to a solid phase substrate either though an affinity tag or otherwise. The SCT may be isolated from copied SCT and the copied SCT may then be detected, either by an amplification detection or by direct detection. For example in some variations the copies of the sequence conversion template (cSCTs) may be analyzed via digital PCR (e.g., directed to the SSR region as described above).

[0216] FIG. 2B is another variation of the method described in FIG. 2A, in which the same primer-coupled target-specific probes may be used to generate an SC Primer as described above, however the primer may hybridize to a complementary SC region of a circular (rather than linear, as shown in FIG. 2A) sequence conversion template (SCT). As shown in FIG. 2B, the SC primer may hybridize to the circular, single-stranded SCT to create a copy of the sequence conversion template (cSCT), which (depending on the assay conditions) may have multiple copies of the SSR as the single primer drives replication. This may provide some amplification of the detection.

[0217] In the variation shown in FIG. 2B the SC primer region of the primer-coupled target-specific probe (PCTSP) includes an affinity tag. Thus, the copies of the cyclic template (the copies of the circular sequence conversion template) each incorporate the affinity marker at the end incorporating the SC Primer (in FIG. 2B, the 5’ end). The affinity -tagged cSCT may then be separated from the circular sequence conversion templates and assayed, e.g., in a digital PCR assay.

[0218] In general, the methods and compositions described herein may allow for the conversion of one oligonucleotide sequence into another one, including converting several oligonucleotide sequences into one sequence. Examples of this may include converting RNA into DNA, converting long sequences into short sequences, short sequences into long sequences, converting a first sequence into another sequence, increasing the apparent copy number of an analyte, and the like.

[0219] In general the engineered SSR sequence may be designated such that it has desirable properties that the target measurand sequence does not have. Thus, these methods and compositions may simplify (e.g., convert, translate) many sequences into few, e.g., consolidating them into a single sequence. As described herein, this may change the length of a target sequence to a length making it detectable or easier to detect. For example, PCR amplicons typically need to have a certain length to accommodate forward and backward primer and, in the case of TaqMan PCR, an additional probe binding region is needed.Existing technologies for detecting polynucleotides are often limited by the size of the polynucleotide, typically requiring a target region length of about 100 bases or longer, preventing shorter DNA or RNA targets (e.g., of e.g. 25 bases or smaller) from being easily detected. The methods and compositions described herein may enable detection of such targets.

[0220] An SCP may be synthesized in any appropriate manner. SCP probes can be made, for example by coupling several different multi-mers that have been pre-manufactured. For example, it might be advantageous to add the S SR as a single building block, or several building blocks, since it would be constant, and the chemical linkage does not have to be a nucleotide or DNA. It might include any other chemical linker. From a structural perspective the SCP does not need to be 100% oligonucleotide.

[0221] Another variation of a sequence conversion method is shown FIG. 5A. In this example, the method includes a sequence conversion probe (SCP) that is a polynucleotide including a target-specific region (TSR) and a signal specific region (SSR). The SSR may be a circularizable SSR. The SSR includes a first engineered polynucleotide marker including one or more primer regions (shown in this example as a forward primer, FB, but it may be a reverse primer, RP). The 5’ end of the SSR may include a ligation blocker, such as 5 ’OH, 5’ biotin, 5’ inverted bases, 5’ digoxin. 5’ D-bases. 3’ PO4, 3’ biotin, 3’ inverted bases, 3’ D- bases. In FIG. 5 A the ligation blocker on the 5’ end of the SCP is shown as 5’D. The 3’ end of the SCP includes one (or more) biotin.

[0222] In any of the methods described herein a plurality of SCPs may be used together. The plurality of SCPs may include a plurality of different TSRs configured to hybridize to different target measurands, while each of the SCP in this (first) plurality may include the same SSR, as described above. In FIG. 5A, the SSR is referred to as a “short SSR” because it may later be annealed to an SSR extension probe, as described below. The SSR may include an abasic or other region to prevent transcription (shown by black circled functional region). One or both ends of the SSR (short SSR) may be configured to hybridize to a linker or lock probe, as shown in FIGS. 5B and 5C, respectively.

[0223] In FIG. 5A, the SCP is added to a sample mixture to be tested for the presence of one (or in some examples, more) target measurand. As described herein, the target measurand may be a genomic (e.g., chromosomal) target, and the mixture may include a sample to be tested. If a target measurand corresponding to the TSR is present in the sample, the SCP may be hybridized (via annealing) so that the TSR region hybridizes to the target measurand, shown in this example and a genomic target measurand; any appropriate measurand may be used, as described above. A nuclease (such as, but not limited to, a type Ils restrictionenzyme) may be used to cut the SCP, releasing the cut SSR (cSSR). Optionally, the sample may be cleaned using streptavidin (e.g., magnetic streptavidin beads) to remove the uncut SCP and the cut TSR, as shown. The resulting cSSR (or “short cSSR”) may then be processed as shown in FIG. 5B (linear product) or FIG. 5C (circular product).

[0224] Optionally, in some examples an SSR extension probe may be added to the end (e.g., the 3’ end) of the short cSSR. In FIG. 5B the SSR extension probe is added, along with a lock probe (which may also be referred to as a linker probe) to ligate the SSR extension probe to the 3’ end of the short cSSR, in order to form the final amplicon, that may be detected. The SSR extension probe may include a second primer region (e.g., including a reverser primer, RP, or a forward primer, FP), shown including a reverse primer in FIG. 5B. The 3’ end of the SSR extension probe in FIG. 5B includes a ligation blocker (shown here as 3’P*) to prevent improper / undesirable ligation. In this example, the lock probe hybridizes to both the short cSSR and the SSR extension probe. The resulting final amplicon may then be amplified using primers directed to the first and second primer regions, as shown. FIG. 5B shows a linear final amplicon. Alternatively, in some examples the final amplicon may be circular, as shown in FIG. 5C.

[0225] In FIG. 5C, as in FIG. 5B, an SSR extension probe may be added to the end of the cut (“short”) SSR (short cSSR). In this example, the cSSR includes a first primer region (including a FP or a RP), and a probe region. A transcription interruption region (shown as a rectangle) may be present between the first primer region and the 5’ end of the SSR, such as an abasic region. The 5’ end of this example SSR also includes a ligation blocker. As in FIG. 5B an SSR extension probe is also included. The lock probe in any of these examples may also be a ligation blocker on one or both ends (as shown in FIGS. 5B and 5C). The short cSSR may be circularized with the lock probe so that the SSR extension probe is ligated into the resulting circularized cSSR (cirSSR). The cirSSR may then be detected as described above, forming a final amplicon between the first (e.g. forward) and second (e.g., reverse) primer regions, as shown.

[0226] The use of an SSR extension probe is optional, and may be used to dramatically reduce background, which may be particularly helpful in some examples. In general, the SSR extension probe may include an appropriate number of nucleotides (e.g., between 6 and 40 or more), and the sequence may be specific to the second primer region.

[0227] The workflow shown in FIG. IE may be used with the technique illustrated in FIGS. 5A-5C illustrated above. The method shown in FIG. IE may incorporate the general method described in FIGS. 5A-5C, in which the sequence conversion probe is configured to be cut to release a short cut SSR (short cSSR) that may be annealed to an SSR extensionprobe using a lock probe. The resulting SSR may be kept linear, or it may be circularized by the lock probe. The lock probe may be generic to all of the cut SSRs (e.g., the 3’ and 5’ end regions of the SSRs having different engineered identification (probe) regions may be the same); alternatively, different cut SSRs may have different lock probes that may be used (e.g., as part of a pool of lock probes). EXAMPLES

[0228] The sequence conversion methods described herein (e.g., using the SCP probes described) may be used for and / or as part of a variety of different techniques. For example, these methods may be used with dPCR for applications including (but not limited to): detection of aneuploidy by digital chromosome counting using cffDNA, somatic mutation panels for oncology, syndromic pathogen panels, etc.

[0229] For example, the somatic mutation panels for oncology may be beneficial to detect a number of known cancer driver somatic mutations in oncogenes, such as EGFR, KRAS, BRAF to help guide therapy selection or screen for cancer. Such mutations are known in the literature and can be found in various databases (e.g. COSMIC). One would hybridize SCPs to mutation sites of interest from gene A (e.g. EGFR) with multiple SCP probes each with unique TSRs but all with a common SSR (e.g. with a probe binding site for a FAM- labeled probe). The TSRs would be perfectly matched to the mutations of interest (e.g. EGFR L858R, T790M, etc.) but would be mismatched to the wild type gene. As a result, only the SCPs bound to the perfectly matched mutant sites would be cleaved by the nuclease. The multiple SCPs for a second gene (gene B, e.g. KRAS) would similarly have unique TSRs for the mutations of interest in gene B but would have a common SSR (e.g. with a probe binding site for a HEX-labeled probe). One would amplify and detect the resulting SSRs in dPCR with as few as one primer pair and a probe specifically labeled for each gene.

