Methods and compositions for increasing detection of structural variants

Abstract

The present disclosure provides compositions and methods for enhancing hybridization and / or ligation events by the addition of additives to hybridization and / or ligation reactions in an assay. The addition of small molecule additives including mono-ols, diols, triols and polyols to hybridization and / or ligation reactions using single stranded DNA templates demonstrates successful separation of structural variants, such as copy number variants, in nucleic acid molecules compared to the absence of a small molecule additive in an assay.

Description

WSGR Docket No. 64100-751.601METHODS AND COMPOSITIONS FOR INCREASING DETECTION OF STRUCTURAL VARIANTSCROSS-REFERENCE

[0001] The present application claims the benefit of United States Provisional Patent Application Serial No. 63 / 749,244, filed on January 24, 2025, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Nucleic acid hybridization refers to the process where two complementary single stranded nucleic acid molecules can bind or anneal together to form a double stranded nucleic acid structure through specific nucleotide base pairing of A-T / U and C / G through hydrogen bonding. It is a fundamental process for replication and gene expression and one of the key techniques for detecting, analyzing and isolating specific sequences in the biological sciences. Nucleic acid ligation refers to the enzymatic process in which nucleic acid fragments, or ends of nucleic acid fragments, are covalently joined together by the formation of a phosphodiester bond between the 3’ hydroxyl group of one nucleotide and the 5’ phosphate group of another nucleotide. Ligation is catalyzed by enzymes known as ligases, which play a critical role in biological and laboratory processes, including nucleic acid replication, repair, recombination and the construction of recombinant nucleic acid molecules (e.g., molecular cloning).

[0003] Additives to hybridization and / or ligation reactions are compositions that may enhance the efficiency and specificity of one or more of the hybridization processes and / or the ligation process, for example by facilitating the processes or by improving the quality of the nucleic acids being hybridized and / or ligated. Existing additives include small molecules like PEG which can promote the close proximity of nucleic acids ends and can stabilize the ligation reaction. Other agents, such as salts, may be included in a hybridization and / or ligation reaction to optimize the hybridization events or ligation events, or enhance the activity of a ligase enzyme. In some cases, additives like DMSO or BSA can be used to relax secondary structures of nucleic acids which can interfere with hybridization and / or ligation efficiency. Additives can also be used to protect against exonuclease or endonuclease activity, which can further enhance the success rate of hybridization and / or ligation reactions. The additives collectively work by, for example, improving nucleic acid stability or enzymatic stability, promoting efficient nucleic acid alignment for hybridization and / or ligation and minimizing unwanted side reactions.WSGR Docket No. 64100-751.601

[0004] However, additives to a hybridization and / or ligation reaction may not produce the same effects or enhancements in all reactions, as there can be variability based on reaction conditions, nucleic acid sequences, and the like.SUMMARY

[0005] Aspects disclosed herein provide compositions comprising an additive, wherein the additive comprises an alcohol, a triol or a polyol, or a combination thereof, and wherein the additive enhances intramolecular hybridization reactions, intermolecular hybridization reactions, or ligation reactions, or a combination thereof, of one or more nucleic acid molecules in a reaction. In some embodiments, the alcohol is selected from the group consisting of methanol, ethanol and isopropanol. In some embodiments, the alcohol comprises the methanol, and wherein the methanol is at a concentration of 750 mM to 1 M in the reaction. In some embodiments, the alcohol comprises the ethanol, and wherein the ethanol is at a concentration of 750 mM to 1 M in the reaction. In some embodiments, the alcohol comprises the isopropanol, and wherein the isopropanol is at a concentration of 250 mM to 1 M in the reaction. In some embodiments, the triol is selected from the group consisting of glycerol, 1,2,4 heptanetri ol, and 1,2,6 hexanetriol. In some embodiments, the triol comprises the glycerol, and wherein the glycerol is at a concentration of 200 mM to 2 M in the reaction. In some embodiments, the triol comprises the 1,2,4 heptanetri ol, and wherein the 1,2,4 heptanetri ol is at a concentration of 250 mM to 1 M in the reaction. In some embodiments, the triol comprises the 1,2,6 hexanetri ol, and wherein the 1,2,6 hexanetriol is at a concentration of 75 mM to 500 mM in the reaction. In some embodiments, the polyol is selected from the group consisting of meso-erythritol, D-sorbitol and trehalose. In some embodiments, the polyol comprises the meso-erythritol, and wherein the meso-erythritol is at a concentration of 500 mM to 1 M in the reaction. In some embodiments, the polyol comprises the D-sorbitol, and wherein the D-sorbitol is at a concentration of 100 mM to 1 M in the reaction. In some embodiments, the polyol comprises the trehalose, and wherein the trehalose is at a concentration of 250 mM to 500 mM in the reaction. In some embodiments, the composition enhances the calling of structural variants at one or more locations in a nucleic acid sample. In some embodiments, the additive comprises two or more of the alcohol, the triol, and the polyol. In some embodiments, the additive comprises the alcohol, the triol, and the polyol. In some embodiments, the additive enhances intramolecular ligation of the one or more nucleic acid molecules in the reaction. In some embodiments, the additive enhances intermolecular ligation of the one or more nucleic acid molecules in the reaction.WSGR Docket No. 64100-751.601

[0006] Aspects disclosed herein provide methods of enhancing hybridization reactions, ligation reactions, or a combination thereof, between the one or more nucleic acid molecules, comprising: a) providing the any one of the composition disclosed herein and the one or more nucleic acid molecules; and b) hybridizing and ligating the one or more nucleic acid molecules together by one or more intramolecular or intermolecular ligation reactions, wherein hybridization reactions, ligation reactions, or the combination thereof, are enhanced compared to a hybridization reaction, a ligation reaction, or a combination thereof, performed in the absence of any one of the compositions disclosed herein. In some embodiments, the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetriol, meso-erythritol, D-sorbitol and trehalose.

[0007] Aspects disclosed herein provide kits comprising: one or more recognition elements, and a reagent comprising any one of the additives disclosed herein. In some embodiments, the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetriol, 1,2,6 hexanetriol, meso-erythritol, D-sorbitol and trehalose.

[0008] Aspects disclosed herein provide methods for determining a structural variant of a target of interest, comprising: a) providing an additive to a hybridization reaction, a ligation reaction, or a combination thereof, wherein the additive is one or more of an alcohol, a triol or a polyol, and wherein the hybridization reaction or the ligation reaction is performed in an assay for determining the structural variant of the target of interest, and b) determining a presence of the structural variant of the target of interest. In some embodiments, the structural variant is a copy number variant. In some embodiments, the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, meso-erythritol, D-sorbitol and trehalose. In some embodiments, the additive is the methanol, and wherein the methanol is at a concentration of 750 mM to 1 M. In some embodiments, the additive is the ethanol, and wherein the ethanol is at a concentration of 750 mM to 1 M. In some embodiments, the additive is the isopropanol, and wherein the isopropanol is at a concentration of 250 mM to 1 M. In some embodiments, the additive is the glycerol, and wherein the glycerol is at a concentration of 200 mM to 2 M. In some embodiments, the additive is the 1,2,4 heptanetri ol, and wherein the 1,2,4 heptanetri ol is at a concentration of 250 mM to 1 M. In some embodiments, the additive is the 1,2,6 hexanetriol, and wherein the 1,2,6 hexanetri ol is at a concentration of 75 mM to 500 mM. In some embodiments, the additive is the meso-erythritol, and wherein the meso-erythritol is at a concentration of 500 mM to 1 M. In some embodiments, the additive is the D-sorbitol, and wherein the D-sorbitol is at a concentration of 100 mM to 1 M. In some embodiments, the additive is the trehalose, and wherein the trehalose is at aWSGR Docket No. 64100-751.601concentration of 250 mM to 500 mM. In some embodiments, the target of interest is from a pharmacogenomic gene. In some embodiments, the pharmacogenomic gene is a CYP gene. In some embodiments, the CYP gene comprises one or more CYP2D6 structural variants. In some embodiments, the CYP2D6 structural variant comprises CYP2D6 *68. In some embodiments, the one or more CYP2D6 structural variants comprises one or more of CYP2D6 *13, CYP2D6 *36, CYP2D6 *61, CYP2D6 *63, CYP2D6 *68, or CYP2D6 *83. In some embodiments, the target of interest is from a chromosome for noninvasive prenatal testing comprising one or more structural variants. In some embodiments, the chromosome is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. In some embodiments, the chromosome is chromosome 13. In some embodiments, the chromosome is chromosome 18. In some embodiments, the chromosome is chromosome 21. In some embodiments, the chromosome is chromosome X. In some embodiments, the chromosome is chromosome Y.