[0230] Any of the methods and kits described herein may be used for minimal residual disease monitoring. Minimal residual disease (MRD) refers to the small number of cancer cells or cell-free circulating DNA derived from such cancer cells that remain in the body after treatment. The number of remaining cells may be so small that they do not cause any physical signs or symptoms and often cannot even be detected through traditional methods, such as viewing cells under a microscope and / or by tracking abnormal serum proteins in the blood. A MRD positive test result means that residual (remaining) disease was detected. A negative result means that residual disease was not detected. The methods and kit described herein may therefore be used to detect markers of cancer cells or cell-free circulating DNA from minimal residual disease. After treating cancer, any remaining cancer cells in the body can become active and start to multiply, causing a relapse of the disease. Detecting MRD mayindicate that the treatment was not completely effective or that the treatment was incomplete. Minimal residual disease may be present after treatment because not all of the cancer cells responded to the therapy, or because the cancer cells became resistant to the medications used.

[0231] Any of the methods and system (e.g., kits) described herein may be used for detecting transplant rejection. For example, the methods described herein may detect donor- derived cell -free DNA and may therefore track DNA markers from the organ donor that appear in the blood (or other tissues or fluids) of the transplant recipient. For example, injured or dying cells from the donor organ release donor DNA fragments into the bloodstream, and higher amounts of donor DNA indicate a higher risk for transplant rejection in the recipient. The methods and systems (e.g., kits) described herein can be used to detect relative amounts of donor and patient DNA, similar to the methods for detecting fetal and maternal DNA described herein. Current tests used to detect heart transplant rejection rely on frequent and painful biopsies of heart tissue. Those biopsies run the risk of damaging the heart and are limited by their invasiveness (ability to obtain tissue samples) and reliability for detecting acute rejection. Acute, or rapid, rejection tends to happen in the first three to six months after transplantation as opposed to chronic rejection, which occurs after many years. For example, blood samples may be monitored the patients for signs of acute rejection using the assays described herein.

[0232] These methods and compositions may be configured as syndromic pathogen panels. Such panels may be beneficial to detect groups of pathogenic bacteria, yeast and / or fungi with SCP probes in conjunction with detection (e.g., by dPCR). For example, multiple enteric pathogens causing gastrointestinal distress could be detected with SCPs including one or more target sequences (e.g. 16S RNA) in one or more bacteria (e.g. Campylobacter, C. difficile, etc.) that could lead to the same symptoms (e.g. acute diarrhea) and that are treated in the same manner (e.g. same classes of antibiotics). The SCPs would have multiple unique TSRs but share a common SSR for common treatment modality (Fam for the antibiotic class A; Hex for antibiotic class B).Example 1

[0233] In one example, the methods described herein may be used to identify the ploidy of a genetic sample.

[0234] Traditional methods for using dPCR to detect aneuploidy require a large number of primers and probes to achieve statistical significance.

[0235] For example, these methods may be used to identify target measurands (e.g., 100 to 200 target measurands) on each of chromosomes 21, 18 and 13 each. This information(e.g., the number of identified target measurands from each chromosome) may be used to determine the ploidy of each of these chromosomes. In some variations the method may include determining the length of each TSR with a ribose to hybridize at 70°C plus minus 10°C that will not dissociate after cleavage. Ribose may be placed as close to the 3' end as is possible to minimize cycling / amplification. The method may also optionally include determining for the SSR amplification part as an ideal amplicon. The primers for the C1C18 / C13 may all be identical, and probes to each of C21, C18 and C13 may be standard TaqMan probes specific for C21, C18 and C13. Each Signal Conversion probe (SCP) may have an affinity tag at one end, such as, but not limited to biotin at the 5' end. Assuming 200 target measurands each for C21 / 13 / 18 and a 100,000 well or droplet array, the method may include getting 200 different SCPs for C21, 200 different SCPs for Cl 8, and getting 200 different SCPs for C13. Probes may be mixed at 1 : 1 : 1 ratio. There should be now roughly 600 SCPs in equimolar ratio. The total amount may be determined experimentally but can be expected to be at least 10-fold higher than the target DNA concentration. A typical concentration of an individual probe could be in the range of 5pM 500pM. Incubation time, temperature, and buffer condition may impact the optimal conditions. The nuclease (e.g., RNase H2) can be added before, together or after addition of SCP, however for some nucleases (e.g. restriction enzymes) it is often preferred to be added last. For example, restriction enzymes need to be added after SCP hybridization otherwise they could destroy the target DNA allele before the SCP can bind. Once the reaction has been completed, uncleaved SCP and cTSR will be removed with streptavidin bound to a solid phase (e.g. beads, column, magnetic beads, etc.). The supernatant or an aliquot may be mixed with the PCR master mix for the digital PCR reaction and run following standard protocols known in the art.

[0236] The number of positive T 21, T 18 and T 13 counts may then be compared and based on mathematical transformations (e.g. difference, ratio or other) to determine if the result is normal or abnormal.Example 2: Sequence Conversion Method for NIPT (detection of Trisomy 13, 18 and 21) using restriction enzyme

[0237] As mentioned above, in some variations a restriction endonuclease (“restriction enzyme”) may be used. For example, in some variations the ploidy of a sample of genetic material including chromosomal material may be determined using the methods described herein.

[0238] In any of the methods described herein in which the nuclease is used to separate the SSR from the SCP, the restriction enzyme to be used may be determined and therestriction site may be engineered into the SCP. For example, selection criterion for determining a restriction enzyme may be based on the specific needs of the methodology used, and may include the recognition site being approximately ~6 bases long (other length restriction sites work as well); the enzyme begin stable and effective at above 45°C. The enzyme chosen may be selected because it can be heat inactivated. In some variations, the recognition site may be found in the potential targets’ sequences with sufficient frequency (ideally >500) on each of chromosome 13, 18 and 21, to support assay statistical requirements (e.g., the number of probes per chromosome).

[0239] In designing the SCPs, the identified restriction sites may be extended to the target-specific region (TSR) sequences. For example, the method may include identifying 200 or more sequence targets on chromosome 21, 18 and 13 each based on the chosen restriction enzyme. Possible candidate restriction enzymes may include BsmI (NEB cat# R0134L), BsmBI-v2 (NEB cat# R0739S), BspQI (NEB cat# R0712S), Bsal (NEB cat# R0535S), BsaI-HF-v2 (NEB cat# R3733S) or BssHII (NEB cat# R0199L). Alleles with the selected restriction enzyme recognition site sequence may be identified for use on chromosomes 13, 18 and 21. To determine the 5’-end for the target-specific region the Tm of the probe may be defined. Since many of these restriction enzyme work well above 45°C (the reaction temperature), the TSR should ideally be fully bound at the reaction temperature.Therefore, a Tm of the probe above 55°C may be used as a target. Based on this Tm in 50mM NaCl, a TSR probe length of 10 40 bases may be ideal, depending on GC content. The TSR binding sequence may be extended (using bioinformatic techniques) for more than 6 bases downstream (direction 3’). The sequence upstream (direction 5’) may be extended as needed to achieve the desired Tm. Unique sequences may be identified: sequences that are found on chromosomes other than the intended chromosome with an identity (or homology) of more than 80% may be discarded, especially those carrying the enzyme’s recognition site. Sequences binding to common SNPs (e.g. >~1% frequency in the human population) in the restriction site may be filtered out. The method may also include ensuring the remaining sequences do not overlap with each other.

[0240] As used herein, when comparing polynucleotide sequences, two sequences are said to be “identical” if the sequence of nucleotides in each of the two sequences is the same when the sequences are aligned for maximum correspondence. The percentage identity between two nucleotide sequences as described herein may be determined according to art- accepted practices and criteria, for instance, the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990).

[0241] The sequences for the Signal Specific Region (SSR) may also be determined. For example, by identifying (using bioinformatics) 3 oligo sequences not found in the human genome or human pathogens long enough to harbor a PCR amplicon. The 3 sequences may be synthetic (have very little homology or identity with the human genome), may contain two universal primer binding sites (one at the 5 ’-end, one at the 3 ’-end of the SSR) same for all three oligonucleotides, the oligo may contain a TaqMan probe binding site specific for Chromosome 13, 18 or 21 between the primer sites.

[0242] The final Sequence Conversion Probe (SCP) design may be determined by concatenating each of 200 identified Target-specific Regions (TSRs) for Chromosome 13, 18 and 21 with their respective universal Signal Specific Region (Cl 3 SSR, Cl 8 SSR and C21 SSR). The TSR may be at the 5’ end and will be synthesized with a 5’ biotin tag; the SSR is at the 3’ end. This results in 200 each chromosome specific Sequence Conversion Probes. The approximately 600 HPLC purified probes may be generated, providing, for example, approximately 200 identified SCPs each for Chromosomes 13, 18 and 21.

[0243] Primers and probes may be optimized for dPCR cycling conditions with an extension temperature of 58°C. For example, the primer Tm may be 58 60°C, the probe Tm may be 68 70°C. The first probe (e.g., probe 1, C21) may include a reporter 1 (e.g., FAM) and a Quencher (e.g., Iowa Black). The second probe (probe 2, Cl 8) may include a Reporter 2 (e.g., HEX) and a Quencher (e.g., Iowa Black). The third probe (probe 3, C13) may include a reporter 3 (e.g., Cy5), and a Quencher (e.g., Iowa Black). The primers and probe for dPCR may be optimized to be compatible with the Bio Rad ddPCR Supermix for Probes (Catalog number: 186-3010 for 500 reactions).

[0244] The method may include: (1) maternal and fetal cfDNA Extraction, (2) sequence Conversion Reaction, (3) cleanup of sequence conversion reaction, (4) partitioning (e.g. droplet generation) of cSSRs, (5) dPCR and (6) data analysis.

[0245] Sample collection and DNA extraction may include isolating plasma (e.g., within five days from blood draw). An average of 9.5 mL whole blood may be collected in cell-free DNA BCT® blood collection tubes (Streck) via a double centrifugation protocol consisting of a first centrifugation step at 1342 x g for 30 minutes, transfer of the plasma fraction to a secondary tube and a second centrifugation step at 2267 x g for 20 minutes. Plasma is stored at -80 C until further processing.

[0246] Extract cfDNA from 1 mL of plasma of a pregnant female as described by the manufacturer. Reagents: Maxwell® RSC ccfDNA Plasma Kit (Catalog number: AS 1480); Instrument: ProMega Maxwell® RSC 48 Instrument (AS8500). Elute cfDNA in 50 pL elution buffer. Minimize salt carry over.Sequence conversion reaction

[0247] 50 [il cfDNA eluate is transferred to a microtiter plate and 10 pL of SCP solution is added which contains 5 500 pM (final) locus-specific SCP probe (3 300 nM total SCP probe concentration for 600 SCP probes. Probes are at equimolar concentration). Denature at 95°C for 60 sec, then lower temperature to 50 65°C. Add and mix lOpL of Restriction Enzyme Buffer (containing 1 10 U BsaI-HF-v2 (New England Biolabs), 7 pL of lOx CutSmart Buffer Cat number B7204S). Incubation time and probe concentration at 50 65°C is optimized following guidelines by Zhang et al (Nat Chem. 2018 Jan; 10(1): 91-98.) for 5 500 pM probe (individual SCP) at 0.5 24 hours. Temperature, concentrations and time interact with each other and allow a number of solutions to accommodate user requirements.

[0248] Cleanup of sequence conversion reaction may include, once the reaction has been completed, removal of any uncleaved SCP and cTSR by binding with streptavidin bound to a solid phase (e.g. beads, column, magnetic beads). The cSSR may be partitioned for dPCR. For example, the supernatant from the cleanup of the sequence conversion reaction may be mixed with the dPCR master mix and any additional reagents as required for the specific dPCR system (e.g. oil for droplet generation for the BioRad QX ONE Droplet Digital PCR System). The number of partitions is selected such that the required statistical significance for the intended use of the assay can be achieved. The dPCR workflow, including droplet generation, is described in “QX ONE Droplet Digital PCR System and QX ONE Software User Guide”.

[0249] Digital PCR (dPCR) may be performed on the resulting SSRs. For example, a Bio-Rad QX ONE Droplet Digital PCR System (M / N 12006536) may be used with PX1 PCR Plate Sealer (1814000), and Supermix for Probes (Biorad Catalog number: 186-3010 for 500 reactions).

[0250] Table 2, below illustrates the volumes required of 10 pM stock solutions for forward primer, reverse primer, and probe to achieve final concentrations of 900 nM primer concentration and 250 nM probe concentration for one reaction:

[0251] Note that if higher sample input is needed, reduce volume of Molecular Grade Water. The total volume accommodates the dead volume for pipetting.

[0252] Table 3 Shows an example of a thermal cycling protocol for dPCR that may be used:

[0253] The number of positive chromosome 21, 18 and 13 counts (e.g. empty vs occupied partitions, using Poisson distribution to determine absolute copy numbers) may be compared and based on mathematical transformations (e.g. difference, ratio or other) to ascertain if the risk for aneuploidy of chromosome 21, 18 or 13 of a particular sample is increased. Further risk factors like maternal age, being carriers of the genetic translocation, having had one child with an aneuploidy etc. can be used in combination with the assay result to generate a risk score.DESIGN AND SELECTION OF SCPs

[0254] Described herein are methods for designing and selecting the sequence conversion probes (SCPs) that may be used with any of the Sequence Conversion Reactions described and illustrated herein (including but not limited to the assay illustrated in FIG. 1 A). In general, the Sequence Conversion Reactions described herein use a Sequence Conversion Probe (SCP), which is includes a Target Specific Region (TSR), which hybridizes to a specific target sequence and a Signal Specific Region (SSR), which is a synthetic sequence not found in the pool of target sequences (e.g., is not in the genome being examined). The SCP typically includes a chemically labile site or recognition site for a nuclease between the TSR and SSR, that is configured so that the SSR may be cleaved from the TSR only when the TSR is bound to the specific target sequence. As mentioned above, an optional affinity tag such as biotin may be present at either the 5’ or 3’ end, preferably at the 3’ end which may include more than one affinity tag molecule.

[0255] For example, a sequence conversion assay may start with an extracted subject (or pooled subjects) sample, e.g., of cell free DNA (cfDNA). SCP probes are then added toextracted cfDNA, which may be heated to denature the double-stranded cfDNA and cooled to allow specific annealing of SCP probes to target alleles within the cfDNA. Nuclease (e.g., restriction enzyme) is then added, and incubated to cleave the hybridized SCP probes at the pre-determined position, releasing the SSR portion of the SCP probe (cut SSR, or cSSR) only from those SCP probes whose TSR regions have hybridized to target regions of the cfDNA. A lock probe and ligase may then be added to circularize the cSSR. Optionally non-circular ssDNA and dsDNA may be digested; this step is not necessary, particularly where the “primer away” amplicon configuration (as shown in FIGS. 1C and 4C-4D) is utilized. The circularized SSR may optionally be amplified. The supernatant containing circular cSSR may be used in a dPCR detection reaction. For example, the inverse orientation of the forward and reverse primer and probe binding sites on the SSR may ensure that only circularized SSRs (cirSSRs) can be amplified.

[0256] Thus, described herein are methods of forming sequence conversion probes (SCPs) that may be used to form a functional SCP. These SCPs may work in any of the sequence conversion assay. Although specific examples of sequences for SCPs, and the SSRs and TSRs that form them are provided, for example in SEQ ID Nos. 1-5 and 8-75, it should be understood that the methods described herein teach and enable one of skill in the art to make SCPs that will work with the sequence conversion assays described herein and are not limited to a particular sequence. SCPs having virtually any sequence that conforms to the properties described herein for selecting and combining the TSR and SSR will work.

[0257] For example, an SCP may generally include a target-specific region (TSR) extending between 15 and 80 base pairs (bp), wherein a polynucleotide sequence of the TSR is at least 80% identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, etc.) to only a single region of one chromosome within a target (e.g., human) genome. In some cases the polynucleotide sequence of the TSR may be between 15 and 50 bp, between 15 and 45 bp, between 15 and 35 bp, between 15 and 30 bp, etc. Preferably the TSR is between 15 and 35 bp. As will be described in greater detail below, the TSR is homologous (e.g., having a signification identity) to only a single region of one chromosome within a target genome, so that the TSR will bind with reasonable affinity to only a single site within the genome. As described in greater detail, the TSR may be selected so that it occurs on a known chromosome (e.g., chromosome 21) in the genome. Although the examples described herein are specific to assays of human genomes, it should be understood that these assays may be directed to targets for other genomes (including other animal genomes, plant genomes, etc.).