[0009] Aspects disclosed herein provide systems for determining one or more structural variants of a target of interest, comprising: a) an assay module comprising a hybridization reaction or a ligation reaction, or a combination thereof, wherein the hybridization reaction or the ligation reaction, or the combination thereof, comprises an additive, wherein the additive comprises one or more of an alcohol, a triol, or a polyol, or a combination thereof, and wherein the additive is configured to enhance intramolecular hybridization, intermolecular hybridization, or ligation events, or a combination thereof, of the one or more nucleic acid molecules; and b) an analysis module configured to determine a presence of the one or more structural variants of the target of interest based on products generated from the assay module. In some embodiments, the one or more structural variants comprise one or more copy number variants. In some embodiments, the assay module further comprises an amplification reaction. In some embodiments, the assay module comprises a substantially simultaneous hybridization and ligation reaction comprising a ligase and the additive, and wherein the analysis module comprises a detection reaction. In some embodiments, the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, meso-erythritol, D-sorbitol and trehalose. In some embodiments, the analysis module comprises detection reagents. In some embodiments, the detection reagents comprise fluorescently labeled oligonucleotides.WSGR Docket No. 64100-751.601INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings.

[0012] FIGs. 1A-B show representative positive and negative control graphs; FIG. 1A) propanediol, an additive reported to enhance a ligation reaction, served as the positive control additive and was added to an assay for CNV calling, and FIG. IB) water served as the negative control additive which was added to an assay for CNV calling.

[0013] FIG. 2 shows an exemplary graph when using methanol as an additive in an assay for CNV calling.

[0014] FIG. 3 shows an exemplary graph when using ethanol as an additive in an assay for CNV calling.

[0015] FIG. 4 shows an exemplary graph when using isopropanol as an additive in an assay for CNV calling.

[0016] FIGs. 5A-B show exemplary graphs when using glycerol as an additive in an assay for CNV calling, FIG. 5A) glycerol at 0.5 M, and FIG. 5B) glycerol at 1.5 M.

[0017] FIG. 6 shows an exemplary graph when using 1,2,4 butanetri ol as an additive in an assay for CNV calling.

[0018] FIG. 7 shows an exemplary graph when using 1,2,6 hexanetri ol as an additive in an assay for CNV calling.

[0019] FIG. 8 shows an exemplary graph when using meso-erythritol as an additive in an assay for CNV calling.

[0020] FIG. 9 shows an exemplary graph when using D-sorbitol as an additive in an assay for CNV calling.WSGR Docket No. 64100-751.601

[0021] FIG. 10 shows an exemplary graph when using trehalose as an additive in an assay for CNV calling.

[0022] FIG. 11 shows an illustration of a recognition element that can be used to evaluate the ability of an additive for enhancing a hybridization and / or ligation reaction.

[0023] FIG. 12 shows an illustration of an exemplary assay workflow where recognition elements are ligated, circularized and amplified in anticipation of identifying the presence or absence of a copy number variant from a sample.

[0024] FIG. 13 shows the target CNV gene locations for CYP2D6 and CYP2D7 and the recognition element 5’ and 3’ complementary sequences for hybridizing with the target gene location sequences used in the evaluation of the additives.DETAILED DESCRIPTION

[0025] The present disclosure provides for the inclusion of additives in hybridization and / or ligation reactions to enhance the hybridization and / or ligation of intermolecular and intramolecular nucleic acid molecules, as evidenced by an increase in sensitivity in detecting nucleic acid variants, in particular structural variants such as copy number variants. Ligation dependent nucleic acid assays used to detect variants, such as used to identify copy number variants in pharmacogenomics targets or NIPT related targets, can suffer from a lack of sensitivity, which can be due to decreased chemical or enzymatic efficiencies in the assays. Suboptimal hybridization and / or ligation can reduce the yield of assay specific ligated nucleic acid molecules, which can lead to a decrease in assay sensitivity, for example unreliable detection of low-abundance or borderline variants. A decrease in assay sensitivity can result in, for example, under-detection of clinically significant variants, increased variability between samples and reduced confidence in, assay result interpretations which can be critical for informed therapeutic decision making.

[0026] The additives disclosed herein can positively influence probe abundance variability. Probe abundance variability, with regards to the present disclosure, refers to the potential for the inconsistent distribution of signals across different amplification products from the same target region, or when compared across different samples. For example, high probe abundance variability can be considered as background noise which can result in the failure of an assay to accurately detect real DNA copy number variants. For example, the target CNV signal that is desired can be swamped out by the background signal noise. In some embodiments, in the assay ligation steps described herein, the target nucleic acid molecules and the ends of the recognition elements can hybridize, which can lead to ligation of the proximal ends of the recognitionWSGR Docket No. 64100-751.601elements as they are hybridized to their target nucleic acid molecules, thereby generating circularized recognition elements which can be amplified. In some embodiments, if either the hybridization or the ligation is inefficient or inconsistent, the amount, or abundance, of the circularized recognition elements which are amplifiable can vary significantly. It is contemplated that the additives as described herein can help solve the problem of hybridization and / or ligation inconsistencies which can lead to probe abundance variability.

[0027] In some embodiments, it is contemplated that the additives described herein may increase hybridization efficiency. For example, it is contemplated that the additives may reduce the thermal stability of GC pairing, while stabilizing AT pairing, during hybridization of two nucleic acid molecules. One practical effect can include a more consistent Tm across all the hybridizing nucleic acid molecules, which can lead to uniform ligation across multiple different target hybridization events.

[0028] In some embodiments, it is contemplated that the additives described herein may decrease the generation of secondary structures. For example, secondary structures can form in hybridized nucleic acid molecules, such as nucleic acid folding which can result in complex shapes such as hairpin structures. In some embodiments, if secondary structures occlude the site of ligation, a ligase enzyme may not be able to access the 5’ and 3’ ends of the recognition element and can, therefore, be not be able to ligate the ends to generate a circularized recognition element. In some embodiments, it is contemplated that the additives as described herein can act as denaturants. For example, it is contemplated that the additives can lower the energy that may be required to break the internal hydrogen bonds of, for example, secondary structures that can hinder access of a ligase to the ends of a hybridized recognition element, thereby relaxing the secondary structure to allow for infiltration of a ligase and ligation of the ends of the recognition element.

[0029] In some embodiments, it is contemplated that the additives described herein may promote molecular crowding of the nucleic acid molecules. For example, in some embodiments, a ligation event comprises a multi-step enzymatic reaction wherein the ligase has to come in contact with proximal 5’ phosphorylated and 3’ hydroxylated ends of one or more nucleic acid molecules. In the present disclosure, in some embodiments, these ends are exemplified by the 5’ and 3’ ends of a recognition element that is hybridized to a target nucleic acid molecule. In dilute reactions, wherein the ligase may be in low abundance or the nucleic acid molecule to be ligated is in low abundance, the ligation events may be greatly decreased. It is contemplated that the additives as described herein may act as crowding agents, for example by taking up physical space in the reaction, in effect crowding or causing the ligase and its target to be closer togetherWSGR Docket No. 64100-751.601in the reaction. As such, it is contemplated that the additives as described herein may increase the local concentration of the reactants, thereby allowing for more efficient ligation reactions that may additionally be more resistant to minor fluctuations in reaction temperatures or salt concentrations.

[0030] Regardless of the mechanism, as exemplified in at least Examples 2-5, the additives described herein can reduce probe abundance variability in an assay leading to an increase in sensitivity for calling structural variants, such as CNVs, in a target of interest, as compared to an assay without one or more of the disclosed additives.

[0031] The present disclosure provides for additives that can be added to hybridization and / or ligation reactions to enhance the hybridization and / or ligation reactions in an assay.Enhancing the hybridization and / or ligation reactions in assays of a workflow in determining an outcome of an assay can reduce the variability of an assay, for example detectable probe (e.g., amplification product) abundance variability, and lead to a more successful assay outcome. The present disclosure demonstrates the ability of additives to a hybridization and ligation reaction, where hybridization of two or more nucleic acid molecules precedes ligation, that can increase the success of separation of nucleic acid copy number variants when added to an assay, as compared to an assay where there is no additive present. By increasing the success of copy number variant separations, the success of calling one or more copy number variants of a chromosome in a sample can be increased.

[0032] By increasing hybridization and / or ligation events, sensitivity of an assay can be increased. Such an increase in sensitivity can provide for gains in, for example, personalized medicine, diagnosis and treatment. Personalized medicine can tailor diagnosis to one individual based on their genetic makeup, environment and lifestyle for enhancing disease prevention, diagnosis and treatment. By analyzing an individual’s genetic profile to, for example, predict drug response and disease risk by way of pharmacogenomics, diagnosticians and clinicians can better select medications and doses leading to improved patient outcomes, fewer side effects, and more effective care, for example, in cancer diagnosis and treatment.