[0258] The TSR is selected explicitly to include within the sequence of the TSR a restriction site for a type Ils restriction enzyme. Example of type Ils restriction enzymes that may be used are provided herein. In some examples the cut site for the restriction enzyme may be at or near the first end of the TSR. The TSR may have a GC content of greater than 50%. In general, the restriction site for the restriction enzyme refers to the polynucleotide sequence which forms the cognate restriction site recognized by the restriction enzyme.

[0259] As mentioned, the TSR probe is specific to a given chromosome and occurs only once in that chromosome. Typically, multiple instances of SCPs are included that have the same SSR but different TSRs, as described above. All of the TSRs within a set of SCPs (including a variety of different TSR probes) may have the same restriction site. The restriction site (the restriction site for a type Ils restriction enzyme) may be positioned in approximately the same position in all of the TSRs of the set of SPCs. For example, the site may be positioned with the cut site within 8 bp (e.g., within 7 bp, within 6 bp, within 5 bp, within 4 pb, within 3 bp, within 2 bp, within 1 bp, etc.) of the 3’ end of the TSR where the TSR is coupled to the SSR. For example, all of the TSRs may have the restriction site within about 10 bp downstream and upstream of the 5’ and 3’ ends of the TSR, respectively. The site may be adjacent to the end (e.g., the 5’ end) of the TSR where the TSR is coupled to the SSR. In some examples all of the TSRs of the set of TSRs within the set of SCPs have approximately the same size (e.g., about 13 bp, about 14 bp, about 15 bp, about 16 bp, about 17 bp, about 18 bp, about 19 bp, about 20 bp, about 21 bp, about 22 bp, about 23 bp, about 24 bp, about 25 bp, about 26 bp, about 27 bp, about 28 bp, between about 13-50 bp, between about 15-40 bp, between about 15-35 bp, between about 15-30 bp, between about 15-25 bp, etc.).

[0260] The different TSRs of a set of SCPs are each unique and non-overlapping; the TSRs of a set of SCPs do not overlap with each other (e.g., all hybridize to different, nonoverlapping regions of the genome.

[0261] In general, the GC content of each TSR is greater than 50%, and the Tm (melting temperature) for each TSR is typically within a set range (e.g., greater than 55 degrees C, greater than 56 degrees C, greater than 57 degrees C, greater than 58 degrees C, greater than 59 degrees C, between about 55-65 degrees C, between about 57-67 degrees C, between about 57-65 degrees C, etc.).

[0262] Finally, as will be described in greater detail below, each TRS probe may be selected to minimize their predicted secondary structure. The secondary structure may be calculated or approximated by calculating a minimum free energy (MFE) in kcal / mol and determining if the restriction site for a type Ils restriction enzyme within the TSR prove is notpart of a hairpin loop or otherwise blocked or disrupted by second structure. This determination may also or alternatively be made at the SCP stage, once the putative TSR has been combined with the SSR.

[0263] The sequence-specific region (SSR) may have a polynucleotide sequence extending greater than 40 bp (e.g., greater than 45 bp, greater than 50 bp, greater than 55 bp, between 40 and 100 bp, between 40-90 bp, between 40-80 bp, between 50-100 bp, between 50-90 bp, between 50-80 bp, etc.). The polynucleotide sequence of the SSR sequence does not occur in the target genome. For example, the SSR may have a sequence that is engineered and compared to the target genome (e.g., the human genome, including and non- chromosomal DNA, such as mitochondrial DNA) to confirm that it does not appear within the sequence of the target genome (so that it would not hybridize to the target genome). The SSR may have a CG content of greater than 50%.

[0264] In general, the end of the TSR nearest to the cut site of the type Ils restriction enzyme region (e.g., a 5’ end) may be joined to the SSR. As already mentioned above, the cognate restriction site is not part of a hairpin structure in the assembled SCP.

[0265] In general the SSR of each SCP in a set of SCPs may be the same polynucleotide sequence and may share a set of forward and backward primers that hybridize with high selectively and efficiency. For example, the SSR may include a forward primer region and a reverse primer region.

[0266] The SSR may include one or more regions that prevent or block enzymatic extension (e.g., polymerase) based on a modification of the polynucleotide sequence. For example, any of the SSRs may include a forward primer region and a reverse primer region and an abasic region between the forward primer region and the reverse primer region.

[0267] An abasic region (also referred to as an abasic site or an AP site, or apurinic / apyrimidinic site) may occur naturally by hydrolysis of nucleoside residues in DNA to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. These sites may be included in engineered polynucleotides, including the SSRs described herein. For example, a 1,2'-Dideoxyribose modification can be used to insert a single base space into a DNA oligonucleotide sequence, which replicates the loss of base pairing ability by a nucleotide, which can occur naturally through depurination or other mechanisms. A C3 Spacer phosphoramidite (iSpC3) can be used as a spacer arm in an oligonucleotide. The compounds may be added in multiple additions when a longer spacer is required. 3'-Spacer C3 CPG or phosphoramidite may also act as a blocker of exonuclease and polymerase and may be used to introduce a stable abasic site within an oligonucleotide. For example, dSpacer CE Phosphoramidite (5'-O-Dimethoxytrityl-r,2'-Dideoxyribose-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) may be used. dSpacer is also referred to as an abasic site, tetrahydrofuran (THF), or apurinic / apyrimidinic (AP) site. It can be incorporated into an oligonucleotide sequence internally, or at the 5' or 3' end of a sequence. Any number of spacers may be used to form the abasic region / site (e.g., 1, 2, 3, 4, 5, etc.).

[0268] FIG. 3 A schematically illustrates one example of an SCP as described herein. In this example, the SCP includes an SSR and a TSR. The SSR in this example is similar to that shown in SEQ ID No. 4, but includes an abasic region between a reverse primer region and a forward primer region. For example, the SSR may be constructed by connecting the polynucleotide region before the abasic region (SEQ ID NO. 2) with the polynucleotide region after the abasic region (SEQ ID NO. 5), in which the abasic region links the two polynucleotide regions. This SSR is then coupled to a TSR (e.g., SEQ ID NO. 75), which was constructed as described herein from a unique BspQI site on human chromosome 21. The SSR also includes two probe regions that may hybridize to a linker polynucleotide to circularize the SSR after it has been cut, as described above.

[0269] FIG. 3B schematically illustrates another example of an SPC as described herein, using an SSR region that is configured as in SEQ ID NO. 78 jointed to SEQ ID NO. 79 by an abasic site.

[0270] The SSR identified in SEQ ID NO. 1 shows one example of an SSR; alternative SSRs are shown in SEQ ID NOS. 2-5. For example, SEQ ID NO. 2 shows an SSR portion. This portion may be combined with the SSR portion of SEQ ID NO. 3 or SEQ ID NO. 5 (e.g., connected by an abasic region as shown in FIG. 3 A). As mentioned, any of the identified SSRs may be modified to include a short region that disrupts replication over, particularly between opposite-facing (away facing) forward and reverse polynucleotides for later detection amplification. As illustrated in FIGS. 26-4D, an SSR may (once cut from the TSR) be circularized as described previously and assayed using the general primers (forward and reverse primers) that, once linearized, allow amplification, as described above.

[0271] For example, detection may be performed using digital PCR (dPCR). In some examples the system may include TaqMan probes. The TaqMan probe site may be engineered on the SSR so that the TaqMan probe does not interfere with the method described herein. For example, TaqMan probes may be positioned at the 3’ end of the SSR, rather than at the SSR 5’ end or straddling the cut site, both of which may interfere with the method. For example, if the TaqMan probe straddles the cut site, the TaqMan probe may also act to bridge the forward and reverse primers, resulting in undesired amplification (and therefore undesired PCR signal). Alternatively, it has been found that positioning the TaqMan probe site at the SSR 5’ end may result in sequestering of the TaqMan probe.Further Examples

[0272] The various methods and steps described herein were examined using sample primers and template as described herein.