[0033] In the present application, the term “enhancer” or “enhances” or “to enhance” reflects the ability of a disclosed additive to have a positive effect on a hybridization and / or ligation reaction, the positive effect being an increase in sensitivity for determining the presence of a structural variant, such as a copy number variant, in a nucleic acid sample. The disclosed additives can serve to increase the sensitivity of an assay in identifying the presence of a structural variant as compared in an assay without a disclosed additive, wherein the structural variant may not have been previously identifiable.WSGR Docket No. 64100-751.601

[0034] In some embodiments, the addition of an additive during a hybridization and / or ligation reaction can increase the success of an assay in determining, for example, the presence of a chromosomal copy number variant, or CNV. However, while CNV separation is reported in this disclosure as an illustration of the benefits of the addition of an additive in a hybridization and / or ligation reaction, it is contemplated that any assay where an increase in hybridization and / or ligation reactions would benefit would also be covered by this disclosure. For example, the addition of an additive in a nucleic acid hybridization and / or ligation reaction may enhance detection of nucleic acid variants, or low frequency variants, and the like. As such, a skilled artisan would understand that the additives listed herein can be used for any assay where an increased efficiency of the hybridization and / or ligation of two nucleic acid fragments is desired. In some embodiments, additives disclosed herein that can enhance hybridization and / or ligation of nucleic acids includes one or more alcohols, triols and polyols. In some embodiments, the additive includes alcohols. In some embodiments, the additive includes triols. In some embodiments, the additive includes polyols. In some embodiments, the additive includes alcohols, triols, and polyols. In some embodiments, additives as disclosed herein comprise alcohols comprising one to five carbons. In some embodiments, additives as disclosed herein comprise straight or branched triols or polyols, comprising two to twenty carbons.

[0035] The present disclosure provides compositions for enhancing hybridization and / or ligation reactions in an assay, by way of example to achieve successful separation of copy number variants, or CNVs, in one or more chromosomes from a sample as representative of increased hybridization and / or ligation reactions. To demonstrate additive effect on reaction enhancement, a pharmacogenomic highly polymorphic gene locus, Cytochrome P4502D6, or CYP2D6, was evaluated in assays with and without the experimental additives. The CYP2D6 pharmacogene was chosen because of its structural variants, which can cause challenges in CNV detection, and because of its importance in drug metabolism which can impact drug efficacy and safety in personalized medicine. CYP2D6 activity in drug metabolism can range, person-to-person, from complete absence to increased activity, as such is arguably one of the most important drug metabolizing enzymes with the inter-individual activity variation having significant clinical consequences. As such, the study of CYP2D6 as one of the most complex and clinically relevant pharmacogene was leveraged in evaluation of the disclosed additives.

[0036] The CYP2D6 gene is one of the most clinically important pharmacogenes, responsible for the metabolism of approximately 20-25% of commonly prescribed drugs, including antidepressants, antipsychotics, opioids, beta-blockers, and tamoxifen. In addition to single-nucleotide variants, structural variation at the CYP2D6 locus plays a major role inWSGR Docket No. 64100-751.601determining enzyme activity. Among the most complex and clinically relevant forms of structural variation are CYP2D6 hybrid genes, which arise from recombination events between CYP2D6 and its highly homologous pseudogene, CYP2D7.

[0037] CYP2D6 hybrid genes are generated through unequal crossover or gene conversion events during meiosis, facilitated by the high sequence similarity between CYP2D6 and CYP2D7. These recombination events can produce chimeric genes that contain segments from both loci, such as a CYP2D6 gene with a CYP2D7-derived exon or promoter region. Depending on the breakpoint and structure, hybrids may encode a nonfunctional enzyme, reduced function, or in rare cases altered expression. Well-known examples include CYP2D636*, CYP2D6 13*, and CYP2D668*, and more rare structural variants include CYP2D6 *61, CYP2D6 *63, and CYP2D6 *83. Many of the CYP2D6 structural variants are population-specific and can often occur in tandem with gene duplications or deletions.

[0038] In pharmacogenomics, CYP2D6 hybrids are particularly important because they can confound genotype-to-phenotype predictions if not accurately identified. Standard genotyping assays that target only a limited set of variants may misclassify hybrids as normal-function alleles or simple copy number changes, leading to incorrect metabolizer status assignments. This can have direct clinical consequences, such as inappropriate drug selection or dosing, increased risk of adverse drug reactions, or therapeutic failure especially for drugs with narrow therapeutic windows or strong CYP2D6 dependence.

[0039] Accurate identification of CYP2D6 hybrids in a DNA sample is therefore critical for reliable clinical interpretation. As pharmacogenomic testing becomes more integrated into routine clinical care, the ability to detect and correctly interpret CYP2D6 hybrid alleles is essential for delivering precise, safe, and effective personalized therapy.

[0040] It is contemplated that the disclosed additives for enhancing hybridization and / or ligation reactions would also be useful for identifying the presence of other structural variants in a sample, for example those that are also important in pharmacogenomics or other areas of study. Beyond the pharmacogene CYP2D6, other pharmacogenomically genes harbor clinically important hybrid or chimeric structural variants which may be difficult to identify, for example as arising from recombination with nearby paralogs or pseudogenes. For example, CYP2A6:CYP2A7 hybrids can affect drug metabolism that may alter lung cancer risk.CYP2B6:CYP2B7P1 hybrids can affect drug metabolism that can affect central nervous system toxicity. HLA class I and II hybrid alleles generated by interlocus recombination can affect the prediction of adverse drug reactions and hypersensitivity. UGT2B17:UGT2B15 structural hybrids can affect drug clearance. GSTM1:GSTM1 hybrids can impact the detoxifications ofWSGR Docket No. 64100-751.601chemotherapeutic drugs. Collectively, hybrid structural variants underscore the importance of resolving complex genomic architecture in pharmacogenomic testing, and a failure to detect them can lead to inaccurate phenotype predication and suboptimal clinical decision making.

[0041] Another example where compositions for enhancing hybridization and / or ligation reactions in an assay to achieve separation of CNVs, where separation may be difficult or problematic to resolve, may be useful is for non-invasive prenatal testing, or NIPT. Testing for NIPT includes identifying copy number variants (either gains or losses in copy number) in maternal and fetal DNA, examples including, but not limited to, trisomies such as trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), other chromosomal duplications or deletions such as monosomy X (Turner syndrome), XXY (Klinefelter syndrome), XXX (Triple X syndrome), XYY (Jacobs syndrome), chromosomal microdeletions and microduplications such as DiGeorge syndrome, Prader-Willi syndrome, Angelman syndrome, Cri du chat syndrome and Wolf-Hirschhom syndrome. The enhancement of hybridization and / or ligation reactions in an assay could result in increased discrimination in NIPT variant detection in an assay, thereby increasing the sensitivity of an assay in detection of potential fetal diseases and conditions.

[0042] An additive that is added to an assay wherein the additive increases the amount of hybridization and / or ligation reactions that would lead to successful CNV separation and ultimately more accurate CNV calls compared to an assay without any additive is considered an additive with the ability to enhance the specificity of CNV determinations. An additive with the ability to enhance copy number variant separation could then be considered one that might increase the ability to successfully call copy number variants in a sample. In some embodiments, an additive that is added to an assay wherein the additive does not increase the ability to successfully separate CNVs can be discarded from consideration as not having the ability to enhance the calling of CNVs in a sample. To demonstrate, FIGs. 1A-B show examples of two graphs demonstrating the effect of an additive on CNV separation of four target sites in a CYP2D6 hybrid gene. FIG.1A includes propanediol as a positive control, and FIG.1B includes water as a negative control. Propanediol, which has been reported to increase ligation reactions of nucleic acids in assays, shows successful separation of CNVs at the four different target sites such that successfully calling of CNVs at the four sites of the CYP2D6 gene is possible, whereas water is not able to successfully separate CNVs at any of the four sites, thereby making accurate calling of CNVs at the four sites of the CYP2D6 gene highly improbable.

[0043] In some embodiments, an additive comprises an alcohol. In some embodiments, an additive comprises a mono-ol. A mono-ol is a chemical compound that comprises one hydroxylWSGR Docket No. 64100-751.601group (-OH) attached to one carbon atom. In some embodiments, a mono-ol which may be used as an additive to enhance a hybridization and / or ligation reaction includes, but is not limited to, methanol, ethanol, and 2-propanol (isopropanol, propan-2-ol, isopropyl alcohol). In some embodiments, an additive comprises methanol. In some embodiments, an additive comprises ethanol. In some embodiments, an additive comprises 2-propanol. In some embodiments, an additive comprises isopropanol. In some embodiments, an additive comprises propan-2-ol. In some embodiments, an additive comprises isopropyl alcohol.