[0273] A two-part assay with an SCP, including Linear Ligation and qPCR was also performed, similar to FIGS. 5A and 5B, described above. This two-part SCP assay, a reverse primer binding site replaces the TSR following removal of the TSR by nuclease cleavage. The primer binding region, also termed the SSR extension probe, can be joined to the short cSSR by one ligation event to generate a linear single-stranded SSR (see, e.g., FIG. 5B), or by two ligation events to generate a covalently closed circular single-stranded SSR (see, e.g., FIG. 5C). The resulting SSR may be detected by qPCR. An example of a single ligation event to produce a linear SSR was performed as described herein. For example, 2e4 copy / ml of Short SSR (SCR0146, SEQ ID No. 97) and 0.5nM SSR extension probe with or without 3’ Phosphate (SCR0142, SEQ ID No. 95, or SCR0142a, SEQ ID No. 96) were ligated in the presence of 0.5nM Lock Probe (SCR0147, SEQ ID No. 98) by treatment with Hi-T4 ligase (60 U; NEB cat #M2622) for 70 minutes at 50°C in 50mM Tris-HCl, 20 mM MgC12, 1.5 mM ATP. The ligation was followed by a 10 min heat inactivation step at 65°C. The ligation product was then amplified and detected by real-time PCR using TaqMan Fast Advanced MasterMix (ThermoFisher cat # 4444556), 200 nM forward and reverse primers (SSR1_FP, SEQ ID No. 86; SSR1 RP, SEQ ID No. 87; IDT) and 100 nM TaqMan probe SCR0078 (SEQ ID No. 88; IDT) by denaturation at 95°C for 20” followed by 40 cycles of 95°C for 1” and 60°C for 20” QuantStudio Real Time PCR System (ThermoFisher Scientific).Tandem TSR SCPs

[0274] Also described herein are SCPs having two component TSRs separating the signal specific region (SSR), that may be cleaved using a type Ils restriction enzyme having a cut site that is at or near an end of each TSR (e.g., TSR 1 and TSR2) that flank the SSR. These SCPs may be referred to herein as tandem TSR SCPs (T-TSR SCPs) or equivalently as bridging SCPs, and may be used in place of the SCPs described above (non-tandem SCPs) in any of the assays, methods and kits.

[0275] Tandem TSR SSRs may be particularly helpful to reduce background that may arise in any of the assays and methods described herein. An unwanted ligation event is hypothesized to be caused by oligonucleotide “impurities” in an SCP reagent. By impurities is meant: (i) shorter fragments with one having the same composition than the product of the digestion of SCP / TSR by restriction enzyme (e.g., BspQI) and / or (ii) SCP with altered basessusceptible to hydrolysis post-synthesis and post-purification, revealing again the same product than the restriction enzyme (e.g., BspQI) reaction with the SCP and TSR. For example, FIG. 6A show an SCP as described above, including a TSR region (in this case, in the 5’ end) and a SSR region. FIG. 6B illustrates the hypothesized composition of the chemically synthesized SCP products which may include a tiny fraction of “contaminant” cSSR as a putative impurity of the chemical synthesis. Even if the full-length SCP represents 99.99% of the molecular mass of the SCP reagent, a 0.01% “contamination” of the SCP product with a shorter SCP coincidentally harboring the composition set for the ligation substrate leads to the unwanted ligation and current SCP workflow outcome background. This is because the concentration of an SCP required to drive efficient annealing to genomic DNA and subsequent restriction digest leads to a very high molar ratio of SCP to gDNA (e.g. 10A5:l).

[0276] Treatment of the chemically synthesized SCP by phosphatases (e.g. calf intestine phosphatase, CIP) can reduce the level of background due to unwanted ligation. Presumably, the CIP removes the 5 ’-phospate necessary for ligation, that is present in the impurities described above. However, in some cases it may be desirable to reduce the background even further.

[0277] One alternative is to produce the SCP by means of enzymatic process, rather than chemical synthesis (e.g. using solid phase phosphoramidite methods) with a DNA polymerase. This may include asymmetric PCR, direct primer extension, or rolling circle amplification. Such methods would not produce DNA with chemical adducts susceptible to depurination and hydrolysis.

[0278] A second alternative is to design the SCP with two TSR portions, one on each end of the SCP and requiring two restriction digest events to generate the ligateable product, a so- called tandem TSR SCP (T-TSR SCP).

[0279] FIG. 7A shows a first example of a T-TSR SCP including a first target specific region (TSR1) and a second target specific region (TSR2) flanking an SSR on both ends (Tandem TSR SCP). In this configuration, shorter chemical synthesis by-products would not be “ligateable” as illustrated in FIG. 7B (showing that the resulting by-product is not suitable to the lock-probe-based ligation reaction). Moreover, the frequency of damaged bases leading to the ligateable product (cSSR), would be present at XA2 (where X=frequency of relevant impurities).

[0280] FIG. 8 illustrates hybridization of a T-TSR SCP to a target polynucleotide (e.g., chromosome). In this example, full hybridization occurs when TSR1 and TSR2 (collectivelya “bridged TSR” that is separated by the SSR, forming the full SCP) hybridize to the target region. NICKING ENDONUCLEASES

[0281] As mentioned above, any of the methods and compositions (including kits) described herein may be configured for use with a nicking endonuclease, particularly when using tandem TSR SCPs. Nicking enzymes (either naturally occurring or engineered) cut only one strand of the double stranded DNA substrate. In particular, the methods and compositions, including the tandem TSR SCPs described herein may be configured to nick the SCP in order to cut and release the SSR without cutting the target measurand. By leaving the target measurand relatively intact, the methods and compositions described herein may amplify the signal by producing multiple SSRs (e.g., cSSRs) from the same target measurand. For example, any of these methods and compositions may include the use of a nicking enzyme to enable cycling such that one target DNA may lead to cutting of multiple SCPs, i.e. a cut SCP, releasing a cSSR, may be dissociated from the target measurand, allowing another SCP to hybridize to the target measurand (e.g., target DNA), so that it may be nicked to release the cut SSR, and so on. Cycling with a nicking enzyme may enable amplification of the signal generating cSSR.

[0282] For example, a series of nicking endonucleases, including Nt.AlwI, Nt.BsmAI, Nt.BstNBI and Nt.BspQI have been tested and validated. FIG. 9 schematically illustrates a tandem SCP in which both TSRs (e.g., TSR1 and TSR2) include an enzyme recognition site for a nicking enzyme (e.g., TSR1 may include a recognition site for Nt.ALwl or Nt.BstNBI; TSR2 may include a recognition site for Nt.BspQI or Nt.BsmAI in this example). The SSR is similar to those described above and may be explicitly engineered so as not to hybridize to the human genetic material, but may include one or more primers.

[0283] Full SCP workflow experiments have been performed with Human chromosome 21 specific tandem TSR SCP with Nt.BstNBLNt.BspQI endonucleases. The particular example of a Nt.BstNBLNt.BspQI SCP design similar to that shown in FIG. 9 has been validated with two other Human chromosomes, chromosome #21 SCP and three Human chromosome #X SCPs. The use of sex chromosome targets together with non-sex chromosome targets may allow for facile contriving of chromosomal aneuploidies at varying ratios, as described above. In general, the nicking endonucleases described herein hydrolyze only the upper strand of the SCP-polynucleotide duplex, then leaving the targeted polynucleotide (measurand) uncleaved. This property allows to repeat the annealing-digestion reactions, providing a linear amplification via cycling method referred to as cycling, as shown schematically in FIG. 10.

[0284] In this example, the method includes mixing the measurand genetic material and the tandem TSR SCP(s) 4301, then hybridizing the SCP(s) to the measurand, by heating and cooling 4303. Once hybridized, the SCP(s) may be nicked using the pair of nicking enzymes as described above 4305. The steps of hybridizing and nicking / cutting (4304 and 4305) may be repeated n times 4307 to amplify the cut SSR (cSSR) signal. In any of these methods, mixing 4301 may include mixing with an excess of SCP. Once this linear amplification is complete, the cSSRs resulting maybe circularized by ligating the cSSRs with a lock probe specific to the cSSR 4309. In some cases a cassette may be inserted, as described herein. Thereafter the cSSRs (and therefore the measurand) may be detected by any of a variety of different techniques, e.g., optionally (as shown in FIG. 10), using a pair of primers on the cSSR(s) that are arranged in FIG. 10 in a “primer away” configuration so that amplification (e.g., by PCR) is possible only once the cSSR has been circularized 4311. Alternatively, the method may include a dU Ligation Inversion, as described in greater detail below.

[0285] The method described in FIG. 10 has been validated using a variety of different pairs of TSRS, and a variety of different nicking enzymes. For example, FIG. 11 is a graph showing example results for a variety of different concentration of tandem TSR SCP types, following the method illustrated in FIG. 10, showing that cycling by nicking the SCP to allow re-use of the measurand results in significantly greater signal. Similarly, FIG. 12 is a graph showing the benefit of cycling as compared to non-cycling with various concentrations of TSR. FIG. 13 shows an example of the effect of both cycling (e.g., 15 cycles vs. no cycling) and concentration of the target measurand (referred to as “TSR” in FIGS. 44-48). In FIGS. 12 and 13, the sample results show that the method allows quantification of 50 fM target measurand (e.g., polynucleotides) with only one SCP.

[0286] The SCP workflow with the Nt.BstNBI-Nt.BspQI SCR0593 SCP has been examined using human genomic DNA, as shown in FIG. 14. The final bar (“At DNA”) in FIG. 14 refers to genomic DNA derived from the plant Arabidopsis thaliana; the extremely low signal on this non-human genomic DNA demonstrates the specificity of the SCP designs and SCP workflow.

[0287] In any of these methods and apparatuses, the SCP workflow may be used to detect multiple different (e.g., 2 or more, 3 or more, etc.) human chromosomes, including chromosome #21. In FIG. 15 an example of detecting a portion of chromosome #21 is shown. Thus, an SCP as described herein including tandem TSRs arranged and configured as described herein may result in detection of human genomic DNA.

[0288] In any of these examples, as shown in FIG. 16, additional amplification may be used during any of these steps, in addition to, or instead of cycling to re-use the measurand,as described above. For example, rolling circle amplification (RCA) may also or alternatively be used. Rolling circle amplification may be used after the cut SSR is circularized. As shown in FIG. 13, rolling circle amplification may be performed after the ligation using a cSSR specific primer and a polymerase, such as the phi29 DNA Polymerase. The use of rolling circle amplification enhances the SCR workflow read-out. RCA (as well as the prior ligation step) may potentially be performed in the partitions of a dPCR instrument. RCA may be used for detection exclusively, or may be combined with dPCR.

[0289] In any of the methods described herein the cSSR may be removed to reduce the background, for example, by digesting with a nicking enzyme. For example, if rolling amplification is used, as just described, the circularized SSR may be cut (e.g., by nicking) without cutting the product of the rolling amplification, before detecting the first engineered polynucleotide marker. The nicked SSR may be further digested to reduce background.

[0290] For example, in some testing cases, the SCP workflow were performed with tandem TSR SCPs, cycling using nicking enzymes as described above, for example, using endonucleases (e.g., Nt.BstNBI-Nt.BspQI) with SCPs having a pair of flanking (e.g., tandem) TSRs each with a restriction site for the nicking endonucleases. Both the cycling procedure in which excess SCP is re-annealed to the target measurand after digestion with the endonucleases and the post-ligation RCA step have been shown to increase the sensitivity of the SCR workflow. Thus, either or both cycling (e.g., reusing the measurand) and / or RCA may be used for amplification. These amplification steps may further increase the Signal-to- Noise ratio (SNR) of the assays described herein.

[0291] In general, the methods and compositions (e.g., kits) described herein may modified to enhance sensitivity and reduce background, particularly for use in quantitative PCR in which sensitivity and low background are particularly critical. In particular, these methods and apparatuses may include the be configured to enhance the specificity of ligation and circularization of the cleaved SSR in the methods described above.

[0292] For example, in any of these methods and compositions the lock probe may be configured to increase both ligation specificity as well as amplification specificity of downstream amplification, including (but not limited to) rolling amplification. FIG. 17A illustrates the use of a 3’ block to prevent extension of the lock probe. For example, the 3’ end may include a phosphate to prevent extension. Alternatively or additionally, the 3’ end may include an overhang (e.g., a “flap”) region that is configured to prevent extension, as shown in FIG. 17B. This 3’ overhang region does not hybridize to the cleaved SSR, a shown.

[0293] In any of these cases a lock cassette may be used and ligated to the cut ends of the cSSR. This is illustrated schematically in FIG. 17C. in this example, the lock probe (whichmay also be blocked at the 3’ end and / or may include a flap / overhang), may be hybridized to the cleaved SSR (cSSR) as shown in the upper left. A cassette (referred to equivalently as a cassette oligo and / or a cassette sequence) may be added and hybridized to the lock probe. The cassette has a sequence that is complimentary to the gap region of the lock probe (e.g., the region between the hybridizing 3’ and 5’ ends. A ligase may then be used to ligate the cassette (hybridized to the already-hybridized lock probe) to the ends of the cSRR, as shown, resulting in a fully circularized SSR. In some cases a primer for amplification (which may be used, e.g., as shown in FIG. 26) may be formed by the ligation, e.g., by the joining of the cSSR end (3’ or 5’) and the cassette.

[0294] Surprisingly, the use of a cassette in combination with the lock probe may, particularly in combination with dU ligation inversion, as described below, dramatically reduce the background when used with qPCR. This is illustrated in FIG. 17D showing a comparison of variations in SCP reaction workflows. In general, FIG. 17D shows that the use of cassette ligation may increase the specificity and reduce background. For example, samples that include cSSR (numbers 2 and 4, showing both one- and two-TSR versions of the SCP) resulted in significantly lower Ct, representing non-b ackground signal as compared with ligase-only controls. The use of cassettes with the lock probe in these cases, when ligated with Hi-T4 ligase resulted in a clear signal by qPCR that was not present without the cassette and ligation.

[0295] Any of the methods described herein may also alternatively use dU and a Uracil- DNA glycosylase (e.g., Uracil-N-glycosylase). FIG. 18A schematically illustrates an example of a method that incorporates some of the features described herein that may reduce the background and amplify the signal when using an SCP as described herein. In this example, the SSR is configured to include dUs (e.g., some or all of the dT’s in the SSR are replaced with DU’s (dUTPs) that may allow UNG cleanup during the method. Thus, background may be suppressed, and particularly the background that arises as a result of extension from unligated oligonucleotide probe using uracil-N-glycosylase prior to amplification (see also FIG. 21, below). Digestion of the sequences with uracil-N-glycosylase (also referred to as “UNG”) breaks the SCP (e.g., the SSR) into fragments to prevent amplification. Thus, In FIG. 18A, the SCP, and in particular the SSR, may include dUTPs incorporated into the sequence. During the processing step, following circularization (e.g., using a lock probe, as shown in FIG. 18 A, or using a padlock primer (e.g., padlock extension primer), the circularized SSR may then be amplified. In FIG. 18A a polymerase such as Phi29 (RCA polymerase) may be used to form long RCA products that incorporate dTs rather than dUs. Following this amplification step the material may then be treated to digest an dU containingmaterial by UDG / UNG cleanup, resulting in an RCA product that is otherwise ready for PCR amplification and has a much lower background.

[0296] Any of these methods may include the use of dU and UDG / UNG as part of a dU ligation inversion step that may linearize and ‘invert’ the circularized SSR that has been circularized with a cassette and lock probe, as illustrated and described above. This is shown in FIG. 18B an SCP may include a region of one or more deoxyuridines (dUs) on the SSR region of the SCP; the dU region may be positioned between the forward and reverse primer regions (e.g., binding sites for forward, FP, and reverse, RP, primers) and may be approximately midway through the SSR region. The dU region may be cut in a relatively precise manner by the use of an uracil-N-glycosylase (as part of the UDG / UNG cleanup step). When performed on the circularized cut-out SSR, as shown in FIG. 18B, the result is a partial inversion of the original SSR, so that the forward and reverse primers, which in the original SSR and the circularized SSR (shown in FIG. 18B, top) have a primer orientation in which the primers are directed away from each other (limiting cycling replication during amplification), are instead oriented towards each other, allowing amplification by PCR. As will be described in greater detail below, this inversion, in combination with the use of a lock cartridge, may result in a surprisingly low background, sufficient to allow robust digital PCR to be performed, including in particular on low copy signals.

[0297] FIG. 19 illustrates an alternative configuration for processing and detection of cleaved SSR as part of a detection step. In FIG. 19 the cleaved SSR is circularized using a lock probe that may optionally be a gap-filling lock probe. The probe may be blocked on one end (e.g., the 3’ end to prevent extension of the lock probe. Polymerase activity may extend the SSR 3’ end to fill in the gap, and ligase may be used to circularize the SSR. An exonuclese may remove the lock probe and the resulting circularized SSR may then be ready for preamplification and amplification.

[0298] FIG. 20 outlines an overall method for using a sequence conversion probe (SCP) to detect and amplify a very low-copy signal that may be particularly appropriate for digital PCR as described herein. In this example, the sequence conversion probe (SCP) includes a TSR that is configured to bind to cfDNA to form a BspQI cut site (restriction site, R), allowing release of a sequence specific region (SSR) that includes qPCR primers for downstream detection. The first (annealing) step hybridizes the cFDAN to the TSR, followed by a second, digestion, step with the restriction enzyme, e.g., BspQI, as described above. The cut SSR includes a forward and reverse set of primers (qPCR primers). The cut SSR may then be circularized to form circular SSR (cSSR), e.g., using a lock probe, and (as shown in step 4), an exonuclease may be used to clean up the reaction. In this example, the ligationthen allows primer-away amplification for PCR, including for quantitative PCR (or dPCR readout).