[0044] In some embodiments, an additive comprises a diol. A diol is a chemical compound that comprises two hydroxy groups attached to one or more carbon atoms within a molecule. In some embodiments, a diol which may be used as a hybridization and / or ligation enhancer includes, but is not limited to, propane- 1,2-diol (1,2 propanediol), propane-1, 3 -diol (1,3 propanediol), and butane-l,3-diol (1,3 butanediol). In some embodiments, an additive comprises 1,2 propanediol. In some embodiments, an additive comprises 1,3 propanediol. In some embodiments, an additive comprises 1,3 butanediol.

[0045] In some embodiments, an additive comprises a triol. A triol is a chemical compound that comprises three hydroxy groups attached to one or more carbons within a molecule. In some embodiments, a triol which may be used as a hybridization and / or ligation enhancer includes, but is not limited to, propane- 1,2, 3 -tri ol (glycerol), butane- 1, 2, 4-triol (1,2,4 butanetri ol), hexane-1,2,6-triol (1,2,6, hexanetriol), 1,2,3-heptanetriol, and 2-hydroxymethyl-tetrahydro-pyran-3,4,5-triol. In some embodiments, an additive comprises glycerol. In some embodiments, an additive comprises 1,2,4 butanetriol. In some embodiments, an additive comprises 1,2,6 hexanetriol.

[0046] In some embodiments, an additive comprises a polyol. A polyol is a compound with multiple hydroxyl groups attached to one or more carbons within a molecule. In some embodiments, a polyol which may be used as a hybridization and / or ligation enhancer includes, but is not limited to, butane-l,2,3,4-triol (meso-erythritol), 2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol (trehalose), (2S, 3R, 4R, 5R)-hexane-l,2,3,4,5,6-hexol (sorbitol, glucitol), and a D-enantiomer of (2S, 3R, 4R, 5R)-hexane-l,2,3,4,5,6-hexol (D-sorbitol, D-glucitol). In some embodiments, an additive comprises meso-erythritol. In some embodiments, an additive comprises trehalose. In some embodiments, an additive comprises D-sorbitol.

[0047] In some embodiments, one or more additives as disclosed herein can be included in an assay comprising a preparation of nucleic acid molecules and one or more ligases thereby enhancing ligation of one or more of the nucleic acid molecules. In some embodiments, the additives described herein can enhance or increase the degree or amount of ligation of aWSGR Docket No. 64100-751.601phosphorylated 5’ end and a hydroxyl 3’ end of one or more nucleic acid molecules by a ligase enzyme. In some embodiments, a ligase enzyme may be used to join together two or more nucleic acid molecules. In some embodiments, a ligate enzyme may be used to join together the end of one nucleic acid molecule. A ligase which can be used in conjunction with an additive as disclosed herein in a ligation reaction comprises a thermolabile ligase or a thermostable ligase, or derivatives thereof. A ligase which can be used in an assay with an additive as disclosed herein includes, for example, ATP-dependent ligases, NAD+-dependent DNA ligases, NAD+-dependent RNA ligases, and single stranded nucleotide ligases, or derivatives thereof. A ligase which can be used in an assay with an additive as disclosed herein includes prokaryotic or eukaryotic derived ligases, or derivatives thereof. A ligase which can be used in an assay with an additive as disclosed herein includes fusion ligases and ligases where one or more nucleotides or amino acids has been mutated. For example, a fusion ligase may include a ligase wherein the 5’ terminus is fused to a DNA binding domain, or a ligase wherein the catalytic domain comprises one or more mutations thereby modifying the performance of the ligase. Examples of ligases that can be used in conjunction with an additive as described herein include, but are not limited to, T4 DNA ligase, Ampligase® DNA ligase, E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Sso7 DNA ligase, Sso7 Taq DNA ligase, Sso7-E. coli DNA ligase, Sso7 Ampligase DNA ligase, T4 RNA ligase, T4 RNA ligase 1, or T4 RNA ligase 2.

[0048] The ability of the additives as disclosed herein to enhance a hybridization and / or ligation reaction is demonstrated at least in part by the disclosed Examples. Assays used to evaluate additives and their effect on increasing the sensitivity of structural variant calling are described herein. However, it is understood that any assay that has the ability to determine increases in structural variant calling sensitivity is also contemplated herein, and the present disclosure is not limited to any particular assay or embodiment disclosed in that regard.

[0049] The assay methods used to evaluate the ability of a compound to increase sensitivity in structural variant calling includes a single stranded DNA called a “recognition element”. In some embodiments, the methods include providing one or more recognition elements to an assay for identifying the presence of a target molecule in a sample, in this instance for example, the presence of a 1 copy number variant in a sample. In some embodiments, each recognition element of the one or more recognition elements comprises target recognition regions. In some embodiments, the target recognition regions of the recognition elements comprise one or more nucleic acid sequence(s) configured to hybridize to a target nucleic acid molecule, by way of example in this disclosure the target includes four different CNV locations in a CYP2D6 gene.WSGR Docket No. 64100-751.601As shown in FIG. 11, an exemplary recognition element comprises two target recognition regions, one at a 5’ end and another at a 3’ end of the recognition element, which are complementary to sequences in the target of interest (e.g., CYP2D6 gene targets of interest). In some embodiments, a recognition element further comprises a code. In FIG. 11, the example of the code comprises four nucleic acid segments. In some embodiments, the code can be used as a surrogate or proxy for the target molecule, thereby providing an indirect detection of the presence (or absence) of a copy number variant in a sample by detection of the code with a labeled detection reagent or by sequencing. The sequencing may comprise sequence by synthesis, nanopore sequencing, or any other next generation sequencing methodology.

[0050] Upon hybridization to a target molecule of interest, the two ends (e.g. the 5’ end and the 3’ end) of the recognition element are brought into proximity, generating a padlock probe configuration for subsequent circularization by ligation. The recognition element ends can be ligated together if hybridization occurs, thereby generating circular recognition elements that are indicative of the hybridization event and hence the presence of the target of interest. Without a target of interest, hybridization, ligation and circularization is not expected to occur. For the present evaluation, the recognition element is utilized in conjunction with an additive to identify whether the hybridization events and / or ligation enzymatic events can be enhanced, thereby leading to successful separation of a CNV in a location in the CYP2D6 gene, and whether sensitivity enhancement can occur concurrently for multiple CNV locations leading to successful CNV separation at multiple locations in a gene in a multiplex reaction. In some embodiments, success in CNV separation can lead to more accurate CNV calls by a bioinformatics pipeline.

[0051] FIG. 12 shows an example of a workflow in which the recognition element is used to evaluate an additive. The exemplary workflow can be divided into three steps, the assay step of the workflow, the detection step of the workflow and the decoding step of the workflow.

[0052] For the assay step of the workflow, in some embodiments, a recognition element is generated such that the 5’ and 3’ ends are complementary to a location on a target nucleic acid molecule of interest. The recognition element further comprises a code that is unique for the recognition element and its target and therefore can be used to uniquely identify whether successful CNV separation is present at the particular site on the target of interest. If the sequence is present in the sample, the target sequence can hybridize to its complementary sequences in the recognition element, bringing the two ends of the recognition element into proximity for a ligation reaction to occur. If the target locus sequence is not present, then there is expected to be no or minimal hybridization and no ligation. After hybridization, a ligationWSGR Docket No. 64100-751.601reaction can be performed. In the present disclosure, the ligation reaction is performed in the presence of an additive, or propanediol (positive control) or water (negative control).

[0053] FIGs. 1A-1B show examples of representative positive and negative control graphs demonstrating the effect of how propanediol as a positive control provides an increase in targeted ligation reactions, using CNV separation as the method to evaluate the effect of an additive. FIG. 1A shows an example of the positive control (0.8 M propanediol) which demonstrates good separation of the copy numbers. FIG. IB conversely shows an example of the negative control of water, demonstrating that without an additive there is not enough separation of the copy numbers and any CNV calls would be highly improbable.

[0054] After ligating the recognition elements, remaining single stranded recognition elements and target sequences can be optionally removed from the reaction by exonuclease treatment. In some embodiments, amplification of the ligated recognition elements is performed, for example by rolling circle amplification (RCA), for generating concatemeric amplification products, wherein the concatemer contains multiple copies of a circularized ligated recognition element, including associated codes, target recognition regions, and any other functional sequences that are included in the circularized and ligated recognition element.

[0055] In some embodiments, the amplification products can be queried for the presence of the code, which serves as a proxy for the presence of the target CNV. The example in FIG. 12 shows the detection of the concatemeric amplification products by using detectable nucleic acid complexes that identify the presence of code sequences. In some embodiments, the detection complexes can hybridize to the code sequences, or a portion thereof, and allow for imaging of the hybridization events, the image events of which can be decoded by using a soft decision decoding method, for example. In some embodiments, another alternative to detecting the presence of a code, and thereby the target CNV, is by sequencing the concatemeric amplification products.