[0299] FIG. 21 illustrates some of the key improvements to this general technique. All or some of these improvements may be used. These improvements may generally be used to provide a much lower background; as discussed above, this significantly lower background may permit reliable digital PCR (dPCR) readout. In addition to the use of dUTPs in the SCP (and therefore the SSR) which may allow for downstream UNG / UDG cleanup (e.g., in a step after amplification, e.g., rolling circle amplification, but prior to digital PCR readout) to reduce the background. In some examples, prior to UNG / UGD cleanup stage, rolling circle amplification may be used.

[0300] Alternatively or additionally, any of the methods and compositions described herein may include padlock primer extension (P-LEX). In P-LEX a forward primer may be used with a padlock primer and the SSR (e.g., cut SSR) may include a Taqman probe sequence for detection (e.g., via digital PCR). In FIG. 22, the SSR, once cut following successful hybridization of the TSR region, e.g., using a restriction endonuclease such as BspQI, may then be circularized using an appropriate padlock primer and a forward primer pair. Note that the circularized “circularization complex” in when using these techniques does not have to be ligated; the lock primer (“padlock primer”) may circularize the ends of the cut SSR and allow amplification of the cut SSR using the padlock primer. This may reduce the background in the assy.

[0301] However, not all padlock (forward and / or reverse) probes are appropriate. For example, FIGS. 23A-23B illustrates important considerations in configuring the SSR and associated primers. FIG. 23 A shows a full-length SCP, and illustrates that the addition of the P-LEX primers (e.g., forward and reverse primers, and / or Taqman / detection primer) to the full length SCP does not result in binding and circularization or extension of the full length SCP. In this case, the TSR prevent circularization, and the padlock primer binding to the SSR is very unstable, as the primer is engineered to have a very low melting temperature (Tm) (e.g., less than between about 15-20 degrees C). In addition, the forward primer (FP) may be configured so that it cannot bind to the original SSR, as it may have the same sequence as the top stand of the SCP. Thus, the method cannot result in padlock extension during amplification (e.g., PCR).

[0302] Once the SSR is separated (cut) from the SCP, as shown in FIG. 23B, the addition of the P-LEX primers can circularize the SSR, as the binding of the primer in this configuration is highly stable (having a higher overall Tm. The forward primer is also engineered to bind to the product of the padlock primer binding to the cut SCP. In any ofthese methods and compositions, the forward primer is configured so that it does not dimerize with padlock primer. In general, it may be particularly advantageous to design the padlock primers so that the padlock primer has a 3’ overlap extending 6 bases or more (e.g., 7 bp, 8 bp, 9 bp, 10 bp, etc.). These primers may be configured to prevent problems that may result from degradation by the polymerase 5’ nuclease activity. FIGS. 24A-24B illustrate examples of the sequence of one example of an SSR sequence, as well as associated primers, including putative forward primers. In some cases this sequence and other sequences are shifted. In particular, the forward primer (fp) may be separated from and non-overlapping with the region forming the ends of the SSR. These probes may be referred to herein as ‘no ligation’ probes, as the SSRs may be circularized without disrupting the detection. FIG. 24B also illustrates one example of sequences and specific sequence changes. Examples of different (and improved) forward primer sequences, reverse primer sequences, and probes as shown and described.

[0303] Thus, the spacing between the forward primer and the extension primer (or reverse primer) may be between 5-9 base pairs. In some cases the amplification protocol include a pre-anneal step (e.g., 95 to 60C holding for 30 mins) and PCR (65C extension) step. The primer may be “walked” experimentally, as illustrated in FIGD. 25 A-25D. In these examples, the amount of overlap (spacing) may be varied and the resulting amplification may be monitored. In FIGS. 25A the use of a positive control (“826”) at different concentrations is shown in comparison in FIGS. 25A-25D. At 8 bp of overlap, the test sequence (“593”) best maps to an increase in sensitivity and activity. In any of these examples the concentration of the reverse primer (RP) may be configured favor intra-molecular binding (400 nM here).

[0304] Thus, in general, these methods an apparatuses may include a padlock primer in a reverse orientation relative to the SSR; the methods described herein may not operate properly unless the padlock primer is in the reverse orientation, in order to prevent nonspecific binding n extension. The padlock primer may be configured to that the 3' end of the padlock primer has an overlap length that is 8-9 bp in length of lower (e.g., in some cases the padlock primer has an overlap that is less than 8 bp (e.g., less than 8 bp, etc.). At about 6bp or overlap (e.g., above 7 bp), amplification on SCP may be seen; below about 6bp, no background may be seen. The predicted Tm for the padlock primer’s 3' overlap Tm is predicted to be about 15-20°C. dU Ligation Inversion

[0305] In some cases a method for performing a very low-noise sequence conversion assay may include cutting only SSR regions that are bound to target, to remove them from the TSR region(s), then circularizing the cut SSRs, and then cutting them (e.g., using a dU regionand an Uracil-DNA glycosylase) to partially invert the circular SSR and linearize it. In any of these methods, a lock probe, lock cassette and released SSR, which includes a dU region between a forward and reverse primer that are oriented away from each other in the circularized SSR, may be used to generate a PCR amplifiable sequence that includes a probe or marker (e.g., engineered polynucleotide marker) allowing detection, including in particular, detection by highly sensitive digital PCR techniques.

[0306] For example, FIG. 26 schematically illustrates one example of a dU ligation inversion technique that may be used to detect a probe or maker of a cut SSR with very low noise. In FIG. 26, the method may include binding / hybri dizing the TS1 and, in some cases, TS2, probe regions of the SCP to target sequences, then nicking or cutting the SCPs that are bound to release the SSR regions. In some examples, as shown in FIG. 26, the SSR regions may have a dU region between a forward primer (FP) and a reverse primer (RP). The cut SSR may be circularized by the use of a lock probe as well as a cassette that hybridizes to the lock probe and is ligated (e.g., using ligase) to circularize the cut SSR, the ligation product in FIG. 26.

[0307] Thereafter the circularized SSR may be again linearized, but by cutting a different region of the SSR, the dU region, by cutting between the forward and reverse primers. This changes the orientation of these primers, as shown in FIG. 26, bottom right. The use of dU and a Uracil-DNA glycosylase may therefore result in lengths of the SSR that include a probe or marker region that may be amplified by the use of PCR with the forward and reverse primers. In contrast SCPs that are not hybridized (if the complementary region is not present in the sample) will be cleaved into component parts that cannot be amplified exponentially by PCR and therefore detected by digital PCR.

[0308] In practice this technique, using dU ligation inversion with a cassette, may result in a significant reduction in background, which is particularly important when performing digital PCR. FIGS. 27 A and 27B illustrate the improvements in the background of digital PCR assay when using dU ligation inversion as described above. FIG. 27A is a graph showing the detection of signal for a variety of different conditions. In this graph, the earliest case (using dU ligation inversion, “+ TSR” is the first to advance, following by the other conditions, including conditions for which the target (TSR) binding is not present (No TSR, Lig only, PCR ctls). FIG. 27B is a table summarizing the data from FIG. 27A.

[0309] FIGS. 28A and 28B also graphically illustrate the during a full workflow, in which the target polynucleotide may be a very small signal (e.g., a low probability event). FIGS. 28A-28B show examples (FIG. 28A) or summaries (FIG. 28B) of the use of dU ligation inversion resulting in significantly lower background. The plots shown in FIG. 28A showexamples of the results of a standard workflow (showing FAM florescence and a workflow in which a restriction enzyme was added during the process. The results are also quantified and shown the Table of FIG. 28B.

[0310] Finally, although the methods and apparatuses described herein may include a pair of TSRs or a single TSR. In some cases it may be particularly beneficial to use a pair of TSRs rather than a single TSR region. This benefit is illustrated in FIG. 29, showing that the 2-TSR SCP background is much lower (the plot is ‘cleaner’) than the 1-TSR SCP background This was surprising, since the qPCR background signal was the same. The two TSR SCPs had a ligation efficiency that was high at 76%, with the background only covering 7%.

[0311] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0312] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as

[0313] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations ofthe device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0314] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0315] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0316] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive, and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.