[0056] The workflow exemplified herein to evaluate the effect of an additive on a hybridization and / or ligation reaction is an example of one workflow that can be leveraged to evaluate an additive’s ability to increase hybridization and / or ligation reactions in an assay. A skilled artisan will appreciate the myriad of other workflows that can also be used for evaluation of an additive. The workflow described herein is demonstrated in more detail in one or more patent applications, for example in PCT patent application serial numbers PCT / US2024 / 062056, PCT / US2024 / 038497, PCT / US2024 / 060535, and PCT / US2024 / 038517, all of which are incorporated herein by reference in their entireties.WSGR Docket No. 64100-751.601

[0057] The present disclosure provides additives to enzymatic reactions that may enhance the enzymatic reaction, thereby enhancing an assay outcome. In particular, the present disclosure provides additives to a ligation reaction that may enhance the ligation reactions in a reaction, thereby enhancing the sensitivity of data output resulting from an assay in which a ligation reaction is part of the assay.

[0058] Ligases are known in the biological sciences for, for examplejoining gaps that can form in nucleic acids during normal cellular replication, repair and recombination events. DNA ligases catalyze the formation of phosphodiester bonds between adjacent nucleotides, one of which comprises a hydroxyl group and the other a phosphate group. Some ligases utilize ATP hydrolysis and inorganic pyrophosphate for effecting the ligation of the two adjacent nucleotides, however, some ligases use nicotinamide adenine dinucleotide, or NAD, in lieu of ATP as a cofactor and release AMP and nicotinamide mononucleotide (NMN) as a result of phosphodiester bond formation between two nucleotides. DNA ligases may form covalent linkages at a nick in double stranded DNA or when joining sticky nucleic acid molecules or blunt ended nucleic acid molecules. The present application, while exemplifying DNA ligases, contemplates that the novel aspects of the disclosure would also be suited from use with RNA ligases.

[0059] There are a myriad of ligases which can be used in the methods and compositions described herein. Bacterially derived ligase enzymes include, but are not limited to, DNA ligase from E. coli or T. aquaticus. Bacteriophage derived ligase enzymes include, but are not limited to, T4 DNA ligase and T4 RNA ligase. Mammalian derived ligase enzymes include, but are not limited to, DNA ligase 1, DNA ligase 3 and DNA ligase 4.

[0060] Ligases that can be used in methods and compositions of the present disclosure include heat labile ligases such as ligases not derived from thermophilic bacteria. For example, heat labile ligases may be inactivated above 37°C, as such heating a heat labile enzyme denatures the enzyme. However, ligases that are derived from thermophilic bacteria, such as ligases found in Thermus aquaticus, can withstand high temperatures up to 95°C for more than an hour. The exceptional thermostability of ligases from thermophilic bacterial permit for extremely high hybridization stringency and ligation specificity. Heat labile ligases include, but are not limited to, E. coli DNA ligase, T4 DNA ligase, T4 RNA ligase.

[0061] The present disclosure provides additives in the form of small molecules for use in enhancing the abundance of copy number variants in a sample. In some embodiments, small molecule additives include straight or branched mono-ol, diol, triol and polyol compounds comprising 2 to 20 carbons. In some embodiments, the additives enhance ligation of the 5’ andWSGR Docket No. 64100-751.6013’ ends of a recognition element as it is hybridized to target sequences. In some embodiments, additives useful in methods for determining CNVs comprise an alcohol such as methanol, ethanol, and isopropanol, or combinations thereof. In some embodiments, additives useful in methods for determining CNVs comprise a triol such as glycerol, 1,2,4 heptanetri ol, 1,2,4 butanetri ol, and 1,2,6 hexanetri ol, or combinations thereof. In some embodiments, additives useful in methods for determining CNVs comprises a polyol such as meso-erythritol, trehalose and D-sorbitol, or combinations thereof. In some embodiments, additives as described herein can be combined for enhancing the determination of CNVs in a sample, for example one or more alcohols, one or more triols, one or more polyols, or any combination thereof, can be combined in an assay.

[0062] In some embodiments, methanol can be added to an assay to increase hybridization and / or ligation reactions between nucleic acid molecules and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a methanol concentration of about 750 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a methanol concentration of about 750 mM, of about 800 mM, of about 850 mM, or about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the methanol comprises a concentration of greater than or equal to 600 mM, 620 mM, 640 mM, 660 mM, 680 mM, 700 mM, 720 mM, 740 mM, 760 mM, 780 mM, 800 mM, 820 mM, 840 mM, 860 mM, 880 mM, 900 mM, 920 mM, 940 mM, 960 mM, 980 mM, 1,000 mM, 1,020 mM, 1,040 mM, 1,060 mM, 1,080 mM, 1,100 mM, 1,200 mM, 1,300 mM, 1,400 mM, or 1,500 mM. In some embodiments, the methanol comprises a concentration of less than or equal to 600 mM, 620 mM, 640 mM, 660 mM, 680 mM, 700 mM, 720 mM, 740 mM, 760 mM, 780 mM, 800 mM, 820 mM, 840 mM, 860 mM, 880 mM, 900 mM, 920 mM, 940 mM, 960 mM, 980 mM, 1,000 mM, 1,020 mM, 1,040 mM, 1,060 mM, 1,080 mM, 1,100 mM, 1,200 mM, 1,300 mM, 1,400 mM, or 1,500 mM.

[0063] In some embodiments, ethanol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, an ethanol concentration of about 750 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, an ethanol concentration of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine theWSGR Docket No. 64100-751.601presence of a CNV in a sample. In some embodiments, the ethanol comprises a concentration of greater than or equal to 600 mM, 620 mM, 640 mM, 660 mM, 680 mM, 700 mM, 720 mM, 740 mM, 760 mM, 780 mM, 800 mM, 820 mM, 840 mM, 860 mM, 880 mM, 900 mM, 920 mM, 940 mM, 960 mM, 980 mM, 1,000 mM, 1,020 mM, 1,040 mM, 1,060 mM, 1,080 mM, 1,100 mM, 1,200 mM, 1,300 mM, 1,400 mM, or 1,500 mM. In some embodiments, the ethanol comprises a concentration of less than or equal to 600 mM, 620 mM, 640 mM, 660 mM, 680 mM, 700 mM, 720 mM, 740 mM, 760 mM, 780 mM, 800 mM, 820 mM, 840 mM, 860 mM, 880 mM, 900 mM, 920 mM, 940 mM, 960 mM, 980 mM, 1,000 mM, 1,020 mM, 1,040 mM, 1,060 mM, 1,080 mM, 1,100 mM, 1,200 mM, 1,300 mM, 1,400 mM, or 1,500 mM.

[0064] In some embodiments, isopropanol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, an isopropanol concentration of about 250 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, an isopropanol concentration of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, of about 500 mM, of about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the isopropanol comprises a concentration of greater than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM. In some embodiments, the isopropanol comprises a concentration of less than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM.

[0065] In some embodiments, glycerol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a glycerol concentration of about 200 mM to about 2 M can be added to an assay to increase the ability of the assay toWSGR Docket No. 64100-751.601determine the presence of a CNV in a sample. In some embodiments, a glycerol concentration of about 200 mM, of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, of about 500 mM, of about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, of about 1 M, of about 1.5 M, or of about 2 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the glycerol comprises a concentration of greater than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, 1,250 mM, 1,300 mM, 1,350 mM, 1,400 mM, 1,450 mM, 1,500 mM, 1,550 mM, 1,600 mM, 1,650 mM, 1,700 mM, 1,750 mM, 1,800 mM, 1,850 mM, 1,900 mM, 1,950 mM, 2,000 mM, 2,050 mM, 2,100 mM, 2,150 mM, 2,200 mM, 2,250 mM, 2,300 mM, 2,350 mM, 2,400 mM, 2,450 mM, or 2,500 mM. In some embodiments, the glycerol comprises a concentration of less than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, 1,250 mM, 1,300 mM, 1,350 mM, 1,400 mM, 1,450 mM, 1,500 mM, 1,550 mM, 1,600 mM, 1,650 mM, 1,700 mM, 1,750 mM, 1,800 mM, 1,850 mM, 1,900 mM, 1,950 mM, 2,000 mM, 2,050 mM, 2,100 mM, 2,150 mM, 2,200 mM, 2,250 mM, 2,300 mM, 2,350 mM, 2,400 mM, 2,450 mM, or 2,500 mM.

[0066] In some embodiments, 1,2,4 heptanetri ol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a glycerol concentration of about 250 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a 1,2,4 heptanetriol concentration of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, of about 500 mM, of about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the 1,2,4WSGR Docket No. 64100-751.601heptanetriol comprises a concentration of greater than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM. In some embodiments, the 1,2,4 heptanetri ol comprises a concentration of less than or equal to 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM.