[0317] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. Forexample, if the value " 10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0318] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0319] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of theabove embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including one or more target-specific regions (TSRs), and a signal specific region (SSR), the SSR further comprising a forward primer region and a reverse primer region that are oriented away from each other and a dU region comprising one or more deoxyuridines between the forward and reverse primer regions, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSR to a lock probe and to a cassette sequence to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence between the 3’ hybridization site and the 5’ hybridization site to form a circular SSR; digesting the circular SSR with a Uracil-DNA glycosylase to form an inverted cSSR; amplifying a probe sequence on the inverted cSSR using a forward primer binding to the forward primer region and a reverse primer binding on the reverse primer region; and detecting the probe sequence.

2. The method of claim 1, wherein each SCP comprises two TSRs.

3. The method of any of claims 1-2, wherein the lock probe has a flap region at a 3’ end of the lock probe that does not hybridize to the cSSR.

4. The method of any of claims 1-3, wherein the lock probe has a phosphate at a 3' end of the lock probe to prevent extension by a DNA polymerase.

5. The method of any of claims 1-4, wherein the probe sequence spans a junction between the cSSR and the cassette sequence.

6. The method of any of claims 1-5, wherein amplifying the probe sequence comprises amplifying the probe sequence extending at least partially across the cassette sequence.

7. The method of any of claims 1-6, wherein the lock probe binds to the 3’ hybridization site comprising 1 or more base pairs on the 3’ end of the cSSR.

8. The method of any of claims 1-7, wherein the lock probe binding to the 3’ hybridization site has a low melting temperature of less than 20 degrees C to prevent binding to the lock probe binding to the sequence conversion probe.

9. The method of any of claims 1-8, wherein the forward primer region is at least 4 base pairs from the 5’ end of the cSSR and at least 1 base pairs from the 3’ end of the cSSR.

10. The method of any of claims 1-9, wherein each SCP comprises a pair of target-specific regions (TSRs), TSR1 and TSR2 positioned at either ends of the SSR, wherein hybridizing the plurality of different TSRs comprises hybridizing TSR1 and TSR2 to the one or more target measurand within the sample mixture, and wherein cutting the SCPs comprises cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs).

11. The method of any of claims 1-10, wherein detecting comprises performing digital PCR.

12. The method of any of claims 1-11, wherein combining further comprises combining a second plurality of SCPs with the sample mixture, wherein the second plurality of SCPs comprises a plurality of different TSRs configured to hybridize to different target measurands, and wherein each SCP of the second plurality of SCPs includes a same SSR that is different from the SSR of the first plurality of SCPs.

13. The method of claim 12, wherein the plurality of different TSRs of the first plurality of SCPs that are configured to hybridize to different target measurands are configured to hybridize to a first chromosome and the plurality of different TSRs of the second plurality ofSCPs that are configured to hybridize to different target measurands are configured to hybridize to a second chromosome.

14. The method of any of claims 1-13, wherein cutting SCPs to release cSSRs comprises nicking the SCPs without cutting the one or more target measurand.

15. The method of claim 14, further comprising repeating the steps of hybridizing the plurality of TSRs to the one or more target measurand within the sample mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs.

16. A method, comprising: combining a first plurality of sequence conversion probes (SCPs) with a sample mixture including one or more target measurand, wherein each SCP comprises a polynucleotide including two target-specific regions (TSRs), and a circularizable signal specific region (SSR), wherein the circularizable SSR comprises one or more deoxyuridines between a forward primer region and a reverse primer region wherein the forward primer region is oriented away from the second primer region, the circularizable SSR further comprising a first engineered polynucleotide marker, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the plurality of different TSRs to the one or more target measurand within the sample mixture; cutting SCPs having TSRs that are hybridized to the one or more target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe and hybridizing a cassette sequence to the lock probe to produce circularization complexes, wherein the lock probe binds to a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence to the 3’ end of the cSSR and to the 5’ end of the cSSR to form a circular SSR; cutting the circular SSR with a Uracil-DNA glycosylase to form an inverted cSSR;amplifying the first engineered polynucleotide marker from the inverted cSSR using a forward primer binding to the forward primer region and a reverse primer binding to the reverse primer region; and detecting the first engineered polynucleotide marker.

17. The method of claim 16, wherein the Uracil-DNA glycosylase comprise a Uracil-N- glycosylase (UDG).

18. The method of any of claims 16-17, further wherein a 3’ end of the lock probe is blocked to prevent extension.

19. The method of any of claims 16-18, wherein the 3’ end of the lock probe is blocked to prevent extension by an overhang flap region that does not hybridize to the circularized cSSR.

20. The method of any of claims 16-19 wherein each SCP comprises a pair of target-specific regions (TSRs), TSR1 and TSR2 positioned at either ends of the SSR, wherein hybridizing the plurality of different TSRs comprises hybridizing TSR1 and TSR2 to the one or more target measurand within the sample mixture, and wherein cutting the SCPs comprises cutting SCPs having TSRls and TSR2s that are each hybridized to the one or more target measurands to release cut SSRs (cSSRs).

21. The method of any of claims 16-20, wherein detecting comprises detecting the first engineered polynucleotide marker by performing digital PCR.

22. The method of any of claims 16-21, wherein combining further comprises combining a second plurality of SCPs with the sample mixture, wherein the second plurality of SCPs comprises a plurality of different TSRs configured to hybridize to different target measurands, and wherein each SCP of the second plurality of SCPs includes a same SSR that is different from the SSR of the first plurality of SCPs.

23. The method of any of claims 16-22, wherein the plurality of different TSRs of the first plurality of SCPs that are configured to hybridize to different target measurands are configured to hybridize to a first chromosome and the plurality of different TSRs of the second plurality of SCPs that are configured to hybridize to different target measurands are configured to hybridize to a second chromosome.

24. The method of any of claims 16-23, wherein cutting SCPs to release cSSRs comprises nicking the SCPs without cutting the one or more target measurand.

25. The method of claim 24, further comprising repeating the steps of hybridizing the plurality of TSRs to the one or more target measurand within the sample mixture and cutting SCPs to release cSSRs to amplify the number of cSSRs.

26. The method of any of claims 16-25, wherein cutting the circular SSR with the Uracil- DNA glycosylase further comprising cutting any linear SCP.

27. A kit configured to perform the method of any of claims 1-26.

28. A method of determining a fetal aneuploidy, the method comprising: combining a mixture of maternal and fetal genetic material including a plurality of target measurands with a plurality of sets of sequence conversion probes (SCPs), wherein each set of SCPs comprises a plurality of SCPs, further wherein each SCP comprises: a first polynucleotide target-specific region (TSR1) and a second polynucleotide target-specific region (TSR2); a circularizable signal specific region (SSR) between TSR1 and TSR2, wherein the circularizable SSR comprises one or more deoxyuridines between a first primer region and a second primer region wherein the first primer region is oriented away from the second primer region, the circularizable SSR further comprising a first engineered polynucleotide marker, further wherein the first plurality of SCPs comprise a plurality of different TSRs configured to hybridize to different target measurands, wherein each SCP of the first plurality of SCPs includes the same SSR; further wherein the first plurality of SCPs comprise a plurality of different TSRls and TSR2s configured to hybridize to different target measurands on a same chromosome, wherein each SCP of the first plurality of SCPs includes the same SSR; hybridizing the TSRls and TSR2s to target measurands in the mixture; cutting SCPs having TSRs that are hybridized to the target measurands to release cut SSRs (cSSRs); hybridizing the cSSRs to a lock probe and hybridizing a cassette sequence to the lock probe to produce circularization complexes, wherein the lock probe bindsto a 3’ hybridization site on a 3’ end of the cSSR and a 5’ hybridization site on a 5’ end of the cSSR; ligating the cassette sequence to the 3’ end of the cSSR and to the 5’ end of the cSSR to form a circular SSR; cutting the circular SSR with a Uracil-DNA glycosylase to form an inverted cSSR; distributing the inverted cSSRs into a plurality of reaction samples at a dilution such that at least some of the reaction samples contain inverted cSSRs and some do not contain inverted cSSRs; amplifying the first engineered polynucleotide marker from the inverted cSSRs using the forward primer binding to the forward primer region and the reverse primer binding to the reverse primer region; detecting the first engineered polynucleotide marker; analyzing the engineered polynucleotide markers of the inverted cSSRs present or absent in individual reaction samples to provide a first number of binary results representing presence or absence of a presumably euploid fetal chromosome in the reaction samples and a second number of binary results representing presence or absence of a presumably aneuploid fetal chromosome in the reaction samples; and determining the presence or absence of a fetal aneuploidy by comparing said first number with said second number.

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