[0067] In some embodiments, 1,2,6 hexanetri ol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a 1,2,6 hexanetri ol concentration of about 75 mM to about 800 mM can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a 1,2,6 hexanetriol concentration of about 75 mM, of about 100 mM, of about 150 mM, of about 200 mM, of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, of about 500 mM, of about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, or of about 800 mM can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the 1,2,6 hexanetriol comprises a concentration of greater than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM. In some embodiments, the 1,2,6 hexanetri ol comprises a concentration of less than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000WSGR Docket No. 64100-751.601mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM.

[0068] In some embodiments, meso-erythritol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a mesoerythritol concentration of about 500 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a meso-erythritol concentration of about 500 mM, of about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the mesoerythritol comprises a concentration of greater than or equal to 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, 1,250 mM, 1,300 mM, 1,350 mM, 1,400 mM, 1,450 mM, 1,500 mM, 1,550 mM, 1,600 mM, 1,650 mM, 1,700 mM, 1,750 mM, 1,800 mM, 1,850 mM, 1,900 mM, 1,950 mM, 2,000 mM, 2,050 mM, 2,100 mM, 2,150 mM, or 2,200 mM. In some embodiments, the meso-erythritol comprises a concentration of less than or equal to 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, 1,250 mM, 1,300 mM, 1,350 mM, 1,400 mM, 1,450 mM, 1,500 mM, 1,550 mM, 1,600 mM, 1,650 mM, 1,700 mM, 1,750 mM, 1,800 mM, 1,850 mM, 1,900 mM, 1,950 mM, 2,000 mM, 2,050 mM, 2,100 mM, 2,150 mM, or 2,200 mM.

[0069] In some embodiments, D-sorbitol can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a D-sorbitol concentration of about 100 mM to about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a D-sorbitol concentration of about 100 mM, of about 150 mM, of about 200 mM, of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, of about 500 mM, ofWSGR Docket No. 64100-751.601about 550 mM, of about 600 mM, of about 650 mM, of about 700 mM, of about 750 mM, of about 800 mM, of about 850 mM, of about 900 mM, of about 950 mM, or of about 1 M can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the D-sorbitol comprises a concentration of greater than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM. In some embodiments, the D-sorbitol comprises a concentration of less than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, 1,200 mM, 1,225 mM, or 1,250 mM.

[0070] In some embodiments, trehalose can be added to an assay to increase hybridization and / or ligation reactions and thereby increase the sensitivity of an assay to determine the presence of a copy number variant in a sample. In some embodiments, a trehalose concentration of about 100 mM to about 500 mM can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, a trehalose concentration of about 100 mM, of about 150 mM, of about 200 mM, of about 250 mM, of about 300 mM, of about 350 mM, of about 400 mM, of about 450 mM, or of about 500 mM can be added to an assay to increase the ability of the assay to determine the presence of a CNV in a sample. In some embodiments, the trehalose comprises a concentration of greater than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, or 1,200 mM. In some embodiments, the trehalose comprises a concentration of less than or equal to 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750WSGR Docket No. 64100-751.601mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 975 mM, 1,000 mM, 1,025 mM, 1,050 mM, 1,075 mM, 1,100 mM, 1,125 mM, 1,150 mM, 1,175 mM, or 1,200 mM.

[0071] In some embodiments, use of an additive for enhancing a hybridization and / or ligation reaction increases the sensitivity of identifying one or more CYP2D6 structural variants in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *68 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *4 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *13 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *36 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *61 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *63 structural variant in a DNA sample from an individual. In some embodiments, use of an additive increases the sensitivity of identifying the presence of CYP2D6 *83 structural variant in a DNA sample from an individual. In some embodiments, use of an additive for enhancing a hybridization and / or ligation reaction increases the sensitivity of identifying the presence of one or more structural variants of one or more target genes of interest.

[0072] The present application provides for kits for performing the assays as described herein. A kit as described herein could be used in increasing the sensitivity in calling variants that might be present in a sample. In some embodiments, a kit described herein could be used for identifying structural variants important in determining the presence of fetal diseases, such as identified during NIPT. In some embodiments, a kit described herein could be used for identifying structural variants of pharmacogenomics targets useful in personalized medicine initiatives. In some embodiments, a kit comprises one or more recognition elements, wherein each of the one or more recognition elements hybridizes to a target nucleic acid. In some embodiments a kit comprises one or more recognition elements, wherein two or more of the one or more recognition elements hybridizes to the same target nucleic acid. In some embodiments, the one or more recognition elements comprises a unique code that is specific to a target nucleic acid. In some embodiments, a kit further comprises a reaction reagent comprising an additive for enhancing hybridization and / or ligation. In some embodiments, the additive of the reactionWSGR Docket No. 64100-751.601reagent of a kit is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, meso-erythritol, D-sorbitol and trehalose, or a combination thereof. In some embodiments, a kit further comprises a ligase. In some embodiments, a kit further comprises a polymerase. In some embodiments, a kit further comprises a polymerase buffer. In some embodiments, a kit further comprises one or more exonucleases as described herein. In some embodiments, a kit of the present disclosure comprises one or more reagents for performing one or more of a ligation reaction, an amplification reaction, or an exonuclease reaction. In some embodiments, a kit comprises instructions for performing an assay comprising a reaction reagent comprising an additive as described herein.EXAMPLES

[0073] Example 1-Assay for additive evaluation

[0074] This example presents a general exemplary workflow used in experiments where an additive was evaluated for its ability to enhance sensitivity in CNV calling. In this example, a positive control of propanediol at an 800 mM concentration and a negative control using water were run in conjunction with the test experiments evaluating an additive as described herein.

[0075] The general workflow is exemplified in FIG. 12. The example general workflow comprises combining a sample comprising target nucleic acids with one or more recognition elements that include complementary ends to one or more target nucleic acid sequences in the target nucleic acids, wherein each recognition elements further comprises a code that is unique to the recognition element ends, and can be used as an indirect method for determining the presence of the target nucleic acid. The example general workflow comprises simultaneously, or substantially simultaneously, hybridizing the target nucleic acids and the recognition elements and ligating the ends of the recognition elements, thereby generating circularized and ligated recognition elements. The example general workflow may include exonuclease treating the circularized recognition elements to remove linear nucleic acid molecules, for example, unhybridized target nucleic acids and unhybridized recognition elements. The example general workflow comprises amplifying the circularized and ligated recognition elements. The example general workflow comprises detecting the code of the amplified recognition elements. The example general workflow comprises decoding the detection output to determine whether copy number variants are present and sufficiently separated to make a call as to whether a copy number variant is present in the target nucleic acids.

[0076] In this example, the target nucleic acid samples used included pharmacogenomic target structural variants, for example CYP2D6 *68 star allele gene targets of interest. TheWSGR Docket No. 64100-751.601samples were obtained from the Coriell Institute for Medical Research. Four locations in CYP2D6 *68 were chosen as targets of interest as the hybrid variant gene is clinically relevant in pharmacogenomics. CYP2D6 gene is important in its metabolism of a subset of clinical drugs as previously described, whereas the *68 star allele hybrid variant is nonfunctional and cannot metabolize the drugs, as such it is a clinically relevant pharmacogene target of interest. For example, the Intron 1 up stream target in CYP2D6 *68 has a copy number of three, whereas the Intron l_Intron4, Intron4_Exon9 and Exon9_downstream targets all have a copy number of two, and their copy number pattern, 3-2-2-2, respectively are indicative of CYP2D6 being star allele *68. The presence of the four targets in CYP2D6 *68 can be difficult to determine due to the hybrid nature of CYP2D6 *68 gene. As the four CYP2D6 *68 targets are clinically relevant it is important to be able to identify with specificity the copy number variation. In this example, CYP2D7 pseudogene is used as the internal well control (“spacer”) for each of the CNV targets of CYP2D6 as there are no copy number variants associated with CYP2D7. As such, in this example, the CNV count should be two alleles, or the normal allelic complement for any given location on the CYP2D7 gene. As such, in this example, it was determined that the best way to identify an additive of interest was to use them in a real world scenario for identifying whether they had an impact on identifying clinically relevant copy number variants in the CYP2D6 hybrid gene.

[0077] In this example, the DNA samples (5 ng) were combined with a reaction mixture that included 0.5 nM of each recognition element, a varied concentration of additive depending on the additive being evaluated (or propanediol as the positive control and water as the negative control), Ampligase and ligase buffer, and water to a total reaction volume 20 pL. The targets of interest and their complementary recognition element 5’ and 3’ end sequences are shown in the example of FIG. 13 (human genome version used was GRCh38), as such 17 different recognition elements targeting different target sequences of interest were combined and used for each different DNA sample. In this example, CYP2D7 served as the normal control (“spacer”) with a copy number count of 2. The reactions were mixed and incubated for concurrent hybridization and ligation; 95°C / 5 min., 60°C / 20 min. followed by 95°C / 2 min. for six cycles, 95°C / I min. followed by placing on ice thereby generating circularized and ligated recognition elements.

[0078] An endonuclease reagent master mix, including Exonuclease I, Exonuclease III, an exonuclease buffer and water, was added to each sample. The exonuclease digestion reactions were incubated at 37°C for 30 min. followed by an enzyme inactivation at 95°C for 5 min. After the exonuclease digestion, the samples were placed on ice.WSGR Docket No. 64100-751.601

[0079] Following exonuclease digestion, the reactions were transferred to wells of a 96 well plate that was pre-treated with a compound for nucleic acid immobilization and further included an optically clear bottom layer for subsequent detection. After transfer, the plate was sealed and incubated at 42°C for 1 hr.

[0080] Following incubation, the circularized and ligated recognition elements were amplified by rolling circle amplification. An amplification master mix reagent comprised 1 mM each dNTP, EquiPhi DNA polymerase and EquiPhi DNA polymerase buffer, 10 nM of an amplification primer, 1 mM DTT and water for a total well dispense volume of 50 pL. The 50 uL amplification reagent was added to each well, and the amplification reactions were incubated at 42°C for 2 hrs. The amplification reactions were washed several times with TE buffer, incubated with 0.1 N NaOH for 1 min., washed several times with TE buffer, and the amplification products were left in TE buffer for subsequent detection.

[0081] Detection reagents were generated that included a hybridization buffer, 0.5 pM of an anchor oligonucleotide, 0.5 pM of a fluorescently labeled detection oligonucleotide, a DNA blocker and water for a total well dispense volume of 50 pL. Detection reagent was added to each well, and the reactions were incubated at room temperature for 15 min. and washed several times to remove any unhybridized anchor and detection oligonucleotides. The wells were imaged on a fluorescent imager (in these experiments a RAPTOR fluorescent imaging instrument was used for detection and decoding). Each well was queried eight times, thereby generating a fluorescent profile indicative of the unique code for each of the amplified recognition elements in each of the wells. In this example, four different fluorescently labeled detection oligonucleotides were used in conjunction with their complementary anchor oligonucleotides. A fluorescent moiety AZdye532 (ex / em 532 / 554 nM)), AZdye568 (ex / em 578 / 602 nM), AZdye647 (ex / em 648 / 671 nM) or AZdye680 (ex / em 678 / 701 nM) was attached to a detection oligonucleotide such that four colors were used for detection of code sequences, and the final fluorescent profile built for each code following multiple queries, which was then decoded, was built for each amplified recognition element.

[0082] After the amplified recognition element detection cycles were completed and a fluorescent profile was generated, the fluorescent profiles were bioinformatically decoded using a soft decision decoding pipeline and matched to the profile for the codes used in the recognition elements and the resulting data was used to determine whether an additive could result in increased sensitivity for sufficient separation of CNVs for a given CNV target wherein CNV calling was obtained.WSGR Docket No. 64100-751.601Example 2-Determining which additive to evaluate

[0083] In this example, to help identify compounds and their concentrations that might be useful as additives that would result in successful separation of CNVs akin to that of propanediol (positive control), molecular beacon assays were performed using different compounds with concentrations ranging from 25 mM to 1 M of each compound. In this example, the target melting temperature range (Tm) was from approximately 72°C to approximately 78°C, and compounds which fell within the target Tmrange in molecular beacon assays provided a range of potential target compounds and their potential concentration range for further experimentation. Based on the molecular beacon assay, a concentration for testing was chosen, details found in Table 1.Table 1-Ligation enhancers, potential range of concentration and concentration testedExample 3-Evaluation of mono-ols as ligation enhancers

[0084] In this example, several mono-ols were evaluated as additives fortheir ability to increase the specificity of CNV calling. The alcohols methanol, ethanol and isopropanol were evaluated as representative compounds from this group.

[0085] The assay as described in Example 1 herein was followed, including 800 mM of either methanol, ethanol or isopropanol as the additive during the hybridization and ligation reactions. The Coriell samples tested and their expected copy number count is shown in Table 2. For each expected copy number count, two samples were tested.WSGR Docket No. 64100-751.601Table 2- Samples tested and their expected copy number count

[0086] Data from the evaluation of methanol, ethanol and isopropanol as additives is shown in the examples of FIG. 2, FIG. 3 and FIG. 4, respectively. The positive control and negative control performed are shown in the example of FIGs. 1A-1B for each of the alcohols tested. In this example, the controls demonstrated that, with the positive control (propanediol) the CNV counts are well separated and CNV calls can be made with confidence, whereas with the negative control (water) separation of the CNV counts are poor and as such CNV calls cannot be made with any confidence. As shown in the example of FIG. 2, the addition of methanol as an additive resulted in increased sensitivity and separation of all loci CNV counts and the copy numbers for the samples matched their expected copy number. The same can be seen for ethanol (FIG. 3) and isopropanol (FIG. 4). The intrawell CYP2D7 control showed a copy number of 2, as expected.

[0087] Therefore, in this example, it was determined that a plurality of mono-ols were successful as additives to enhance hybridization and / or ligation reactions thereby increasing sensitivity in calling structural variants as evidenced by their ability to successfully separate different copy number variants.Example 4-Evaluation of triols as additives

[0088] In this example, several triols were evaluated for their ability to enhance specificity in CNV calling. The triols glycerol, 1,2,4 heptanetri ol, and 1,2,6 hexanetri ol were evaluated as representative compounds from this group.

[0089] The assay as described in Example 1 herein was followed, including 500 mM of glycerol, 1.5 M glycerol, 800 mM of 1,2,4 heptanetriol, or 300 mM of 1,2,6 hexanetriol as the additive. The Coriell samples tested and their expected copy number count for the different triols is shown in Table 3, 4 and 5.WSGR Docket No. 64100-751.601Table 3-Samples tested and their expected copy number count using glycerolTable 4-Samples tested and their expected copy number count using 1,2,4 butanetriolTable 5-Samples tested and their expected copy number count using 1,2,6 hexanetriol<<

[0090] Data from the evaluation of glycerol, 1,2,4 butanediol and 1,2,6 hexanetri ol as additives is shown in the example of FIGs. FIG. 5A, 5B, 6 and 7, respectively. The positive control and negative control performed are shown in the example of FIG. 1 for each of the triols tested. In this example, the controls demonstrated that with the positive control (propanediol) the CNV counts were well separated and CNV calls can be made with confidence, whereas with the negative control (water) separation of the CNV counts were poor and as such CNV calls cannot be made with any confidence. As shown in the example of FIGs. 5A-5B, the addition ofWSGR Docket No. 64100-751.601glycerol at either 500 mM or 1.5 M, respectively, as an additive resulted in increased sensitivity and separation of all copy number variants and the copy numbers for the samples matched their expected copy number. The same can be seen for 1,2,4 butanediol (FIG. 6) and 1,2,6 hexanetri ol (FIG. 7). In this example, the intrawell CYP2D7 control showed a copy number of 2 as expected.

[0091] Therefore, in this example, it was determined that a plurality of triols were successful as additives to hybridization and / or ligation reactions to increase the sensitivity of calling structural variants as evidenced by their ability to successfully separate different copy number variants.Example 5-Evaluation of polyols as additives

[0092] In this example, several polyols were evaluated for their ability to serve as an additive to enhance hybridization and / or ligation as described in Example 1. The polyols mesoerythritol, D-sorbitol, and trehalose were evaluated as representative compounds from this group.

[0093] The assay as described in Example 1 herein was followed, including 500 mM of meso-erythritol, 500 mM of D-sorbitol, or 250 mM of trehalose as the additive. The Coriell samples were tested and their expected copy number count for experiments including mesoerythritol and D-sorbitol is found in Table 5, while Coriell samples and expected copy number counts for experiments including trehalose are shown in Table 6.Table 6-Samples tested and their expected copy number count for trehalose

[0094] Data from the evaluation of meso-erythritol, D-sorbitol and trehalose as additives is shown in the examples of FIG. 8, FIG. 9 and FIG. 10, respectively. The positive control and negative control performed as shown in the example of FIG. 1 for each of the polyols tested. In this example, the controls demonstrated that with a positive control (propanediol) the CNVWSGR Docket No. 64100-751.601counts were well separated and CNV calls can be made with confidence, whereas with the negative control (water) separation of the CNV counts were poor and as such CNV calls cannot be made with any confidence. As shown in the example of FIG. 8, the addition of mesoerythritol as an additive resulted in increased sensitivity and separation of all copy number variant counts and the copy numbers for the samples matched their expected copy number. The same can be seen for D-sorbitol (FIG. 9) and trehalose (FIG. 10). In this example, the intrawell CYP2D7 control showed a copy number of 2 as expected.

[0095] Therefore, in this example, it was determined that a plurality of polyols were successful as additives to hybridization and / or ligation reactions in increasing sensitivity and structural variant calling as evidenced by their ability to successfully separate different copy number variants.

[0096] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0097] Various modifications and variations of the disclosed methods, compositions and uses as disclosed herein will be apparent to the skilled person without departing from the scope and spirit of the inventive concepts. Although methods have been disclosed in connection with specific preferred aspects or embodiments, it should be understood that methods, compositions and systems as claimed should not be unduly limited to such specific aspects or embodiments. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. WSGR Docket No. 64100-751.601CLAIMS1. A composition comprising an additive, wherein the additive comprises an alcohol, a triol or a polyol, or a combination thereof, and wherein the additive enhances intramolecular hybridization reactions, intermolecular hybridization reactions, or ligation reactions, or a combination thereof, of one or more nucleic acid molecules in a reaction.

2. The composition of claim 1, wherein the alcohol is selected from the group consisting of methanol, ethanol and isopropanol.

3. The composition of claim 2, wherein the alcohol comprises the methanol, and wherein the methanol is at a concentration of 750 mM to 1 M in the reaction.

4. The composition of claim 2, wherein the alcohol comprises the ethanol, and wherein the ethanol is at a concentration of 750 mM to 1 M in the reaction.

5. The composition of claim 2, wherein the alcohol comprises the isopropanol, and wherein the isopropanol is at a concentration of 250 mM to 1 M in the reaction.

6. The composition of claim 1, wherein the triol is selected from the group consisting of glycerol, 1,2,4 heptanetri ol, and 1,2,6 hexanetri ol.

7. The composition of claim 6, wherein the triol comprises the glycerol, and wherein the glycerol is at a concentration of 200 mM to 2 M in the reaction.

8. The composition of claim 6, wherein the triol comprises the 1,2,4 heptanetriol, and wherein the 1,2,4 heptanetri ol is at a concentration of 250 mM to 1 M in the reaction.

9. The composition of claim 6, wherein the triol comprises the 1,2,6 hexanetri ol, and wherein the 1,2,6 hexanetriol is at a concentration of 75 mM to 500 mM in the reaction.

10. The composition of claim 1, wherein the polyol is selected from the group consisting of meso-erythritol, D-sorbitol and trehalose.WSGR Docket No. 64100-751.60111. The composition of claim 10, wherein the polyol comprises the meso-erythritol, and wherein the meso-erythritol is at a concentration of 500 mM to 1 M in the reaction.

12. The composition of claim 10, wherein the polyol comprises the D-sorbitol, and wherein the D-sorbitol is at a concentration of 100 mM to 1 M in the reaction.

13. The composition of claim 10, wherein the polyol comprises the trehalose, and wherein the trehalose is at a concentration of 250 mM to 500 mM in the reaction.

14. The composition of any one of claims 1-13, wherein the composition enhances the calling of structural variants at one or more locations in a nucleic acid sample.

15. The composition of claim 1, wherein the additive comprises two or more of the alcohol, the triol, and the polyol.

16. The composition of claim 1, wherein the additive comprises the alcohol, the triol, and the polyol.

17. The composition of claim 1, wherein the additive enhances intramolecular ligation of the one or more nucleic acid molecules in the reaction.

18. The composition of claim 1, wherein the additive enhances interm olecular ligation of the one or more nucleic acid molecules in the reaction.

19. A method of enhancing hybridization reactions, ligation reactions, or a combination thereof, between the one or more nucleic acid molecules, comprising:a) providing the composition of claim 1 and the one or more nucleic acid molecules; andb) hybridizing and ligating the one or more nucleic acid molecules together by one or more intramolecular or intermolecular ligation reactions, wherein hybridization reactions, ligation reactions, or the combination thereof, are enhanced compared to a hybridization reaction, a ligation reaction, or a combination thereof, performed in the absence of the composition of claim 1.WSGR Docket No. 64100-751.60120. The method of claim 19, wherein the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetriol, mesoerythritol, D-sorbitol and trehalose.

21. A kit comprising:a) one or more recognition elements, andb) a reagent comprising the additive of claim 1.

22. The kit of claim 21, wherein the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, meso-erythritol, D- sorbitol and trehalose.

23. A method for determining a structural variant of a target of interest, comprising:a) providing an additive to a hybridization reaction, a ligation reaction, or a combination thereof, wherein the additive is one or more of an alcohol, a triol or a polyol, and wherein the hybridization reaction or the ligation reaction is performed in an assay for determining the structural variant of the target of interest, andb) determining a presence of the structural variant of the target of interest.

24. The method of claim 23, wherein the structural variant is a copy number variant.

25. The method of claim 23, wherein the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, mesoerythritol, D-sorbitol and trehalose.

26. The method of claim 25, wherein the additive is the methanol, and wherein the methanol is at a concentration of 750 mM to 1 M.

27. The method of claim 25, wherein the additive is the ethanol, and wherein the ethanol is at a concentration of 750 mM to 1 M.

28. The method of claim 25, wherein the additive is the isopropanol, and wherein the isopropanol is at a concentration of 250 mM to 1 M.WSGR Docket No. 64100-751.60129. The method of claim 25, wherein the additive is the glycerol, and wherein the glycerol is at a concentration of 200 mM to 2 M.

30. The method of claim 25, wherein the additive is the 1,2,4 heptanetriol, and wherein the 1,2,4 heptanetriol is at a concentration of 250 mM to 1 M.

31. The method of claim 25, wherein the additive is the 1,2,6 hexanetriol, and wherein the 1,2,6 hexanetriol is at a concentration of 75 mM to 500 mM.

32. The method of claim 25, wherein the additive is the meso-erythritol, and wherein the mesoerythritol is at a concentration of 500 mM to 1 M.

33. The method of claim 25, wherein the additive is the D-sorbitol, and wherein the D-sorbitol is at a concentration of 100 mM to 1 M.

34. The method of claim 25, wherein the additive is the trehalose, and wherein the trehalose is at a concentration of 250 mM to 500 mM.

35. The method of claim 23, wherein the target of interest is from a pharmacogenomic gene.

36. The method of claim 35, wherein the pharmacogenomic gene is a CYP gene.

37. The method of claim 36, wherein the CYP gene comprises one or more CYP2D6 structural variants.

38. The method of claim 37, wherein the CYP2D6 structural variant comprises CYP2D6 *68.

39. The method of claim 37, wherein the one or more CYP2D6 structural variants comprises one or more of CYP2D6 *13, CYP2D6 *36, CYP2D6 *61, CYP2D6 *63, CYP2D6 *68, or CYP2D6 *83.

40. The method of claim 23, wherein the target of interest is from a chromosome for noninvasive prenatal testing comprising one or more structural variants.WSGR Docket No. 64100-751.60141. The method of claim 40, wherein the chromosome is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.

42. The method of claim 41, wherein the chromosome is chromosome 13.

43. The method of claim 41, wherein the chromosome is chromosome 18.

44. The method of claim 41, wherein the chromosome is chromosome 21.

45. The method of claim 41, wherein the chromosome is chromosome X.

46. The method of claim 41, wherein the chromosome is chromosome Y.

47. A system for determining one or more structural variants of a target of interest, comprising:a) an assay module comprising a hybridization reaction or a ligation reaction, or a combination thereof, wherein the hybridization reaction or the ligation reaction, or the combination thereof, comprises an additive, wherein the additive comprises one or more of an alcohol, a triol, or a polyol, or a combination thereof, and wherein the additive is configured to enhance intramolecular hybridization, intermolecular hybridization, or ligation events, or a combination thereof, of the one or more nucleic acid molecules; andb) an analysis module configured to determine a presence of the one or more structural variants of the target of interest based on products generated from the assay module.

48. The system of claim 47, wherein the one or more structural variants comprise one or more copy number variants.

49. The system of claim 47, wherein the assay module further comprises an amplification reaction.

50. The system of claim 47, wherein the assay module comprises a substantially simultaneous hybridization and ligation reaction comprising a ligase and the additive, and wherein the analysis module comprises a detection reaction.WSGR Docket No. 64100-751.60151. The system of claim 47, wherein the additive is selected from the group consisting of methanol, ethanol, isopropanol, glycerol, 1,2,4 butanetri ol, 1,2,6 hexanetri ol, mesoerythritol, D-sorbitol and trehalose.

52. The system of claim 47, wherein the analysis module comprises detection reagents.

53. The system of claim 52, wherein the detection reagents comprise fluorescently labeled oligonucleotides.

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